AN EVIDENCE-BASED APPROACH TO THE CONTROL OF FELINE PANLEUKOPENIA, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN SHELTER CATS

Transcription

1 AN EVIDENCE-BASED APPROACH TO THE CONTROL OF FELINE PANLEUKOPENIA, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN SHELTER CATS By BRIAN ANTHONY DIGANGI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA

2 2011 Brian Anthony DiGangi 2

3 To all those working tirelessly to enhance the health and welfare of shelter animals 3

4 ACKNOWLEDGMENTS I thank Alachua County Animal Services, Biogal Galed Laboratories, Jacksonville Animal Care and Protective Services, Jacksonville Humane Society, Maddie s Fund, Merial Veterinary Scholars Program, New York State Animal Health Diagnostic Center, and Sweetbay Foundation for their generous support of these studies. I thank my committee members: Dr. Julie Levy for her efficient and thorough editing, Dr. Brenda Griffin for her unwavering support and encouragement, and Dr. Jeff Abbott for going with the flow. I am grateful to Dr. Cynda Crawford for her advice and support along the way and Dr. Natalie Isaza for her patience and understanding as I completed these studies. I thank Michael Crandall, Patty Dingman, Katie Green, and Sylvia Tucker for their assistance with data collection and organization. Finally, thanks to Mom, Dad and Dana for demonstrating their support and genuine interest in all my educational pursuits. 4

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS... 4 LIST OF TABLES... 7 LIST OF FIGURES... 8 LIST OF ABBREVIATIONS... 9 ABSTRACT INTRODUCTION Feline Panleukopenia (FPV) Disease Overview Risk Factors and Significance to Animal Shelters Feline Herpesvirus-1 (FHV) and Feline Calicivirus (FCV) Disease Overview Risk Factors and Significance to Animal Shelters Vaccination against FPV, FHV, and FCV Vaccination Protocols for Shelter Cats Vaccine Failure Vaccine Type Serologic Surveys of Cats Use of Serology Feline Panleukopenia Feline Herpesvirus-1 and Feline Calicivirus Outbreak Management Definition and Initial Response Individual Animal Risk Assessment Summary and Objectives EFFECTS OF MATERNALLY DERIVED ANTIBODIES (MDA) ON SEROLOGIC RESPONSES TO VACCINATION IN KITTENS Background Materials and Methods Animals Vaccination Serological Testing Statistical Analysis Results MDA Titer Groups Serum Anti-FPV Antibody Titers Serum Anti-FHV Antibody Titers Serum Anti-FCV Antibody Titers

6 Serum Anti-Rabies Antibody Titers Discussion PREVALENCE OF PROTECTIVE ANTIBODY TITERS (PAT) FOR FELINE PANLEUKPENIA VIRUS, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN CATS ENTERING FLORIDA ANIMAL SHELTERS Background Materials and Methods Study Sites Animals Serological Testing Statistical Analysis Results Cats Factors Associated with PAT Discussion DETECTION OF PROTECTIVE ANTIBODY TITERS AGAINST FELINE PANLEUKOPENIA, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN CATS USING A POINT-OF-CARE ELISA Background Materials and Methods Samples Serological Testing Statistical Analysis Results Pilot Testing of Canine and Feline Assays Definitive Testing of Feline Assay Discussion CONCLUSIONS AND SUMMARY Effect of MDA on Serological Response to Vaccination Prevalence and Risk Factors for PAT Detection of PAT Creating an Evidence-Based Plan for Disease Control Disease Prevention Outbreak Response LIST OF REFERENCES BIOGRAPHICAL SKETCH

7 LIST OF TABLES Table page 1-1 Professional veterinary medical guidelines for vaccination against feline panleukopenia (FPV), feline herpesvirus-1 (FHV) and feline calicivirus (FCV) Advantages and disadvantages of modified- live virus (MLV) versus inactivated (IA) vaccinations for FPV, FHV, and FCV Prevalence of protective antibody titers (PAT) against FPV, FHV, and FCV in various feline populations Median (range) serum antibody titers against FPV, FHV, and FCV in specificpathogen-free kittens Demographics of 418 cats and kittens admitted to 3 animal shelters in north Florida Results of univariate logistic regression analysis of factors associated with PAT against FPV Results of univariate logistic regression analysis of factors associated with PAT against FHV Results of univariate logistic regression analysis of factors associated with PAT against FCV Results of multivariate analysis of factors associated with PAT against FPV, FCV, and FHV Results of 2 semi-quantitative point-of-care assays for the detection of PAT Performance of a feline point-of-care assay for the detection of PAT against FPV, FHV, and FCV Calculated positive and negative predictive values of a feline point-of-care enzyme-linked immunosorbent assay (ELISA) for 3 different prevalence levels of FPV, FHV, and FCV Percent of concordant results and strength of agreement of a point-of-care assay for the detection of PAT against FPV, FHV, and FCV Risk factors for PAT against FPV, FHV, and FCV for cats entering animal shelters Presumed risk factors for infection with FPV, FHV, and FCV for cats in animal shelters

8 LIST OF FIGURES Figure page 1-1 Components of the epidemiological triad that should be considered in response to an infectious disease outbreak in an animal shelter Proportion of specific-pathogen-free kittens with protective antibody titers (PAT) against feline panleukopenia (FPV) Proportion of specific-pathogen-free kittens with PAT against feline herpesvirus-1 (FHV) Proportion of specific-pathogen-free kittens with PAT against feline calicivirus (FCV) Distribution of antibody titers for FPV (A), FHV (B), and FCV (C) A feline point-of-care enzyme-linked immunosorbent assay (ELISA) kit for the determination of PAT against FPV. FHV, and FCV Evidence-based individual risk assessment protocol for cats entering animal shelters during an outbreak of FPV

9 LIST OF ABBREVIATIONS BCS CI ELISA FCV FeLV FHV FIV FPV HI IA MDA MLV NPV OR PAT PPV REF SC SN URTD Body condition score Confidence interval Enzyme-linked immunosorbent assay Feline calicivirus Feline leukemia virus Feline herpesvirus-1 Feline immunodeficiency virus Feline panleukopenia virus Hemagglutination inhibition Inactivated Maternally-derived antibodies Modified-live virus Negative predictive value Odds ratio Protective antibody titer Positive predictive value Referent Subcutaneous Serum neutralization Upper respiratory tract disease 9

