Molecular Methods in Milk Quality

Transcription

1 Molecular Methods in Milk Quality Proceedings of a Symposium to celebrate the opening of the new Ithaca facilities of Quality Milk Production Services Edited by R. N. Zadoks Ithaca, NY September 30, October QMPS is a program within the Animal Health Diagnostic Center, a partnership between the NYS Department of Agriculture and Markets and the College of Veterinary Medicine at Cornell University - 1 -

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3 TABLE OF CONTENTS Welcome 5 Y. H. Schukken, R. N. González, R. N. Zadoks Symposium Sponsors 6 Symposium Program 7 Molecular Methods in Food Safety 9 K. J. Boor Molecular Methods And Mastitis Research With Particular Reference To Streptococcus uberis S. P. Oliver and B. E. Gillespie 13 Monitoring Antimicrobial Resistance in USA Agriculture 19 P. F. Cray Molecular Methods in Antimicrobial Resistance of Mastitis Pathogens 20 L. L. Tikofsky, R. N. Zadoks, I. Loch Molecular Methods on Dairy Farms: Case Studies 31 R. N. Zadoks Molecular Medicine, A Reality Coming Through 40 V. Kapur Decoding The MAP Genome 41 V. Kapur MLST And Antimicrobial Resistance Of Salmonella 42 S. Alcaine, S. Sukhnanand, L. D. Warnick, W.-L. Su, P. McDonough, M. Wiedmann Listeria monocytogenes Contains Two Species-Like Evolutionary Lineages And Subtypes With Reduced Invasiveness. K. K. Nightingale, K. Windham, and M. Wiedmann 46 MLST of Streptococcus uberis 49 R. N. Zadoks, Y. H. Schukken and M. Wiedmann. Real-time PCR in milk Food Safety in Times of War and Peace 52 J. Karns - 3 -

4 TABLE OF CONTENTS, ctd. Past, Present And Future Applications Of Bulk Tank Milk Analysis To Assess Milk Quality And Herd Health Status B. Jayarao 55 PCR Applications In Food Safety Research 74 S. P. Oliver and B. E. Gillespie Polymerase Chain Reaction For Detection Of Mycoplasma Bovis In Clinical Samples S. Klaessig Bovine Infection Of Coxiella Burnetii (Q Fever) In U.S. Dairy Herds: Use Of Conventional And Real-Time PCR For Detection Of Coxiella Burnetii In Milk S. Kim Diagnostic Strategies For Bovine Viral Diarrhea Virus 88 E. Dubovi Speaker Biographies and Contact Information 91 Acknowledgements

5 WELCOME On behalf of Quality Milk Production Services, we would like to welcome you to the two day symposium on "Molecular Methods for Milk Quality." We hope you will share our enthusiasm for this program of outstanding speakers and are pleased to have you in our audience. Thank you for your interest, participation and support. From the beginning of the "Genomics Initiative" at Cornell University and other universities around the world, there has been a promise of new opportunities. The publication of the human genome was a major step in this process. Subsequently, genomes of many organisms that are important to animal and public health have been deciphered and published. The genomes of Mycobacterium avium spp. paratuberculosis, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Listeria monocytogenes and several other species have been fully sequenced. We are now actively pursuing the opportunities provided by these genomic developments and are putting them to work towards better understanding of the biology of foodborne, zoonotic and bovine diseases, particularly mammary gland diseases. With the opening of our Molecular Laboratory we hope to greatly improve the service we can provide to the dairy industry in these areas, to the benefit of cows, producers, processors and consumers alike. This two day program brings a variety of opportunities and applications that will highlight the new directions in which research and the diagnostic process are moving. You will hear of developments in Milk Quality and Food Safety research at Cornell University, the Agricultural Research Service of the USDA and several other prominent research institutes in the U.S.A. The significance of genome sequencing in diagnostics and disease prevention as well as the use of molecular methods in the fields of monitoring and improvement of animal health, milk quality, food safety and antimicrobial resistance of mastitis pathogens will be discussed along with their practical application to real life problem solving on dairy farms. We hope this symposium will provide you with information, inspiration and the opportunity to connect with colleagues and that the impact of this event will last well beyond the two days that you will be our guest. Again, thank you for you attendance and please enjoy this symposium. Ynte H. Schukken Rubén N. González Ruth N. Zadoks Ithaca, NY September

6 WE THANKS OUR SPONSORS FOR THEIR FINANCIAL SUPPORT OF THIS SYMPOSIUM In alphabetical order: Fort Dodge A division of - 6 -

7 SYMPOSIUM PROGRAM Thursday, September 29, 2004 Ramada Inn, Ithaca, NY 09:30 Registration and Coffee 10:00 Introduction and Welcome - Dr. Rubén Gozález 10:15 Molecular Methods in Food Safety Dr. Kathryn Boor 11:00 Molecular Methods in Streptococcus uberis mastitis Dr. Stephen Oliver 11:30 Monitoring Antimicrobial Resistance in USA Agriculture Dr. Paula Cray 12:15 Lunch 01:30 Molecular Methods in Antimicrobial Resistance of Mastitis Pathogens Dr. Linda Tikofsky 02:00 Molecular Methods on Dairy Farms: Case Studies Dr. Ruth Zadoks 2:30 Break 03:00 Molecular Medicine A Reality Coming Through Dr. Vivek Kapur 04:00 Conclusion - Dr. Rubén Gozález 04:30 Official Opening of QMPS Laboratory 22 Thornwood Drive, Ithaca Friday, October 1 College of Veterinary Medicine, Cornell University 09:00 Decoding the MAP genome Dr. Vivek Kapur 10:00 MLST and antimicrobial resistance of Salmonella Dr. Martin Wiedmann 10:30 Break 11:00 MLST of Listeria monocytogenes: a tale of two lineages Ms. Kendra Nightingale 11:30 MLST of Streptococcus uberis: a tale of no lineages Dr. Ruth Zadoks 12:00 Lunch 01:00 Real-time PCR in milk: Food safety in times of war and peace Dr. Jeffrey Karns 01:45 Past, present and future applications of bulk tank milk analysis to assess milk quality and herd health status Dr. Bhushan Jayarao 02:15 PCR applications in food safety research Dr. Stephen Oliver 2:45 Break 3:15 PCR for Detection of Mycoplasma bovis Ms. Suzanne Klaessig 3:30 Bovine Infection of Coxiella burnetii (Q fever) in U.S. Dairy Herds Dr. Sung Kim 4:00 Bovine viral diarrhoea virus detecion in milk Dr. Ed Dubovi 4:30 Discussion Dr. Ynte Schukken 5:00 Refreshments - 7 -

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9 MOLECULAR METHODS IN FOOD SAFETY Kathryn J. Boor Department of Food Science Cornell University, Ithaca, NY ABSTRACT DNA-based detection and subtyping methods offer improvements in detection and characterization of foodborne pathogens beyond classical plating and phenotypic methods. The past 5 years have yielded advancements in development of sensitive, rapid, automated, and increasingly easy-to-use molecular detection and subtyping methods for a variety of different foodborne pathogens. This summary highlights key aspects of different DNA-based detection and subtyping methods and their applications for foodborne pathogens. DNA-BASED DETECTION METHODS FOR FOODBORNE PATHOGENS Detection methods currently applied for foodborne pathogens are predominantly based on nucleic-acid hybridization or polymerase chain reaction. These methods can be designed to detect either DNA or mrna. While detection of DNA is often technically more straightforward than that of mrna, the stability of DNA leads to the possibility that DNA-based detection methods may yield positive results from non-viable and/or inactivated pathogens. Detection of non-viable and inactivated pathogens thus represents a major concern in the direct application of PCR methods, especially those not designed to include an enrichment step. mrna, on the other hand, is less stable than DNA, and has thus has potential as a target for more specific detection of viable pathogen populations. Nucleic-acid hybridization-based methods. Nucleic acid hybridization methods have been used as relatively rapid screening strategies for identification of foodborne pathogens. These methods also can be used to detect target pathogens in enrichment media. Many commercial nucleic-acid hybridization-based detection methods use a dip-stick format, e.g., the GenTrack assay. rrna is often used as the target for these detection methods, since it provides a high level of sensitivity due to the presence of a high number of target copies (>1,000) in a single bacterial cell. A disadvantage of rrna-based detection methods lies in the limited specificity of these assays due to the fact that closely related species (e.g., the pathogen L. monocytogenes and the closely related non-pathogenic species L. innocua) can share highly similar rrna sequences, which do not allow their differentiation. Polymerase chain reaction (PCR). PCR has been used as a research tool for more than 15 years, and also has been developed into a tool for rapid and specific detection of foodborne pathogens. While early PCR assays for foodborne pathogen detection were generally developed by research laboratories and were not suitably developed for routine detection, over the last 3-5 years, multiple companies have introduced commercial PCR systems for detection of foodborne pathogens such as Listeria monocytogenes, E. coli O157:H7, and Salmonella. These methods often allow superior specificity to traditional biochemical identification methods. To date, all commercial PCR assays still require pre-enrichment steps to achieve appropriate sensitivity. These pre-enrichment protocols also greatly reduce the risk of false positive results due to detection of killed organisms, mainly due to the sample dilution step inherent to these protocols. Another critical component of these commercial assays is the inclusion of an internal positive control that indicates PCR failures, e.g., through carry over of PCR inhibitors. While most early PCR commercial assays used detection of PCR products by agarose gel electrophoresis as output, - 9 -

10 at least some commercial assays currently available use a real-time format with either a 5 nuclease probe or a SYBR Green-based detection of PCR products. These formats negate the need for agarose gel electrophoresis and further speed up completion of PCR-based assays. DNA-BASED SUBTYPING METHODS FOR FOODBORNE PATHOGENS The use of subtyping methods to differentiate strains (or subtypes) of bacterial, viral, and parasitic pathogens has important applications for more rapid, precise, and efficient foodborne disease surveillance, outbreak detection, and source tracking throughout the food chain. Differentiation of bacterial foodborne pathogens beyond the species level also provides exciting opportunities to better understand the biology of bacterial strains and subtypes, including differences in their ability to cause human foodborne disease. In the context of subtyping, the terms subtyping, strain typing, and fingerprinting are often used interchangeably. All of these terms describe the process of differentiating bacterial isolates beyond the species or subspecies level. The term fingerprinting can be somewhat misleading when used in this context, however, since bacterial subtyping differs significantly from fingerprinting of humans. Importantly, asexual reproduction in bacteria allows for the parallel existence of virtually identical organisms. Bacterial subtyping is used to characterize two or more distinct isolates with the goal of determining their (ancestral) relationship. For example, in outbreak investigations, the goal of subtyping bacterial isolates is to probe the likelihood that two or more isolates share a very recent (days to weeks, perhaps months) common ancestor. Fingerprinting of humans, on the other hand, is used to characterize and track a single specific individual (Wiedmann, 2002b). The choice of an appropriate subtyping method (or methods) depends on the intended application and the goal of the exercise. Commonly used criteria for evaluating subtyping methods include (i) discriminatory ability; (ii) cost; (iii) standardization and reproducibility; (iv) automation and ease of use; (v) speed; and (vi) applicability of a given subtyping method to different bacterial species (Wiedmann, 2002a; de Boer and Beumer, 1999). The discriminatory ability of a subtyping method can be characterized using Simpson s Index of Discrimination, which quantifies the probability that two unrelated strains will be characterized as different subtypes. No single subtyping method will perform optimally with regard to all of these criteria. The intended application of subtyping will determine the relative importance of each criterion. For example, a food testing laboratory that subtypes a limited number of isolates representing a variety of different foodborne pathogens (e.g., Escherichia coli O157:H7; Listeria monocytogenes, Salmonella) will have different requirements for a subtyping method than a national or international subtyping network or reference laboratory that needs to subtype a large number (>1,000) of isolates of one specific pathogen. In general, bacterial subtyping methods can be divided into (i) phenotype-based, and (ii) molecular, genetic or DNA-based methods (Olive and Bean, 1999; Wiedmann, 2002b). Commonly used phenotype-based strain typing methods for bacterial pathogens include serotyping, biotyping, phage typing, and multilocus enzyme electrophoresis. While a variety of shortcomings and concerns may be associated with different phenotype-based strain typing methods, these methods are still regularly used and have some utility for characterization of bacterial foodborne pathogens. Phenotype-based methods may lack discriminatory power and reproducibility. Furthermore, a considerable proportion of bacterial isolates may be untypable with some of these methods. To overcome these issues and to provide improved strain differentiation, molecular subtyping methods, which are based on the microbial genotypes, have been developed (Wiedmann, 2002b). The widespread development of multiple DNA-based subtyping methods has dramatically improved our ability to differentiate subtypes of bacterial foodborne pathogens. Commonly used DNA-based subtyping approaches for bacterial pathogens include plasmid

11 profiling, Pulsed-Field Gel Electrophoresis (PFGE), ribotyping, Amplified Fragment Length Polymorphism (AFLP), random amplification of polymorphic DNA (RAPD) as well as other PCR-based subtyping methods (Olive and Bean, 1999; Wiedmann, 2002a). Increasingly, DNAsequencing based methods, such as multilocus sequence typing (MLST) are also being developed. Many DNA-based methods are superior to classical methods (e.g., serotyping) in several respects. For example, DNA-based subtyping methods often provide more sensitive strain discrimination and a higher level of standardization and reproducibility as compared to phenotype-based methods. The use of multiple subtyping methods often improves subtype discrimination and may thus be appropriate for certain applications and specifically for epidemiological outbreak investigations. Key aspects of selected and commonly used molecular subtyping methods are summarized below. For a more comprehensive review the reader is referred to one of the many review articles on bacterial subtyping methods (Olive and Bean, 1999; de Boer and Beumer, 1999; van Belkum et al., 2001). Pulsed-Field Gel Electrophoresis (PFGE). PFGE characterizes bacteria into subtypes (sometimes referred to as pulsotypes ) by generating DNA banding patterns after restriction digestion of the bacterial genomic DNA. Specifically, complete bacterial DNA is purified and subsequently cut into diagnostic DNA fragments using restriction enzymes, which cut DNA where a specific short DNA sequence is present. Restriction enzymes are chosen such that they cut DNA only rarely to yield between approximately 8 and 25 large DNA bands ranging from kb (Wiedmann, 2002a). Since DNA fragments this large cannot be separated by standard gel electrophoresis techniques, a specific electrophoresis technique using alternating electric fields needs to be used for size separation of the resulting DNA fragments (i.e., pulsed-field gel electrophoresis), which will subsequently be visualized as DNA banding patterns. DNA banding patterns for different bacterial isolates are compared to differentiate distinct bacterial subtypes from those that share identical (or very similar) DNA fragment patterns. The CDC and state health departments in the US have developed a national network (PulseNet) to rapidly exchange standardized PFGE subtype data for isolates of foodborne pathogens (Swaminathan et al., 2001). PFGE subtyping shows a high level of discrimination for many foodborne bacterial pathogens and thus is often considered the current gold standard for discriminatory ability. It is important to realize, however, that PFGE (as well as other subtyping methods) may also sometimes detect small genetic differences (e.g., 2-3 different bands) that may not be epidemiologically significant (Tenover et al., 1995). On the other hand, the detection of an identical PFGE type (or a subtype determined by another method) in two samples (e.g., a food sample and a sample from a clinically affected human) does not necessarily imply a causal relationship or a link between these two isolates. Rather, in outbreak investigations, molecular subtyping information needs to be analyzed in conjunction with epidemiological data to determine causal relationships between two or more isolates. Ribotyping. Ribotyping is another DNA based subtyping method in which bacterial DNA is initially cut into fragments using restriction enzymes. While PFGE uses restriction enzymes that cut the bacterial DNA in very few large pieces, the initial DNA digestion for ribotyping cuts DNA into many (> ) smaller pieces (approximately 1 to 30 kb). These DNA fragments are separated by size through agarose gel electrophoresis and a subsequent Southern blot step uses DNA probes to specifically label and detect the DNA fragments that contain the bacterial genes encoding the ribosomal RNA (rrna). The resulting DNA banding patterns are thus based on only those DNA fragments that contain the rrna genes. A completely automated, standardized system for ribotyping (the RiboPrinter Microbial Characterization system) has been developed by Qualicon-DuPont (Wilmington, DE) and is commercially available (Wiedmann, 2002a; Bruce, 1996)

12 DNA sequencing-based subtyping. DNA sequencing of one or more selected bacterial genes represents another genetic subtyping method. Multilocus sequence typing (MLST) refers to a molecular subtyping approach that uses DNA sequencing of multiple genes or gene fragments to differentiate bacterial subtypes and to determine the genetic relatedness of isolates. MLST often refers to sequencing of multiple housekeeping genes (Spratt, 1999), but sequencing of multiple virulence genes can also be used as a subtyping method (Wiedmann, 2002a). A major advantage of this approach is that sequence data are considerably less ambiguous (Spratt, 1999) and easier to interpret than banding pattern-based subtypes obtained through the other DNA-based subtyping approaches described above. The development of internet accessible databases for MLST information (such as the MLST database at will also facilitate global, large scale surveillance and tracking of bacterial foodborne pathogens (Spratt, 1999). DNA sequencing data also provide an opportunity to reconstruct ancestral and evolutionary relationships among bacterial isolates, allowing scientists to further probe the evolutionary biology and the ecology of foodborne pathogens. MLST-based approaches for subtyping of bacterial foodborne pathogens are still in the early developmental stages and optimal target genes are still being defined for the different bacteria of interest. REFERENCES Bruce J. Automated system rapidly identifies and characterizes microorganisms in food. Food Technol 1996;50: De Boer E, Beumer R. Methodology for detection and typing of foodborne microorganisms. Int J Food Microbiol 1999;50: Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 1999;37: Spratt BG. Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the internet. Curr Opin Microbiol 1999;2: Swaminathan B, Barrett T, Hunter S, et al. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 2001;7: Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33: Van Belkum A, Struelens M, de Visser A, et al. Role of genomic typing in taxonomy, evolutionary genetics, and microbial epidemiology. Clin Microbiol Rev 2001;14: Wiedmann M. Molecular subtyping methods for Listeria monocytogenes. J AOAC Int 2002a;85: Wiedmann M. Subtyping technologies for bacterial foodborne pathogens. Nutr Rev 2002b;60:

13 MOLECULAR METHODS AND MASTITIS RESEARCH WITH PARTICULAR REFERENCE TO STREPTOCOCCUS UBERIS S. P. Oliver and B. E. Gillespie Food Safety Center of Excellence and the Department of Animal Science The University of Tennessee, Knoxville, TN Introduction Advances in molecular biology in the last decade or so have brought exciting new technology that can be used to solve complex problems. Utilization of molecular techniques such as the polymerase chain reaction (PCR), restriction fragment length polymorphism, real-time PCR, multiplex PCR, pulsed-field gel electrophoresis, ribotyping, single nucleotide polymorphisms, genomics, proteomics, DNA sequencing, and cloning are used more and more frequently in many research laboratories in the United States and throughout the world. Use of these techniques may facilitate the discovery of more effective methods for the prevention, control and detection of diseases affecting food producing animals. The purpose of this communication is to describe how the Mastitis/Food Safety Research Program at The University of Tennessee utilizes molecular-based techniques in our research approach with particular reference to our research on Streptococcus uberis. Identification of Disease Susceptible and Resistant Dairy Cows Novel approaches are currently being developed and utilized to determine what genetic factors are involved in disease resistance. Identification of such factors will be critical for developing strategies for eradicating or reducing the incidence of disease. Selection of dairy cows for enhanced disease resistance without compromising production traits is a very appealing concept that until the last decade was primarily a theoretical fantasy. However, excellent molecular techniques have been developed resulting in the identification of new genetic markers that have been used to identify and characterize genes responsible for production traits and host immunity. Major histocompatibility complex (MHC) genes, also called bovine lymphocyte antigens or BoLA, have received much recent attention because of their involvement in host immunity. Significant associations have been made with some infectious diseases of cattle and BoLA genes. There is strong evidence indicating that BoLA genes are important in resistance or susceptibility to diseases such as mastitis, retained placenta and cystic ovarian disease in dairy cattle. For example, one BoLA-DRB gene pattern in a study of 106 Holstein cows was associated with resistance to Staphylococcus aureus mastitis. Results of our research on BoLA-DRB3.2 gene fingerprinting of Jersey cows at The University of Tennessee Dairy Experiment Station were published by Gillespie et al. (1999a). Jersey cows (n=172) were genotyped for the BoLA-DRB3.2 allele using polymerase chain reaction and restriction fragment length polymorphism analysis. Bovine DNA was isolated from aliquots of whole blood. A two step polymerase chain reaction followed by digestion with restriction endonucleases RsaI, BstyI, and HaeIII was conducted on the DNA from Jersey cattle. Twenty-four BoLA-DRB3.2 alleles were identified with frequencies ranging from 0.3 to 22.9%. Thirteen allele types were similar to those reported previously; eleven were new allele types that have not been reported previously. Allele types reported previously include: BoLA-DRB3.2*2, *8, *10, *15, *17, *20, *21, *22, *23, *25, *28, *36, and *37. Their frequencies were 0.3%, 11.3%, 22.9%, 13.6%, 5.5%, 3.7%, 10.7%, 3.5%, 0.9%, 0.3%, 4.7%, 9.3%, and 0.9%, respectively. Of the new allele types detected, *ibe occurred at the highest frequency (6.1%) in Jersey cows from this herd. The six most frequently isolated alleles (BoLA-DRB3. *8, *10, *15, *21, *36 and *ibe) accounted for about 74 %

14 of the alleles in the population of this herd. Results of our study demonstrated that the BoLA- DRB3.2 locus is highly polymorphic in Jersey cattle. Thus, the BoLA gene may not be the best candidate for determining a relationship between genotype and mastitis susceptibility or resistance in Jersey cows. A genetic marker associated with inflammatory responses is also being evaluated. One potential marker is CXCR2, a chemokine receptor required for neutrophil migration to infection sites, which contains single nucleotide polymorphisms (SNP) within the gene. In a study by Youngerman et al. (2004a), single nucleotide polymorphisms (SNPs) and resulting haplotypes in the bovine CXCR2 gene were identified as a potential target for a genetic marker for mastitis susceptibility. A 311-bp segment of the bovine CXCR2 gene was amplified and sequenced. Five SNPs at positions 612, 684, 777, 858, and 861 were expressed in both Holstein and Jersey dairy cattle. Four SNPs resulted in synonymous substitutions, while a nonsynonymous switch at position 777 (G to C) resulted in a glutamine to histidine substitution at amino acid residue 245. The five polymorphisms generated ten distinct haplotypes. Six haplotypes were common between the two breeds, while Holsteins and Jerseys each uniquely expressed two haplotypes. Of the six common haplotypes, two represented 83% of the Jersey population; whereas four of these haplotypes represented 95% of the Holstein population. The association of CXCR2 SNP genotypes with subclinical and clinical mastitis was evaluated by Youngerman et al. (2004b). Thirty-seven Holstein and 42 Jersey cows that completed at least 2 full lactations were used. A significant association was detected between CXCR2 SNP +777 genotype and percentages of subclinical mastitis cases in Holsteins. Holsteins expressing genotype GG had decreased percentages of subclinical mastitis, but genotype CC cows had increased percentages of subclinical mastitis. Significant differences in clinical mastitis incidence were not detected between genotypes for either breed. This approach of genetically identifying mastitis resistant cows may represent an effective means of marker-assisted selection for mastitis and other inflammatory diseases involving neutrophils. The initial work is encouraging and several studies are ongoing in this exciting research area. Identifying host mechanisms that contribute to mastitis resistance is difficult due to variability observed with an outbred population. Progress towards identifying these mechanisms could be made more quickly with cows that are genetically similar. New techniques such as cloning now offer a similar opportunity to mastitis researchers. A team at UT scientists led by Drs. Lannett Edwards and Neal Schrick have successfully cloned Jersey dairy cows from mastitis susceptible cows and mastitis resistant cows (Pighetti et al., 2003). The mastitis susceptible cow has been chronically infected with Strep. uberis for about 7 lactations in spite of numerous attempts to eliminate the infection. Some of these heifers are currently of breeding age, some have been breed and some have calved and are now in early lactation ( ~taescomm/utcloneproject). By having a unique set of genetically identical animals, it is possible to develop our understanding of what contributes to mastitis resistance or susceptibility under different management schemes, vaccination protocols, or stress-situations, without the added complication of genetic variation. Our first step towards identifying these mechanisms is to determine if differences in blood leukocyte profiles exist in comparison to age-matched herdmates. Future research will be conducted to determine if immune responsiveness of clones from mastitis susceptible animals are less than those of herdmates, thus contributing to susceptibility. Once identified, more basic research altering conditions of the entire animal can begin to dissect the mechanisms that contribute to susceptibility or resistance to mastitis or other diseases of diary cattle. Identification of such factors could lead to improved selection strategies and/or novel approaches for eradicating or reducing incidence of mastitis and other diseases impacting dairy cows

15 The Streptococcus uberis Story Streptococcus uberis is an important cause of mastitis in dairy cows -- particularly during the dry period, the period around calving, and during early lactation -- that is not controlled effectively by current mastitis control practices. Many Strep. uberis intramammary infections (IMI) that originate during the nonlactating period and near calving result in clinical and subclinical mastitis during early lactation. Control programs for reducing Strep. uberis IMI should focus on periods adjacent to the nonlactating period where opportunities exist to develop strategies to reduce the impact of Strep. uberis infections in the dairy herd. We began our Strep. uberis research journey in the early 1990 s. Earlier research in England demonstrated the presence of two Strep. uberis genotypes designated types I and II. Subsequent research from England determined the nucleotide sequences of 16S ribosomal RNA of Strep. uberis genotypes I and II and showed that the two genotypes were phylogenetically distinct and proposed that Strep. uberis genotype II be designated Streptococcus parauberis. However, differentiation of Strep. uberis from Strep. parauberis was only possible by DNA hybridization or 16S rrna sequencing, since cultural, morphological, biochemical and serological characteristics of the two closely related species are indistinguishable. A technique was developed by Jayarao et al. (1991) for differentiating Strep. uberis from Strep. parauberis based on DNA fingerprinting. Results of those studies demonstrated that the predominant organism isolated from infected mammary glands was Strep. uberis and that Strep. parauberis occurred infrequently. This method was also used for species identification and differentiation of bacteria of bovine origin. Using the PCR reaction, oligonucleotide primers complementary to 16S rrna genes have been used to amplify the 16S ribosomal gene fragment from bacterial genomic DNA. Characteristic 16S rdna fingerprint patterns have been used to correctly identify 11 different Enterococcus and Streptococcus species (Jayarao et al., 1992). Research from our laboratory has focused extensively on development of in vivo and in vitro models to study host-pathogen interactions, and on identification and characterization of virulence factors associated with the pathogenesis of Strep. uberis mastitis and other environmental streptococci (Oliver et al., 1998a). We have shown that Strep. uberis was able to adhere to epithelial cells and that was followed by internalization into the host cell via exploitation of host cell machinery. Our lab demonstrated that Strep. uberis used host elements like extracellular matrix proteins to achieve increased adherence, probably utilizing these as a molecular bridge to attach to host cell membranes. Another of these host cell factors appears to be lactoferrin (LF), a whey protein found in milk. Use of molecular biology tools such as proteomics, genomics and bioinformatics has led to the discovery of a novel protein produced by Strep. uberis referred to as Streptococcus uberis Adhesion Molecule or SUAM. We have conducted numerous studies on SUAM and a brief summary of these studies follows. Collectively, experiments from our laboratory have provided evidence that: (1) Strep. uberis produces SUAM (Fang and Oliver, 1999), (2) SUAM bound to LF in milk (Fang et al., 2000), (3) binding of LF through SUAM enhanced adherence of Strep. uberis to bovine mammary epithelial cells (Fang et al., 2000). Lactoferrin may function as a bridging molecule between Strep. uberis and bovine mammary epithelial cells facilitating adherence of this important mastitis pathogen to host cells, (4) SUAM in the absence of LF influenced adherence to and internalization of Strep. uberis into bovine mammary epithelial cells, (5) SUAM was isolated, purified and sequenced, (6) a SUAM-like protein was identified in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae (Park et al., 2002a), (7) SUAM-like proteins produced by Strep. dysgalactiae subsp. dysgalactiae bound to bovine LF similar to what we observed with Strep. uberis (Park et al., 2002b), (8) antibodies against SUAM (whole protein) and to a synthetic peptide (pepsuam)