10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science AN EVIDENCE-BASED APPROACH TO THE CONTROL OF FELINE PANLEUKOPENIA, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN SHELTER CATS Chair: Julie K. Levy Major: Veterinary Medical Sciences By Brian Anthony DiGangi May 2011 Infections with feline panleukopenia (FPV), feline herpesvirus-1 (FHV), and feline calicivirus (FCV) threaten the health and welfare of cats in animal shelters, representing a significant cause of morbidity and mortality in this population. This situation contrasts with that for cats in the general pet population where infections with FHV and FCV are generally minor nuisances and seldom threaten an individual cat s life and FPV infection is rare. Despite the importance of these diseases to the millions of cats residing in animal shelters in the United States, there are significant gaps in our knowledge pertaining to their control in the shelter setting. A series of experiments was undertaken to enhance the ability of shelter veterinarians and staff to make evidence-based decisions when managing FPV, FHV, and FCV in populations of cats. Vaccination represents the cornerstone of control of FPV, FHV, and FCV; however, successful immunization is influenced by a number of factors. In particular, maternally-derived antibody (MDA) interference and the type of vaccine product administered are known to effect vaccine response. The first experiment described in this work evaluated the impact of MDA interference on vaccination using modified-live 10

11 virus (MLV) versus inactivated (IA) products in specific-pathogen-free kittens in order to provide evidence upon which the most effective vaccination protocols can be based. The data reported indicate that induction of protective antibody titers (PAT) in naïve animals is most efficiently achieved when MLV products are used in the face of low MDA, and when the initial vaccination protocol is extended beyond 14 weeks of age. Serology can be a useful tool for the evaluation of the prevalence of cats at risk for disease and the need for vaccination, particularly for FPV, but little to no serological data pertaining specifically to cats entering animal shelters have previously been published. The second experiment sought to provide objective evidence of risk of infection with FPV, FHV, and FCV based on antibody titer assessment at the time of admission to animal shelters. The data indicate that 59.1%, 89.5%, and 63.6% of 418 cats entering Florida animal shelters lack PAT against FPV, FHV, and FCV, respectively, and that various animal-related factors (such as age, neuter status, and origin) can be correlated with the likelihood of the presence of PAT. Finally, although vaccination history and the determination of PAT may be among the most important components of individual animal risk assessment during disease outbreak response, current methods of determining PAT are not practical for regular use in most animal shelters. This is due to the need for commercial diagnostic laboratory analysis and the associated high costs and delays in result reporting. The third experiment evaluates the use of a point-of-care enzyme-linked immunosorbent assay (ELISA) for determination of PAT against FPV, FHV, and FCV in a population of shelter cats. The data indicate that the ELISA s high positive predictive value (96%) may allow for limited utility in specific shelter populations, but it s low sensitivity (49%) is an 11

12 obstacle to widespread use. In general, titer determination remains an expensive and time-consuming process for most animal shelters. The experiments described in this work enhance our knowledge and understanding of the management of FPV, FHV and FCV infections. An example of how this information can be utilized to create an evidence-based response plan to an outbreak of these diseases in an animal shelter is presented. The collection of data presented herein provide evidence upon which shelter medicine practitioners can build objective protocols for the prevention and control of FPV, FHV, and FCV in shelter cats. 12

13 CHAPTER 1 INTRODUCTION Feline Panleukopenia Disease Overview Feline panleukopenia (FPV) is a non-enveloped, single-stranded DNA virus that can cause disease in all feline species. Fortunately, due to widespread vaccination, the majority of infections in adult cats are subclinical. In contrast, FPV infection is often severe in susceptible individuals with highest morbidity and mortality in kittens between 3 and 5 months of age (Greene and Addie 2006). FPV can be shed in all body secretions, particularly feces. The viral shedding period is typically 1-2 days, although it can persist as long as 6 weeks following recovery from clinical illness (Greene and Addie 2006). In utero transmission is possible, but the virus most significant means of transmission is via fomites and mechanical vectors contaminated with feces (such as litter boxes, clothing, shoes, hands, and food/ water bowls). The virus has incredible persistence in the environment and is resistant to common disinfectants, allowing for direct transmission in cats living in a contaminated environment (Greene and Addie 2006). After an incubation period ranging from 2 to 14 days (Tuzio 2009), FPV targets tissues with the greatest rate of mitotic activity resulting in severe systemic disease. In utero infection can result in infertility, fetal death, fetal resorption, abortion, optic nerve atrophy, retinopathy, hydrancephaly and cerebellar hypoplasia. Infection after birth may result in lymphoid tissue necrosis followed by proliferation, thymic involution and degeneration, damaged intestinal mucosal crypt cells and shortening of intestinal villi resulting in bacterial translocation and malabsorptive diarrhea. Neutropenia results first 13

14 from the exudation of neutrophils into infected gastrointestinal tissue and is followed by panleukopenia resulting from direct bone marrow suppression by the virus. Secondary bacterial infections are common and can be followed by endotoxemia, sepsis and, occasionally, disseminated intravascular coagulation. Myocarditis and cardiomyopathy have also been identified in kittens born to queens exposed to FPV during pregnancy (Greene and Addie 2006). Finally, acute death may occur in infected individuals without any preceding clinical signs. Presumptive diagnosis of FPV infection is most commonly made based on suggestive clinical signs in the presence of leukopenia (Greene and Addie 2006). An increasingly popular method of FPV diagnosis is via the use of a fecal enzyme-linked immunosorbent assay (ELISA) for canine parvovirus, which has been validated for use in cats (Abd-Eldaim, Beall et al. 2009; Tuzio 2009). Serology indicating a fourfold rise in antibody titers as well as virus isolation and fecal polymerase chain reaction may also be utilized (Schunck, Kraft et al. 1995; Greene and Addie 2006). Segmental enteritis of the jejunum and ileum is a common necropsy finding (Greene and Addie 2006). As with most viral infections, therapy for FPV relies on symptomatic and supportive care. Antiemetics, antimicrobials, fluid therapy including blood or plasma transfusion, and vitamin therapy as well as immune serum, immunostimulants, and antiviral drugs have been utilized in the treatment of FPV infection (Greene and Addie 2006; Tuzio 2009). Risk Factors and Significance to Animal Shelters Factors associated with increased risk of disease from FPV in pet cats include being less than 1 year of age and having an incomplete vaccination history (Kruse, Unterer et al. 2010). Studies evaluating risk factors for cats in animal shelters are 14

15 scarce, but it has been demonstrated, not surprisingly, that the likelihood of infection and death from FPV increases with decreasing age (Cave, Thompson et al. 2002). In contrast to the case for the pet cat population, FPV is a constant and serious threat to the health and welfare of cats in animal shelters. This is because animal shelters frequently house animals under stressful conditions that make exposure and transmission likely. For example, high density housing and frequent introduction of susceptible cats are common in many facilities. In addition, sanitation and preventive health care protocols are often inadequate, further increasing the risk of disease. Such deficiencies create optimal situations for the introduction and maintenance of FPV and shelter staff members frequently unwittingly facilitate the transmission of infection throughout the population. Outbreaks of FPV in shelters have been reported across the country and are frequently managed by depopulation of both clinically diseased and healthy exposed cats (Pappas 2005; Patterson, Reese et al. 2007; King 2010). Feline Herpesvirus-1 and Feline Calicivirus Disease Overview Feline herpesvirus-1 (FHV) and feline calicivirus (FCV) are the most common viral agents resulting in upper respiratory tract disease (URTD) in cats, with as many as 86% and 77% of some cat populations demonstrating serologic evidence of previous exposure to FHV and FCV, respectively (Goto, Horimoto et al. 1981). FHV is a doublestranded DNA, α-herpesvirus with a glycoprotein-lipid envelope. FCV is a nonenveloped, single-stranded RNA virus (Gaskell 2006). Both viruses are widespread in the feline population and are primarily transmitted via fomite contact with nasal, oral and conjunctival secretions. Direct cat-to-cat and macrodroplet transmission are also common (Scarlett 2009). Control of these infections is difficult due to their epidemiology 15

16 as well the fact that vaccination only provides partial immunity. Active FHV infection is always followed by a latent infection that may result in intermittent viral shedding. Reactivation of clinical disease may occur, particularly during times of stress. Numerous viral strains of FCV exist with varying levels of pathogenicity and infection results in a chronic carrier state in a large proportion of cats. FHV is easily destroyed by common disinfectants and can only survive up to 18 hours outside of the host (Povey 1979; Gaskell 2006). In contrast, FCV is frequently resistant to disinfection, and can survive up to 28 days outside of the host (Doultree, Druce et al. 1999; Gaskell 2006). Clinical signs of both FHV and FCV are similar and generally result in mild, selflimiting illness in most healthy pet cats. Incubation periods for FHV and FCV can vary considerably, although 2-6 days for FHV and 1-2 days for FCV is typical (Foley and Bannasch 2004). Depression, sneezing, inappetance, pyrexia, ocular and nasal discharge and conjunctivitis are common. Oral ulceration may also occur. Keratitis is a clinical finding unique to FHV, while primary viral pneumonia and lameness are occasionally seen with FCV infection (Gaskell 2006). Diagnosis of URTD is most commonly based on the presence of distinctive clinical signs. Virus isolation, fluorescent antibody evaluation of conjunctival smears, and PCR may be warranted to confirm the diagnosis when increases in disease frequency or severity are evident (Gaskell and Povey 1982; Gaskell 2006; Scarlett 2009). In addition, bacterial cultures may be used to identify the causative agent of concurrent bacterial infections. Due to their relatively high cost and technical nature, these diagnostic tests are seldom utilized in animal shelters as a routine component of an infectious disease management plan. Treatment of URTD relies on symptomatic and 16

17 supportive care and administration of antimicrobials for the control of secondary bacterial infections. Vaccination, stress reduction and prevention of transmission through careful biosecurity are the mainstays of minimizing morbidity and severity of clinical disease from FHV and FCV (Scarlett 2009). Risk Factors and Significance to Animal Shelters A variety of risk factors for URTD have been identified. Factors such as young age, a recent visit to a boarding facility, lapses in vaccination history, living in a research colony and living with dogs with respiratory disease were all found to increase the risk of URTD (Binns, Dawson et al. 2000). Cats that come into contact with cats outside of their home may be at greater risk for FHV infection (Sykes, Anderson et al. 1999). Regarding animal shelter populations, it has been demonstrated that increasing length of stay is correlated with URTD, particularly for kittens (Dinnage, Scarlett et al. 2009). Despite these findings, it has been suggested that such cat-related factors are not as important as facility-related factors (e.g., housing density, separation of intake and isolation areas) in assessing the risk of URTD in animal shelters (Edwards, Coyne et al. 2008). As in the case of FPV, the environment of an animal shelter is ideal for the introduction, transmission and maintenance of FHV and FCV in the population, thus these infections frequently become endemic (Scarlett 2009). Whereas these infections are generally relatively minor nuisances in pet cats, they frequently become severe and even life-threatening in the shelter environment. This is because disease severity increases during times of stress and shelters commonly lack adequate resources for treatment, resulting in the euthanasia of sick cats. 17

18 Vaccination against FPV, FHV, and FCV Vaccination Protocols for Shelter Cats According to professional veterinary medical guidelines, vaccination is the cornerstone for prevention of disease caused by FPV, FHV and FCV. Vaccination protocols should be tailored according to an individual cat s risk and cats residing in animal shelters are considered to be at great risk of acquiring these diseases (Table 1-1) (Richards, Elston et al. 2006; Griffin 2009). In fact, the risk is so great, that shelter cats should be vaccinated even in the face of visible signs of illness and pregnancy (Richards, Elston et al. 2006). The potential benefits of vaccinating such individuals outweigh the risks posed by the administration of vaccine products. The rapid onset of immunity after administration of vaccine products supports their use shortly before or at admission to the shelter environment (Brun, Chappuis et al. 1979; Lappin, Sebring et al. 2006; Jas, Aeberle et al. 2009; Lappin, Veir et al. 2009). Vaccine Failure A number of environmental and physical factors have been implicated in cases of failed immunization. Among these are concurrent illness, exposure to high numbers of pathogens, improper vaccine handling or administration, and vaccination at the time of anesthesia or surgery (Richards, Elston et al. 2006). While some of these concerns are legitimate, others have been disproven. In the case of perioperative vaccination, kittens and feral cats have been shown to mount a significant immune response even when undergoing anesthetic and surgical procedures (Fischer, Quest et al. 2007; Reese, Patterson et al. 2008). MDA interference is a particularly well-documented cause of vaccine failure in kittens up to 14 weeks of age (Richards, Elston et al. 2006). The presence of MDA 18