16 encompassing 15 amino acids of the N-terminus of SUAM cross-reacted with homologous proteins present in other strains of Strep. uberis demonstrating the ubiquity of SUAM across all strains of Strep. uberis evaluated, (9) pepsuam and SUAM antibodies cross-reacted with Strep. agalactiae, Strep. dysgalactiae subsp. dysgalactiae, and Streptococcus pyogenes, (10) antibodies directed against pepsuam inhibited adherence to and internalization of Strep. uberis into bovine mammary epithelial cells suggesting that pepsuam is biologically active. In addition, we have determined the theoretical DNA sequence of SUAM and confirmed this by PCR and restriction digests. Further confirmation of the theoretical SUAM sequence was obtained when the SUAM gene from the mastitis pathogen Strep. uberis UT888 was amplified, cloned and sequenced. Sequence analysis demonstrated that UT888 SUAM has 99% sequence identity to the theoretical SUAM identified in the Sanger Strep. uberis genomic database by homology to the reverse translated peptide sequence. When the SUAM DNA sequence was compared to GeneBank (NCBI nr GeneBank), no homologies as an entire gene were found demonstrating that SUAM is a unique Strep. uberis protein. We hypothesize that SUAM plays a critical role in the pathogenesis of streptococcal mastitis by facilitating bacterial adherence to bovine mammary epithelial cells. Our hypothesis is that Strep. uberis expresses SUAM and uses LF in milk and/or on the epithelial cell surface to adhere to mammary epithelial cells. Nucleic Acid-Based Methods for Mastitis Pathogen Detection Detection and subtyping of bacteria for epidemiological evaluation has been made possible by randomly amplified polymorphic DNA (RAPD) fingerprinting. We have used this technique to identify Streptococcus species (Gillespie et al., 1997; Gillespie et al., 2004) and other mastitis pathogens (Jayarao et al., 1996); detect new and persistent Strep. uberis and Streptococcus dysgalactiae subsp. dysgalactiae IMI in dairy cows (Oliver et al., 1998b). Using phenotypic methods of streptococcal identification, these new IMI would not have been detected. RAPD fingerprinting has also been used for confirmation of Strep. uberis after intramammary challenge with Strep. uberis and identified new Strep. uberis infections in challenged quarters. Subtyping of Strep. uberis and Strep. dysgalactiae by RAPD fingerprinting demonstrated isolates from New Zealand were distinct from isolates from the USA Gillespie et al., 1998). RAPD fingerprinting has been used to study the possibility of Staphylococcus aureus transmission by horn flies to heifers (Owens et al., 1998; Gillespie et al., 1999b). This technique is also useful in antibiotic efficacy studies in indicating new IMI or persistent IMI following antibiotic therapy. Molecular techniques described herein can aide mastitis researchers in identification of bacteria and subtyping of bacteria isolates for epidemiological applications, identification of genetic markers associated with disease susceptibility or resistance, and could aid in selection of dairy cattle that are more or less susceptible to mastitis. Application of these molecular techniques will allow dairy researchers greater flexibility to explore their area of scientific interest at the molecular level and may expedite discoveries leading to more effective methods for the control of mastitis and other diseases affecting dairy cows. References Fang, W. and S. P. Oliver Identification of lactoferrin-binding proteins in bovine mastitiscausing Streptococcus uberis. FEMS Microbiol. Lett. 176: Fang, W., R. A. Almeida and S. P. Oliver Effects of lactoferrin and milk on adherence of Streptococcus uberis to bovine mammary epithelial cells. Am. J. Vet. Res. 61:

17 Gillespie, B. E., B. M. Jayarao, and S. P. Oliver Identification of Streptococcus species by randomly amplified polymorphic DNA fingerprinting. J. Dairy Sci. 80: Gillespie, B. E., B. M. Jayarao, J. W. Pankey, and S. P. Oliver Subtyping of Streptococcus uberis and Streptococcus dysgalactiae isolated from bovine mammary glands by DNA fingerprinting. J. Vet. Med. B 45: Gillespie, B. E., B. M. Jayarao, H. H. Dowlen, and S. P. Oliver. 1999a. Analysis and frequency of bovine lymphocyte antigen DRB3.2 alleles in Jersey cows. J. Dairy Sci. 82: Gillespie, B. E., W. E. Owens, S. C. Nickerson, and S. P. Oliver. 1999b. Deoxyribonucleic acid fingerprinting of Staphylococcus aureus from heifer mammary secretions and from horn flies. J. Dairy Sci. 82: Gillespie, B. E., and S. P. Oliver Comparison of an automated ribotyping system, pulsedfield gel electrophoresis and randomly amplified DNA fingerprinting for differentiation of Streptococcus uberis strains. Biotechnology 3: Jayarao, B. M., J. J. E. Dore, Jr., G. A. Baumbach, K. R. Matthews, and S. P. Oliver Differentiation of Streptococcus uberis from Streptococcus parauberis by polymerase chain reaction and restriction fragment length polymorphism analysis of 16S ribosomal DNA. J. Clin. Micro. 29: Jayarao, B. M., J. J. E. Dore, Jr., and S. P. Oliver Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin. J. Clin Microbiol. 30: Jayarao, B. M., B. E. Gillespie, and S. P. Oliver Application of randomly amplified polymorphic DNA fingerprinting for species identification of bacteria isolated from bovine milk. J. Food Prot. 59: Oliver, S. P., R. A. Almeida, and L. F. Calvinho. 1998a. Virulence factors of Streptococcus uberis isolated from cows with mastitis. J. Vet. Med. B 45: Oliver, S. P., B. E. Gillespie and B. M. Jayarao. 1998b. Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting. FEMS Microbiol. Lett. 160: Owens, W. E., S. P. Oliver, B. E. Gillespie, C. H. Ray, and S. C. Nickerson The role of horn flies (Haemetobia irritans) in Staphylococcus aureus-induced mastitis in dairy heifers. Am. J. Vet. Res. 59: Park, H. M., R. A. Almeida, and S. P. Oliver. 2002a. Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis. FEMS Microbiol. Lett. 207: Park, H. M., R. A. Almeida, D. A. Luther, and S. P. Oliver. 2002b. Binding of bovine lactoferrin to Streptococcus dysgalactiae subsp. dysgalactiae isolated from cows with mastitis. FEMS Microbiol. Lett. 208:

18 Pighetti, G. M., J. L. Edwards, F. N. Schrick, A. M. Saxton, C. J. Davies, and S. P. Oliver Cloning adult dairy cows: A viable new tool in the fight against mastitis. In: Proc. National Mastitis Council, pp Youngerman, S.M., A.M. Saxton, and G.M. Pighetti. 2004a. Identification of single nucleotide polymorphisms, haplotypes and their frequencies within the bovine IL-8 receptor locus in Jersey and Holstein cattle. Immunogenetics. 56: Youngerman, S. M., A. M. Saxton, S. P. Oliver, and G. M. Pighetti. 2004b. Analysis of bovine CXCR2 polymorphisms with subclinical and clinical mastitis incidence in Holstein and Jersey cattle. J. Dairy Sci. 87:

19 MONITORING ANTIMICROBIAL RESISTANCE IN USA AGRICULTURE Dr. Paula J. Fedorka-Cray Antimicrobial Resistance Research Unit USDA-ARS, Athens, GA National Antimicrobial Resistance Monitoring System CD with NARMS update 2003 here

20 MOLECULAR METHODS IN ANTIMICROBIAL RESISTANCE OF MASTITIS PATHOGENS Linda L. Tikofsky, Ruth N. Zadoks, and Irene Loch Department of Food Science and Quality Milk Production Services Cornell University, Ithaca, NY Introduction Bovine mastitis is the single most common cause for antibiotic use on dairy farms. Antibiotics are used for treatment of clinical and subclinical mastitis during lactation and at dry-off. In the United States, treatment of all quarters of all cows at dry off with a broad-spectrum antibiotic, regardless of infection status is recommended. Although the FDA has restricted the use of many drugs previously used in dairy cattle, many others are still available from veterinarians, feed stores, dairy suppliers, and the internet. Data from a survey performed by the Animal Health Institute in 1998 found that approximately 17 of the 50 million pounds of antibiotics produced in the US each year are used in animals 2. Administration of these drugs is often performed by people unfamiliar with basic principles of pharmacology and therapeutic regimens and may be guided by the concept If some is good, more is probably better rather than by evidence based recommendations. In practice, this can result in undesirable treamtent choices, such as If drug A isn t having an effect after a day, maybe I should switch to drug B, and if that doesn t work within a day, what about drug C, even though I m really not supposed to use it in cows? Or maybe a combination of drugs?. Since it is known that selective pressure from antibiotics can influence the development and transfer of antimicrobial resistance, there is concern that antibiotic use in animals may create a reservoir of resistant bacteria or resistance genes that can be transferred to humans via food products, direct human-animal contact or farm effluents. Current scientific evidence does not support a widespread, emerging resistance to antibiotics among mastitis pathogens 12. However, information on the relationship between antibiotic use patterns and their influence on antimicrobial resistance on dairy farms is limited and continued monitoring is needed. Here at Quality Milk Production Services (QMPS) we have been exploring the question of antimicrobial susceptibility and resistance in mastitis bacteria for years. Questions we have attempted to answer through various studies include the following: Does antimicrobial resistance exist in the mastitis bacteria isolated from New York dairy herds and is it changing over time? What happens to antimicrobial susceptibility in herds that don t use antibiotics? How does antimicrobial use influence the development or acquisition of antimicrobial resistance by bacteria on the dairy farm? What resistance mechanisms exist in streptococci and staphylococci isolated from bovine milk samples? In this contribution, answers to the above questions will be discussed, and new questions will be raised, many of which we hope to address with techniques available to us in the QMPS new Molecular Laboratory

21 Does antimicrobial resistance exist in the mastitis bacteria isolated from New York dairy herds and is it changing over time? QMPS exists to address issues of udder health and milk quality for the dairy farmers of New York and surrounding states. Over 200,000 milk samples are submitted to the laboratories of QMPS for bacterial culture each year. Figure 1 depicts the changing prevalence of various mastitis causing bacteria cultured from milk samples taken at herd surveys over the past ten years at QMPS. Culture results for cow and quarter milk samples - central QMPS laboratory 20.0% 18.0% 16.0% 14.0% S. agalactiae Strep. spp. S. aureus E. coli Klebsiella % of samples 12.0% 10.0% 8.0% 6.0% 4.0% 2.0% 0.0% 1992 Average 1993 Average 1994 Average 1995 Average 1996 Average 1997 Average 1998 Average 1999 Average 2000 Average 2001 Average 2002 Average Figure 1: Culture results from quarter and composite milk samples taken at QMPS herd surveys over the past decade. Results show a decrease in prevalence of Streptococcus agalactiae, relatively stable prevalence of Staph. aureus, especially in the last six years, and an increasing prevalence of cultures positive for Streptococcus spp. The number of coliform-positive samples in this graph is low, because coliforms are mostly found in clinical mastitis and Figure 1 represents milk samples taken at herd surveys, most of which are not from cows with clinical mastitis. In addition to data on prevalence of bacteria, data on susceptibility patterns of bacterial isolates are available. In contrast to the prevalence data that were summarized in Figure 1, the susceptibility data come from samples that were submitted by veterinarians throughout New York State. This population represents mostly clinical mastitis samples. The data base covers approximately 3,400 isolates tested by QMPS between 1985 and 2000 by means of the Kirby- Bauer agar disk diffusion method. These tests are performed and interpreted according to NCCLS standards 11. Zones of inhibition (lack of growth) of the bacteria are measured around various antibiotic impregnated disks. Based on the diameters of the inhibition zones, bacteria are classified as resistant, intermediately resistant or susceptible. For statistical analysis of these records, two categories were used: susceptible and intermediate/resistant. Statistical analysis was done using chi-square analysis, and logistic regression to evaluate changes in antibiotic susceptibility through the years. Between 1985 and 2000, there was a significant decrease in the susceptibility of Streptococcus spp. to ampicillin, cloxacillin, penicillin, erythromycin, pirlimycin, and tetracycline. Erythromycin

22 and pirlimycin are available as lactating cow preparations. Tetracycline is used as an intravenous antibiotic, as prophylactic in milk replacers and may also (but rarely and off-label) be used as an intramammary infusion for dry cows. Susceptibility of Streptococcus spp. to both amoxicillin and cephalothin remained stable at 89% and 98%, respectively. Susceptibility of Strep spp. Susceptibility of Staph aureus % susceptible Years since 1985 ampcillin penicillin % susceptible Years since 1985 amp pen Figure 2. Examples of changes in antimicrobial susceptibility results to ampicillin (amp) and penicillin (pen) observed for Streptococcus spp. and Staphylococcus aureus. The trend for Staph. aureus is very different than the trend for Streptococcus spp. Where Streptococcus spp. showed a decrease in susceptibility, Staph. aureus showed significant increases in susceptibility to both ampicillin and penicillin (Figure 2). Susceptibility to amoxicillin (94%) and cephalothin (98%) remained stable. Susceptibility to cloxacillin appeared to decrease but the trend was not significant. Staph. aureus susceptibility to erythromycin, pirlimycin, and tetracycline did not change significantly over time either. These analyses were performed for all major mastitis bacteria (Staph aureus, Streptococcus spp., Staphylococcus spp., E.coli, and Klebsiella spp.) and results are summarized in Table 1. Similar results have been found in other mastitis research laboratories 5,9. Table 1: Changes in antimicrobial susceptibility over time (proportion susceptible) for selected milk isolates cultured at QMPS, 1985 to Antibiotic Staph. aureus Strep spp. E.coli Klebsiella Ampicillin I* D* D* NC Amoxicillin NC NC - - Cephalosporin NC NC D* NC (Cl)oxacillin NC D* - - Erythromycin NC D* - - Gentamycin - - NC NC Penicillin I* D* - - Pencillin-novobiocin NC D* - - Pirlimycin NC D* - - Spectinomycin NC NC D* D* Streptomycin - - D* NC Sulfathiazole - - D* D* TMP- sulfa - - NC NC Tetracycline NC D* NC D* *p < 0.05 I = increased D = decreased NC = no change

23 In 1975, Davidson performed a similar analysis that covered 10 years of QMPS data on antimicrobial resistance 3. The contrast between the 1975 and 1999 data, a 25 year span, is striking. Davidson found that 95% of Streptococcus spp. test were susceptible to ampicillin in 1975 while our data indicates as little as 26% susceptibility of Streptococcus spp. in By contrast, only 49% of Staph. aureus isolates tested in 1975 were susceptible to ampicillin; in 1999, the overall susceptibility to ampicillin was 79%. These changes in susceptibility may reflect changes in mastitis treatments used over the years. Whatever the underlying cause for the observed changes may be, the data suggest that long-term trends in antimicrobial resistance may be different from trends measured over a limited number of years. The apparent decline in susceptibility of Streptococcus spp. to commonly used antibiotics deserves further study. The term Streptococcus spp. refers to a group of species, and the actual species within this group may differ from each other in prevalence over the years and in antimicrobial susceptibility. For example, Strep. uberis has been found to be less susceptible to erythromycin, tetracycline, and streptomycin than Streptococcus dysgalactiae and Strep. uberis is currently the most common Streptococcus found in NYS bulk tank milk 17. As a whole, QMPS data that were collected over the years show that the development of bacterial resistance to antibiotics is of concern and warrants careful monitoring, and that results that are averaged across years or bacterial species may not convey sufficient information on changes in susceptibility patterns. What happens to antimicrobial susceptibility in herds that don t use antibiotics? Organic livestock production practices are distinguished by the limited use of synthetic medications including antibiotics. In 2002, the USDA established national standards on organic agriculture production and handling. Use of non-therapeutic antibiotics and growth promoters is prohibited under USDA organic livestock production standards, unless animal welfare is compromised 15. As a result antibiotic use on organic dairy farms is rare or infrequent, in contrast to the situation on most conventional farms. In 2001, a cross-sectional study of antibiotic susceptibility patterns for Staph. aureus isolated from bovine milk samples from organic and conventional dairy herds in New York and Vermont was performed. The antimicrobial susceptibility patterns for 144 isolates from 22 organic herds and 117 isolates from 16 conventional farms were compared. Antibiotic susceptibility testing was performed as described for isolates from clinical samples. Antibiotics were chosen based on their activity against Gram positive cocci and included ampicillin, cephalothin, erythromycin, novobiocin, oxacillin, penicillin, penicillin-novobiocin, pirlimycin, tetracycline and vancomycin. Many of these antibiotics are routinely employed in dairy herd health practices. Further details on methods and statistical analyses have been described previously 14. Using a categorical comparison (susceptible vs. resistant), percent susceptible for cephalothin, oxacillin, novobiocin, penicillin-novobiocin, and pirlimycin approached 100% and no differences were found between organic and conventional isolates. For the remaining antibiotics, conventional herds had fewer isolates in the susceptible range than organic herds: ampicillin (61.5% vs. 80.5%), penicillin (65.8% vs. 79.9%) and tetracycline (87.2% vs. 99.3%). Percent susceptible for erythromycin did not differ significantly and was low for conventional as well as organic herds (49.5% vs. 55.5%). Results are summarized in Table

24 Table 2. Comparison of susceptible and resistant isolates by category for organic and conventional herds. Conventional n = 117 Organic n = 144 Antibiotic Susceptible Resistant Susceptible Resistant Significance Ampicillin p = Cephalothin Oxacillin Erythromycin Novobiocin Penicillin Penicillinnovobiocin Pirlimycin Tetracycline No difference No difference No difference No difference p = No difference No difference p = When results were compared on a continuous scale, that is on the basis of zone of growth inhibition in millimeters, significant differences between organic and conventional herds were observed for ampicillin, cephalothin, oxacillin, penicillin, penicillin-novobiocin, pirlimycin, and tetracycline. Most conventional and organic isolates fell within the susceptible category, but within that category the distribution of zone diameters for isolates from the conventional herds is shifted to the left (smaller zone diameters) compared to that of the organic herds (Figures 3 and 4), implying that Staph. aureus isolates from conventional herds exhibit decreased antibiotic susceptibility when compared to those from organic herds N Conventional Organic Zone diameter (mm) Figure 3. Kirby-Bauer zone diameters (mm) for pirlimycin in Staphylococcus aureus from organic and conventional herds. Arrow indicates breakpoint for susceptibility

25 N conventional organic Zone diameter (mm) Figure 4. Kirby-Bauer zone diameters (mm) for oxacillin in Staphylococcus aureus from organic and conventional herds. Arrow indicates breakpoint for susceptibility. In this study, most isolates tested expressed antibiotic susceptibilities well within the sensitive category. Overall this is good news since the antibiotics currently being used do not appear to be putting overwhelming pressure on mastitis bacteria and creating resistance. However, the subtle differences in degree of susceptibility warrant further study which can be accomplished through the implementation of molecular diagnostics. Several hypotheses can be investigated: Are different strains of Staph. aureus populating organic and conventional dairy herds? Are the resistance genes present in Staph aureus from organic and conventional herds but does antibiotic use turn them on? Are the strains of Staph aureus similar on both organic and conventional herds but does antibiotic use encourage the acquisition of resistance genes from other farm bacteria? QMPS is prepared to answer these questions. All Staph. aureus isolates from this study have been preserved on Microbeads and frozen at C for molecular typing in the future. Strain differences within and between herds and routes of pathogen transmission (cow-to-cow transmission or infection from multiple environmental sources) can be evaluated. This will contribute to a better understanding of the impact of antimicrobial usage on antimicrobial sensitivity, and can lead to changes in management to prevent infection in the future. How does antimicrobial use influence the development or acquisition of antimicrobial resistance by bacteria on the dairy farm? Cross-sectional comparisons of bacterial isolates between herds with no antibiotic usage and herds with routine antibiotic usages showed subtle but significant differences. To gain more insight into mechanisms that could underlie such differences, a longitudinal study was performed in which isolates were compared before ( no antibiotic usage ) and after ( routine antibiotic usage ) dry cow therapy with intramammary antibiotics. Details of the field study, that was designed as a Staph. aureus treatment trial, have been published 4. In this section, we will focus on analysis of the antimicrobial susceptibility of the Staph. aureus isolates. Briefly, approximately 300 different isolates of Staph.aureus from seventy-five dairy herds in Canada were derived from a year-long study of the efficacy of tilmicosin as a dry cow treatment. Cows infected with Staph. aureus in at least one quarter were randomly assigned to be treated

26 with either a beta-lactam antibiotic, 500 mg benzathine cloxacillin (DryClox, Ayerst Laboratory, Guelph, Ontario, Canada) or with 1500 mg of tilmicosin phosphate, a broad spectrum macrolide antibiotic (supplied by Provel, Division of Eli Lilly Canada, Inc, Guelph, Ontario, Canada). For all Staph. aureus isolates, minimum inhibitory concentrations (MIC) for multiple antibiotics were determined by broth microdilution (Sensititre, Westlake, Ohio). Data were analyzed in a manner similar to the comparison of isolates from organic and conventional farms. Sixty-four pairs of isolates (before and after treatment) from the same quarter of the same cow were available for additional study. Twenty-six of the pairs showed a shift in MIC values for one of the Macrolide-Lincosamide-Streptogramin (MLS) antibiotics before and after treatment. Twelve of the pairs exhibiting the shift, representing eight cows from five herds were selected for molecular typing. Strain typing was performed with restriction enzyme EcoRI at Cornell University s Laboratory for Molecular Typing using the RiboPrinter Microbial Characterization (Qualicon, Wilmington, DE USA). Isolates from cows receiving treatment with tilmicosin exhibited statistically significant changes in resistance patterns for the MLS antibiotics (tilmicosin, erythromycin, pirlimycin) and tetracycline (Figure 5). No differences were observed for penicillin, oxacillin, ceftiofur, sulfadimethoxine, penicillin-novobiocin, and cephalothin. Significant increases in the proportion of resistant isolates after the dry period were likewise observed for tilmicosin, erythromycin, and pirlimycin. No significant differences were observed in either MIC patterns or proportion resistant for Staph. aureus isolates from cows receiving the cloxacillin treatment. Cephalothin Tetracycline Penicillin/Novobiocin After Calving Before Dry Off Pirlimycin Erythromycin Ceftiofur Tilmicosin Oxacillin Penicillin Percent of Isolates Resistant Figure 5: Percent of Staphylococcus aureus isolates resistant to specified antibiotics before (cross-hatched bars) and after (black bars) treatment with tilmicosin at dry-off From the twelve pairs of isolates submitted for molecular characterization, five ribotypes (169-5, , , , ) were identified (Figure 6). Ribotype was present in seven cows from five herds. Each of the other ribotypes was present in one cow per herd in four separate herds. In herd 52, there were two ribotypes present; herd 53 had one cow with the same strain in two quarters, and herd 62 had one strain infecting three separate cows. In all cases,

27 isolates before and after treatment were the same ribotype and were thus likely to represent persistent infections rather than acquisition of a new infection. Molecular Typing RH RH RH RH RH RH RH RH-6 Figure 6. Examples of riboprint patterns for four pairs of Staph. aureus isolates collected before and after treatment with Tilmicoson at dry off. Isolate identification is show on the right hand side and strains (1106-1, , 169-5, ) on the left hand side. Members of the MLS family of antibiotics are widely used in both animal and human medicine. The primary resistance mechanism, coded for by various erm genes, is ribosomal methylation, which decreases binding of the macrolide. Resistance in staphylococci is the result of expression of primarily the erma or ermc genes genes 6,8. The majority of erm genes identified in bovine Staph aureus are erma 1. Coagulase negative staphylococci are a large reservoir of both erma and ermc, which are present on mobile genetic elements (transposons and plasmids, respectively). Expression of erm genes generally results in high levels of resistance to multiple members of the macrolide family. Efflux pump activity is a another macrolide resistance mechanism that has been identified in streptococci and staphylococci and that results from the expression of mefa/e and msra, respectively. Efflux pumps may confer a low, medium, or high resistance to macrolide antibiotics and resistance levels may differ between different members of the MLS group and can also be induced by exposure to antibiotics16. In this study, strains from the same quarter of the same cow were indistinguishable before and after treatment. The change in MIC values after exposure to tilmicosin but not after exposure to cloxacillin suggests induction by the antibiotic even after a single exposure. This increase in resistance may be due to acquisition of resistance genes from the environment, e.g. coagulase negative staphylococci, or up-regulation of an existing mechanism, such as an efflux pump. Further molecular investigation into the presence resistance genes in these isolates is planned. What resistance mechanisms exist in streptococci isolated from bovine milk samples? In a recent study, we explored which genes encode macrolide resistance in streptococcal isolates from bovine milk. Strep. uberis and Strep. dysgalactiae isolates from composite milk and bulk tank samples from herds in the Southwestern USA were tested for the presence of macrolide resistance genes belonging to the classses of ribosomal methylaseas (erma/tr, ermb, ermc) and efflux