19 during vaccination may result in elimination of the vaccine antigen, preventing effective immunization. In fact, it has been reported that effective immunization of kittens against FPV may be prevented when MDA titers as low as 10 are present (Greene and Addie 2006). The initial level of MDA is also thought to influence the duration of MDA interference and a wide variation exists with MDA interference up to 16 weeks of age for FPV (Scott, Csiza et al. 1970), 10 weeks of age for FHV (Gaskell and Povey 1982; Johnson and Povey 1985), and 14 weeks of age for FCV (Johnson and Povey 1983). These variables can have a substantial impact on the duration of the vaccination protocol that is chosen for an individual or group of cats. In some cases, vaccine failure may be perceived as a result of misunderstanding the intended effects of certain vaccine products. Vaccination against FPV produces sterilizing immunity and prevents the development of clinical signs associated with infection with FPV. In contrast, vaccination against FHV and FCV are only capable of inducing partial immunity, reducing the severity and duration of clinical signs, but not preventing infection (Gaskell 2006; Greene and Addie 2006; Richards, Elston et al. 2006). Finally, it is important to recognize that a very small proportion of individuals will fail to respond to individual antigens or to vaccination in general (i.e., non-responders). Vaccine Type The advantages and disadvantages of inactivated (IA) versus modified-live virus (MLV) vaccines have been well described (Table 1-2) (Greene and Addie 2006). Both types of vaccines are effective in preventing disease (in the case of FPV) and reducing the severity of disease (in the case of FHV and FCV) (Johnson and Povey 1985; Greene and Addie 2006). In general, MLV vaccines provide a more rapid onset of immunity and are better able to overcome MDA interference than IA products (Gaskell 19

20 and Povey 1982; Greene and Addie 2006; Richards, Elston et al. 2006; Lappin and Jensen 2007). As such, MLV vaccines are the preferred type of vaccination in high-risk situations such as in animal shelters (Greene and Schultz 2006; Richards, Elston et al. 2006; Griffin 2009). In the absence of MDA, protective titers are usually achieved after a single priming dose of a MLV product (Greene and Addie 2006; Fischer, Quest et al. 2007). Even so, a second priming vaccination is still commonly recommended to enhance the anamnestic response (Richards, Elston et al. 2006; Griffin 2009). Although IA vaccinations against FPV, FHV, and FCV are commonly reported to require two injections to induce a titer equivalent to that attained following a single injection of a MLV product (Greene and Addie 2006), data from a population of previously unvaccinated feral cats demonstrated induction of protective immunity and equivalent titers after only one injection of an IA or MLV product (Fischer, Quest et al. 2007). Studies investigating the effects of MDA on the serological responses of cats vaccinated with both MLV and IA products against FPV, FHV and FCV are needed in order to better characterize the onset of immunity following vaccination and to optimize vaccination protocols for shelters. Serologic Surveys of Cats Use of Serology Detection of serum antibody titers against FPV, FHV and FCV has been utilized to estimate disease prevalence in various cat populations (Greene and Addie 2006; Fischer, Quest et al. 2007; Tuzio 2009). In addition, titer measurement can be used as evidence of previous infection and in evaluating the onset, magnitude, and duration of response to vaccination. In addition, FPV titers have been used in the assessment of individual animal risk during disease outbreaks in animal shelters. In these cases, cats 20

21 without clinical signs of disease that have high titers against FPV are presumed to be at little to no risk of disease (Larson, Newbury et al. 2009). The antibody titer sufficient to prevent disease when challenged by an infectious dose of a virus is commonly referred to as a protective antibody titer (PAT); however, the precise numerical titer reported as PAT varies by laboratory and methodology (Greene and Schultz 2006). Caution must be taken when using serum antibody titers as indications of absolute protection against disease their presence does not ensure protection and their absence does not indicate susceptibility in every case (Greene and Schultz 2006). The method of antibody measurement (i.e., serum neutralization [SN] vs. hemagglutination inhibition [HI] vs. ELISA vs. immunofluorescent antibody), the type of infection (i.e., extracellular vs. intracellular), and the type of antibody measured (i.e., IgG vs. IgA vs. IgM) all influence the degree of correlation between a titer value and protection against disease (Greene and Schultz 2006). Despite these limitations, studies have demonstrated that serum antibody titers can be a useful measurement of protection against FPV, FHV and FCV (Scott and Geissinger 1999; Lappin, Andrews et al. 2002). Feline Panleukopenia Serologic studies have demonstrated the ubiquitous nature of FPV in the feline population of the United States, particularly in unvaccinated free-roaming strays (Greene and Addie 2006; Fischer, Quest et al. 2007; Tuzio 2009). Detectable FPV antibodies have been demonstrated in 31 to 58% of laboratory and healthy household cats, although the vaccination history of these cats is unknown (Goto, Horimoto et al. 1981; Tsuchihashi and Soma 2006). Similarly, a Wisconsin animal shelter reported PAT against FPV in 48% of cats, though it is unknown if PAT was the result of vaccination or 21

22 previous exposure (Table 1-3) (Tuzio 2009). Large-scale studies of the prevalence of PAT in cats upon admission to animal shelters have not been reported previously despite the potential value of such information in the development of rational disease control strategies. Such information could provide insight into the optimal vaccination protocols for shelter cats as well as assessment of individual animal risk during a FPV outbreak. Feline Herpesvirus-1 and Feline Calicivirus In the general healthy feline population, prevalence of detectable antibody titers ranges from 21 to 86% for FHV and from 64 to 77% for FCV (Table 1-3) (Goto, Horimoto et al. 1981; Fischer, Quest et al. 2007). No data demonstrating the prevalence of PAT in animal shelters have been reported. As in the case of FPV, this information may be useful in the development of optimal vaccination protocols for shelter cats. However, in contrast to FPV, the correlation of antibody titers against FHV and FCV to immunity in individual cats is not always clear cut. The development of chronic carrier states and partial immunity associated with FHV and FCV infection can confound titer interpretation. Outbreak Management Definition and Initial Response An infectious disease outbreak is defined as an increase in the frequency and/or severity of a given disease in a population. As previously described, outbreaks of FPV are common in animal shelters. When FPV is introduced to a susceptible population of cats, it typically results in high morbidity and mortality, prompting immediate recognition of the need for a strategic response to limit its transmission. In contrast, FHV and FCV are often endemic in animal shelters (Scarlett 2009), resulting in a lack of urgency 22