28 pumps (mefa/e). The isolates in this study originated from herds that used macrolide antibiotics for mastitis control. All streptococcal isolates tested positive for the presence of the ermb gene by means of PCR, while erma/tr, ermc, and mefa/e were not detected. PCR amplicons were subsequently sequenced and the streptococcal isolates were further characterized by automated ribotyping with PvuII. Results are summarized in Figure 7, which shows a phylogenetic tree of ermb alleles (a, b and c) and includes information on herd of origin (numbers 1-6), ribotype (letters A-N), species (SUB = Strep. uberis; SDY = Strep. dysgalactiae), and isolate identification (starting with Z3). Alleles a and b were both associated with very high phenotypic resistance to erythromycin and pirlimycin (MIC 64 for both macrolides), while allele c was associated with slightly lower resistance (MIC 64 for erythromycin and 16 for pirlimycin.) 1 - A - SUB - z C - SUB - z K - SUB - z K - SUB - z K - SUB - z K - SUB - z M - SDY - z M - SDY - z M - SDY - z M - SDY - z3101 Alelle a Very high resistance 2 - F - SUB - z F - SUB - z N - SUB - z N - SUB - z3102 Allele b Very high resistance 2 - E - SUB - z L - SUB - z3094 Allele c High resistance Figure 7. Relatedness of ermb alleles from Streptococcus uberis (SUB) and Streptococcus dysgalactiae (SDY) from bovine milk. Numbers (1 through 6) indicate herds. Letters (A through N) indicate strains based on ribotyping. Isolates are identified by numbers starting with z3. Isolates with allele a or b had MIC 64 for erythromycin and pirlimycin. Isolates with allele c had MIC 64 for erythromycin and 16 for pirlimycin. Figure 7 shows that: (i) phenotypic resistance profiles are associated with specific genotypic profiles (allele c encodes lower resistance than allele a or b ; (ii) S. uberis isolates from different herds and belonging to different strains may carry the same ermb allele (e.g. strains F and N from herds 2 and 6); and (iii) within a herd, multiple alleles of the ermb gene may occur (herd 2, strains E and F, alleles b and c ; herd 4, strains L and K, alleles a and c ). Furthermore, it can be seen that the same ermb allele was found in Strep. uberis and in Strep. dysgalactiae. The fact that the same allele for a resistance gene can be found in multiple streptococcal strains and species suggests that horizontal transfer of the resistance gene may have taken place. To assess whether other bacterial species might carry the same resistance alleles, data from our study were compared to DNA-sequence data that are available via de World Wide Web ( (Figure 8)

29 Enterococcus hirae -source unknown Arcanobacterium.pyogenes -cow Bacillus cereus soil Clostridium perfingens source unknown Strep. gallolyticus pigeon, human Strep. uberis - z cow Allele b Lactobacillus reuteri source unknown Strep. uberis - z cow Allele a Enterococcus faecium source unknown Strep. uberis z cow Staph. intermedius -dog Allele c Figure 8. Genetic relatedness of ermb alleles from Streptococcus uberis and other bacteria, originating from bovine milk and from a variety of host species and sources, respectively. Comparison of ermb sequence data from Strep. uberis with ermb sequence data from other bacterial species showed that the same alleles can be found in streptococci that infect other host species, and in other genera of bacteria, including bacteria isolated from soil. This reinforces the idea that horizontal gene transfer may occur, and adds the possibility of interspecies gene transfer 7 which could potentially occur between different bacteria carried by the same cow or in the dairy farm environment. Summary and Conclusion Strep. pneumoniae -human Strep. agalactiae cow Strep. agalactiae cow Data collected by QMPS over the past decades show that the population of mastitis bacteria changes over time. This is true for the occurrence of bacterial species and for the occurrence of antimicrobial resistance within a bacterial species. For some bacterial species and antimicrobials, susceptibility increased over the years, while it decreased for others. Staph. aureus isolates from milk samples from organic dairy farms show higher levels of susceptibility to antibiotics than Staph. aureus isolates from conventional farms, although most isolates from both types of farms would be considered susceptible under current guidelines. While dry-cow treatment with betalactam antibiotics did not cause a shift in antimicrobial susceptibility among Staph. aureus isolates, treatment with tilmicosin, a member of the MLS group of antibiotics, causes a significant loss of susceptibility. Resistance to MLS antibiotics also occurs in streptococci from bovine milk, where it is encoded by alleles of the ermb gene. The same alleles are also found in other bacterial species, including bacteria from humans and soil. This suggests that horizontal transfer of resistance genes has occurred, and implies that such transfer may continue to happen, especially if use of antimicrobials on dairy farms creates an environment that favors the survival of resistant bacteria. Such transfer may lead to uptake of resistance genes by mastitis pathogens so that treatment of cows becomes unsuccessful, or it may lead to transfer of resistance genes by mastitis pathogens to other bacteria, including those that may reach and/or affect humans. Currently, antimicrobial resistance is not a major problem in mastitis pathogens or the dairy industry, but antimicrobial use is associated with differences and changes in antimicrobial susceptibility,

30 specifically susceptibility of mastitis-causing streptococci and staphylococci. Hence, monitoring of antimicrobial resistance and use of management and treatment strategies that minimize the risk of development of antimicrobial resistance continue to be necessary. References 1. Aarestrup FM, Agerso Y, Ahrens P, Jørgensen JC, Madsen M, Jensen LB Antimicrobial susceptibility and presence of resistance genes in staphylococci from poultry. Vet Microbiol. 74: Animal Health Institute. Survey on Antibiotic Use in Humans and Animals in Feb (www. AHI.com) 3. Davidson JN. Antibiotic Resistance Patterns of Bovine Mastitis Pathogens. Proc. 19 th National Mastitis Council Meeting Dingwell RM, Leslie KE, Duffield TF, Schukken YH, DesCoteaux, Keefe GP, Kelton DF, Lissemore KD, Shewfelt W, Dick P, Bagg R Efficacy of intramammary tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period. J Dairy Sci. 86: Erskine RJ, Walker RD, Bolin CA., Bartlett PC, White DG Trends in Antibacterial Susceptibility of Mastitis Pathogens During a Seven-Year Period. J Dairy Sci. 85: Khan SA, Nawasz MS, Khan AA, Cerniglia CE Transfer of erythromycin resistance from poultry to human clinical strains of Staphylococcus aureus. J. Clin Microbiol. 38: Kimpe A, Descostere A, Martel A, Devriese A, Haesebrouck F Phenotypic and genetic characterization of resistance against macrolides and lincosamides in Streptococcus gallolyticus strains isolated from pigeons and humans. Microb Drug Resist. 9 Suppl1:S35-S Lina G, Quaglia A, Reverdy M, LeClercq R, VanDenNesch F, Etienne J Distribution of genes encoding resistance to macrolides, lincosamides and streptogramins among staphylococci. Antimicrob. Agents Chemother. 43: Makovec JA and Ruegg PL Results of milk samples submitted for microbiological examination in Wisconsin from 1994 to J Dairy Sci. 86: Martel A, Meulenaere V, Devriese LS, Decostere A, and Haesebrouck F Macrolide and lincosamide resistance in the gram-positive nasal and tonsillar flora of pigs. Microb. Drug Resist. 9: NCCLS Performance Standards of Antimicrobial Disk Susceptibility Tests. 6 th edition. Vol. 17. Jan National Mastitis Council Research Committee Report. Bovine Mastitis Pathogens and Trends in Resistance to Antibacterial Drugs Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L Detection of erythromycinresistant determinants by PCR. Antimicrob.Agents Chemother. 40: Tikofsky LL, Barlow JW, Santisteban. C, Schukken YH A Comparison of Antimicrobial Susceptibility Patterns for Staphylococcus aureus from Conventional and Organic Farms. Microb. Drug Resist. 9 Suppl. 1:S USDA USDA National Standards on Organic Agricultural Production and handling. ( last accessed October 7, 2002) 16. Van Bambeke F, Balzi E, Tulkens PM Antibiotic efflux pumps. Biochem Pharm. 60: Zadoks RN, González RN, Boor KJ, Schukken YH Mastitis-Causing Streptococci are Important Contributors to Bacterial Counts in Raw Bulk Tank Milk. Journal of Food Protection. In press

31 MOLECULAR METHODS ON DAIRY FARMS: CASE STUDIES Ruth N. Zadoks Department of Food Science and Quality Milk Production Services Cornell University, Ithaca, NY Introduction How does use of molecular methods contribute to improvement of udder health and milk quality on dairy farms? Why would a farmer or a veterinarian consider their use? Why pay for it? Because it provides information that regular bacterial culture can t provide. Molecular typing, also called strain typing or DNA-fingerprinting, gives a level of detail and insight that is not available with traditional culture methods. It provides answers to riddles that can t be solved otherwise. It provides the data to convince people of things they are reluctant to believe because it goes against the common knowledge of the day. By molecular methods we mean methods to identify and characterize bacteria based on detection of their DNA. The fact that molecular methods are DNA-based sets them apart from bacterial culture methods, where it is the growth of bacteria that is observed. Molecular methods can be used to detect the presence of bacterial species (e.g. E. coli, Klebsiella, Staphylococcus aureus and other staphylococci, Streptococcus agalactiae and other streptococci, etc.), bacterial strains (groups of bacteria within a species that have a specific characteristic in common, compare to breeds within animal species), or specific genes (e.g. genes for resistance to antibiotics). In this contribution, examples of on-farm applications of molecular methods will be given. All examples are from work that has been done with real-world dairy farms to address their mastitis or milk quality concerns. Questions that we answered with molecular methods include: I have had a closed and Streptococcus agalactiae-free herd for years and now the lab found Strep. agalactiae in my milk. How can that be? What is the cause of the high bacteria count in my bulk tank milk? Can I get rid of environmental streptococci? My milking hygiene is perfect, and yet my bulk tank somatic cell count is too high. What else can I do to lower the SCC so that I can get a quality premium? For questions about any of these examples, to discuss whether your milk quality questions could be answered with molecular methods, or to submit samples, please contact Ruth Zadoks at QMPS, phone (607) , or by rz26@cornell.edu. How can milk from a Streptococcus agalactiae-free herd be positive for Strep. ag.? Streptococcus agalactiae is a contagious pathogen of dairy cows. It spreads very easily between cows, mostly during milking, and causes high somatic cell counts (SCC). Because the mastitis that is caused by Strep. agalactiae is often subclinical (invisible), it may go unnoticed until there are so many cows with high SCC that the bulk tank SCC rises and even exceeds PMO limits (750,000 cells/ml). In a Strep. agalactiae eradication program, it is extremely important to detect all infected cows. To show how dangerous and contagious Strep. agalactiae can be, here is an example: QMPS worked with a herd of approximately 700 cows and bulk tank SCC around 300,000 cells/m. The herd was expanded with 140 purchased animals, and within two months the bulk tank SCC shot up to 700,000 cells/ml. Whole herd milk culture revealed that 68 cows

32 were infected with Strep. agalactiae, which had probably been brought into the herd by the new cows. It took 18 months to control the disease, or rather, to get to the point where the disease appeared to be under control. With only 3 cases left, control measures started to lapse. Within five months, the number of infected cows was back up to 26 and bulk tank SCC was on the rise again. Strep. agalactiae is very common in people, although it is mostly known by a different name: group B streptococcus or GBS. A large proportion of people carry GBS or Strep. agalactiae in their gastrointestinal or urogenital tract 10. Both men and women can be carriers, usually without any signs or symptoms. The danger lies mostly in infection of children at birth (for more information see GBS is also considered an emerging pathogen in immunocompromised adults and the elderly. Dogs and cats can be carriers of Strep. agalactiae 14, but this not very common. Do cows and people have the same Strep. agalactiae? This question has been studied by a number of research groups 3,11, including Cornell University's Food Safety Laboratory (FSL) in collaboration with QMPS. In 2002, 52 human and 52 bovine Strep. agalactiae isolates were collected in New York State. For each of these isolates, strain typing was performed by several methods, including DNA sequencing and ribotyping. Based on these methods, it could be shown that people and cows each have their own strains of Strep. agalactiae (Figure 1; based on 13 ). Although one clone of S. agalactiae that may affect people has arisen from a bovine ancestor at some point in the past 1, there is no evidence that S. agalactiae from bovine milk currently presents a risk to human health. FSL S3-027 FSL S3-048 ( S-8) FSL S3-062 ( S-7) FSL S3-043 ( S-7) FSL S3-034 ( S-5) FSL S3-030 ( S-4) FSL S3-026 FSL C1-487 ( S-1) FSL S3-012 ( S-5) FSL S3-008 ( S-5) FSL S3-018 ( S-5) FSL C1-496 ( S-2) FSL F2-338 ( S-4) FSL F2-347 ( S-1) FSL S3-009 ( S-5) Figure 1. Molecular typing results (DNA-sequencing of hyaluronidase gene) for Streptococcus agalactiae: Evolutionary tree showing the relatedness of isolates from cows and humans 13. Bovine and human strains of Strep. agalactiae form separate groups. So what about Strep. agalactiae in closed dairy herds? How can a bulk tank sample test positive for Strep. agalactiae when all the cows are Strep. agalactiae-free according to the herd-survey? And how can a cow test positive in a closed herd that has been free for years? And how can it be only one

33 cow? Does that imply it is the very start of a major mastitis outbreak? Based on reports that milkers may infect their animals with Staph. aureus 2, we postulated that people might infect their cows with Strep. agalactiae. After all, many healthy people are carriers of Strep. agalactiae. If a human were the source of Strep. agalactiae that could also explain how a bulk tank can test positive while none of the cows does. Ribotyping confirmed our suspicions: in a herd where only the bulk tank tested positive, the Strep. agalactiae that was identified belonged to a human type. In three herds where only one cow tested positive, each Strep. agalactiae case was caused by a human type. The human types of Strep. agalactiae are not specifically adapted to survival in the cow. They may cause clinical mastitis, which is unusual for Strep. agalactiae in cows, and the infections may not last long, again unusual for Strep. agalactiae in cows 8. Additional work in our laboratories has shown that human and bovine Strep. agalactiae also differ in their resistance to antimicrobials, with higher resistance levels in human than in bovine isolates 4. Dogs and cats, by the way, also carry the human rather than the bovine strains 14. In summary: there are exceptions to the Strep. agalactiae rules. If you find Strep. agalactiae in a closed herd or in a herd with bulk tank SCC below 200,000 cells/ml, with only one cow testing positive, possibly manifesting as a clinical mastitis, and resistant to antibiotics (mostly tetracycline and macrolides), or maybe only in the bulk tank milk and not in any cows at all, that Strep. agalactiae may belong to a human type. To be on the safe side, treatment of cows infected with human strains is recommended. That will most likely be the end of it, and an outbreak of mastitis is unlikely to occur. What is the cause of the high bacteria count in my bulk tank milk? Bacteria counts of bulk tank milk are monitored routinely. The PMO limit is 100,000 cfu/ml (cfu = colony forming units), but ideally bacteria counts do not exceed 10,000. If they do, there may be a problem with milking time hygiene, equipment cleaning, milk cooling or cow health 5,7,12. Sometimes, people look for the explanation in the field they are most familiar with. A veterinarian may be more likely to focus on cows, while an equipment expert may be more likely to focus on cleaning and cooling. Alternatively, people may try to shift the blame to the field that is not their area of expertise, which contributes as little to a solution as focusing on the wrong cause. Collaborative work between FSL and QMPS has shown that high bulk tank counts are most likely to be caused by Streptococci, specifically Strep. uberis, and coliform bacteria, predominantly E. coli 6. Both Strep. uberis and E. coli are considered to be environmental pathogens, i.e. bacteria that are common in the environment of the cow. Because the bacteria are common in the environment, their presence in bulk tank milk is often attributed to poor milking time hygiene or poor equipment cleaning 5,12. If environmental contamination was the source of a high bulk tank bacterial count, the population in the bulk tank milk would be expected to reflect the high heterogeneity of the environmental bacterial population. In other words: we would expect a large variety of bacterial strains in the tank if the bacteria came from the environment. If a cow has mastitis, she is usually infected with one strain of bacteria. Hence, if a cow with mastitis were the source of a high bacteria count in bulk tank milk, one would expect to find the strain from the cow to be the dominant strain in the tank. To determine whether cows could be the source of high counts of streptococci or E. coli in bulk tank milk, we used molecular methods 1) to explore the diversity of the bacterial population in the bulk tank milk; and 2) to compare the strains from cows with mastitis to the strains found in the bulk tank. Table 1 shows two examples of results for Streptococcus uberis, the most common non-agalactiae streptococcus

34 Table 1. Molecular typing results (ribotyping) for Streptococcus uberis from bulk tank milk isolates and from all Strep. uberis infected cows in each herd. Farm Source Ribotype RiboPrinter TM pattern A Bulk Tank Bulk Tank Bulk Tank Cow 1 Cow E Bulk Tank Bulk Tank Bulk Tank Cow For five dairy herds, multiple Strep. uberis isolates were available from the bulk tank milk so that the strain diversity in the bulk tank could be assessed. For all five herds, a dominant strain was identified. For herd A, this was strain and for herd E, this was strain The presence of a dominant strains shows that a point source was more likely to be the cause of high bulk tank count than environmental contamination. The bulk tank milk sample had been taken on the day that a whole herd survey was performed so that individual results were available for all cows that had contributed to the bulk tank milk. All cows were tested for environmental streptococci, and the species and strain of the mastitis-causing bacteria was determined. In each of the five herds, a cow was shown to shed the strain that was found in the bulk tank milk. Thus, cows with mastitis were the most likely source of high bacteria counts in those herds. After seeing the results shown above for S. uberis, one of the QMPS veterinarians brought in some client samples. A whole herd survey for the client s herd had just been completed. Among the results was a high bacteria count in the bulk tank milk, caused by E. coli, and one cow that was infected with E. coli. The veterinarian and the farmer discussed the possibility of the cow being the source of the bulk tank count. Although that seemed plausible, it went against everything the farmer had always been told about high coliform counts in milk. Therefore, we decided to use strain typing to show whether the bacteria in the bulk tank belonged to one or multiple strains, and also whether the strain from the infected cow was found in the bulk tank. Again, a dominant strain was found in the bulk tank, and the same strain was indeed found in the cow (Figure 2). With those results, veterinarian and farmer were now convinced that the cow had been the cause, and the bulk tank bacterial count problem could be resolved by dealing with the cow, BTM C without adjustments to milking procedures, equipment cleaning or cooling installations. Figure 2. Molecular typing results (RAPD-PCR) for three bulk tank milk (BTM) isolates and one cow isolate (C) of E. coli from a herd with high coliform counts. Left hand lane shows molecular size marker. No other cows were infected with E. coli in this herd

35 Can I get rid of environmental streptococci? So cows may be the source of bacteria in bulk tank milk. But where do the cows get their bacteria? If the source of environmental bacteria could be identified, targeted intervention measures might be possible, such as use of different bedding material. Some studies have suggested that specific animals in the herd may be carriers of Strep. uberis in their gastro-intestinal tract 9. Fecal dispersal of the bacteria in the environment by such carrier animals may put the rest of the herd at risk of mastitis. If such animals could be identified, environmental contamination and subsequent environmental mastitis could potentially be prevented. To find out whether specific cows or specific environmental sites could be pinned down as the source of Strep. uberis on a dairy farm, we did a 10-month study in central New York. Every month, we collected samples from feed (hay, haylage, grass/pasture greens), water (trough, ponds, streams), lying areas (bedding from stalls or body imprints in pasture), and gathering/traffic areas (doorway, around watering trough, under stand of shade providing trees). In addition, fecal and milk samples were collected from cows at dry-off and at calving. Molecular methods were used to determine whether Strep. uberis was present in environmental, fecal and milk samples, and also to explore which strains were present in the different sources. Strep. uberis was found in 63% of environmental samples, 25% of fecal samples and 4% of milk samples. Many environmental sites and fecal samples harbored multiple different strains of Strep. uberis (example shown in Figure 3). Figure 3. Molecular typing results (ribotyping) for Strep. uberis from a soil sample. Among eight isolates from one sample, five strains (indicated by RiboGroups on the left hand side) were identified. Close to 90% of soil samples, two-thirds of grazing matter, and 40% of water samples tested positive for the presence of Strep. uberis. The strains of Strep. uberis that were found in environmental samples did not differ from those in fecal samples or milk samples. Thus, any environmental site could potentially be a source of Strep. uberis infection and there is no particular site that could be targeted for preventive efforts. Targeting fecal shedders did not seem to be an option either: many animals tested positive for Strep. uberis at some point in time, but very few animals tested positive repeatedly, and persistent fecal shedders were not identified. For this herd, promoting cow health and mastitis resistance through measures such as adequate nutrition and selective breeding is a more promising road to mastitis prevention than elimination of potential sources of infection. What can I do to lower my bulk tank SCC? A farmer in Vermont struggled to lower his bulk tank somatic cell count (BMSCC). The current BMSCC level was around 300,000 cells/ml and the farmer felt he did everything he possibly could to prevent transmission of mastitis pathogens during milking. Still, the target of lowering BMSCC to 200,000 cells/ml and the associated quality premium seemed elusive. To add insult to injury, one of his advisors insisted that something must be wrong with his milking time hygiene for the BMSCC to remain that high

36 Through a different advisor, the farmer heard about the use of molecular methods for milk quality improvement. As shown in Figure 4, molecular methods can be used to determine whether mastitis cases are caused by one strain that is spread from cow to cow as contagious pathogen (contagious, dotted black circle), or by a variety of strains that come from the cow s environment (environmental, thin black ellipse) Contagious 10 number of infected quarters 8 6 Environmental B A C D F G I S. uberis strain J L M P Q farm visit Figure 4. Molecular typing results (RAPD) for Streptococcus uberis isolates from a dairy herd. Samples were collected from all quarters of all cows at 3-weekly farm visit. Molecular typing was performed for isolates from all udder quarters (strains identified by letters). For each visit, the number of quarters infected with a specific strain was determined (vertical axis). Adapted from 15. To help the farmer solve his BMSCC problem, a combination of approaches was used: a herd visit, inspection of DHIA data, culture of cow milk samples, and molecular methods. During the herd visit, milking time hygiene seemed impeccable. Inspection of DHIA data on somatic cell counts of individual cows showed that most cows were healthy, new infections were rare and mostly occurred at calving, most cows with high SCC had chronic mastitis, and few cows cured (Figure 5). Routine culture results showed that environmental streptococci were the most common mastitis pathogens. They were found in eight milk samples. Molecular methods (species-specific PCR) showed that two isolates belonged to the species Strep. dysgalactiae and six isolates belonged to the species Strep. uberis. Because two species were involved, at least two sources of infection had played a role and not all infections were the result of transmission in the milking parlor

37 Figure 5. DHIA data for linear score of individual cows. Horizontal and vertical axis show previous and current score (PLS and LS), respectively. Dividing lines indicate LS=4 and PLS = 4. Values above 4 are considered too high. Based on the combination of PLS and LS, cows can be categorized as healthy (PLS and LS low), new cases (PLS low, LS high), chronic infections (PLS and LS high) or cures (PLS high, LS low). New cases Healthy Chronics Cures Subsequently, molecular methods were used to differentiate Strep. uberis at the strain level. Among six isolates, originating from six quarters of four cows, four strains were identified, with one strain per cow (Figure 6). Clearly, with each cow having her own species or her own strain of streptococcus, the infections were not due to cow-to-cow transmission during milking, but rather to independent infections of each cow with bacteria from a variety of sources. Thus, herd inspection, SCC data and molecular results all confirmed the herd owner s notion that his milking time hygiene was excellent. In this herd, room for improvement was to be found in prevention of new infections around calving and in detection and treatment or culling of chronically infected cows. M M A B B C C D E F A B B C C D E F I II III IV RF LF LF LH Figure 6. Molecular typing (RAPD) of six Streptococcus uberis isolates (identified by numbers) from four cows (indentified by roman numerals) with strain designation (letters A-F). Typing performed in duplicate. Molecular markers (M), positive (+) and negative (-) controls are included. RF = right front quarter, LF = left front quater, LH = left hind quarter

38 Conclusion and future prospects This paper covered a few examples of herd health and milk quality issues that were addressed with the help of molecular methods. Many more applications are conceivable, including testing of raw milk for presence of food borne pathogens before consumption, detection of potential fecal carriage of Klebsiella by dairy cows resulting in contamination of bedding material that was originally Klebsiella free, monitoring for presence of antimicrobial resistance genes, and evaluation of the success of treatment with differentiation between non-cures and re-infections of cured quarters. Molecular methods have been used with incredible success in research laboratories for the past 15 years. Now that techniques are becoming increasingly user-friendly and affordable, they are slowly starting to make their way into the diagnostic laboratory. Although their routine implementation still faces challenges, for example in terms of cost recovery and turn-around times, it is inevitable that in another few years, molecular methods will be among the routine tools used in diagnostic laboratories, mastitis management, and the promotion of udder health and milk quality. References 1. Bisharat, N., D. W. Crook, J. Leigh, R. M. Harding, P. N. Ward, T. J. Coffey, M. C. Maiden, T. Peto, and N. Jones Hyperinvasive neonatal group B streptococcus has arisen from a bovine ancestor. J. Clin. Microbiol. 42: Devriese, L. A. and J. Hommez Epidemiology of methicillin-resistant Staphylococcus aureus in dairy herds. Res. Vet. Sci. 19: Dmitriev, A., E. Shakleina, L. Tkacikova, I. Mikula, and A. Totolian Genetic heterogeneity of the pathogenic potentials of human and bovine group B streptococci. Folia Microbiol. (Praha) 47: Dogan, B., Y. H. Schukken, C. Santisteban, and K. J. Boor Serotype distributions and antimicrobial resistances of Streptococcus agalactiae isolates from bovine or human origin. In preparation. 5. Farnsworth, R. J Microbiologic examination of bulk tank milk. Vet. Clin. North Am. Food Anim Pract. 9: Hayes, M. C., R. D. Ralyea, S. C. Murphy, N. R. Carey, J. M. Scarlett, and K. J. Boor Identification and characterization of elevated microbial counts in bulk tank raw milk. J. Dairy Sci. 84: Jayarao, B. M. and D. R. Wolfgang Bulk-tank milk analysis. A useful tool for improving milk quality and herd udder health. Vet. Clin. North Am. Food Anim Pract. 19:75-92, vi. 8. Jensen, N. E Experimental bovine group-b streptococcal mastitis induced by strains of human and bovine origin. Nord. Vet. Med. 34: Kruze, J. and A. J. Bramley Sources of Streptococcus uberis in the dairy herd. II. Evidence of colonization of the bovine intestine by Str. uberis. J. Dairy Res. 49:

39 10. Manning, S. D., P. Tallman, C. J. Baker, B. Gillespie, C. F. Marrs, and B. Foxman Determinants of co-colonization with group B streptococcus among heterosexual college couples. Epidemiology 13: Martinez, G., J. Harel, R. Higgins, S. Lacouture, D. Daignault, and M. Gottschalk Characterization of Streptococcus agalactiae isolates of bovine and human origin by randomly amplified polymorphic DNA analysis. J. Clin. Microbiol. 38: Saran, A Disinfection in the dairy parlour. Rev. Sci. Tech. 14: Sukhnanand, S., B. Dogan, M. O. Ayodele, R. N. Zadoks, M. P. J. Craver, N. B. Dumas, Y. H. Schukken, K. J. Boor, and M. Wiedmann Molecular subtyping and population genetics of bovine and human Streptococcus agalactiae isolates. J. Clin. Microbiol. Manuscript JCM Yildirim, A. O., C. Lammler, R. Weiss, and P. Kopp Pheno- and genotypic properties of streptococci of serological group B of canine and feline origin. FEMS Microbiol. Lett. 212: Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H. Schukken Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol. Infect. 130:

40 MICROBIAL PATHOGENOMICS, A REALITY COMING THROUGH Vivek Kapur Department of Microbiology University of Minnesota, St. Paul, MN Microbial pathogenomics is an emerging discipline that is concerned with the application of genomics based approaches to the study of biological processes associated with infectious agents and the disease they cause. The rapid progress in genomics technologies has provided an important tool for the study of infectious diseases during the twenty-first century. These microbial pathogenomics investigations have provided important insights on pathogen and host biology as well as on host-pathogen interactions. For instance, the availability of the human genome sequence along with the complete genomic sequences of nearly all of the major pathogens associated with disease in humans has provided key missing insights on the basic mechanisms of infectious disease pathogenesis (5, 7). In addition, the development of methods for detection of gene expression profiles using microarays tools for studying how large numbers of genes interact with each other and how a cell s regulatory networks control vast batteries of genes simultaneously, or for high-throughput genotyping (such as SNP analysis), have added considerably to the repertoire in the toolkit of the modern day infectious disease researcher. Finally, the rapidly emerging fields of computational molecular biology and bioinformatics that together deal with the development and application of computational tools and approaches for expanding the use of biological, medical, and health-related data, including tools to acquire, store, organize, archive, analyze, or visualize such data, have proved to be essential for the extraction of biological insights and knowledge from the vast quantities of information generated during a typical microbial pathogenomics experiment. Recent research activities in my laboratory have focused on functional genomics and proteomics applications relating to microbe-host interactions. Our group has participated in the complete genome sequencing of various microbial pathogens (for e.g. 1, 6, 8, 16) as well as functional genomics investigations for these agents including genome-scale studies of host-pathogen interactions using custom and Affymterix-based microarrays and other sophisticated tools for pathogen and host gene expression analysis (1-4,6,8-19). A list of microbial pathogen genomes that have been completely sequenced in our Laboratory at the Microbial Pathogen Size (Mbp) Brucella abortus 2.1 Cryptosporidium parvum 9.1 Lawsonia intracellularis 1.8 Mycobacterium paratuberculosis 4.8 Pasteurella multocida 2.2 Staphylococcus aureus RF Staphylococcus aureus TSS 2.5 Streptococcus pyogenes 1.9 University of Minnesota along with the size of the genome is provided in the adjacent table. Taken together, these sequencing and functional genomics studies have led to the appreciation of the commonalities and particularities amongst microbial pathogens and the strategies they adopt to successfully infect, colonize, infect, and cause disease in their hosts. What are these commonalities? What, if anything, of practical utility has resulted from these microbial pathogenomics investigations? My presentation at the symposium will highlight the results of recent investigations carried out in our laboratory in an attempt to answer these and related questions. -40A - --

41 Literature Cited. 1. Abrahamsen, M. S., T. J. Templeton, S. Enomoto, J. E. Abrahante, G. Zhu, C. A. Lancto, M. Deng, C. Liu, G. Widmer, S. Tzipori, G. A. Buck, P. Xu, A. T. Bankier, P. H. Dear, B. A. Konfortov, H. F. Spriggs, L. Iyer, V. Anantharaman, L. Aravind, and V. Kapur Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304: Baechler, E. C., F. M. Batliwalla, G. Karypis, P. M. Gaffney, W. A. Ortmann, K. J. Espe, K. B. Shark, W. J. Grande, K. M. Hughes, V. Kapur, P. K. Gregersen, and T. W. Behrens Interferoninducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A 100: Boyce, J. D., I. Wilkie, M. Harper, M. L. Paustian, V. Kapur, and B. Adler Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host. Infect Immun 70: Boyce, J. D., I. Wilkie, M. Harper, M. L. Paustian, V. Kapur, and B. Adler Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera. Microbes Infect 6: Cummings, C. A., and D. A. Relman Using DNA microarrays to study host-microbe interactions. Emerg Infect Dis 6: Herron, L. L., R. Chakravarty, C. Dwan, J. R. Fitzgerald, J. M. Musser, E. Retzel, and V. Kapur Genome sequence survey identifies unique sequences and key virulence genes with unusual rates of amino Acid substitution in bovine Staphylococcus aureus. Infect Immun 70: Manger, I. D., and D. A. Relman How the host 'sees' pathogens: global gene expression responses to infection. Curr Opin Immunol 12: May, B. J., Q. Zhang, L. L. Li, M. L. Paustian, T. S. Whittam, and V. Kapur Complete genomic sequence of Pasteurella multocida, Pm70. Proc Natl Acad Sci U S A 98: Munir, S., and V. Kapur Regulation of host cell transcriptional physiology by the avian pneumovirus provides key insights into host-pathogen interactions. J Virol 77: Munir, S., and V. Kapur Transcriptional analysis of the response of poultry species to respiratory pathogens. Poult Sci 82: Munir, S., S. Singh, K. Kaur, and V. Kapur Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells. Biol Proced Online 6: Paustian, M. L., B. J. May, D. Cao, D. Boley, and V. Kapur Transcriptional response of Pasteurella multocida to defined iron sources. J Bacteriol 184: Paustian, M. L., B. J. May, and V. Kapur Pasteurella multocida gene expression in response to iron limitation. Infect Immun 69: Paustian, M. L., B. J. May, and V. Kapur Transcriptional response of Pasteurella multocida to nutrient limitation. J Bacteriol 184: Scamurra, R. W., D. J. Miller, L. Dahl, M. Abrahamsen, V. Kapur, S. M. Wahl, E. C. Milner, and E. N. Janoff Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells. J Immunol 164: Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee, G. L. Sylva, D. E. Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins, S. B. Beres, D. S. Campbell, T. M. Smith, Q. Zhang, V. Kapur, J. A. Daly, L. G. Veasy, and J. M. Musser Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc Natl Acad Sci U S A 99: Stratmann, J., B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host. Infect Immun 72: Yarwood, J. M., J. K. McCormick, M. L. Paustian, V. Kapur, and P. M. Schlievert Repression of the Staphylococcus aureus accessory gene regulator in serum and in vivo. J Bacteriol 184: Yarwood, J. M., J. K. McCormick, M. L. Paustian, P. M. Orwin, V. Kapur, and P. M. Schlievert Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands. J Biol Chem 277: B 41 --

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43 DECODING THE MAP GENOME Vivek Kapur Department of Microbiology University of Minnesota, St. Paul, MN Selected Recent Publications Motiwala AS, Amonsin A, Strother M, Manning EJ, Kapur V, Sreevatsan S. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species. J Clin Microbiol Apr;42(4): Amonsin A, Li LL, Zhang Q, Bannantine JP, Motiwala AS, Sreevatsan S, Kapur V. Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains. J Clin Microbiol Apr;42(4): Stratmann J, Strommenger B, Goethe R, Dohmann K, Gerlach GF, Stevenson K, Li, LL, Zhang Q, Kapur V, Bull TJ. A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host. Infect Immun Mar;72(3): Bannantine JP, Hansen JK, Paustian ML, Amonsin A, Li LL, Stabel JR, Kapur V. Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol Jan;42(1): Dohmann K, Strommenger B, Stevenson K, de Juan L, Stratmann J, Kapur V, Bull TJ, Gerlach GF. Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates. J Clin Microbiol Nov;41(11): Bannantine JP, Zhang Q, Li LL, Kapur V. Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates. BMC Microbiol May 9;3(1):10. Motiwala AS, Strother M, Amonsin A, Byrum B, Naser SA, Stabel JR, Shulaw WP, Bannantine JP, Kapur V, Sreevatsan S. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis. J Clin Microbiol May;41(5):

44 MLST AND ANTIMICROBIAL RESISTANCE OF SALMONELLA Sam Alcaine 1, Sharinne Sukhnanand 1, Lorin D. Warnick 2, Wan-Lin Su 1, Patrick McDonough 2, and Martin Wiedmann 1 Department of Food Science, 2 Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 1 INTRODUCTION Salmonella is an important zoonotic pathogen. According to the Centers for Disease Control and Prevention (CDC), an estimated 1.4 million cases of human disease due to nontyphoidal salmonellosis occur annually in the U.S. (Mead et al., 1999). Within the genus Salmonella, almost 2,500 serotypes can be differentiated using the standard Kauffman-White scheme. Serotypes within subspecies Ι (enterica) are responsible for the vast majority of salmonellosis infections in warm-blooded animals. These serotypes differ widely in a variety of features, most notably, their host range and the severity and type of disease typically caused by them. For example, Salmonella serotype Typhimurium causes gastroenteritis in a multitude of hosts, whereas Salmonella serotype Dublin causes enteric fever and induces abortion primarily in cattle. While the overall number of salmonellosis cases has been decreasing, there has been a rise in the number of antibiotic resistant isolates encountered. For example, while the incidence of the two most common serotypes found in humans, Typhimurium and Enteritidis, has fallen 23% and 35%, respectively, from 1997 to 2002, the incidence of Salmonella Newport, a serotype recently associated multi-drug resistance, has risen 165% in the same period (CDC, 2002). These resistant strains pose a serious risk to human and animal populations. It has been speculated that the rise of these strains may be linked to improper administration of antibiotics in hospitals and/or use of antibiotics in livestock. Children under the age of 5 account for a quarter of all salmonellosis cases (CDC, 2002), and represent a vulnerable portion of the population. Of particular concern is the appearance of Salmonella strains resistant to ceftriaxone, the drug of choice for treatment of invasive salmonellosis cases in children. Ceftriaxone is closely related to ceftiofur, a 3 rd generation cephalosporin with widespread use in cattle herds. Beef and dairy products account for 10% of reported food borne Salmonella outbreaks (CDC, 2002), and concerns have been raised on possible transmission of antibiotic resistant Salmonella, including ones resistant to ceftiofur, from cattle to people (Fey et al., 2004). While serotyping has been widely used to differentiate Salmonella subtypes, this method has limited discriminatory power and does not reveal the genetic relationships of strains within the same or different serotypes. More discriminatory methods for subtyping of Salmonella isolates include phage typing as well as pulsed field gel electrophoresis (PFGE). Multi-Locus Enzyme Electrophoresis (MLEE) has also been used successfully to subtype Salmonella isolates and to study the evolution and population genetics of various Salmonella serotypes (e.g., Selander et al., 1990). However, MLEE is technically difficult and hard to standardize between laboratories and thus does not represent a subtyping method suitable for routine surveillance. The advent of automated DNA sequencing technology has led to the development and implementation of DNA sequence-based subtyping techniques, such as Multi-Locus Sequence Typing (MLST). MLST is based on the concepts of MLEE except that allelic types are determined from nucleotide sequences of housekeeping genes rather than by the electrophoretic mobilities of the enzymes they encode (Maiden et al., 1998). One key advantage of MLST over MLEE and other banding pattern-based subtyping techniques is that the sequence data generated is non-ambiguous and can

45 be readily compared between laboratories, thus facilitating global, large-scale surveillance (Maiden et al., 1998). The changing epidemiology of Salmonella infections and the emergence of new Salmonella strains (e.g., multi-drug resistant Salmonella Typhimurium DT 104 and multi-drug resistant Salmonella Newport) make it imperative to develop new Salmonella subtyping methods that not only allow for sensitive subtype discrimination, but also provide data that can be used for evolutionary analyses of Salmonella. In addition, molecular subtyping methods for Salmonella should also allow for serotype prediction, thus obviating the need for maintenance of specialized serotype reagents for Salmonella. Thus, our goal was to develop an MLST scheme for Salmonella enterica serotypes that (i) provides sensitive subtype discrimination, (ii) reliably predicts Salmonella serotypes and (iii) provides data that can be used for evolutionary analyses. We subsequently applied the MLST scheme we developed to probe the emergence and transmission of ceftiofur resistant Salmonella on dairy farms. MLST DEVELOPMENT The results of our efforts to develop a Salmonella MLST scheme have been submitted for publication (Sukhnanand et al., 2004). A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multi-locus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes (panb, fima, acek, mdh, icda, manb, and span). A total of 8 MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for Typhimurium isolates. Since manb, fima, and mdh allowed for highest subtype discrimination among the initial 25 isolates, we choose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This three-gene sequence typing scheme allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported, evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella. Three-gene sequence typing also supports that Salmonella serotypes represent both monophyletic and polyphyletic lineages. EVOLUTION AND POPULATION GENETICS OF CEFTIOFUR RESISTANT SALMONELLA Our studies on the application of the three-gene sequence typing scheme described above to study the evolution and transmission of ceftiofur resistant Salmonella are currently being prepared for submission (Alcaine et al., 2004). Both ceftiofur resistant and sensitive isolates from eight farms, which had previously been found to be characterized by the presence of ceftiofur resistant Salmonella strains (Warnick et al., 2003), were analyzed for genetic relatedness (using the three-gene sequence typing scheme described above) as well as for the presence of class I integrons and CMY-2. Ceftiofur susceptibility was determined using an automated broth dilution method (Sensititre, Trek Diagnostics). It should be noted that cut-offs for classifying Salmonella isolates as sensitive or resistant to ceftiofur have not been validated. For this study, the breakpoints were those typically used for National Antimicrobial Resistance Monitoring System

46 reports. CMY-2 is a beta-lactamase gene associated with broad resistance to cephalosporins, which has been shown to confer resistance to ceftiofur. Class I integrons are mobile genetic elements, that tend to carry and transmit antibiotic resistance genes. The isolates grouped into six sequence types (STs), three of which contained isolates with ceftiofur resistance. Each ST contained isolates from multiple farms, and included both resistant and sensitive isolates. CMY- 2 was found in all except three isolates that had ceftiofur resistance, and DNA sequencing showed that the CMY-2 genes carried in all isolates were 100% identical, suggesting horizontal gene transfer of CMY-2. Furthermore, on the farm level, each farm contained only one Salmonella ST that had resistance, though not all isolates of the same ST type on the farm had resistance, suggesting recent acquisition or loss of resistance. Presence of class I integrons and CMY-2 were not correlated, suggesting that CMY-2 is not carried in an integron, consistent with other studies that have shown CMY-2 to be associated with a plasmid. Overall, our data are consistent with a model that both horizontal and vertical transfer contribute to spread of ceftiofur resistant Salmonella among cattle. SUMMARY AND CONCLUSIONS Using an initial collection of 25 Salmonella isolates, we developed a seven-gene MLST scheme targeting a combination of housekeeping and virulence genes. Based on the initial data obtained with this seven-gene MLST scheme, we chose three genes with the highest discriminatory ability to develop and apply a more economical three-gene sequence typing scheme using 66 Salmonella isolates, representing 12 serotypes. Our results show that (i) a three-gene sequence typing scheme allows for serotype prediction and for limited subtype discrimination within serotypes, and (ii) Salmonella serotypes represent both monophyletic and polyphyletic lineages. Application of the three-gene sequence typing scheme furthermore allowed us to initially probe the evolution and transmission of ceftiofur resistant Salmonella. Our preliminary data are consistent with a model that both horizontal and vertical transfer contribute to spread of ceftiofur resistant Salmonella among cattle. On-going studies on larger sets of human and animal Salmonella isolates are designed to provide further insight into the evolution and transmission of Salmonella (with a particular focus on antibiotic resistant strains) among and between humans and farm animals. REFERENCES Alcaine, S., L. D. Warnick, P. McDonough, K. J. Boor, and M. Wiedmann Ceftiofurresistant Salmonella represent multiple widely distributed subtypes that evolved by independent horizontal acquisition of CMY-2. J. Clin. Microbiol. (in preparation) Centers for Disease Control and Prevention Salmonella Annual Summary Fey P.D., T. J. Safranek, M. E. Rupp, E. F. Dunne, E. Ribot, P. C. Iwen, P. A. Bradford, F. J. Angulo, and S. H. Hinrichs Ceftriaxone-resistant Salmonella infection acquired by a child from cattle. N. Engl. J. Med. 342: Maiden, M. C. J., J. A. Bygraves, E. Feil, G. Morelli, J. E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D. A. Caugant, I. M. Feavers, M. Achtman, and B. G. Spratt Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Nat. Acad. Sci. USA 95:

47 Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe Food-related illness and death in the United States. Emerg. Infect. Dis. 5: Selander, R. K., P. Beltran, N. H. Smith, R. Helmuth, F. A. Rubin, D. J. Kopecko, K. Ferris, B. D. Tall, A. Cravioto, and J. M. Musser Evolutionary genetic relationships of clones of Salmonella serovars that cause human typhoid and other enteric fevers. Infect. Immun. 58: Sukhnanand, S., S. Alcaine, W.-L. Su, J. Hof, M. P. J. Craver, L. D. Warnick, P. McDonough, K. J. Boor, and M. Wiedmann DNA sequence-based subtyping and evolutionary analysis of selected Salmonella enterica serotypes. J. Clin. Microbiol. (submitted 9/16/04; JCM ). Warnick, L. D., K. Kanistanon, P. L. McDonough, and L. Power Effect of previous antimicrobial treatment on fecal shedding of Salmonella enterica subsp. enterica serogroup B in New York dairy herds with recent clinical salmonellosis. Prev. Vet. Med. 56:

48 LISTERIA MONOCYTOGENES CONTAINS TWO SPECIES-LIKE EVOLUTIONARY LINEAGES AND SUBTYPES WITH REDUCED INVASIVENESS Kendra K. Nightingale, Katy Windham, and Martin Wiedmann Department of Food Science Cornell University, Ithaca, NY Background and significance. Listeria monocytogenes is a facultative intracellular human foodborne and animal pathogen that may cause severe invasive disease in immunocompromised individuals (Schlech, 2000). Clinical manifestations of invasive human listeriosis include meningitis, encephalitis, late-term spontaneous abortion, and septicemia. While invasive listeriosis is a rare disease with an estimated frequency of 2 to 15 cases/million population, the case mortality for this disease is very high (13-34%) (Roberts and Wiedmann, 2003). Apparently healthy individuals infected by L. monocytogenes may experience a mild noninvasive form of listeriosis characterized by gastrointestinal illness (Roberts and Wiedmann, 2003). The vast majority of human listeriosis infections (99%) are thought to be foodborne (Mead et al., 1999). Listeriosis is a systemic bacterial infection characterized by diffusion of L. monocytogenes from the intestinal lumen to the central nervous system and the placenta (Lecuit et al., 2001). A systemic infection requires that bacteria be internalized both by professional phagocytes such as macrophages and induce their own uptake in non-professional phagocytes including epithelial, endothelial and hepatic cells (Braun et al., 2000). Internalin (InlA) is a surface protein encoded by inla that facilitates the entry of L. monocytogenes into epithelial cells that express specific E- cadherin alleles, the receptor for InlA. Because E-cadherin is expressed in various intestinal cells, InlA may be critical for L. monocytogenes to cross the intestinal barrier as well as later on in infection (Lecuit et al., 2004). Researchers have shown that InlA is sufficient to promote internalization L. monocytogenes into several cell lines expressing human E-cadherin (Lecuit et al., 1997). Traditionally, L. monocytogenes has been differentiated into 13 serotypes; however, only four of these serotypes (1/2a, 1/2b, 1/2c and 4b), however, have been reported to cause the majority (98%) of human listeriosis cases (Doumith et al., 2004). Molecular subtyping methods (e.g., automated ribotyping and pulsed field gel electrophoresis) allow for more sensitive discrimination of L. monocytogenes subtypes as compared to phenotypic methods (e.g., serotyping) and have provided an initial understanding of the population structure of this pathogen (Wiedmann, 2002). Data generated with most molecular subtyping methods have shown that L. monocytogenes isolates can be grouped into two major genetic divisions or lineages, termed lineage I and lineage II (Wiedmann, 2002). Interestingly, previous reports have shown a statistically significant epidemiological associated between the isolation lineage I strains from human clinical listeriosis cases as compared to their isolation from contaminated foods (Gray et al., 2004). On the other hand, lineage II appears to contain two distinct subpopulations one of which has been associated with isolation from contaminated food samples while to other appears to be associated with isolation from human listeriosis cases (Gray et al., 2004). Most epidemiological and population genetics studies on L. monocytogenes have used DNA banding pattern-based subtyping methods (e.g., pulsed-field gel electrophoresis and ribotyping). However, these methods are difficult to standardize and data are not easily compared between laboratories. A reliable high-throughput universal subtyping method such as multilocus sequence typing (MLST) is needed to study the molecular epidemiology and evolution of L. monocytogenes. Similar to multilocus enzyme electrophoresis, MLST surveys several loci

49 expected to operate under neutral genetic variation (Dingle et al., 2001; Maiden et al., 1998). MLST typically targets conserved loci such as housekeeping genes that diversify slowly and are not heavily influenced by evolutionary forces other than point mutation (i.e., positive selection and recombination). However, including virulence genes in a MLST scheme may provide an enhanced ability to differentiate isolates and allow researchers to make inferences on the evolution of virulence in L. monocytogenes clonal groups (Cai et al., 2002). L. monocytogenes contains two species-like evolutionary lineages. We assembled a representative geographically matched set of 120 L. monocytogenes isolates from humans and animals with clinical listeriosis as well as from foods to analyze the genetic diversity, population genetics, and evolution of this L. monocytogenes with a specific focus on those molecular subtypes associated with foodborne disease transmission. Partial sequencing of four housekeeping (gap, prs, purm and ribc), one stress-response (sigb), and two virulence (acta and inla) genes revealed between 11 (gap) and 33 (inla) allelic types (unique combination of polymorphisms). acta, ribc, and purm demonstrated the highest levels of nucleotide diversity (π > 0.05). Based on a concatenated data set, 52 unique sequence types (unique combination of allelic types) were differentiated among the 120 L. monocytogenes isolates characterized. Further analyses showed that acta and inla may be affected by positive selection and that these virulence genes along with hypervariable housekeeping loci ribc and purm may have a history of intragenic recombination. Molecular phylogenies of all seven genes, inferred by maximum likelihood methods, indicated that L. monocytogenes contains two deeply-separated evolutionary lineages. Lineage I appears to be highly clonal which may be explained by this lineage experiencing a population bottleneck, while lineage II shows greater diversity and evidence of ancient horizontal gene transfer events. Nucleotide distance within evolutionary lineage was much lower than that observed between lineages suggesting a barrier for genetic exchange between lineages. Additionally, alleles were not shared between lineage I and II with respect to all genes except gap for which a single lineage II isolate showed a lineage I allele. Our data show that (i) L. monocytogenes is a highly diverse species with at least two deeply-separated species-like evolutionary lineages, which differ in their population structure, and (ii) horizontal gene transfer as well as positive selection contributed to the evolution of L. monocytogenes currently present in the food system. L. monocytogenes contains molecular subtypes with reduced invasiveness. DNA sequencing of the C-terminal region of InlA (ca. 270 codons) for L. monocytogenes revealed three unique nonsense mutations upstream of the membrane anchor resulting in a truncated form of InlA. These truncated InlA alleles were observed in 6 L. monocytogenes strains (assigned by automated EcoRI ribotyping) including DUP-1052A and DUP (mutation type 1); DUP-1025A and DUP-1031A (mutation type 2); and DUP-1046B and DUP-1062A (mutation type 3). Searches of the PathogenTracker ( database which contains epidemiological information for more than 5000 L. monocytogenes isolates from human and animal clinical listeriosis cases, food, and various environments were performed to probe the distribution of truncated InlA strains. Results indicated that truncated InlA strains are more commonly isolated from food as compared to their isolation from human listeriosis cases. A PCR-RFLP assay and inla sequencing were used to determine prevalence of these three observed unique nonsense mutations in human clinical and food isolates representing L. monocytogenes strains shown to harbor a truncated form of InlA. Results showed that nonsense mutation were present in ca. 40% L. monocytogenes isolates representing truncated InlA strains resulting from mutation types 1 (DUP-1052A and DUP-16635A) and 2 (DUP-1025A and DUP-1031A). Further, mutation type 3 was detected in 100% of DUP-1046B and DUP-1062A isolates. Nonsense mutations were observed more frequently in L. monocytogenes isolates from food as

50 compared to human clinical cases. A Caco-2 cell invasion assay showed that truncated InlA strains have significantly (P<0.0001) reduced invasiveness as compared to full-length InlA strains. Our data indicate that specific L. monocytogenes subpopulations commonly isolated from contaminated food carry a truncated InlA and thus show reduced virulence in human intestinal epithelial cells. References Braun, L., B. Ghebrehiwet, and P. Cossart gc1q/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes. EMBO. 19: Doumith, M., C. Cazalet, N. Simones, L. Frangeul, C. Jacquet, F. Kunst, P. Martin, P. Cossart, P. Glaser and C. Buchrieser New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infect. Immun. 72: Gray, M. J., R. N. Zadoks, E. D. Fortes, B. Dogan, S. Cai, Y. Chen, V. N. Scott, D. E. Gombas, K. J. Boor, and M. Wiedmann Food and human isolates of Listeria monocytogenes form distinct but overlapping populations. Appl. Environ. Microbiol. (accepted 6/21/04; AEM ). Jonquieres, R., H. Biern, J. Mengaud and P. Cossart The inla gene of Listeria monocytogenes LO28 harbors a nonsense mutation resulting in release of Internalin. Infect. Immun. 66: Lecuit, M., S. Vandormael-Pournin, J. Lefort, M. Huerre, P. Gounon, C. Dupuy, C. Babinet, and P. Cossart A transgenic model for listeriosis: role of Internalin in crossing the intestinal barrier. Science. 292: Lecuit, M., H. Ohayon, L. Braun, J. Mengaud, and P. Cossart Internalin of Listeria monocytogenes with an intact leucine-rich repeat is sufficient to promote internalization. Infect. Immun. 65: Mead, P. S., L. Slutsker, V. Dietz, L. F. McCraig, J. S. Bresee, C. Shapiro, P. M. Griffin and R. V. Tauxe Food-related illness and death in the United States. Emerg. Infect. Dis. 5: Roberts, A. J. and M. Wiedmann Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis. Cell Mol. Life Sci. 60: Wiedmann, M Molecular subtyping methods for Listeria monocytogenes. J. AOAC Int. 85:

51 MULTI-LOCUS SEQUENCE TYPING OF STREPTOCOCCUS UBERIS SHOWS RETICULATE EVOLUTION BETWEEN HOUSEKEEPING GENES AND POSITIVE SELECTION IN A PUTATIVE VACCINE TARGET Ruth N. Zadoks 1, 2, Ynte H. Schukken 2 and Martin Wiedmann 1. 1 Department of Food Science, 2 Quality Milk Production Services Cornell University, Ithaca, NY. Background. Streptococcus uberis is an important udder pathogen of dairy cows. For years, a vaccine has been sought after as a means to protect cows from S. uberis mastitis, with GapC and PauA as the main vaccine targets 9,12, but no field-trials demonstrating vaccine efficacy have been published yet. Improvement of S. uberis control programs has been hampered by insufficient knowledge of the epidemiology and pathophysiology of infections. Knowledge of strain characteristics associated with patterns of transmission, infection or cure could contribute to improvement of herd and cow specific recommendations with respect to mastitis treatment and control, potentially reducing the current reliance on use of antimicrobials as primary control strategy. While DNA banding pattern based typing methods have contributed to insights in the epidemiology of S. uberis mastitis 10,11,13, they have limitations in terms of typeability, discriminatory power, and reproducibility. For several streptococcal species, including S. agalactiae, S. pneumoniae, S. pyogenes, and S. suis, banding pattern based typing methods have been superseded by multilocus sequence typing (MLST), which provides more standardized and informative strain typing data than banding pattern based methods 1,2,4,8. We developed a multilocus sequence typing scheme for S. uberis, and used phylogenetic analyses to explore the evolutionary mechanisms behind S. uberis diversity. MLST is more discriminatory than DNA banding pattern based strain typing and provides taxonomic information. Among 50 S. uberis isolates from the USA (n=30) and The Netherlands (n=20), 35 ribotypes (index of discrimination D = 0.973), and 12 (cpn60), 10 (gap), 14 (oppf), 11 (paua), 9 (sod) and 11 (tuf) alleles were identified, with a total of 39 sequence types (D = 0.989). One strain of S. parauberis was included in the analysis. S. parauberis did not yield amplicons of oppf or paua, and had unique alleles for the remaining four housekeeping genes. In addition to being more discriminatory than banding pattern based methods, MLST differentiated within ribogroups or RAPD-patterns in accordance with epidemiological data (herd of origin, time of collection). Comparison of DNA sequences for four housekeeping genes (cpn60, gap, sod, tuf) showed that S. uberis isolate FSL Z1-015 was only distantly related to the remaining S. uberis isolates and showed higher homology with S. parauberis then with S. uberis. Based on sequencing of the rnp and rpob genes, isolate FSL Z1-015 was subsequently confirmed to belong to the species S. uberis. Isolates with the same MLST type as FSL Z1-015 have been recovered from environmental samples in farm environments and this group appears to constitute a subtaxon within S. uberis with as yet unexplored epidemiological and pathophysiological characteristics. Reticulate evolution occurs within and between genes of S. uberis. Evolution of three housekeeping genes (gap, sod, tuf) could be represented by a bifurcating tree, while reticulate evolution was detected in two housekeeping genes (cpn60, oppf) and a virulence gene (paua). In addition, there was reticulate evolution between genes so that a core phylogeny for S. uberis evolution could not be constructed. Reticulate evolution between genes also plays an important role in overall sequence diversity for other streptococcus species, such as S. pyogenes, group C streptococci (GCS) and group G streptococci (GGS), while only a limited number of housekeeping genes show evidence of within-gene reticulate evolution in these species 6. Despite

52 widespread occurrence of reticulate evolution between genes, clonal groups may be preserved in some streptococcal species. These clonal complexes may be niche specific, as has been shown for S. pyogenes 5 and S. suis 8. To cover the full spectrum of genetic diversity within S. uberis, isolates from a variety of niches, including the mammary gland and environmental sources, should be included in additional population genetic studies. Evolution of paua differs from the evolution of housekeeping genes. Unlike the other genes included in our MLST scheme, paua is a virulence gene and it is not uncommon for virulence genes to be present in a proportion of strains only. Absence of a paua amplicon was observed in 2 of 50 isolates in our study (4.0%) and in 4 of 130 S. uberis isolates (3.1%) in a study from Germany 7. Average G+C content was 0.39 for housekeeping genes and 0.34 for paua, suggesting acquisition of paua through horizontal transfer. The average d N /d S ratio was 0.07 for housekeeping genes and 1.2 for paua, indicating positive selection in paua, which encodes a streptokinase (plasminogen activator A) that is a putative vaccine target 9. Some of the observed non-synonymous mutations in paua are likely to be inconsequential, while other nonsynonymous substitutions may affect the conformation and/or functionality of the protein. Differences between S. uberis isolates in plasminogen activity have been reported 3, but it remains to be elucidated whether any of the amino acid polymorphisms in PauA have an impact on its functionality. Conclusion and Outlook. MLST-based typing of S. uberis was superior to banding pattern-based methods in terms of discriminatory ability, concordance with epidemiological data, and quantitative information regarding relatedness of isolates and taxonomic groups. Reticulate evolution contributes to a limited extent to genetic variability within the genes covered by our typing scheme, but plays a major role in overall sequence variability of S. uberis. Reticulate evolution may provide S. uberis with the ability to diversify in response to control measures taken to reduce the incidence of S. uberis infections in dairy cattle. The evolution of the virulence gene paua, which encodes a putative vaccine target, differed from the evolution of housekeeping genes and was subject to positive selection. In the development and evaluation of vaccines, creation of sub-unit vaccines targeting conserved epitopes and continued monitoring of evolution of vaccine targets should be considered. References 1. Enright, M. C. and B. G. Spratt A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144 ( Pt 11): Enright, M. C., B. G. Spratt, A. Kalia, J. H. Cross, and D. E. Bessen Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect. Immun. 69: Johnsen, L. B., K. Poulsen, M. Kilian, and T. E. Petersen Purification and cloning of a streptokinase from Streptococcus uberis. Infect. Immun. 67: Jones, N., J. F. Bohnsack, S. Takahashi, K. A. Oliver, M. S. Chan, F. Kunst, P. Glaser, C. Rusniok, D. W. Crook, R. M. Harding, N. Bisharat, and B. G. Spratt Multilocus sequence typing system for group B streptococcus. J. Clin. Microbiol. 41:

53 5. Kalia, A. and D. E. Bessen Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations. J. Bacteriol. 186: Kalia, A., M. C. Enright, B. G. Spratt, and D. E. Bessen Directional gene movement from human-pathogenic to commensal-like streptococci. Infect. Immun. 69: Khan, I. U., A. A. Hassan, A. Abdulmawjood, C. Lammler, W. Wolter, and M. Zschock Identification and epidemiological characterization of Streptococcus uberis isolated from bovine mastitis using conventional and molecular methods. J. Vet. Sci. 4: King, S. J., J. A. Leigh, P. J. Heath, I. Luque, C. Tarradas, C. G. Dowson, and A. M. Whatmore Development of a Multilocus Sequence Typing Scheme for the Pig Pathogen Streptococcus suis: Identification of Virulent Clones and Potential Capsular Serotype Exchange. J. Clin. Microbiol. 40: Leigh, J. A., J. M. Finch, T. R. Field, N. C. Real, A. Winter, A. W. Walton, and S. M. Hodgkinson Vaccination with the plasminogen activator from Streptococcus uberis induces an inhibitory response and protects against experimental infection in the dairy cow. Vaccine 17: McDougall, S., T. J. Parkinson, M. Leyland, F. M. Anniss, and S. G. Fenwick Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk. J. Dairy Sci. 87: Oliver, S. P., B. E. Gillespie, and B. M. Jayarao Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting. FEMS Microbiol. Lett. 160: Perez-Casal, J., T. Prysliak, and A. A. Potter A GapC chimera retains the properties of the Streptococcus uberis wild-type GapC protein. Protein Expr. Purif. 33: Zadoks, R. N., B. E. Gillespie, H. W. Barkema, O. C. Sampimon, S. P. Oliver, and Y. H. Schukken Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol. Infect. 130:

54 REAL-TIME PCR IN MILK FOOD SAFETY IN TIMES OF WAR AND PEACE Jeffrey S. Karns Environmental Microbial Safety Lab USDA/ARS, Beltsville, MD In October 2001 the USA was rocked by a terrorist attack in which spores of Bacillus anthracis were distributed through the U. S. Postal system, killing five people due to inhalation anthrax, causing cutaneous anthrax in several others, and causing a fair degree of economic disruption. It instantly became clear that, as a nation, we were unprepared to deal with such an unconscionable act, lacking (at least in the civilian arena) the necessary methods for quick, reliable detection of agents that might be used for biological terror in a large number of samples. Our laboratory responded to the needs of the USDA and several other Federal Agencies in the Washington, D.C., area by setting up a mobile laboratory in which real-time PCR assays were used to screen over 4,500 environmental samples for the presence of B. anthracis. None of the PCR reactions run on DNA extracted directly from the air or surface swabs were positive. Tests with spiked samples indicated that several thousand spores would have to be present in the samples to be detected by PCR, partly due to the small volume of sample added to the PCR reaction and partly because of the dirty condition of the samples. In this case, the real value of the real time PCR assay was rapid and positive identification of suspect colonies as B. anthracis, saving days of confirmatory culture and serological testing. Analysis of possible methods that bioterrorists might use indicated that the nation s food supply might be a means for the delivery of agents to a susceptible public. Milk might be a target for B. anthracis contamination because there are several stages in the production process at which the spores might be added and because the spores would survive the pasteurization process. Although gastrointestinal anthrax is less deadly than inhalation anthrax, such an attack would likely shatter public confidence in the milk supply. We were able to use a commercial kit for realtime PCR detection of B. anthracis to detect 8 spores per reaction (2,500 spores/ml milk). The spores tended to concentrate in the cream fraction when the milk was subjected to low-speed centrifugation, an observation that may be exploited in future experiments to lower the limits of detection of the organism. In other studies we have shown that real-time PCR combined with enrichment culture can be used to detect low levels of Salmonella and pathogenic forms of Escherichia coli in bulk-tank milk. When a commercial kit was used to analyze samples taken as part of the NAHMS 2002 dairy survey, Salmonella contamination was indicated in 101 of 854 samples tested (12%). Conventional enrichment culture followed by plating on selective agar yielded Salmonella in only 26 (3%) of the samples (and 6 of those only upon re-culture after obtaining the PCR results). When the samples were examined for the presence of E. coli using a real-time PCR assay specific for the lacz gene, 826 out of 859 samples were positive (96%). A real-time PCR assay to detect the eaea gene (encoding intimin) indicated that this virulence factor was present in 199 samples (23%). These 199 samples were then tested for the presence of the allele of the tir gene (encoding the translocated intimin receptor) associated with E. coli O157:H7 and 61 were found to be positive (7% of total milk samples). Further examination of these 61 samples with a real-time PCR assay for the detection of rfbo 157 (associated with the production of the O157 antigen) showed that 5 samples potentially contained pathogenic E. coli O157. Culture of the preserved enrichments from these 5 samples resulted in the isolation of a strain of O157 from one sample

55 Real-time PCR assays of the E. coli enrichments also showed that shiga toxin genes (stx1 and stx2) were present in 69 samples (8% of total milk samples). In summary, real-time PCR offers a quick and sensitive alternative to conventional culture techniques for the analysis of the loads of pathogenic bacteria in milk and dairy products. One advantage of real-time PCR over conventional PCR is the time savings gained by eliminating the need to run gels to visualize PCR products. A second advantage is that real-time PCR can be used in a quantitative fashion to obtain information on the actual number of target bacteria in a sample. As the genomes of more pathogenic bacteria are sequenced, providing the information needed to design real-time PCR assays to detect those organisms, we will be able to design the assays needed to assure a safe food supply. Related Publications Gagliardi, J. G. and Karns, J. S Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management practices. Appl. Environ. Microbiol. 66: Shelton, D. R. and Karns, J. S Quantitative detection of Escherichia coli O157 in surface waters using immunomagnetic-electrochemiluminescence (IM-ECL). Applied and Environmental Microbiology 67: Higgins, J.A., M.C. Jenkins, D.R. Shelton, R. Fayer and J.S. Karns Rapid Extraction of DNA from Escherichia coli and Cryptosporidium parvum for Use in PCR. Applied and Environmental Microbiology 67: Gagliardi, J. V. and J.S. Karns Persistence of E. coli O157:H7 in soil and on plant roots. Environmental Microbiology 4: Higgins, J.A., S. Nasarabadi, J.S. Karns, D.R. Shelton, M. Cooper, A. Gbakima, and R.P. Koopman A handheld real-time thermal cycler for bacterial pathogen detection. Biosen. Bioelect. 18: Higgins, J.A., Cooper, M., Schroeder-Tucker, L., Black, S., Miller, D., Karns, J.S., Manthey, E., Breeze, R., and Perdue, M.L A Field Investigation of Bacillus anthracis Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October Appl. Environ. Microbiol. 69: Van Kessel, J. S., Karns, J. S. and Perdue M. L Using portable real-time polymerase chain reaction (RT-PCR) assays to detect Salmonella spp. in raw milk. Journal of Food Protection 66: Perdue, M. L., Karns, J. S., Higgins, J. and Van Kessel, J. S Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk. Journal of Food Protection 66: Shelton, D.R., Van Kessel, J. S., Wachtel, M. R., Belt, K. T. and Karns, J. S Evaluation of parameters affecting detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence. Journal of Microbiological Methods 55:

56 Shelton, D. R., Pachepsky, Y. A., Sadeghi, A. M., Stoudt, W. L., Karns, J. S. and Gburek, W. J Release Rates of Manure-borne Coliform Bacteria from Data on Leaching through Stony Soil. Vadose Zone Journal 2: Van Kessel, J.A.S., Karns, J.S. Gorski, L. McCluskey, B.J. and Perdue M.L Prevalence of Salmonellae, Listeria monocytogenes and Fecal Coliforms in Bulk Tank Milk on U.S. Dairies. Journal of Dairy Science (accepted with revision April 2003). Shelton, D.R., Higgins, J.A., Van Kessel, J.A.S.,Pachepsky, Y., Belt, K. and Karns, J.S Estimation of Viable Escherichia coli O157 In Surface Waters Using Enrichment in Conjunction with Immunological Detection. Journal of Microbiological Methods (Accepted April 2004)

57 PAST, PRESENT AND FUTURE APPLICATIONS OF BULK TANK MILK ANALYSIS TO ASSESS MILK QUALITY AND HERD HEALTH STATUS Bhushan Jayarao Department of Veterinary Science The Pennsylvania State University, University Park, PA Introduction In the United States by late 1950s, collection and storage of farm raw milk gradually shifted from cans to refrigerated bulk tanks (Thomas et al., 1971). With this change the concept of using raw milk in the bulk tank to assess milk quality and mastitis pathogens emerged. Over time, our knowledge on the bacteriology of raw milk, mastitis, and farm management have greatly improved. This coupled with rapid advancement in the areas of biochemical, microbiological and molecular methods has allowed us to formulate strategies to improve milk quality and reduce the incidence of mastitis on dairy herds. Bulk tank is an ideal site to sample, when sampled correctly serves as a unique representative sample of all the cows that were milked prior to sample collection (Jayarao et al., 2004). This paper attempts to provide an overview of different microbiological and molecular assays that have been developed around using BTM, and potential diagnostic tests that could be developed to assess milk quality and herd health status using BTM. Historical perspective on BTM analysis In the early 1960s several studies were conducted to determine the number of somatic cells and the type mastitis pathogens present in BTM. There was a general agreement that analysis of BTM samples provided a fairly accurate picture of mastitis at the herd level (Gray and Schalm, 1960; Spencer and Simon, 1960; and Frank and Pounden, 1963). Around the same time, several researchers utilized BTM samples to establish the relationship between farm management practices and milk quality. Jackson and Clegg (1966) showed that when the bacterial count was less than 20,000 cfu/ml, micrococcus was the predominant organism. With the increase in the bacterial count, gram negative rods and streptococcal organisms usually increased in BTM. The microflora varied considerably between BTM samples collected from different farms. A multi provincial collaborative project was conducted in Canada by Morse et al. (1968a). They reported that BTM bacterial counts ranged from 690 to15,500 cfu/ml. They were of the opinion that improperly washed and dried udders and milk house water with high bacterial counts could lead to high bacterial counts in BTM. In a subsequent study, Morse et al (1968b) observed that high preliminary incubation counts in BTM samples was as result of poor sanitizing procedures and unsatisfactory bulk tank conditions. The use of BTM to identify mastitis pathogens began in California in the late 1970s. Jasper et al. (1979) conducted an extensive study to determine the prevalence of Mycoplasma bovine mastitis in California. Nearly 4% of all dairy herds surveyed had Mycoplasma of potential animal health significance in BTM. The findings of the study showed that BTM could be used for surveying herds with Mycoplasma. Meek and Barnum (1982) investigated the possibility of using BTM somatic cell counts to monitor the level of subclinical mastitis in dairy herds. The findings of the study showed that BTM somatic cell counts did not accurately depict the level of subclinical mastitis in dairy herds. The first comprehensive guide to interpret somatic cells, bacterial counts and mastitis pathogens in BTM was proposed Guterbock and Blackmer (1984). This report was followed by work done by Farnsworth (1993) in Minnesota with emphasis of using BTM culture results for interpreting mastitis and milk quality issues on dairy herds. Subsequent studies conducted over the last decade have shown that examination of BTM is useful for diagnosing multiple problems (current and potential) that might exist in a dairy herd

58 related to milk quality and mastitis pathogens (Godkin and Leslie, 1993; Bray and Shearer, 1996; Jayarao and Wolfgang, 2003; Jayarao et al., 2004). Current outlook on use of BTM for managing milk quality and udder health status The use of BTM analysis has received a lot of attention, especially from veterinarians and dairy health consultants who view milk quality and mastitis as an important part of their consulting service for their clients (Britten and Emerson, 1996; Keeter, 1997; Mickelson et al., 1998). Our knowledge on the bacteriology of raw milk, mastitis, and farm management practices related to milking and milk hygiene has increased considerably, making it possible to formulate strategies to improve milk quality and reduce the incidence of mastitis in dairy herds (Houghtby et al., 1992; Fenlon et al., 1995; Murphy, 1997). More recently, Jayarao et al. (2004) have developed microbiological guidelines for interpreting BTM analysis based on herd size and farm management practices. Review of BTM diagnostic tests Like other diagnostic applications, BTM analysis has its benefits and limitations. The likely circumstances that would augment and or impede BTM analysis and interpretation have been listed in Table 1. To date diagnostic tests reported in literature have focused on enumeration and or detection of; 1) somatic cells, 2) bacterial pathogens (foodborne, animal and zoonotic), 3) bacteria associated with lowering milk quality, 4) viruses of dairy cattle, 5) antibodies in milk against bacterial, viral and gastrointestinal parasites, and 6) substances such as antibiotics, metabolites, drugs, toxins and trace minerals. Diagnostic test that have been developed and used for BTM analysis are listed in Tables 2-5. Bacteria A large majority of the diagnostic tests used currently for detection of bacterial pathogens (foodborne, animal, zoonotic and mastitis) rely quite extensively on bacteriological methods. Use of selective media for enrichment and cultivation followed by rapid identification kits have now become a standard practice in diagnostic laboratories (Table 2). ELISA-based assays that detect antibodies against specific pathogens such as Salmonella, Mycobacterium avium subsp. paratuberculosis, and Coxiella brunetti have been developed. PCR-based assays are available for vast array of pathogens including foodborne, animal and of public health importance. Many of the initial PCR assays relied on pure bacterial cultures and as better DNA isolation techniques and purification matrices were made available, DNA isolation techniques were directly coupled to the PCR assay eliminating the need of culturing the organism. Among mastitis causing pathogens, PCR-based diagnostic test for Mycoplasma was first developed and tested and is now widely used by many diagnostic laboratories. Diagnostic tests based on BTM must be critically evaluated for its reliability, sensitivity, specificity and predictive values. A validated diagnostic test would permit proper interpretation of BTM milk analysis results before implementing changes on the farm. A constant criticism of many of the PCR assays is that information on their sensitivity and specificity are not made available. For example a test that can detect the organism consistently at 100 cfu/ml would be better than an assay that is able to detect the organism at10, 000 cfu/ml. In general, current PCR-based assays have a detection threshold of ,000 cfu/ml. However under circumstances when the number of the organisms is low (example <10 cfu/ml) the PCR assay would fail to detect the target DNA. To overcome this hurdle two unique approaches have been developed. The first approach is based on the use of short selective enrichment (4-12 h) coupled with DNA isolation and PCR assay. Kits that rely on this technique are now available commercially for Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7. The second approach employs selective concentration of the organism present in milk using

59 immunomagnetic beads. Organisms bound to the immunomagnetic beads are then subjected to culture and or PCR analysis. Immunomagnetic bead coupled PCR assays have been developed for many foodborne and animal pathogens such as Mycobacterium avium subsp. paratuberculosis. The concept of detecting multiple pathogens or multiple gene sequences for a given pathogen in a single PCR assay has received a lot of attention. To date there are several multiplex PCR assays have been developed for foodborne pathogens (Tables 2 and 3). As with uniplex PCR assays, multiplex assays have also to contend with optimization of primer concentration, PCR mixture, multiple DNA templates, and stringency of PCR reactions. However standardization of multiplex assay is much more time consuming and generally are less precise as compared to their respective uniplex assays. Recently, in our laboratory we have successfully developed a multiplex PCR assay to detect three contagious mastitis pathogens directly from milk. The sensitivity and specificity of the multiplex PCR for detection of M. bovis was 89%, 97%; S. aureus-67%, 94%; S. agalactiae- 83%, 84%, respectively (Tmanova, 2003). The Gastroenteric Disease Center at Penn State has developed a multiplex PCR assay for simultaneous detection of Salmonella, Listeria monocytogenes and Escherichia coli directly from raw milk. The sensitivity and specificity of the assay ranges from 89-98% and 95-99%, respectively (DebRoy and Jayarao, 2003, unpublished data). The foot and mouth disease in Britain in 2000 and the 9/11 terrorist act has accelerated the development of real time detection methods for potential agents of bio- and agro-terrorism. The Agricultural Research Service at Beltsville has developed real time PCR assays for Bacillus anthracis (Perdue et al., 2003) and Salmonella (Van Kessel et al., 2003) in milk. Khare et al. (2004) have developed a real time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis. A real time PCR assay with high sensitivity and specificity was developed by Sreevatsan et al. (2000) to detect Mycobacterium bovis and Brucella abortus simultaneously. Viruses BTM has been used for detection of antibodies to viruses including bovine viral diarrhea virus (BVDV), bovine leucosis virus, food and mouth disease virus, bovine herpes virus, bovine respiratory syncytial virus, and bovine corona virus using ELISA-based tests (Table 2). Use of BTM to detect BVDV has received considerable attention (Table 2). RT-PCR assay using BTM for detection of BVDV could become a standard test for screening herds for BVDV or prior to purchase of animals. Considerations for developing a diagnostic test using milk from bulk tank Three specific aspects have to be considered before developing a diagnostic test using BTM. They include; 1) the biology and epidemiology of the organism, 2) characteristics of the food matrix that hosts the organism, 3) the technology that will be used for detection, and 4) validation of the test under laboratory and field conditions. Biology and epidemiology of the organism The following factors need to be considered with regard to the biology and epidemiology of the organism: 1) likely pathways through which organisms can gain access into BTM, 2) frequency, type, number and distribution of the organisms in the milk phase, 3) viability of the organism under refrigeration temperatures, 4) ability of the target organism to compete for milk-based nutrients with other organisms and survive for extended period of time in milk, and 5) unique target molecule that can be used to detect the organism (number of targets, location, and factors associated with its expression)

60 Characteristics of the food matrix As compared to other food, milk may appear as a simple mixture of nutrients that is easy to collect, store, handle, process and analyze, in fact it is a complex matrix of nutrients. Milk favors growth and survival of many organisms and also protects the organisms by allowing them to attach to milk proteins and fat globules and evade isolation and detection. Further macrophages in milk have the ability to internalize bacteria such as Staphylococcus aureus, Streptococcus uberis, Salmonella spp., and Listeria monocytogenes. Once within the macrophage, some bacterial pathogens have developed cellular mechanisms to evade their destruction and even multiply within the macrophage. Detection system DNA- and biosensor-based methods are by far the most rapidly growing segment of molecular diagnostics. A detection system needs to take into account the biology of the organism and the characteristics of the food matrix with special reference to factors that would hinder the process of selecting the target molecule. Bickley et al. (1996) showed that calcium in milk could reduce the efficiency of DNA isolation and inhibitory substances in the DNA lysates prepared directly from milk could interfere with the PCR reaction. DNA-based test needs to be rigorously tested and optimized using experimental conditions that would mimic field samples. In other words, a PCR-based assay that is developed using experimentally inoculated milk sample would be more realistic that using pure cultures in broth samples to standardize and optimize the assay. Validation of a diagnostic test The true value of a diagnostic test can be determined after it has been tested using farm BTM or quarter milk samples. A diagnostic test that has been meticulously evaluated for its sensitivity, specificity and predictive values will give the diagnostician a better appreciation of the test (Martin and Meek, 1986). Future prospects of BTM-based diagnostics tests The use of BTM for monitoring herd health has continued to gain popularity and it is expected that many more tests, particularly PCR-based real time assays will be developed. Existing PCR assays will be adapted to real time assays. It in the near future, real time assays kits may become commercially available. Multiplex PCR assays may not gain popularity unless it has a very high level of sensitivity and specificity. There is a widespread notion that microbiological assays are the gold standard to which PCR-based assays need to be compared. This may prove to be one of the constraints for implementing PCR-based assays, as several studies have repeatedly shown that PCR-based assays detect a higher number of positive samples as compared to established culture techniques. Use of concordant analysis or other statistical methods can be used to address this discrepancy (Johnson et al., 2004; Puppe et al., 2004). It may soon be possible to tailor a set of BTM diagnostic assays based on the type investigation that needs to be done. For example, a module consisting of contagious mastitis pathogens, Mycobacterium paratuberculosis and BVDV may be used for screening animals on a herd or animals that are being considered for purchase. BTM analysis is ideally suited for herds that collect milk in a bulk tank. However on many large farms (> 500 cows in milk) one would expect to see more sophisticated milking operations in which cooled milk is directly pumped into a tank of a milk hauler truck. Sampling milk under such situations may be challenging. To overcome some of these issues, milk-line sampling devices have been developed and validated (Godden et al., 2002). One would expect more milk-line devices being employed for sampling milk for analysis. Milk-line sampling device could permit sequential analysis of milk samples collected over time during a milking session and may be valuable for troubleshooting problems related to contagious mastitis, particularly Mycoplasma mastitis. A diagnostic test is not of much value,