23 perceived among shelter staff to take steps to curtail disease. A notable exception occurs with outbreaks involving virulent systemic calicivirus. Unlike most strains of calicivirus, virulent systemic calicivirus strains are associated with both high morbidity and high mortality. Cross-protection from vaccination or exposure to other strains of calicivirus have not been demonstrated and immediate action is required to limit disease spread and mortality (Hurley, Pesavento et al. 2004; Pesavento, MacLachlan et al. 2004; Foley, Hurley et al. 2006; Reynolds, Poulet et al. 2009). Once identified, the response to any disease outbreak in an animal shelter must take into account all components of the epidemiological triad because disease results from a combination of animal, environmental and agent factors, rather than any single variable (Figure 1-1) (Hurley 2005; Hurley 2009). Many animal and environmental factors can be manipulated to decrease the risk of disease, and the mechanisms for doing so have been thoroughly described (Hurley 2005). In contrast, little can be done to alter the agent-related factors. In general, six core responses are required for the management of a disease outbreak in an animal shelter (Hurley 2009). These include: 1. Diagnosis and isolation of diseased animals 2. Identification and management of exposed/at-risk animals 3. Environmental decontamination 4. Protection of newly admitted animals 5. Documentation 6. Communication with staff members, stakeholders, adopters, and the public In particular, effective management of an outbreak relies heavily on the prompt identification of both exposed and at-risk (i.e., susceptible) animals. For this reason, individual animal risk assessment is crucial to limiting morbidity and mortality during an 23

24 outbreak. In the case of FPV, accurate risk assessment of individual cats has the potential to make or break disease control efforts. Individual Animal Risk Assessment Once an outbreak is identified, further response relies principally on the identification and subsequent isolation of animals exposed to the infectious agent of concern that have not yet developed clinical signs of disease (Hurley 2005; Hurley 2009). Determining the likelihood that an individual will become infected if exposed can be done by assessing that individual s risk based on known risk factors. Such risk factor analysis has been utilized in the human medical field for a variety of disease states to aid in clinical and diagnostic decision-making (Antman, Cohen et al. 2000; Mann, Neal et al. 2007; Singh, Gersh et al. 2008). This methodology can be applied to the development of an outbreak response in an animal shelter (Hurley 2009). The proximity to a known infected animal and, therefore, the dose of infectious agent to which it is exposed, the vaccination history, and the age of vaccination of an atrisk animal are important considerations in individual animal risk assessment (Hurley 2009). Additionally, for diseases for which antibody titers correlate with protection against illness (such as FPV), assessment of serum antibody titers is recommended (Hurley 2009). Despite the potential benefits of utilizing serum antibody titers as part of a disease outbreak management plan, titer determination remains cost prohibitive for many animal shelters. In addition, sending titers to commercial reference laboratories for analysis generally requires a large sample size, is expensive, and results typically take several days. Identification of animal factors that are reliably associated with the presence of PAT against FPV could greatly enhance individual risk assessment and animal 24

25 management during an outbreak. In other words, if certain animal characteristics (such as age or previous health care history) were demonstrated to be highly correlated with PAT against FPV, cats with these characteristics could be deemed to be at low risk of disease if exposed during an outbreak, decreasing the need for quarantine or euthanasia. These cats could safely and selectively be admitted to the shelter and directed toward an adoption pathway even in the midst of an outbreak if intake could not be diverted. Summary and Objectives Infection with FPV, FHV, and FCV represents a significant threat to the health and welfare of cats in animal shelters. Despite the importance of effective immunization against these diseases, previously published studies investigating the interaction between MDA and the type of vaccination on serological responses of cats are lacking. Serology can be a useful tool for the evaluation of the proportion of cats at risk for disease, particularly for FPV, but little to no serological data pertaining specifically to cats entering animal shelters have been published. The presence of protective titers and an adequate vaccination history have been cited among the most important criteria for individual animal risk assessment during disease outbreak response in animal shelters; however, little information is available to shelter managers or veterinarians to aid in the identification of cats likely to have PAT. Furthermore, current methods of determining PAT are not practical for regular use in most animal shelters. Validation of in-house, point-of-care tests for titer measurement could enhance the utility of titer testing by providing immediate results, saving time and money. The main focus of this investigation was to develop an evidence-based approach to the control of these important diseases in shelter cats. A series of experiments was 25

26 performed in order to enhance currently available knowledge regarding vaccination of cats as well as to better define the number of susceptible cats entering shelters and to determine factors associated with the presence of PAT upon admission. Specific experimental objectives included the following: 1. Describe the effect of MDA and the type of vaccination (MLV vs. IA) on serological response in kittens in order to better define optimal vaccination strategies 2. Determine the prevalence of PAT and readily identifiable animal factors associated with PAT for cats entering animal shelters in order to facilitate the determination of individual animal risk for infection and/or disease 3. Evaluate 2 point-of-care assays for the detection of PAT against FPV, FHV, and FCV as a practical tool for use in animal shelters. 26

27 Table 1-1. Professional veterinary medical guidelines for vaccination against FPV, FHV and FCV for cats residing in households and animal shelters (Richards, Elston et al. 2006; Griffin 2009). Vaccine Household Pets Feline Panleukopenia, Feline Herpesvirus-1 & Feline Calicivirus Shelter Cats Feline Panleukopenia, Feline Herpesvirus-1 & Feline Calicivirus Kittens (<16 weeks) Begin at 6 weeks Repeat every 3-4 weeks until 16 weeks Begin at 4-6 weeks Repeat every 2 weeks until 16 weeks Adults (>16 weeks) Booster Administer 2 doses 3 to 4 weeks apart Administer 2 doses 2 weeks apart Single dose 1 year following last dose of initial series, then every 3 years Single dose 1 year following last dose of initial series, then every 3 years Table 1-2. Advantages and disadvantages of MLV versus IA vaccinations for FPV, FHV, and FCV. MLV IA Advantages Disadvantages Advantages Disadvantages More rapid onset of immunity than IA No risk to developing fetuses Better able to overcome MDA May cause disease in developing fetuses May cause signs of disease Less susceptive to cold-chain disruption Do not require reconstitution Not capable of causing signs of illness Less able to overcome MDA than MLV Require adjuvant which may increase vaccine reactions, including vaccineassociated sarcoma 27

28 Table 1-3. Prevalence of protective antibody titers against FPV, FHV, and FCV in various feline populations. Note: Specific titer value representing PAT may vary between studies. Source Maximum Prevalence (%) FPV FHV FCV Population Random-source laboratory cats Goto, 1981 Tsuchihashi, 2006 Household pets Fischer, 2007 Feral cats Tuzio, 2009 Feral cats Tuzio, 2009 Animal shelters