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67 Walker, R. L., H. Kinde, R. J. Anderson, and A. E. Brown Comparison of VIDAS enzyme-linked fluorescent immunoassay using Moore swab sampling and conventional culture method for Salmonella detection in bulk tank milk and in-line milk filters in California dairies. Int J Food Microbiol. 67: Warnick, L. D., J. B. Kaneene, P. L. Ruegg, S. J. Wells, C. Fossler, L. Halbert, and A. Campbell Evaluation of herd sampling for Salmonella isolation on midwest and northeast US dairy farms. Prev Vet Med. 60: Wedderkopp, A., U. Stroger, and P. Lind Salmonella Dublin in Danish dairy herds: frequency of change to positive serological status in bulk tank milk ELISA in relation to serostatus of neighbouring farms. Acta Vet Scand. 42: Whyte, P., K. McGill, D. Cowley, R. H. Madden, L. Moran, P. Scates, C. Carroll, A. O'Leary, S. Fanning, J. D. Collins, E. McNamara, J. E. Moore, and M. Cormican Occurrence of Campylobacter in retail foods in Ireland. Int J Food Microbiol. 95: Wichtel, J. J., G. P. Keefe, J. A. Van Leeuwen, E. Spangler, M. A. McNiven, and T. H. Ogilvie The selenium status of dairy herds in Prince Edward Island. Can Vet J. 45: Yang, C., Y. Jiang, K. Huang, C. Zhu, and Y. Yin Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water. FEMS Immunol Med Microbiol.38: Yang, L., Y. Li, C. L. Griffis, and M. G. Johnson Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella Typhimurium. Biosens Bioelectron. 19:

68 Table 1. Benefits and limitations of BTM analysis Benefits Provides a logical approach for troubleshooting herds with multiple milk quality and mastitis related problems. Limitations Does not provide information about milk quality and mastitis at individual cow level. Less expensive than quarter milk sampling the whole herd. Understanding milk quality and mastitis problems in a herd cannot be done effectively using a single BTM sample. BTM analysis can be done in about 96 hours. Information on herd management practices on milking cows, mastitis prevention, milk sanitation and general farm hygiene are required to interpret BTM analysis results. A reliable tool for veterinarians to troubleshoot milk quality and herd level mastitis. Proper interpretation of BTM milk analysis results is critical before implementing changes on the farm. An important component of total herd health management or veterinary practice consultancy services. BTM analysis report becomes documentary evidence of milk quality assurance protocol practiced on the farm. Non invasive technique, when collected properly serves a representative herd sample. Samples have to be analyzed within 36 hours of collection. Samples collected for somatic cell and milk quality cannot be frozen. Samples have to be held at 4 o C till analyzed. Milk is a complex matrix consisting of water, cells, proteins, fats, minerals, and carbohydrates. This inherent nature of the matrix could hinder the isolation of some types of bacteria and viruses that are intracellular in nature. Volume of milk in the bulk tank, the number of organisms and their relative distribution in the milk phase may influence the results of the BTM analysis. Milk held over period of time or subjected to temperature abuse may lead to overgrowth of certain types of bacteria. This could result in: 1) false picture on the bacterial flora in the sample, and 2) hinder in the isolation of organisms that are very few in number. A diagnostic test that has not been validated for its reliability, sensitivity, specificity and predictive value could be a serious limiting factor. Interpretation of results based on an un-validated test could lead to erroneous conclusions. Interpreting BTM analysis results for large herds may be difficult, unless multiple samples or string samples are collected and analyzed

69 Table 2. Use of BTM for detection of foodborne pathogens and pathogens of animal and public health importance. Pathogens in BTM Technique Comments Reference Bacillus anthracis Brucella abortus Campylobacter jejuni Campylobacter spp. Coxiella burnetii Escherichia coli Real time PCR Fluorescence polarization assay Real time PCR Bacterial Culture ELISA Multiplex PCR E. coli O157:H7 Bacterial culture Foodborne Pathogens Foodborne Pathogens Foodborne Pathogens Foodborne Pathogens Bacterial Culture Bacterial Culture Bacterial Culture Bacterial Culture Pasteurization of milk inoculated with B. anthracis spores (2 separate occasions) did not kill all of the spores. The assay had a sensitivity and specificity of 100 and 95.9%, respectively. Surveillance and eradication of B. abortus would be more cost-effective using this test. The whole assay was completed in 60 min with a detection limit of approximately 1 CFU/ml. 27.3% (82/300) of milk samples were positive for C. jejuni. 1.6% BTM samples were positive for Campylobacter. 21% of BTM samples showed serological evidence of C. burnetii infection. A multiplex PCR allowed detection of pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxinproducing E. coli. 2 of 268 (0.75%) BTM samples positive for E. coli O157:H7. Campylobacter jejuni, Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica were detected in 12.3, 4.1, 8.9, and 15.1 % of BTM samples, respectively. Campylobacter jejuni, shiga toxin producing E. coli Listeria monocytogenes, and Salmonella spp., were detected in 0.47, 0.87, 2.7, and 0.17 % of BTM samples, respectively. Campylobacter jejuni, shiga-toxin producing Escherichia coli, Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica were detected in 9.2, 3.8, 4.6, 6.1, and 6.1% of bulk tank milk samples, respectively. Salmonella (n=9 serotypes) was detected in 2.6% of BTM samples. Listeria monocytogenes (n=5 serotypes) was detected in 6.5% of BTM samples. Perdue et al. (2003) Gall et al. (2002) Yang et al. (2003) Whyte et al. (2004) Paiba et al. (1999) Bottero et al. (2004) Murinda et al. (2002) Rohrbach et al. (1992) Steele et al. (1997) Jayarao and Henning (1999) Van Kessel et al. (2004)

70 Table 2. continued Pathogens in BTM Technique Comments Reference Listeria monocytogenes Salmonella Dublin Salmonella spp. Salmonella spp. Salmonella spp. Salmonella spp. Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Mycobacterium bovis Brucella abortus Salmonella and Listeria monocytogenes Bacterial Culture Indirect ELISA VIDAS Salmonella assay Bacterial Culture Bacterial Culture Real Time PCR ELISA Bacterial Culture Bacterial Culture Multiplex PCR assay Multiplex PCR assay Immunomagnetic separation - Multiplex PCR assay (IMS-mPCR) Farm milk contamination with L. monocytogenes was a sporadic event. Low numbers of L. monocytogenes in milk could be due to environmental contamination. Elisa titer values can be used to predict seroconversion status in a herd and for a geographical region VIDAS Salmonella assay using a modified sampling method compared favorably to the conventional culture method. Six of 268 (2.24%) of BTM positive for Salmonella. 1.1% of BTM samples positive for Salmonella. 12.6% milk line filters positive for Salmonella. Real-time PCR system allowed detection of Salmonella in raw milk. The combination of enrichment and real-time PCR techniques yielded results in 24 h. The ELISA test had a sensitivity and specificity of 90%, 97% respectively. S. aureus was isolated from 183 of 300 raw milk samples at a Milk Cooperative in Kenya. 72 of 97 (74.2%) of the isolates produced one or more enterotoxins. The mean counts of S. aureus in BTM ranged from 5,900 to 12,000 cfu/ml. 45 of 105 (42.9%) isolates produced Staphylococcal enterotoxin A, B, C, D or a combination of these toxins. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk. The assay allowed presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h. The multiplex assay provides a highly sensitive, cost effective and economically viable alternative to serological testing. IMS-mPCR was completed within 7 h. A detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens. Meyer-Broseta (2003) Wedderkopp et al. (2001) Walker et al. (2001) Murinda et al. (2002). Warnick et al. (2003) Van Kessel et al. (2003) Grove and Jones (1992) Ombui et al. (1992) Adesiyun et al. (1998) Tamarapu et al. (2001) Sreevatsan et al. (2000) Li et al. (2000)

71 Table 2. continued Parasites and Pathogens Ostertagia ostertagi Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis Mycobacterium avium subsp. paratuberculosis Technique Comments Reference ELISA IS900 PCR assay IS900 PCR assay Immunomagnetic separation - PCR assay (IMS-PCR) Bacterial Culture PCR assay ELISA Immunomagnetic separation real time PCR assay (IMS-rPCR) Indirect ELISA using an O. ostertagi crude antigen was a useful technique for monitoring gastrointestinal parasite burdens in adult dairy cows and perhaps could be used as a potential predictor of response to anthelmintic treatment. 273 (19.7%) of the 1384 BTM samples were positive by IS900 PCR-assay. The prevalence of M. paratuberculosis varied considerably for different regions of Switzerland. M. paratuberculosis can be detected directly from quarter milk and bulk tank milk by IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). Milk samples all cultured negative, but analysis of milk samples by PCR resulted in 68% of herds positive for M. paratuberculosis DNA including 24 of 31 herds with positive fecal cultures and 11 of 21 herds with negative fecal cultures. Contamination of bulk tank milk samples with M. paratuberculosis does occur in seropositive herds, even in some with negative fecal cultures. The technical performance of the ELISA was not sufficient to provide a tool for surveillance. IMS-rPCR allowed detection of M. paratuberculosis from milk. Ten or fewer M. paratuberculosis organisms were consistently detected in milk (2-ml). Sanchez et al (2002) Corti and Stephan (2002) Pillai and Jayarao (2002) Grant et al (2000) Stabel et al (2002) Nielsen et al (2000) Khare et al (2004)

72 Table 3. Use of BTM for determination of milk quality and herd udder health Pathogens in BTM Technique Comments Reference S. agalactiae Bacterial culture BTM milk samples need to be analyzed frequently to identify herds with S. agalactiae infection. Andersen et al. (2003) Mycoplasma Nested PCR assay 100% sensitivity; 99.8% specificity. Baird et al. (1999) Bacterial and somatic cell count (SCC) estimation of BTM when interpreted within Milk Quality and Herd the context of the farm's management Jayarao and Bacterial Culture Udder health practices provided a basis for evaluating Wolfgang (2003) current and potential milk quality and mastitis problems in a herd. Milk quality components were not strongly related to the presence of Mycoplasma spp. Mycoplasma Bacterial Culture in BTM. The presence of other contagious mastitis pathogens was also not related to Fox et al. (2003) Streptococci and Streptococci like organisms Bacterial counts Trichosporon beigelii Staphylococcus aureus Staphylococcus aureus Bacterial Culture Bacterial Culture Culture RPLA ELISA Culture Culture ELISA the presence of Mycoplasma. Edwards modified medium supplemented with colistin sulfate (5 mg/l) and oxolinic acid (2.5 mg/l) was evaluated using BTM samples, the sensitivity and specificity of this medium was observed to be 100 and 87.5%, respectively. The bacterial composition of BTM milk (n= 13 farms) was tested over a 2-wk period to study sudden elevations ("spikes") in the total bacterial count. Twenty standard plate count spikes were observed. S. uberis was the predominant organism in 11 spikes. T beigelii could be associated with high counts in BTM. TSST was detected in 25 of 43(58.1%) isolates from clinical mastitic cow s milk, 79 of 103(76.7%) isolates from subclinical mastistic cow s milk and 85 of 126(67.4%) of farm bulk milk. 37.5% of the isolates of mastitic samples isolates were S. aureus, none of these isolates encoded for enterotoxins or TSST of 13 isolates and 6 of 20 isolates from Washington State and Korea expressed enterotoxins, respectively. Isolates from different geographical locations varied considerably. Sawant et al. (2002) Hayes et al. (2001) Gonzalez et al. (2001) Takeuchi et al. (1998) Lee et al. (1998)

73 Table 3. continued Mastitis pathogens and milk quality indicators Somatic Cells Antibiotic Residues Polymorphonuclear Neutrophils (PMN) Gram Negative Bacteria Mycoplasma spp. Bacterial Counts Prototheca zopfii Staphylococcus aureus Streptococcus agalactiae Streptococcus uberis Streptococcus dysgalactiae Detection of Gram + / - Bacteria Staphylococcus aureus Streptococcus agalactiae Mycoplasma Technique Comments Reference Epidemiological analysis of monthly official state regulatory data IR analysis Bacterial Culture Bacterial Culture Bacterial Culture Bacterial Culture Multiplex PCR Flow cytometry Multiplex PCR SCC values were significantly higher for samples with positive antibiotic residue tests for grade A milk during all 4 yr tested. The SCC values were significantly higher for samples with positive antibiotic residue tests for grade B milk for 3 of 4 yr. the concentration of PMN may be a useful indicator of herd status in bulk tank monitoring schemes. Examination of BTM for coliforms and non-coliform bacteria could provide an indication of current and potential problems associated with milk quality Mycoplasma bovis (243/499; 48.6%) was the most commonly isolated species. Distribution of Mycoplasma spp. varied by year, number of colonies isolated per sample, season, and herd. Bacteriological counts increased at each stage as the milk passed through the milking machine. A strong correlation (0.98) between total and streptococcal counts of the bulk milk was observed. New selective Prototheca enrichment method was developed. Prototheca spp. were recovered from 28 of 787 BTM samples. Regular analysis of BTM using the multiplex PCR assay could be a useful tool for monitoring S. agalactiae, but was of less value for monitoring other organisms. BTM inoculated with Staphylococcus aureus and Escherichia coli can be detected using flow cytometry without precultivation. The sensitivity and specificity of the multiplex PCR for detection of M. bovis was 89%, 97%; S. aureus-67%, 94%; S. agalactiae- 83%, 84%, respectively. Ruegg and Tabone (2001). Kelly et al. (2000) Jayarao and Wang (1999) Kirk et al. (1997) McKinnon et al. (1990) Pore et al. (1987) Phuektes et al. (2003) Holm and Jespersen (2003) Tmanova Lyubov (2003). MS Thesis The Pennsylvania State University

74 Table 4. Use of BTM for detection of viruses of dairy cattle Viruses in BTM Technique Comments Reference Bovine Viral Diarrhea Virus Bovine Leukosis Virus Bovine Leukosis Virus Foot and Mouth Disease Virus Bovine Viral Diarrhea Virus Bovine Herpes Virus-1 Bovine Viral Diarrhea Virus Bovine Viral Diarrhea Virus Bovine Herpes Virus 1(BHV-1) Bovine Respiratory Syncytial Virus (BRSV) Bovine Corona Virus (BCV) Bovine Viral Diarrhea Virus Indirect ELISA ELISA ELISA Liquid phase blocking ELISA RT-PCR assay ELISA Virus isolation RT-PCR ELISA PCR assay Virus isolation The proportion of positive herds in Chile ranged from 71.2 to 83%. BTM samples were tested for BLV for the purpose of eradication and monitoring of BLV in Finland. Examination of pooled milk samples with the ELISA provided a reliable, practical, and economic procedure for identification of BLV-infected herds. Significant overall correlation (R=0.53; n=624) was obtained between serum titers and milk IgG (1) results derived from the modified specific isotype assay (SIA). RT-PCR had high sensitivity and can be used as an alternate technique to current standard methods for detecting BVDV from BTM. The BHV-1 blocking ELISA on BTM allowed detection of seropositive herds. RT-PCR was superior to virus isolation with respect to sensitivity, specificity, and turnaround time. Both methods are recommended for successful detection of the virus. 65 % of the herds had a high level of BTM antibody suggestive of recent infection with BVDV 69% of the herds were BHV-1 antibody-positive and all the herds tested were antibody positive to BRSV and BCV. PCR assay was 14.6 times more sensitive than virus isolation in detecting BVDV RNA in purified milk somatic cells. BVDV RNA was detected in 33 of 136 BTM samples. Melendez and Donovan (2003) Nuotio et al. (2003) Gutierrez et al. (2001) Armstrong and Matthew (2001) Scheibner et al. (2000) Nylin et al. (1999) Renshaw et al. (2000) Paton et al. (2000) Radwan et al. (1995)

75 Table 5. Use of BTM for detection of trace minerals, toxins, off flavors and metabolites. Analytes and Devices Technique Comments Reference Selenium Selenium concentration in BTM Atomic Absorption could be related to levels of SE in Spectophotometry feed. Wichtel et al. (2004) Aflatoxin M (1) 78% of BTM samples had >5ng/L Liquid aflatoxin M (1). None of the samples Chromatography exceeded EU limit of 50ng/L. Roussi et al. (2002). Bulk Tank Milk Urea Nitrogen (BTMUN) Off-flavors Salmonella Typhimurium Beta-lactams Progesterone Staphylococcal enterotoxin B Infra red analysis Organoleptic analysis Microelectrode impedance sensor Surface plasmon resonance (SPR) biosensor Surface plasmon resonance (SPR) biosensor Surface plasmon resonance (SPR) biosensor The BTMUN had good correlation with weighted average of the individual cow MUN levels (CC= 0.91). Poor air quality in the lactating cows barn, baled silage as the main forage, as well as feeding roughage before milking was significantly associated with the incidence of transmitted flavors in BTM. The test could detect 4.8 and 5.4 x 10 5 CFU/ml of Salmonella in 9.3 and 2.2 h, respectively. The technique allowed detection of 1 bacterial cell/sample. The results of the 2 biosensor assays showed good agreement with those of the other assays including Delvotest SP, Penzym S, Beta- STAR, SNAP, and Parallux. The assay could be used in-line in the milk parlor and could be an important tool for reproductive management for detecting heat and predict pregnancy in cattle. The assay showed that SPR biosensor may be a useful tool for real-time analysis of toxin in foods. Arunvipas et al. (2004) Mounchili et al. (2004) Yang et al. (2004) Gustavasson and Sternesjo (2004) Gillis et al. (2002) Rasooly (2001)

76 PCR APPLICATIONS IN FOOD SAFETY RESEARCH S. P. Oliver and B. E. Gillespie Food Safety Center of Excellence and the Department of Animal Science The University of Tennessee, Knoxville, TN Introduction Molecular techniques such as the polymerase chain reaction (PCR), multiplex PCR, and realtime PCR are very useful tools used frequently in many research laboratories in the United States and throughout the world. Use of PCR-based techniques has facilitated the discovery of more effective methods for the detection of foodborne pathogens associated with food-producing animal environments and foodborne pathogens causing disease in humans. These techniques have also been quite useful to delineate virulence factors as well as antimicrobial resistance genes of important foodborne pathogens. The purpose of this communication is to briefly describe some of the PCR techniques in use today, and to demonstrate how these molecular-based techniques are used in our research approach on food safety and foodborne pathogens at The University of Tennessee Food Safety Research Center of Excellence. PCR The PCR method is based on in vitro amplification of target DNA sequences and involves the application of primers (carefully selected oligonucleotides) and heat stable Taq DNA polymerase. With PCR, it is theoretically possible to detect pathogens in the sample (food or environmental) directly but so far this has been done in very few investigations. Major disadvantages of this technique are the inability to distinguish between live and dead cells, the presence of polymerase inhibitors in test samples, and the inability of the method to facilitate further identification. Preenrichment of test samples overcomes most of these problems and is presently needed for detection of specific pathogens in food by PCR. Conventional PCR formats are limited in that they only provide for detection of a single pathogen. However, many clinical diseases manifest in a nonspecific or syndromic fashion thereby necessitating the simultaneous assessment of multiple suspect pathogens. Multiplex PCR Multiplex PCR can be used for specific identification and profiling of several gene sequences from the same pathogen or from a mixture of pathogens simultaneously. The major advantage of multiplex PCR over conventional PCR methods is its cost-effectiveness. It reduces the amounts of reagents such as Taq DNA polymerase used in each assay. It is more applicable to routine diagnostic use and automation and requires less preparation and analysis time than systems in which several pathogens are analyzed individually. In many cases, more than 4 pairs of primers can be used. Real-Time PCR Several types of fluorogenic probes have been used to quantitate bacteria in real-time such as TaqMan and molecular beacons (MBs). TaqMan differs from MBs in that it has no hairpin structure and exploits the 5-3 nucleolytic activity of Taq DNA polymerase to cleave a reporter

77 dye, such as fluorescein, from the 5 end of a labeled linear probe that has hybridized downstream from the forward PCR primer. When this cleavage occurs, the reporter dye is separated from the quencher and fluorescence occurs. Common reporters (5 end) for TaqMan are 6- carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein, or hexachloro-6-carboxyfluorescein, with the quencher dye 6-carboxytetramethylrhodamine (TAMRA) attached 2 or more bases downstream from the reporter. The proximity of the reporter and quencher reduces emission intensity of the reporter until the reporter is cleaved. Molecular beacons (MBs) are hair-pin oligonucleotide probes that fluoresce upon hybridization with complimentary sequences. MBs have been used to construct probes that are useful for realtime detection of nucleic acids of target pathogens. MB probes are based on single-stranded nucleic acid molecules that possess a stem-and-loop structure. The loop portion contains a sequence complimentary to a target gene sequence; the stem is formed by annealing of two complimentary arm sequences that are not related to the target gene sequence. A fluorescent moiety is attached to the end of one arm and a non-fluorescent quenching moiety is attached to the other end. When the two moieties are close together no fluorescence is produced due to the quenching effect. However, when the probe is in proximity to a single stranded target oligonucleotide, it hybridizes with the target and undergoes a spontaneous conformational change that forces the moieties apart resulting in fluorescence. The interaction of TaqMan probes and MBs with their targets is extraordinarily specific. As target strands synthesized in PCR accumulate, the fraction probe bound to targets increases causing a brighter fluorescence. Measurement of fluorescence permits simultaneous quantitation and monitoring progress of the reaction in real-time in the PCR tube. MBs are more sensitive than TaqMan since they can distinguish single base differences thus allowing multiple allele discrimination in the same reaction. TaqMan has been applied to detect food pathogens like Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7. Recently, several studies have successfully applied MBs in a variety of PCR reactions for study of pathogens like enterohemorrhagic E. coli and Salmonella and as few as 2 colony forming units (CFU) can be detected with MB-based PCR assays. Analysis of fluorescence data recorded at each annealing stage using real-time PCR gives a clear profile of the amplification process. The critical cycle (Ct), which is the cycle at which a significant increase in fluorescence is first recorded, increases as the initial number of template DNA molecules decreases. Samples containing low concentrations of template DNA would require more PCR cycles to replicate enough copies to produce a significant fluorescence signal. The Ct is inversely proportional to the logarithm of the initial number of target molecules. These data can be used to formulate a standard quantitation curve for detection of a specific pathogen. Detection of Foodborne Pathogens and Virulence Factor Genes Using Different PCR Formats The need for rapid, sensitive and reproducible techniques for bacterial strain identification is evident in many areas of public health, agriculture, and national security. Bacterial detection methods for differentiating bacterial species and strains are based on both phenotype and genotype. Techniques based on phenotype, such as metabolic studies, serotyping and immunological methods, are not specific enough to completely distinguish among different genera, species and strains of bacteria and are not general enough to apply to a diverse set of pathogens. Additionally, genes may not be expressed under certain cultural conditions. Methods based on the genotype examine differences in DNA sequences and are much more successful in

78 discriminating among different bacterial strains. The most definitive methods in use for bacterial subtyping are restriction fragment length polymorphism (RFLP), PCR-based methods and ribotyping. PCR-based methods require only small quantities of DNA, whereas RFLP requires relatively large amounts of DNA. Unique bacterial DNA-sequences (chromosomal and/or plasmidal) can be used for detection to the genus and in many cases to the species level. Current methods used for routine identification and confirmation of foodborne pathogens such as Campylobacter, Salmonella spp., Listeria monocytogenes and Shiga toxin-producing E. coli (STEC; O157 and non-o157 STEC) are generally slow, inadequate, laborious and non-existent in the case of critical detection of pathogens like non-o157 STEC. Many clinical laboratories do not routinely report non-o157 STEC serotypes since they cannot easily identify these microorganisms. Conventional diagnostic methods are often too cumbersome and timeconsuming to be useful for timely monitoring of foods, especially those with limited shelf-lives. Rapid detection methods could be used effectively for quality control in food processing facilities to rapidly screen incoming ingredients and raw materials. Rapid detection methods allow: (1) timely monitoring of food processing equipment and the immediate environment, (2) brisk corrective action (product recall and release of lots/batches of product for distribution), and (3) faster intervention in the case of threats of disease or potential death, without having to wait several days for results, as in the case of most current microbiological methods. Shiga toxin-producing E. coli are of immense economic and public health significance. STEC O157:H7 are characterized by low infectious doses (1-100 colony-forming units) and are highly pathogenic in humans where they cause serious acute illness and long-term sequelae. Manifestations of illnesses caused by STEC that are linked to production of Shiga toxins include, non-bloody diarrhea, diarrhea-associated hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. Intimin and enterohemolysin are among the prominent ancillary virulence factors elaborated by STEC. There is a general consensus that ruminants are the main source of human pathogenic STEC. An array of food products that include, beef, apple cider, salad, fruits and contaminated well water have been implicated in foodborne disease involving STEC. Sufficient evidence has been presented on the zoonotic nature of bacterial enteric pathogens and the role companion animals play as reservoirs of some human pathogenic STEC serotypes. We used a multiplex PCR format to identify E. coli O157:H7 strains that target common virulence genes encoding Shiga toxin 1 and 2 (stx1 and stx2), enterohemolysin (hly 933 ), intimin (eaea), and flagellar H7 (flicc h7 ) gene sequences (Murinda et al., 2002; 2004a; 2004b; 2004d). The objective of this study was to characterize 400 E. coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease and food products for prevalence of STEC virulence markers. The rationale of the study was that isolates of the same serotype that were obtained from different sources and possessed the same marker profiles could be cross-species transmissible. Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2) and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle and food) isolates were stx-positive. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx-positive. Five of 6 stx-positive O26 and O111 produced Stx1, however, stx-positive O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-o157 stx-positive strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n=105), and pigs, dogs, parrot and iguanas (n=48) were

79 stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle. We have also used a multiplex PCR format to confirm and identify Campylobacter jejuni isolated from the dairy farm environment and from dairy cows (Murinda et al., 2004c). Campylobacter is a leading cause of bacterial foodborne illness in the USA and in many other industrialized countries. This organism is widespread in nature and can be isolated from the gastrointestinal tracts of many animal species, including poultry, freshwater and bulk tank milk. The ubiquity and low infectious dose of Campylobacter makes its presence in the food supply a significant health hazard. It is therefore important to have accurate and reliable methods for isolation and detection of Campylobacter spp. in particular C. jejuni, which is the most common species associated with acute bacterial enteritis. The major disadvantages of the commonly used phenotype-based typing schemes, such as biochemical tests, including serology, are that they are time-consuming, technically demanding and may lead to a high number of untypable strains. Consequently, there is an increasing need for highly sensitive and reliable DNA-based methods for typing C. jejuni. Targets that we used for identification of C. jejuni were the hippuricase gene (hip) and a 23S rrna gene specific for thermophilic Campylobacter. All 265 bulk tank milk samples analyzed were negative for C. jejuni, whereas, 5 of 411 (1.2%) fecal samples tested positive. This is the first report that has used a combination of sequences of the two genes in a multiplex format to identify C. jejuni to the species level. The method described has potential for routine use in the detection of thermophilic Campylobacter in farm environmental samples as well as other samples. This multiplex PCR assay can decrease the time for identification and confirmation of C. jejuni. PCR-ELISA Molecular techniques can also be utilized to serogroup bacterial isolates. We used a polymerase chain reaction-based enzyme linked immunosorbent assay (PCR-ELISA) to identify Salmonella somatic groups B, C1, C2, D and E1 (Gillespie et al., 2003). Salmonella are important foodborne pathogens that are responsible for serious cases of foodborne illness. Salmonella may be transmitted by a wide variety of agricultural products and processed foods. Foods of animal origin such as beef, pork, chicken, eggs, and milk have been shown to carry these pathogens. Salmonellosis is commonly diagnosed in dairy cows and calves, and the presence of Salmonella on dairy farms has been well documented. Several serogroups of this bacterium occur with varying degrees of relevance to human and animal health. Identification of Salmonella is important for surveillance, prevention, and control of foodborne diseases. An accurate and rapid procedure for identification of Salmonella is needed to identify sources, reservoirs, and transfer of these foodborne pathogens through the food chain. However, there are many problems associated with differentiating Salmonella species, subspecies and serovars. Current available screening tests only provide presumptive identification of Salmonella as a group without identification of serogroups. Negative results are considered definitive, but positive results must be confirmed by conventional methods and serology. The concept of targeting gene sequences that encode for species specificity is promising. In Salmonella, the rfb gene clusters are responsible for biosynthesis of the O antigens of Salmonella lipopolysaccharide. Variations among different O antigen structures are manifested in the types of sugar present or arrangement of sugars. This variability provides the basis for serotyping Salmonella into serogroups. This highly polymorphic rfb gene cluster has been targeted as a

80 molecular marker for the organism for detection of Salmonella serovars. In our study (Gillespie et al., 2003), a PCR-ELISA procedure was developed to identify Salmonella serogroups A, B, C1, C2 and D. Primers were selected from the rfb gene cluster, which is responsible for biosynthesis of O antigens of Salmonella lipopolysaccharide. Previously serogrouped Salmonella isolates (n=169) were evaluated by the PCR-ELISA procedure. DNA from all isolates was amplified using the PCR procedure for selected somatic groups and subjected to the ELISA procedure. This technique correctly identified 93% of Salmonella isolates belonging to somatic groups B, C1, C2, D and E1. The sensitivity of this procedure to correctly identify Salmonella somatic groups was 96% and the specificity was 98%. Utilization of this procedure circumvents the need to have Salmonella isolates serogrouped state or regional reference laboratories. Real-Time PCR We have dedicated much time and many resources attempting to develop real-time PCR techniques for detecting pathogens directly from milk. Real-time PCR is a relatively new DNAbased technique that monitors amplification of target DNA in real-time by monitoring florescence. Real-time PCR can be used to quantify bacteria from various samples including milk, feces, food and water. Real-time PCR can be used for processing, detecting and confirming pathogens in multiple samples at one time in a 96-well plate format. Additional post-detection methods are not utilized, therefore, eliminating potential cross-contamination that may occur after amplification. A multiplex real-time PCR method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis directly from milk has been developed (Gillespie and Oliver, 2004). These three mastitis pathogens frequently cause mastitis in dairy cows throughout the world. Targets that we used for the multiplex real-time PCR were a Staph. aureus specific genetic marker, the cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor for Strep. agalactiae, and the plasminogen activator gene for Strep. uberis. A total of 192 quarter milk samples were analyzed by the multiplex real-time PCR assay and conventional microbiological techniques. This technique correctly identified 97.7% of all quarter milk samples and correctly identified 91% of Staph. aureus, 98% of Strep. agalactiae and 100% of Strep. uberis. The overall sensitivity of this procedure to correctly identify Staph. aureus, Strep. agalactiae and Strep. uberis directly from milk was 95.5% and the specificity was 99.6%. Using an enrichment step, the detection limit was one colony forming unit/ml. No cross-reactivity was detected with 53 American Type Culture Collection reference strains representing common mastitis pathogens. Results of this study indicate that the multiplex real-time PCR procedure is a rapid and accurate method for identification of Staph. aureus, Strep. agalactiae and Strep. uberis directly from milk. Manipulation of this multiplex real-time PCR method could be done to include additional or other frequently encountered pathogens found in milk including foodborne pathogens. We have also developed real-time PCR methods for identification of foodborne pathogens in food, dairy environmental samples and dairy cows. Real-time PCR assays utilizing dual labeled probes have been developed to identify E. coli O157:H7 and L. monocytogenes from beef products (Nguyen et al., 2004). Target genes for E. coli O157:H7 and L. monocytogenes were rfbe and hyla, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for (9/10) E. coli O157:H7 and all L. monocytogenes (7/7) strains evaluated. Detection sensitivity ranged from 103 to 104 CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 hours. Approximately CFU/g of

81 L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 hours. Realtime PCR assays used in this study for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive, rapid and appear promising. We have also used SYBR Green in real-time PCR assays to detect C. jejuni from dairy farm environmental samples (Nam et al., 2004b). The melting temperature (T m ) obtained for C. jejuni was 77.5 C. The detection limit for samples spiked with C. jejuni was >103 CFU/ml. However, after a 24 hour enrichment step, the detection limit for samples spiked with C. jejuni was <10 CFU/ml. Eighty-two dairy farm environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding material were analyzed. The SYBR Green real-time PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of these samples being culture positive for C. jejuni. All samples that were positive by standard culture methods were also positive by the SYBR Green real-time PCR assay. A real-time PCR method utilizing SYBR Green I dye and a 119-bp fragment of the inva gene was evaluated for detection of Salmonella spp. in dairy farm environmental samples (Nam et al., 2004a). A total of 240 bacterial strains were evaluated including 124 Salmonella spp. type strains and 116 non-salmonella strains. Only the Salmonella strains tested positive for the inva gene by analyzing the melting temperature (Tm = 79oC) of the amplicon. The detection limit from spiked environmental samples was 103 to 104 CFU/ml in broth without enrichment. The detection limit was reduced to <10 CFU/ml in broth after 18 hours of enrichment. Detection of Antimicrobial Resistance Genes in Veterinary and Foodborne Pathogens Antimicrobials are used extensively in food-producing animals to combat disease and to improve animal performance. On dairy farms, antimicrobials such as tetracyclines, penicillins, and sulfonamides are used to treat or prevent diarrhea and pneumonia, both of which are important diseases in dairy calves. Antimicrobials such as penicillins, cephalosporins, erythromycin and oxytetracyclines are used for treatment and prevention of mastitis, an important disease caused by a variety of Gram-positive and Gram-negative bacteria. Such drugs are often administrated routinely to entire herds to prevent mastitis during the nonlactating period. Benefits of antibiotic use in animal production systems include improved growth and/or feed efficiency, decreased nitrogen excretion and thus reduced environmental impact, decreased pathogen loads, and a lower incidence of disease. In contrast to the above benefits, however, are suggestions that agricultural use of antibiotics may be partly responsible for the emergence of antimicrobial resistant bacteria, which in turn may decrease the efficacy of similar antimicrobials used in human medicine. While investigations have focused on emergence of drug resistant bacteria, persistence of resistant bacteria and effects on human medicine, little information is available with regard to antimicrobial resistance of commensal bacteria and veterinary and foodborne pathogens on dairy production facilities, or management conditions that affect antimicrobial resistance. Information on prevalence of antimicrobial resistance, effects of stressors on the host animal, and the effect of management and environment at the farm level are especially lacking. Furthermore, much of the current available antimicrobial resistance data is derived from evaluating clinical isolates originally obtained from sick animals. Consequently, this information may be biased by several factors, including housing or husbandry conditions, age and condition of animals tested, and previous antibiotic therapies. Because transferable resistance may originate from a variety of bacteria and associated hosts under a number of conditions, it is important that confounding factors are characterized so that more definitive conclusions can be derived

82 The objective of our research is to gain insight into antimicrobial resistance gene flow from commensal bacteria of dairy farms to animal and human pathogenic bacteria. There is only limited information on rates and extent of gene exchange from commensal bacteria to animal and human pathogenic bacteria. We hope to define in detail the predominant resistance constructs in bacterial populations of dairy cows and their environment; and identify reservoirs, how antimicrobial resistance is transferred, and the relationships of antimicrobial use with development of antimicrobial resistance. Molecular tools such as DNA probes and PCR-based detection systems have greatly facilitated the study of the epidemiology of antimicrobial resistance genes and mobile genetic elements (MGE) and their transfer to other bacteria at the genetic level. Antimicrobial resistant commensal bacteria of dairy farms may play a pivotal role in the spread of antimicrobial resistance to pathogens that can cause disease in humans and animals. Since dairy cows are treated with many antimicrobial compounds for prevention and control of different diseases, commensal bacteria of cows and bacteria normally found in the dairy farm environment may acquire antimicrobial resistance. We have used PCR to detect several different antimicrobial genes in a variety of veterinary and foodborne pathogens (Murinda et al., 2004e; Srinivasan et al., 2004a; 2004b; 2004c). Campylobacter jejuni (n=39), Listeria monocytogenes (n=38) and Salmonella spp. (n=12) isolated from dairy farms were evaluated for antimicrobial resistance gene patterns (Srinivasan et al., 2004a). All foodborne pathogens were screened for 21 antibiotic resistance genes using PCR. Campylobacter jejuni (5.1%), L. monocytogenes (31.6%) and Salmonella spp. (100%) contained more than one antibiotic resistance gene. Tetracycline resistant determinant (teta) was found in 15.4%, 31.6% and 100% of C. jejuni, L. monocytogenes and Salmonella spp., respectively; tetc was found only in Salmonella spp.; and tetb, tetc, tete, and tetg were not detected in any of the foodborne pathogens evaluated. The only other antimicrobial resistance gene detected in at least one isolate of each of the foodborne pathogens evaluated was suli. A high frequency of flor (65.8%), pena (36.8%), and stra (34.2%) was found in L. monocytogenes. In Salmonella spp., stra (100%), strb (83.3%), suli (100%), ermb (58.3%) and pena (50%) were amplified and all Salmonella spp. were multi-drug resistant. Results of this study indicate that a high prevalence of foodborne pathogens isolated from the dairy farm environment contain antimicrobial resistance genes. The potential exists for foodborne pathogens carrying antimicrobial resistance genes to acquire additional resistance genes as well as to spread this genetic material to commensal and pathogenic bacteria in the dairy farm environment. In another study (Srinivasan et al., 2004b), antimicrobial resistance gene patterns of 131 E. coli isolated from dairy cows with clinical mastitis were evaluated. All E. coli contained more than one antimicrobial resistance gene. Tetracycline resistance determinants, teta and tetc, were found in 8.4% and 64.1% of isolates, respectively. Other tetracycline resistant determinants (tetb, tetd, tete and tetg) were not observed in any of the isolates studied. Even though many E. coli carried the tetc gene, they were sensitive to tetracycline. Thus, tetracycline MIC data were negatively correlated with the presence of the tetc gene and positively correlated with the presence of teta genes. Streptomycin resistance genes stra (7.6%) and strb (9.9%) and streptomycin-spectinomycin adenyltransferase gene (aada) were found in 77.9% of test isolates. Ampicillin resistance gene (ampc) was the predominant gene (94.7%) found in E. coli from cows with mastitis. Over 99% of E. coli were resistant to ampicillin and this correlated with the presence of the ampc gene. Other resistance genes, pena (49.6%), suli (9.9%) and sulii (8.4%) were observed by PCR. Vancomycin resistance gene, vana, was found in most E. coli (94.7%) but vanb was not present in any of the E. coli evaluated. Only one of 131 E. coli carried the flor gene. None of the isolates carried cmla, aac(3)iv, ermb, erea or ereb. In conclusion, all E. coli from cows with mastitis were multi-drug resistant and carried more than one antimicrobial resistance gene. Escherichia coli causing bovine mastitis may be a reservoir for antimicrobial resistance genes and may play a role in dissemination of antimicrobial resistance

83 genes to other pathogenic and commensal bacteria in the dairy farm environment. However, further research is necessary to substantiate this hypothesis. Enterobacteriaceae isolated from soil samples using standard isolation protocols were screened for antimicrobial resistance (Srinivasan et al., 2004c). Among 36 bacteria isolated from different soils, teta was found in 2 isolates (5.6%), tetb in one isolate (2.8%), flor in 5 isolates (13.9%), stra in 27 isolates (75%), strb in one isolate (2.8%), and no isolates carried cmla. The prevalence of antimicrobial resistance genes was four-fold higher in bacteria isolated from the soil of dairy farms than in bacteria isolated from non-agricultural soils. About 50% of the bacteria isolated from dairy farm soils were multi-drug resistant and carried more than one antimicrobial resistance gene. These soil bacteria could serve as a reservoir for antibiotic resistance genes in the environment and spread to other environmental sources through vertical or horizontal gene transfer mechanisms. PCR amplification of antimicrobial resistance genes in soil metagenomic DNA revealed that all dairy farm soils carried all antimicrobial resistance genes in different combinations whereas only one virgin non-agricultural soil carried only flor. Conclusions PCR-based techniques are very useful tools to study a variety of complex phenomenon and are used frequently in many research laboratories throughout the world. Use of PCR-based techniques have facilitated the discovery of more effective methods for the detection of foodborne pathogens associated with food-producing animal environments and foodborne pathogens causing disease in humans. These techniques have also been quite useful to delineate virulence factors and antimicrobial resistance genes of several important foodborne pathogens The challenges to providing a safe and nutritious food supply are complex because all aspects of food production from farm to fork need to be considered. Given the considerable national/international demand for food safety and the formidable challenges of producing and maintaining a safe food supply, food safety research and educational programs has taken on a new urgency. As the system of food production and distribution changes, the food safety system needs to change with it. A strong science-based approach that addresses all the complex issues involved in continuing to improve food safety and public health is necessary to prevent foodborne illnesses. Not only must research be conducted to solve complex food safety problems, results of that research must be communicated effectively to producers and consumers. Research and educational efforts identifying potential on-farm risk factors will better enable dairy producers to reduce/prevent foodborne pathogen contamination of dairy products leaving the farm. Identification of on-farm reservoirs could aid with implementation of farm-specific pathogen reduction programs. Foodborne pathogens, mastitis, milk quality and dairy food safety are indeed all interrelated. A safe, abundant and nutritious milk and meat supply should be the goal of every dairy producer in the world. References Gillespie, B. E., A. G. Mathew, F. A. Draughon, B. M. Jayarao, and S. P. Oliver A PCR- ELISA technique for detection of somatic group-specific Salmonella spp. J. Food Prot. 66: Gillespie, B. E., and S. P. Oliver Simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis in milk by multiplex real-time polymerase chain reaction. (In preparation)

84 Murinda, S. E., L. T. Nguyen, S. J. Ivey, B. E. Gillespie, R. A. Almeida, and S. P. Oliver Prevalence and molecular characterization of Escherichia coli O157:H7 in bulk tank milk and fecal samples from cull dairy cows: a 12-month survey of dairy farms in East Tennessee. J. Food Prot. 65: Murinda, S. E., L. T. Nguyen, H. M. Nam, R. A. Almeida, S. J. Headrick, and S. P. Oliver. 2004a. Detection of Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in dairy farm environmental samples. Foodborne Pathogens & Disease 1(2) Murinda, S. E., S. D. Batson, L. T. Nguyen,B. E. Gillespie, and S. P. Oliver. 2004b. Phenotypic and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26 strains from North America. Foodborne Pathogens & Disease 1(2): Murinda, S. E., L. T. Nguyen, and S. P. Oliver. 2004c. Problems in isolation of Campylobacter jejuni from frozen-stored raw milk and bovine fecal samples: genetic confirmation of isolates by multiplex PCR. Foodborne Pathogens & Disease 1(3): Murinda, S. E., L. T. Nguyen, T. L. Landers, F. A. Draughon, A. G. Mathew, J. S. Hogan, K. L. Smith, D. D. Hancock, and S. P. Oliver. 2004d. Comparison of Escherichia coli isolates from humans, food, farm and companion animals for presence of Shiga-toxin producing Escherichia coli virulence markers. Foodborne Pathogens & Disease 1(3): Murinda, S. E., P. D. Ebner, L. T. Nguyen, A. G. Mathew, and S. P. Oliver. 2004e. Class 1 integrons, antimicrobial and acid resistance in pathogenic Escherichia coli isolates from dairy cow mastitis milk and feces J. Appl. Microbiol. (Submitted). Nam, H. M., V. Srinivasan, B. E. Gillespie, S. E. Murinda, and S. P. Oliver. 2004a. Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Int. J. Food Microbiol. (Accepted). Nam, H. M., V. Srinivasan, S. E. Murinda and S. P. Oliver. 2004b. Specific detection of Campylobacter jejuni in dairy farm environmental samples using SYBR Green real time PCR. Foodborne Pathogens & Disease (Submitted). Nguyen, L. T., B. E. Gillespie, H. M. Nam, S. E. Murinda, and S. P. Oliver Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction. Foodborne Pathogens & Disease (In press). Srinivasan, V., H. M. Nam, L. T. Nguyen, B. Tamilselvam, S. E. Murinda, and S. P. Oliver. 2004a. Prevalence of antibiotic resistance genes and integrons in foodborne pathogens isolated from dairy farms. In: Proc. Natl. Mastitis Counc. pp Srinivasan, V., B. E. Gillespie, L. T. Nguyen, M. J. Lewis, Y. H. Schukken, and S. P. Oliver. 2004b. Phenotypic and genotypic antibiotic resistance patterns of Escherichia coli isolated from dairy cows with mastitis. In: Proc. Natl. Mastitis Counc. pp

85 Srinivasan, V., B. Tamilselvam, H. M. Nam, L. T. Nguyen, and S. P. Oliver. 2004c. Prevalence of antibiotic resistant bacteria and antibiotic resistance genes in soil from dairy farms. In: Proc. Natl. Mastitis Counc. pp

86 POLYMERASE CHAIN REACTION FOR DETECTION OF MYCOPLASMA BOVIS IN CLINICAL SAMPLES Suzanne Klaessig Department of Population Medicine and Diagnostic Sciences Cornell University, Ithaca, NY Mycoplasmas are parasites widely distributed in nature. Hosts include mammals, plants, reptiles, fish and insects. Over 180 species have been identified thus far (1). Mycoplasmas differ from other bacteria of the class Mollicutes by their minute size (they are the smallest self-replicating organisms), and lack of cell wall. They usually grow symbiotically with their host, often coinfecting with other pathogens. The use of molecular techniques, including the comparison of the highly conserved 16S rrna gene sequence, has greatly expanded the understanding of these organisms. Mycoplasmas can exist both extra- and intracellularly, and have multiple, complex methods of interaction with their host cells. The discovery of variable surface proteins (Vsps) on the outer surface of the plasma membrane has led to many studies of the interaction of mycoplasmas with the immune system. These Vsps may stimulate or help evade the host immune system, and possibly mediate adherence to cells. Mycoplasma bovis is the most important agent of bovine mycoplasmal mastitis, although at least eleven other Mycoplasma and Acholeplasma species have been isolated from milk (2). It can cause both chronic and acute infections, and like most mycoplasmas exhibits organ and tissue specificity, infecting the mucosal surfaces of the respiratory and urogenital tracts, eye, alimentary canal, mammary gland and joints. Cows can be infected at any age and lactational stage (2). Mycoplasma is easily contagious by airborne transmission and direct contact, as well as via milking machines or milker s hands. In lactating cows, it can cause severe clinical mastitis that resists treatment, and also chronic infections with little or no decrease in milk production. As few as one-hundred colony-forming units (cfu) can cause intramammary infection (IMI) (3), and the infected animal can shed the organism for prolonged periods of time. Because vaccines and antibiotics are not available for treatment, current methods for controlling Mycoplasma include culling, segregation and hygienic measures. Identification of the infected animals at an early stage is critical to prevention of transmission in the herd. Diagnosing M. bovis is most commonly done by culture and subsequent serology to identify individual species. Mycoplasmas are notoriously slow to grow and difficult to culture. Traditionally, very complex media have been used for culture, based on beef heart infusion, including peptones, yeast extract and serum. These rich growth media have recently been found to be inhibitory in some cases. Genomic work has shown that Mycoplasmas have few genes involved in biosynthetic pathways, for example, both M. genitalium and M. pneumoniae lack all genes involved in amino acid synthesis. When collecting milk samples for culture they must be kept cool and plated promptly. Overgrowth by other species of bacteria can occur even when antibiotic inhibitors are included in the media. Plates must be viewed daily, both to scan for contaminants and to watch for the distinctive fried egg morphology of the Mycoplasma on solid medium (2). Incubation and observation should continue for 7-10 days before plates are considered negative, and false-negatives are common due to low numbers of organisms in the sample, or the fragility of Mycoplasma itself. The use of DNA-based tests promises to be faster, more sensitive and more specific. The detection of Mycoplasma by polymerase chain reaction (PCR) is based on the in vitro amplification of the highly-conserved 16S rrna gene (3, 4). PCR can amplify the target DNA sequence by as much as five orders of magnitude, thus potentially solving the two largest problems dealing with Mycoplasma: early detection, and low numbers of organisms in the clinical

87 samples. Detection levels as low as 5 cfu/ml in milk samples have been reported (3). Detection of M. bovis from broth cultures or DNA extracts can be carried out on a routine basis, but detection in milk presents a number of problems. The presence of high levels of protein, calcium, and unidentified proteases has an inhibitory effect on amplification. Numerous approaches have been investigated to increase the yield of amplifiable bacterial DNA (5). We report our preliminary results of a PCR procedure for detection of M. bovis DNA using the Pennsylvania State University Animal Diagnostic Laboratory protocol (6). Further work will include comparing methods of DNA extraction from milk for PCR, and PCR detection of other species of Mycoplasma. References (1) Razin S, Yoger D, Naot Y. Molecular Biology and Pathogenicity of Mycoplasmas. Microbiol & Molec Biol. Reviews. Dec 1998: (2) Gonzales R, Wilson D. Mycoplasmal Mastitis in Dairy Herds. Vet Clin Food Anim (19): (3) Pinnow C, Butler J, Sachse K, Hotzel H, Timms L, Rosenbusch R. Detection of Mycoplasma bovis in Preservative-Treated Field Milk Samples. J Dairy Sci 2001 (84): (4) Hirose K, Kawasaki Y, Kotani K, Tanaka A, Abiko K, Ogawa H. Detection of Mycoplasma in Mastitic Milk by PCR Analysis and Culture Method. J Vet Med Sci (6): (5) Hotzel H, Sachse K, Pfutzner H. Rapid Detection of Mycoplasma bovis in Milk Sample and Nasal Swabs Using the Polymerase Chain Reaction. J Applied Bact 1996 (80): (6) The Pennsylvania State University Animal Diagnostic Laboratory. Polymerase Chain Reaction for detection of Mycoplasma in Clinical Samples. SOP

88 BOVINE INFECTION OF COXIELLA BURNETII (Q FEVER) IN THE U.S. DAIRY HERDS: USE OF CONVENTIONAL AND REAL-TIME PCR FOR THE DETECTION OF COXIELLA BURNETII (Q FEVER) IN MILK Sung Kim Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences Cornell University, Ithaca, NY Introduction Q (Query) fever is a ubiquitous zoonosis caused by Coxiella burnetii, an obligate intracellular rickettsial organism. Since its first independent report by Australian and American investigators in 1935, the disease has been reported from all over the world except New Zealand. C. burnetii infections are reported in humans, farm animals, pet animals, wild animals, and arthropods. Among farm animals, dairy cattle, sheep and goats are implicated as the major reservoirs of human C. burnetii infection. Animals are often naturally infected but most animals usually do not show any typical symptoms of C. burnetii infection. C. burnetii could be isolated from the blood, lungs, spleen, and liver in the acute phase. The female uterus and mammary glands are primary sites in the chronic phase of C. burnetii infection. Shedding of C. burnetii into the environment occurs mainly during parturition by birth products, particularly the heavily contaminated placenta. Shedding of C. burnetii in milk by infected dairy cows is also well documented. The clinical signs associated with C. burnetii infection are abortion, significantly in sheep and goats, and reproductive disorders in cattle. Though shedding of C. burnetii in their milk by infected dairy cows has been known, only limited information by some earlier investigators in 1940 s and 1950 s is currently available. Previous studies on the prevalence of Q fever in dairy cattle were mostly based on serological tests, including complement fixation (CF), indirect inmmunofluorescene (IF), and enzymelinked immunosorbent assay (ELISA). Recent seroepidemiological studies for cattle have indicated that C. burnetii antibody seroprevalence in these animals is higher nowadays than 20 or 30 years ago. However, the real prevalence of C. burnetii infection in such animals is still not available, partly due to the lack of surveillance. The objects of this presentation are to increase the awareness of Q fever and to report the prevalence of Q fever in the U.S. dairy herds. Epidemiology and Transmission The primary mode of transmission is by inhalation of infected aerosols with contaminated droplets or dusts from birth products, milk, and excreta of infected animals. Infectious aerosols containing viable organisms can be spread in distance by the wind. Direct contact with infected animals or other contaminated materials and ingestion of contaminated raw milk with the organisms also can be potential routes of transmission. There is relatively little information on the prevalence of the infection. Farm animals, particularly cattle, goats and sheep are considered the primary reservoirs. A California study conducted at 20 dairy herds in 17 counties showed 100% (20/20) of herds had seropositive cows and 82% of 1,052 had serum antibodies to C. burnetii. Of 1,634 whey samples, 51% were positive for antibodies Cows that were seropositive were usually also whey positive. A total of 23% of 840 cows shed C. burnetii in their milk. A sharp increase in the prevalence of Q-fever was noted in Ontario dairy cattle herds between 1964 and 1984 from 2.3% to 66.8%. Wild animals, such as coyotes, foxes, rabbits and deer and pet animals, cats and dogs, are also known to be seropositive, indicating as possible reservoirs. Ticks are also known to transmit the disease

89 PCR as a Study Tool Working with the Q-fever agent based on isolation techniques is not an easy task due to the agent s high infectivity and rigorous compliance requirements in handling a selective agent. Recently polymerase chain reaction (PCR) assays have been used to detect C. burnetii. Tran-PCR assay was developed to detect C. burnetii in milk targeting a transposon-like sequence only found in C. burnetii. Trans-PCR assay detects the Q fever agent, C. burnetii, unlike serological assays which detect antibodies. A real-time PCR was developed in this study to measure numbers of C. burnetii shed in milk. The objective of our study was to conduct a preliminary nation-wide prevalence surveillance of Q-fever in the United States dairy herds based on bulk tank milk testing by PCR. Bovine Herd Infection of Q fever in the U.S. We tested bulk tank milk samples from the dairy herds in the United States over a 3-year period by Trans-PCR. Positive results were confirmed by nested PCR and DNA sequencing. The sequencing results of the 687-bp PCR product were consistent with the published sequence of IS1111 with 100% homology. The overall prevalence of C. burnetii in the United States dairy herds was 94.3% with little variations year to year from 93.2 to 94.7%. When compared the prevalence in New York State and the other states, there were no significant variations indicating the Q-fever infection of the dairy herds was persistent or steady with little temporal and regional variations. Conclusions Bulk tank milk has been used to diagnose dairy herds for several bovine diseases including bovine viral diarrhea. We reports that greater than 90% of the United States dairy herds were infected with Q-fever based on bulk tank milk testing over a 3-year period. This high infection rate did not show temporal and regional variations, suggesting the Q fever infection in the dairy herds is prevalent throughout the United States. Our report of the high prevalence of Q fever in the dairy herds is not surprising to take into a consideration of earlier reports regarding a great increase of bovine infection in North America. An early investigator concluded that Q fever was already endemic throughout the United States, and predicted that the high bovine infection may occur in other parts of the country in a similar fashion as in Southern California where a 98% of the herd infection was reported. Widespread and increasing bovine C. burnetii infection was reported. Frequent chronic Q fever infection of dairy cows could be the most important source of human infection. Though the mode and extent of transmission from bovine infection to human has not been determined, epidemiologic studies indicate that Q fever develops in persons in contact with domestic animals including farmers, veterinarians, slaughterhouse workers, and laboratory personnel working with C. burnetii