29 Animal Signalment Vaccination history Health status Nutrition Stress level Environment Population density Cleaning & disinfection Segregation Air quality Noise Temperature Humidity Light cycles Agent Virulence Dose Route of transmission Carrier states Figure 1-1. Components of the epidemiological triad that should be considered in response to an infectious disease outbreak in an animal shelter (Hurley 2005). 29

30 CHAPTER 2 EFFECTS OF MATERNALLY DERIVED ANTIBODIES ON SEROLOGIC RESPONSES TO VACCINATION IN KITTENS Background FPV, FHV, and FCV are present throughout the United States (Gaskell and Povey 1982; Binns, Dawson et al. 2000; Greene and Addie 2006; Scarlett 2009). Due to the widespread use of vaccines in the general pet cat population, FPV is an infrequent cause of life-threatening gastroenteritis in owned cats. FHV and FCV are more common, but usually result in mild to moderate transient upper respiratory disease. In contrast, these infections are a constant and serious threat to the health and welfare of cats residing in animal shelters, often resulting in death due to disease progression or euthanasia. Outbreaks of FPV in shelters have been reported across the country and are frequently managed by depopulation of both clinically diseased and healthy exposed cats (Pappas 2005; Patterson, Reese et al. 2007; King 2010). The risk of upper respiratory disease and associated euthanasia increases with the length of shelter stay (Edinboro, Janowitz et al. 1999; Dinnage, Scarlett et al. 2009). Because of these threats, a rapid response to vaccination is critical in the high-risk environment of an animal shelter (Richards, Elston et al. 2006; Griffin 2009; Day, Horzinek et al. 2010). By regulation, vaccine licensing trials are carried out in seronegative laboratory cats. In the absence of interfering maternal antibodies, kittens generally develop sterilizing immunity or resistance to clinical disease after 1-3 vaccine doses (Slater and York 1976; Brun, Chappuis et al. 1979; Johnson and Povey 1985; Cocker, Newby et al. 1986; Lappin, Sebring et al. 2006; Patterson, Reese et al. 2007; Jas, Aeberle et al. 2009). In the field, the presence of MDA at the time of initial vaccination substantially reduces response to vaccination, such that a large proportion of kittens with MDA may 30

31 fail to develop protective antibody titers (PAT) by the end of the initial vaccination series (Dawson, Willoughby et al. 2001; Poulet 2007; Reese, Patterson et al. 2008). For pet cats with little chance of exposure to viral infections, the risk associated with a delay in effective immunization is minimal. In contrast, kittens entering an animal shelter may face high-dose exposure to viral pathogens under stressful conditions. Rapid immunization may be life-saving in this environment. It is generally not possible to predict which kittens carry interfering MDA. Variability in MDA levels exists even among individuals from the same litter, resulting in a range of susceptibility to infection and the ability to respond to vaccination. Thus vaccination protocols must be developed to successfully immunize kittens with all levels of MDA. The optimal vaccination protocol to induce rapid immunity in kittens with MDA is unknown. Various authors have proposed that either IA vaccines or MLV vaccines are superior for overcoming MDA (Richards, Elston et al. 2006; Poulet 2007). The purpose of the study reported here is to determine the effect of MDA on the serologic responses to vaccination with either IA or MLV vaccines. Materials and Methods Animals Thirteen specific-pathogen-free domestic shorthair queens were bred to give birth to 16 litters of kittens. Twenty-seven kittens were included in the study. All adult cats and kittens were free of feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies as determined by a point-of-care ELISA kit. 1 1 SNAP FIV/FeLV Combo test, IDEXX Laboratories Inc, Westbrook, ME. 31

32 Food 2 and water were offered free choice throughout the study. Kittens were weaned at 8 weeks of age and placed in group housing. Kittens were identified by use of radiofrequency identification microchips. 3 At the conclusion of the study, the animals were surgically sterilized and adopted. The research protocol was approved by the University of Florida Institutional Animal Care and Use Committee and was conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Vaccination All kittens were vaccinated at 8, 11, and 14 weeks of age against FPV, FHV, FCV, and FeLV. The kittens were randomly selected using a computer-generated random numbers table for vaccination with either a MLV vaccine 4 (n=16) or an IA vaccine 5 (n=11). Both products contained IA FeLV vaccine. The vaccines were administered SC in the left hind limb, distal to the stifle joint, as recommended by the American Association of Feline Practitioners for vaccines containing FeLV. At 14 weeks of age, an IA rabies virus vaccine 6 was administered SC in the right hind limb, distal to the stifle. Serological Testing Blood samples (3 to 5 ml) were obtained from kittens via jugular venipuncture at 8, 9, 11, 14, and 17 weeks of age. On vaccination days, blood was collected 2 Iams Kitten Formula. The Iams Co, Dayton, OH. 3 Microchip identification system, AVID, Folsom, LA 4 Fel-O-Guard Plus 3 Lv-K, Fort Dodge Animal Health, Fort Dodge, IA. 5 Fel-O-Vax PCT, Fort Dodge Animal Health, Overland Park, KS. 6 Rabvac 3 TF, Fort Dodge Animal Health, Fort Dodge, IA. 32

33 immediately prior to vaccination. Blood samples were collected into serum separator tubes, allowed to clot for 30 minutes, and then centrifuged for 20 minutes. Serum was separated and stored at -20º C pending analysis. Serum antibody titers were measured for FPV (hemagglutination inhibition [HI]), FHV-1 (serum neutralization [SN]), and FCV (SN) by a university-affiliated diagnostic laboratory. 7 Minimum protective titers established by the laboratory in which assays were conducted were 40, 16 and 32 for FPV, FHV, and FCV, respectively. Kittens whose antibody titers at 8 weeks were 20, 4, and 8 for FPV, FHV, and FCV respectively were considered to have high MDA levels. These titers were selected based on previous observations regarding responses to vaccination in the authors laboratory (Patterson, Reese et al. 2007; Reese, Patterson et al. 2008). Those whose titers were 10, 0, and 6 for FPV, FHV, and FCV, respectively were considered to have low MDA levels. Serum antibody titers were measured for rabies virus (SN) at 14 and 17 weeks of age by a university-affiliated diagnostic laboratory. 8 Statistical Analysis Proportions of kittens with PAT against FPV, FHV, and FCV in each of the 4 groups (low vs. high MDA and MLV vs. IA vaccine) were compared via the Fisher exact test for each time point. Values of P < 0.05 were considered significant. 7 Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY. 8 Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS. 33