90 DIAGNOSTIC STRATEGIES FOR BOVINE VIRAL DIARRHEA VIRUS Edward J. Dubovi Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences Cornell University, Ithaca, NY The challenge which is still with us over 50 years after the first clinical descriptions of BVDV infections in cattle is to develop effect means to control BVDV infections. To do this, the practitioner must be able to deliver a clear, concise, and consistent message to the producer. This message must include a simple description of the pathogenesis of BVDV, the role management plays in this problem, diagnostic testing programs and the limitations of vaccines in the context of the producer's management philosophy. Control of BVDV will need to include the establishment of best management practices combined with a validated immunization program supported by an active surveillance system. For a good control program for BVDV, there are three essential elements. First, one should determine whether the virus currently exists on the farm or ranch. With the use of vaccines, clinical disease is not necessarily a true indication of the presence of BVDV. If the presence of BVDV is detected, then a strategy for its elimination should be instituted. Once the virus has been eliminated, then a program to keep it out must be developed. Over the past ten years, a variety of diagnostic tests have been developed which can accurately detect the presence of BVDV in an animal or in a herd. The problem is not that good tests are not available. The tests that are available are not being used properly or consistently. Too often practitioners shop around the country looking for the perfect test when the issue is the testing program and not the test. Unfortunately, each farm and ranch presents a set of unique conditions that must be taken into account when a testing strategy is developed. The worst thing to happen is to initiate an expensive testing program when the virus does not exist on the premises. Good evidence of infection is not that the owner or the practitioner thinks that all of the problems on the farm are related to BVDV. From the laboratory perspective, good evidence of infection is the isolation of the virus from an animal on the farm, detection of antibody in unvaccinated animals, and detection of viral nucleic acid or antigen in a clinical sample. For years, the gold standard for BVDV detection was the isolation of the virus. Most diagnostic laboratories are now capable of dealing with BVDV once the problems of contaminated reagents were recognized. However, the existence of the noncytopathic biotype of BVDV was a diagnostic nightmare because quality antisera used for its detection were not available. The first technological breakthrough in the diagnosis of BVDV was the development of monoclonal antibodies in the late 80 s. The antigenic variation of field isolates of BVDV was determined by the Mabs and selected Mabs were then used to detect BVDV in cell cultures and tissue samples. For the first time, a consistent reliable reagent was available that could detect BVDV regardless of its biotype. The second major development with regard to Mabs was the discovery that one Mab, namely 15c5, could be used to detect BVDV in formalin-fixed tissue. Monoclonal antibodies to BVDV had been used for immunohistochemistry detection of viral antigens, but these efforts had been done on frozen sections. A Mab that could detect antigen in formalin-fixed tissues opened the door to many retrospective studies on the involvement of BVDV in clinical events that were not initially linked to BVDV. In particular, animals persistently infected (PI) with BVDV have no lesions in tissue samples that can be detected by standard histological techniques. The IHC technique permitted the detection of these animals and extended the link between PI animals and other clinical presentations. A Mab that works on formalin-fixed tissues also eliminates that need for rapid transit of samples from the field to the laboratory. In countries without rapid courier services, the diagnosis of BVDV was now possible on fixed tissue

91 The third advance with regard to BVDV and Mabs was the development of an antigencapture ELISA (ACE) test for the detection of persistently infected animals using serum as the test substance. ACE tests for BVDV had been developed using whole blood as the sample, but these tests required treatment of the sample prior to testing. A test that could work on serum was a great benefit because the serum samples could also be used for other herd testing programs such as Johne s ELISA tests or BLV. As the ACE test became more widely used, an alternative test became available which was based on an IHC test on skin biopsies. With PI animals, an abundance of antigen exits in the epithelial cells of the skin. IHC test became the test of choice particularly in the beef industry. It soon became evident that the ACE test using a skin biopsy specimen was equivalent to the IHC test. Currently either test is used as a herd screening tool. With the advent of molecular techniques, nucleotide sequencing of BVDV isolates became an important issue with the outbreak of severe clinical disease in Canada in the early 90 s. Along with the realization that there was great genetic diversity among BVDV isolates came the challenge for designing PCR primers that would detect all possible isolates. There are numerous papers describing RT-PCR systems for detecting BVDV and with no national standards, it is up to each lab to establish the validity of their testing system. Standard RT-PCR protocols shifted to nested PCRs to increase sensitivity, but this also increased the chances of false positives. With the advent of real-time PCR systems, the contamination problem became less of an issue since the reaction tubes never had to be processed for product detection. PCR tests for BVDV can be used in any situation where virus isolation is used. If the question is simply is there BVDV in the system, then PCR is a viable alternative to virus isolation. If, however, the question is whether there is any pathogen in the system, then PCR becomes less useful. The analytical sensitivity of PCR permits pooling of serum, milk or whole blood samples when detection of a PI animal in a herd is the goal. However, a positive PCR reaction can be generated from at least three possible events: vaccination of a herd with modifiedlive vaccine, acute infection or a persistent infection. PCR cannot in of itself distinguish these three possibilities. Current Testing Strategies at the AHDL The consensus of opinion is that BVDV maintains itself in the bovine population through the generation of persistently infected animals. Therefore, there is an emphasis on being able to screen herds for the presence of one of these animals in normal cows. Cost is the obvious overriding concern with this testing process. Over the years, the AHDL has modified its approach to herd testing to take advantage of technology where appropriate. Currently in dairy herds, we use several protocols that are somewhat dependent upon the management of the farm. We have championed the use of bulk tank milk testing for detecting PI animals in the lactating herd. If BVDV is detected by this approach, then individual cow testing is necessary. This can be achieved either through pooled PCR testing or by use the ACE test directly on all animals in the herd. This approach has generated considerable cost savings over simply testing all animals individually from the very beginning. References There are numerous references available for all aspects of BVDV. For a good comprehensive starting point, I would recommend: Bovine Viral Diarrhea Virus: Persistence is the Key. (2004). Veterinary Clinics of North America: Food Animal Practice. Vol 20 (1)

92 NOTES

93 Speaker Biographies and Contact Information

94 NOTES

95 Kathryn J. Boor, PhD Associate Professor Cornell Institute of Food Science 413 Stocking Hall Cornell University Ithaca, NY Kathryn Boor earned a BS in Food Science from Cornell University and an M.S. in Food Science from the University of Wisconsin. She spent two years in Kenya, East Africa, working with impoverished farmers to enhance their animal-based food production and preservation systems, then earned her PhD in Microbiology at the University of California, Davis. She established the Food Safety Laboratory as an Assistant Professor in the Department of Food Science at Cornell in Dr. Boor currently also directs Cornell s Milk Quality Improvement Program. She serves on the New York State Interagency Task Force on Food Safety and Security, on the editorial boards for Journal of Food Protection, Applied and Environmental Microbiology, and Foodborne Pathogens and Disease, and as Scientific Advisor to the New York State Cheese Manufacturers Association. Dr. Boor received a 2000 USDA Honor Award as a member of the Listeria Outbreak Working Group, the 2000 Foundation Scholar Award of the American Dairy Science Association, the 2000 Cornell University Constance E. Cook and Alice H. Cook Recognition Award, and the 2002 Samuel Cate Prescott Award for Research from the Institute of Food Technologists. She is also Past President (2000) of the New York State Association of Milk and Food Sanitarians

96 Dr. Paula J. Fedorka-Cray, PhD Microbiologist, Research Leader Richard B. Russell Agricultural Research Center USDA-ARS-RRC-ARRU 950 College Station Road Athens, GA Dr. Fedorka-Cray received her BS from Penn State University (Microbiology), MS from North Dakota State University (Bacteriology), MAS from Johns Hopkins University (Administration) and a Ph.D. from the University of Nebraska Medical School (Veterinary Microbiology). She was the recipient of the Joseph J. Garbarino Achievement Award for Excellence in Agricultural Research awarded by the Animal Health Institute Foundation. She has been employed by the USDA - ARS since 1991 and is a Microbiologist, Research Leader for the Antimicrobial Resistance Research Unit. Her program focuses on antimicrobial resistance in food borne pathogens with an emphasis on Salmonella and Campylobacter. In particular she is interested in the host/pathogen relationship as well as the ecological impact of antimicrobial resistance. She also directs the testing efforts for the veterinary arm of the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS). Antimicrobial Resistance Research Unit The mission of the Antimicrobial Resistance Research Unit is to study Antimicrobial resistance in zoonotic food borne pathogens and commensal bacteria. Epidemiology, microbiology, risk factor analysis, and molecular techniques are used to 1) gain an understanding of the prevalence of resistance among food borne pathogens and factors which may affect the development and persistence of resistance in production facilities and in the environment, 2) study the molecular mechanisms that are associated with the development of resistance, and 3) define the role of commensal bacteria in the development and transfer of resistance. The veterinary arm of the National Antimicrobial Resistance Monitoring System - Enteric Bacteria (NARMS) is also located in our Unit. This program is a multi-agency endeavor involving scientists from the USDA - ARS, FDA - Center for Veterinary Medicine, USDA - FSIS, USDA - APHIS, and the CDC. The goal of the program is to track the development of antimicrobial resistance in veterinary isolates as it arises and disseminate the information to all stakeholders in an attempt to arrest the development and spread of resistance, especially among food borne pathogens. The results generated by these endeavors will enhance our knowledge of antimicrobial resistance and provide the scientific data that is critically needed to direct research among the scientific community and to develop policy in a number of agencies, including the USDA and FDA

97 Edward J. Dubovi, PhD Director Virology Section Animal Health Diagnostic Center Department of Population Medicine and Diagnostic Sciences College of Veterinary Medicine PO Box 785 Cornell University Ithaca, NY Received a Masters degree in Virology from Purdue University in 1968 and a PhD in microbiology from the School of Medicine, University of Pittsburgh in Following postdoctoral training at the University of Virginia and the University of North Carolina, assumed position as Director of the Virology Section, Diagnostic Laboratory, College of Veterinary Medicine at Cornell in Areas of research: Viral diseases of cattle with particular emphasis on bovine viral diarrhea virus and bovine retroviruses; viral diseases of horses with emphasis on equine arteritis virus and equine morbilli-like virus. Within the context of the Department of Population Medicine and Diagnostic Sciences, current efforts also included herd health programs that emphasize biosecurity and effective use of veterinary biologicals

98 Rubén N. González, DVM, PhD Senior Research Associate and Associate Director Quality Milk Production Services Department of Population Medicine and Diagnostic Sciences College of Veterinary Medicine, Cornell University 22 Thornwood Drive Park View Technology Center I Ithaca, New York , USA Tel: (607) Fax: (607) rng1@cornell.edu Dr. González graduated as a Doctor of Veterinary Medicine from the Faculty of Veterinary Sciences, National University of La Plata, Argentina. After working for two years as a veterinary practitioner in a mixed rural practice, he returned to academia as an Assistant Professor of Epidemiology and Infectious Diseases at the Faculty of Agronomy and Veterinary Medicine, National University of Río Cuarto, Argentina. Later, as a Senior Bacteriologist, he joined Argentina's National Institute for Agricultural Technology and the United Nations/FAO Project in Animal Health Argentina 75/072 where he helped to set up and supervise a net of regional veterinary diagnostic laboratories in five provinces in Northwestern Argentina. In 1982, Dr. González came to the U.S. and while pursuing graduate studies, worked as a Post-Graduate Research Associate in the Department of Clinical Pathology and the Cooperative Extension Mastitis Laboratory, School of Veterinary Medicine, University of California at Davis. From this university, Dr. González received both a Master in Preventive Veterinary Medicine (1984) and a PhD in Comparative Pathology (1988). He joined Quality Milk Production Services in Areas of research are epidemiology, diagnosis and control of bovine mastitis, with emphasis in Mycoplasma and other non regular agents of the disease

99 Bhushan Jayarao, PhD Extension Veterinarian/Associate Professor Department of Veterinary Science 111 Henning Bldg The Pennsylvania State University University Park, PA Dr. Jayarao received his Bachelor s degree in Veterinary Medicine from Bombay Veterinary College in In 1982, he received his Master s degree in Veterinary Public Health. During the time between 1980 and 1986, Dr. Jayarao taught Zoonoses, Epidemiology and Veterinary Public Health, as well as practiced companion and food animal medicine. In 1985, Dr. Jayarao attended the University of Veterinary Sciences, Budapest where he received his PhD in Microbiology and Epidemiology in One year later, he joined Dr. Stephen Oliver s mastitis research group at the University of Tennessee. Over the next five years ( ), Dr. Jayarao worked on developing diagnostic tests and understanding the epidemiology of S. uberis IMI. During the same period, Dr. Jayarao earned a degree in Master s in Public Health with specialization in communicable diseases. In 1995, Dr. Jayarao joined the Department of Dairy Science at the South Dakota State University. He was engaged in teaching Microbiology courses to undergraduate and graduate students. At this time, his research program focused primarily on pre-harvest food safety. In July of 1998, Dr. Jayarao joined the department of Veterinary Science at the Pennsylvania State University as an extension Veterinarian. Dr. Jayarao s primary responsibility was to develop an extension program that is focused on infectious diseases and public health. His research appointment allows him to pursue research interests related to diagnosis and detection of infectious diseases, molecular epidemiology of antimicrobial resistance, development of plant-based vaccines and food safety. Dr. Jayarao has been successful in obtaining federal, state and industry funding as principal or co-investigator on 24 peer reviewed external grants equivalent to about $ 1.5 million dollars to support extension and research work at Penn State. In 2002, Dr. Jayarao received the 2002 ADSA West Agro award for his significant contributions in the area of understanding the molecular epidemiology of streptococcal infections in dairy cattle. The same year Dr. Jayarao received early tenure and was promoted to Associate Professor. Currently, Dr. Jayarao has 2 post-doctoral research associates, 3 PhD and 3 master s and 2 honor s students, and 4 technicians in his laboratory. His research group conducts research in the following areas: 1) Epidemiology of antimicrobial resistance in companion and food animals, 2) Plant-based vaccines; development of transgenic plants that encode for bacterial antigens, and 3) Mastitis and Milk Quality Dr. Jayarao has published more than 50 peer reviewed journal articles. Dr. Jayarao is an active member of a number of professional societies, including American Dairy Science Association, American Veterinary Medical Association, Pennsylvania Veterinary Medical Association, American Association of Extension Veterinarians, and National Mastitis Council

100 Vivek Kapur, PhD Professor, Department of Microbiology Dept of Vet Path 300 A Vet Science Bldg University of Minnesota 1971 Commonwealth Avenue St. Paul, MN Education: Veterinary Medicine-UAS, Bangalore, India, 1986 Ph.D. - Pennsylvania State University, University. Park, PA 1991 Post Doc - Baylor College of Medicine, Houston, TX, Research Interests: Microbial Pathogenomics, Host-Pathogen Interactions, Functional Genomics. Research in the Kapur Laboratory seeks to define the basic mechanisms by which pathogenic microbes successfully infect, colonize, and cause disease in their hosts. The research effort is organized along three thematic lines: microbial population genetics; microbial pathogenesis; and host response to infection. A translational research component seeks to translate the results of the investigations into improved diagnostic tests and methods for microbial identification, as well as investigate opportunities for developing new generations of antimicrobial vaccines and therapeutics. Synergistic Activities: Dr. Kapur is also Director of the Advanced Genetic Analysis Center ( and co-director of the Biomedical Genomics Center ( at the University of Minnesota and is a member of various genomics and computational biology related advisory committees at the University, national and international levels

101 Jeffrey S. Karns, PhD Environmental Microbial Safety Laboratory, Agricultural Research Service, U. S. Dept. Of Agriculture, Building 173 BARC-East Baltimore Ave, Beltsville, MD Phone: , FAX: Education 1981 Virginia Commonwealth University, Microbiology; PhD 1975 The Pennsylvania State University, Medical Technology, BS Experience Post-Doc, University of Illinois Health Sciences Center, Chicago, IL Post-Doc, University of Maryland, College Park, MD Research Microbiologist, Pesticide Degradation Lab, USDA/ARS, Beltsville, MD Research Microbiologist, Soil Microbial Systems Lab, USDA/ARS, Beltsville, MD Research Microbiologist, Environmental Microbial Safety Laboratory (formerly Animal Waste Pathogens Lab), USDA/ARS, Beltsville, MD Research Accomplishments Characterized the genes and enzymes responsible for the degradation of numerous agrochemicals. Developed methods for the use of pesticide degrading enzymes for the benefit of agriculture. Developed methods for APHIS to control microbial processes in cattle dipping vats so that the acaricide in the vat was preserved in order to get maximum usage and so that microbial processes could be exploited for waste treatment. The methods have become standard operating procedure for the APHIS-VS Tick Eradication Program. Developed real-time PCR methods for the detection of E. coli O157:H7 in soil and water. Investigated the leaching of the organism in soil. Participated in emergency response to anthrax threat in Washington, DC through preparation and operation of a mobile laboratory. Developed protocols for processing of environmental samples and detection of Bacillus anthracis using commercial real-time PCR kits. Honors and Awards Certificates of Merit for outstanding performance in 1995, 1997, 2000 and Certificates of Merit for superior performance in 1990, 1992, 1998, 1999, 2002 and USDA Group Honor Award for Excellence from the Secretary of Agriculture For emergency activities, communications, initiatives and employee security measures taken in response to Sept. 11, 2001 and the anthrax threat to the Department of Agriculture

102 Sung G. Kim, PhD Research Associate Director, Molecular Diagnostics Laboratory Animal Health Diagnostic Center College of Veterinary Medicine Cornell University P.O. Box 5786 Ithaca, NY Dr. Kim received his Masters and Ph. D. degrees from Cornell University. He did his postdoctoral program with Dr. Carl Batt in the area of molecular evolution studies of bacteriophages with an emphasis on DNA sequencing of phage genomes and antisense RNA technology. Since he joined the Animal Health Diagnostic Laboratory at Cornell s College of Veterinary Medicine in 1994, he has been involved in development and application of molecular diagnostic methods to detect pathogens causing animal diseases including BVD, Johne s disease, Lyme disease, Potomac horse fever, E. coli O157/H7, Q-fever, Erhlichiosis, and avian influenza. His research interest is use of molecular diagnostic tools to assure animal and public health. He is currently section head of Molecular Diagnostics at Cornell Animal Health Diagnostic Laboratory

103 Suzanne C. Klaessig Research Support Specialist Department of Population Medicine and Diagnostic Sciences College of Veterinary Medicine S3-118 Schurman Hall Cornell University Ithaca, NY Suzanne Klaessig has been the supervisor of the Cell Biology Laboratory at Cornell for 15 years. The Cell Biology Laboratory is a fee-for-service lab and a section of Quality Milk Production Services. Her responsibilities include training of graduate students, faculty and undergraduates. Suzanne has multi-dimensional experience in research, with extensive experience in mammalian tissue culture. For 10 years, she has also been a staff participant in the Microinjection Techniques section of the Cell Biology course taught at the Marine Biological Laboratory at Woods Hole

104 Kendra K. Nightingale Ph.D. candidate Department of Food Science 412 Stocking Hall Cornell University Ithaca, NY Kendra Nightingale is a Ph.D. candidate in the Department of Food Science at Cornell University, majoring in Food Science with a concentration in Food Microbiology and pursuing minors in Epidemiology and Microbiology. Kendra is originally from a small farming community in Western Kansas. Her most recent work includes studying the molecular epidemiology, ecology, and evolution of the human foodborne and animal pathogen Listeria monocytogenes. Kendra holds a M.S. degree from Kansas State University in Food Science, where her research evaluated the use of lactoferrin, a milk-derived protein, to decontaminate and extend the shelf-life of beef products. She also holds a B.S. degree, cum laude, from Kansas State University, where she participated in the undergraduate honors program. She is a member of several agricultural honor societies including Phi Tau Sigma, and has been recognized with national graduate research fellowships from Institute of Food Technologists, National Milk Producers Federation, and International Association for Food Industry Suppliers as well as awards from the Department of Food Science at Cornell

105 Stephen P. Oliver, PhD Professor, Department of Animal Science Co-Director Food Safety Center of Excellence University of Tennessee 59 McCord Hall 2640 Morgan Circle Drive Knoxville, Tennessee Principle Duties: Research on mammary gland physiology, immunology, and microbiology with emphasis on development of nonantibiotic approaches for the prevention and control of mastitis in dairy cows; and development and evaluation of strategies to control/reduce foodborne pathogens in food producing animals. Educational Background: PhD 1980, Dairy Science (Lactation Physiology), The Ohio State University MS 1978, Dairy Science (Lactation Physiology), The Ohio State University BS 1976, Animal Science, North Carolina State University Other Organizations, Committees, Awards, Honors, Etc. EDITOR-IN-CHIEF of Foodborne Pathogens & Disease, a new peer-reviewed quarterly international journal published both in print and online by Mary Ann Liebert, Inc. Former Chairman and current member of NMC Research Committee Recipient of the 2003 United States Food and Drug Administration s Group Recognition Award as a member of the Tissue Residues & Strategies for Case Development Organization and Training Team. Recipient of several research awards from the American Dairy Science Association (2002 Land O Lakes Award, 1998 Merck AgVet Dairy Management Research Award, 1992 West Agro Award, 1989 Agway Young Scientist Award) and from The University of Tennessee

106 Ynte Hein Schukken, DVM, PhD Director of the Quality Milk Production Services 22 Thornwood Drive Park View Technology Center I Ithaca, New York , USA Tel: (607) Fax: (607) Professor of Epidemiology and Herd Health Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine Cornell University S3 119 Schurmann Hall yhs2@cornell.edu EDUCATION: DVM, University of Utrecht 1985 M.Sc., Cornell University 1987 Ph.D., University of Utrecht 1990 SPECIALTY CERTIFICATION: RESEARCH INTEREST: Bovine Medicine (Royal College of Dutch Veterinarians) Epidemiology (Dutch National Science Foundation) Udder health in well managed dairy herds. A research and service approach based on understanding epidemiology and pathobiology of the diseases affecting mammary health. Understanding population dynamics of infectious diseases in animal populations. Application of epidemiological, statistical and mathematical methods to animal disease research

107 Linda L. Tikofsky, DVM Senior Research Associate Quality Milk Production Services Department of Population Medicine and Diagnostic Sciences College of Veterinary Medicine, Cornell University 22 Thornwood Drive Park View Technology Center I Ithaca, New York , USA Tel: (607) Fax: (607) lg40@cornell.edu Linda graduated with a DVM degree from the University of Illinois in 1984, completed an internship at the University of Missouri and worked in private practice from 1985 through 1997 prior to joining QMPS. Linda began work at QMPS in December of 1997 as a Post Doctoral Associate and now holds the position of Senior Extension Veterinarian. Linda is a field veterinarian and coordinator of the Milker Training Program. On top of her clinical responsibilities, she conducts research into epidemiology of antimicrobial resistance of Staphylococcus aureus. In recent years, Linda established a network of contacts with the organic dairy community, resulting in a series of workshops for organic and transitioning dairy farmers in 2003, a homeopathic treatment trial for S. aureus mastitis in 2004 and most recently, a $518,000 USDA grant for a four-year project entitled "The Transitioning Dairy: Identifying and Addressing Challenges and Opportunities In Milk Quality and Safety"

108 Martin Wiedmann, DVM, PhD Assistant Professor Department of Food Science 412 Stocking Hall Cornell University Ithaca, NY Dr. Martin Wiedmann received a DVM from the Ludwig-Maximilians University in Munich in 1992 and a doctorate in Veterinary Medicine from same university in In 1997, he received a Ph.D. in Food Science from Cornell University. After a two year postdoc in the Cornell Food safety Laboratory with Professor Kathryn Boor, he joined the Department of Food Science at Cornell as an Assistant Professor in His research interests focus on the molecular biology, epidemiology, and transmission of foodborne and zoonotic pathogens. Dr. Wiedmann has published more than 60 peer-reviewed publications, 8 book chapters and reviews, 7 proceedings articles, more than 80 abstracts, and 2 patents. He has given more than 50 invited presentations. Current research in his laboratory is supported by grants from the National Institutes of Health, the USDA National Research Initiative, the USDA Food Safety Initiative, and the International Life Sciences Institute. He was a member of the Listeria Outbreak Working Group, which was honored by a USDA Secretary s Award for Superior Service in 2000 for the detection of a multistate listeriosis outbreak. He also received the Young Scholars award from the American Dairy Science Association in 2002, the Samuel Cate Prescott Award from Institute of Food Technologists, and most recently the International Life Science Institute (ILSI) North America Future Leaders Award

109 Ruth N. Zadoks, DVM, PhD Research Associate Department of Food Science 401 Stocking Hall Cornell University, Ithaca, NY Phone: (607) Ruth Zadoks is a Research Associate in the Laboratory of Food Safety and the Laboratory of Food Microbiology, Department of Food Science, Cornell University, Ithaca NY, and will be heading the new Molecular Laboratory at Quality Milk Production Services, College of Veterinary Medicine, Cornell University. Ruth obtained her DVM (1995, cum laude), MSc (1998, Veterinary Epidemiology and Herd Health), and PhD (2002, cum laude) degrees from the Faculty of Veterinary Medicine, Utrecht University, the Netherlands where she worked as a bovine clinician, and taught bovine herd health and epidemiology of infectious diseases to undergraduate and graduate students from 1996 to The focus of her PhD work, which was carried out in collaboration with the Department of Population Medicine and Diagnostic Sciences at Cornell University, the University of Tennessee, Knoxville TN, and Washington State University, Pullman, WA, was the epidemiology of streptococcal and staphylococcal mastitis in dairy herds. Both mathematical and molecular approaches were used in those studies of mastitis epidemiology. Her current research at Cornell University deals with the ecology, pathogenesis and consequences of contagious and environmental mastitis in dairy herds. In particular, Dr. Zadoks explores the usefulness of bacterial strain typing methods as diagnostic tools in mastitis control, and studies the epidemiology of streptococcal mastitis and antimicrobial resistance in bovine streptococci

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