34 Results MDA Titer Groups Seventeen kittens were determined to have low MDA and 10 kittens were determined to have high MDA against FPV on the basis of 8-week pre-vaccination antibody titers. Seventeen kittens had low MDA and 10 kittens had high MDA against FHV. Sixteen kittens had low MDA and 11 kittens had high MDA against FCV (Table 2-1). Serum Anti-FPV Antibody Titers Fourteen of 27 kittens (52%) had detectable MDA against FPV at the time of the 8-week pre-vaccination antibody titers and 13 were seronegative. Of the 10 kittens with high MDA, a lower proportion of those that received IA vaccines had PAT at each subsequent time point than those receiving MLV vaccines, with a significant difference observed at 11 weeks of age (P = 0.03) (Figure 2-1). Of the 17 kittens with low MDA, a lower proportion of those that received IA vaccines had PAT than those receiving MLV vaccines at 9 weeks of age (P < 0.01). Of the 11 kittens that received IA vaccines, a lower proportion of kittens with high MDA had PAT than those with low MDA at 11 (P = 0.02) and 14 (P < 0.01) weeks of age. Of the kittens with high MDA, 4 (40%) did not achieve PAT against FPV by 17 weeks of age. Serum Anti-FHV Antibody Titers Ten of 27 kittens (37%) had detectable MDA against FHV at the time of the 8- week pre-vaccination antibody titers; and 17 were seronegative. Of the 11 kittens that received IA vaccines, a lower proportion of kittens with high MDA had PAT than those with low MDA at 14 (P = 0.02) and 17 (P = 0.02) weeks of age (Figure 2-2). Of the kittens with high MDA, 12 (70%) did not achieve PAT against FHV by 17 weeks of age. 34

35 Of the kittens with low MDA, 5 (29%) did not achieve PAT against FHV by 17 weeks of age. Serum Anti-FCV Antibody Titers Twenty-one of 27 kittens (78%) had detectable MDA against FCV at the time of the 8-week pre-vaccination titers and 6 were seronegative. Of the 16 kittens with low MDA, a lower proportion of those that received IA vaccines had PAT than those that received MLV vaccines, with a significant difference observed at 11 weeks of age (P < 0.01) (Figure 2-3). Of the kittens with high MDA, 1 (4%) did not achieve PAT against FCV by 17 weeks of age. Serum Anti-Rabies Antibody Titers Twenty-six of 27 kittens (96%) were seronegative at 14 weeks of age (Table 2-1). One kitten had a titer of 14 prior to being vaccinated and 1 kitten remained seronegative 3 weeks after vaccination. This kitten received a MLV FPV/FHV/FCV vaccination, had high MDA against FPV, FHV, and FCV, and did not achieve PAT against FPV or FHV by 17 weeks of age. The remainder of the kittens had titers greater than that which is considered adequate for international movement of cats (0.5 U/ml) (Kansas State Veterinary Diagnostic Laboratory 2008). Discussion High MDA delayed the induction of PAT for all 3 viruses regardless of the type of vaccine administered. Following vaccination at 8, 11, and 14 weeks, 40%, 70%, and 9% of kittens with high MDA had insufficient titers against FPV, FHV and FCV respectively, at 17 weeks of age. A study of kittens from rescue shelters and breeding catteries with varying MDA levels vaccinated at 6, 9, and 12 weeks of age with an MLV vaccine had similar findings: those with no MDA (at 6 weeks of age) responded to vaccination 35

36 sooner than those with MDA and between 67% and 100% of kittens demonstrated response to vaccination at the end of the study (Dawson, Willoughby et al. 2001). It is likely that the lack of protection in all kittens in both studies is a result of MDA interference. The duration of interference is related to the original level of MDA (Greene and Addie 2006) and a wide variation exists with MDA interference reported up to weeks of age for FPV (Scott, Csiza et al. 1970; Dawson, Willoughby et al. 2001; Reese, Patterson et al. 2008), 2-10 weeks of age for FHV (Gaskell and Povey 1982; Johnson and Povey 1985; Dawson, Willoughby et al. 2001; Reese, Patterson et al. 2008), and weeks of age for FCV (Johnson and Povey 1983; Dawson, Willoughby et al. 2001; Reese, Patterson et al. 2008). The current study suggests that interference may last >14 weeks for each virus. Since individual titer measurement is rarely performed during a kitten s initial vaccination series in practice, these data reinforce the need for vaccination with a MLV product at least through 16 weeks of age as is currently recommended, although the optimal age for concluding the initial vaccine series cannot be determined. MLV FPV vaccines were able to overcome MDA sooner than IA products, regardless of the MDA level for the study population. The majority of kittens vaccinated with low MDA achieved PAT against FPV within 1 week of vaccination with a MLV product and within 2 weeks of vaccination with an IA vaccine, which is consistent with previous findings (Brun, Chappuis et al. 1979; Patterson, Reese et al. 2007; Jas, Aeberle et al. 2009; Lappin, Veir et al. 2009). These findings reinforce the need for vaccination against FPV with an MLV product, particularly in high-risk environments 36

37 where a rapid response is desired. A delay in protection of only a few days can predispose to outbreaks of FPV in animal shelters. Consistent with previous reports (Lappin, Andrews et al. 2002; Mouzin, Lorenzen et al. 2004; Lappin, Veir et al. 2009), vaccination against FHV in the current study was less effective than other vaccine components, regardless of type of product. The IA product was only effective at inducing PAT against FHV in kittens with low MDA. This finding contrasts with those of an earlier study in which IA vaccines were able to induce PAT in the presence of MDA by 8 weeks of age, but titers rose at a slower rate in kittens with MDA than in those without MDA (Johnson and Povey 1985). In that study neither product prevented infection following viral challenge and the severity of clinical signs in kittens receiving a MLV product did not differ from those that received an IA product. Among kittens with low FCV MDA, the MLV vaccine was more effective than the IA vaccine for rapid induction of PAT against FCV. Previous studies have demonstrated similar findings, inducing PAT in kittens with low MDA by 9 weeks of age with the use of MLV vaccines (Dawson, Willoughby et al. 2001). A study evaluating the onset of immunity after 2 injections of an IA product induced protection against challenge by 12 weeks of age in kittens with no MDA (Jas, Aeberle et al. 2009). Neither of those studies compared responses between MLV and IA vaccinations. There is no established protective titer against rabies virus, although any detectable titer is indicative of a humoral immune response (Kansas State Veterinary Diagnostic Laboratory 2008). Interestingly, one kitten had an antibody titer of 14 prior to vaccination. Previous exposure to the virus was highly unlikely, so this may represent cross-reactivity or MDA. The 2 kittens with post-vaccination titers of <700 also 37

38 responded poorly to the IA and MLV vaccines. This may represent a deficiency of the humoral immune response in these individuals at that time. The effect of continued vaccination after 14 weeks was not evaluated in this study, leaving the exact age at which PAT can be established in 100% of kittens unknown. However, combined with the variation in previous reports regarding onset of protection against FPV, FHV, and FCV, the current findings emphasize the need for repeated vaccination in kittens to induce protection, particularly in vulnerable populations of animals and when assessment of MDA level is not practical. In the absence of individual titer measurement, vaccination is most likely to be effective at inducing PAT in a majority of kittens when a MLV product is administered at least through 16 weeks of age, in accordance with current professional guidelines for both pet and shelter cats (Richards, Elston et al. 2006; Poulet 2007; Day, Horzinek et al. 2010). It is possible that some kittens are nonresponders to particular antigens and would not develop PAT regardless of the timing of additional vaccinations. It is also possible that some are especially slow responders, either due to very high levels of MDA or to idiosyncracies of their individual immune responses. In these cases PAT may not develop until after the booster vaccination given a year after the kitten series is completed. From 4% to 44% of kittens failed to develop PAT by 17 weeks of age, with failure highest in kittens receiving the IA vaccine and in kittens with high MDA. Since vaccination was discontinued at 14 weeks of age, the optimal age range for the kitten vaccination series remains unknown. 38

39 Table 2-1. Median (range) serum antibody titers against FPV, FHV, FCV, and rabies in specific-pathogen-free kittens with low and high MDA levels that were vaccinated at 8, 11, and 14 weeks of age with IA and MLV vaccines. Age at determination of serum antiviral antibody titers (wks) MDA Antigen level* Vaccine No IA 8 0 (0-10) 10 (0-20) 80 (0-160) 640 ( ) 1280 ( ) Low MLV 9 0 (0-10) 40 (0-320) 1280 (0-5120) 1280 ( ) 1280 ( ) IA 3 20 (20-40) 20 (10-20) 0 (0-10) 0 (0-0) 20 (10-40) FPV High MLV 7 80 (20-160) 40 (10-80) 40 (10-80) 40 (0-160) 160 (0-640) IA 8 0 (0-0) 0 (0-8) 6 (0-12) 40 (12-128) 48 (8-192) Low MLV 9 0 (0-0) 0 (0-4) 0 (0-0) 12 (0-32) 16 (12-24) IA 3 24 (12-24) 12 (8-16) 6 (6-8) 6 (0-6) 0 (0-4) FHV High MLV 7 8 (4-12) 4 (0-12) 4 (0-8) 4 (0-12) 12 (4-48) IA 6 6 (0-6) 12 (0-16) 16 (6-32) 128 (16-256) 320 (32-768) Low MLV 10 2 (0-4) 24 (12-64) 64 (16-512) 384 ( ) 448 ( ) IA 5 32 (8-2048) 8 (6-4096) 16 (6-2048) 192 ( ) 384 ( ) FCV High MLV 6 14 (8-3072) 56 (8-4096) 432 ( ) 2560 ( ) 1152 ( ) Rabies n/a IA 27 n/a n/a n/a 0 (0-14) 6700 (0-7000) *Low MDA is defined by titers 10, 0, and 6 for FPV, FHV, and FCV, respectively. High MDA is defined by titers 20, 4, and 8 for FPV, FHV, and FCV, respectively. 39

40 Figure 2-1. Proportion of specific-pathogen-free kittens with PAT against FPV following vaccination at 8, 11, and 14 weeks of age with IA or MLV vaccines. A) Kittens (n=17) with low MDA at 8 weeks (range 0-10). B) Kittens (n=10) with high MDA at 8 weeks (range ). *indicates significant difference in proportion of kittens receiving MLV vs. IA vaccines that develop PAT (P <0.05); **indicates significant difference in proportion of kittens with low vs. high MDA levels that develop PAT (P <0.05) 40

41 Figure 2-2. Proportion of specific-pathogen-free kittens with PAT against FHV following vaccination at 8, 11, and 14 weeks with IA or MLV vaccines. A) Kittens (n=17) with low MDA at 8 weeks (range 0). B) Kittens (n=10) with high MDA at 8 weeks (range 4-24). ** indicates significant difference in proportion of kittens with low vs. high MDA levels that develop PAT (P <0.05) 41

42 Figure 2-3. Proportion of specific-pathogen-free kittens with PAT against FCV following vaccination at 8, 11, and 14 weeks with IA or MLV vaccines. A) Kittens (n=16) with low MDA at 8 weeks (range 0-6). B) Kittens (n=11) with high MDA at 8 weeks (range ). * indicates significant difference in proportion of kittens receiving MLV vs. IA vaccines that develop PAT (P <0.05) 42

43 CHAPTER 3 PREVALENCE OF PROTECTIVE ANTIBODY TITERS FOR FELINE PANLEUKPENIA VIRUS, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN CATS ENTERING FLORIDA ANIMAL SHELTERS Background FPV, FHV, and FCV are widespread in the feline population in the United States (Gaskell and Povey 1982; Binns, Dawson et al. 2000; Greene and Addie 2006; Scarlett 2009), particularly in unvaccinated free-roaming strays and cats residing in animal shelters (Edinboro, Janowitz et al. 1999; Bannasch and Foley 2005; Fischer, Quest et al. 2007; Tuzio 2009). In contrast to their significance in privately owned pet cats, these infections are a constant and serious threat to the health and welfare of cats residing in animal shelters, often resulting in death. Outbreaks of FPV are frequently managed by depopulation and it has been demonstrated that increasing time in the shelter environment exacerbates the risk of developing clinical signs of respiratory disease and subsequent euthanasia (Edinboro, Janowitz et al. 1999; Pappas 2005; Dinnage, Scarlett et al. 2009; King 2010). Current guidelines recommend vaccination of all cats 4 weeks of age against FPV, FHV, and FCV at the time of admission to animal shelters (Richards, Elston et al. 2006). However, this practice is frequently considered cost prohibitive, unsafe, or unnecessary, particularly in shelters with a high rate of euthanasia for other measures, such as crowding. In addition, the proportion of cats entering animal shelters that are already protected against disease because of prior vaccination or natural exposure, and thus not benefiting from vaccination at admission, is unknown. Shelter managers are left with little information upon which to make evidence-based decisions regarding allocation of scarce resources for the best cost-benefit outcome. 43

44 Detection of serum antibodies has been utilized as evidence of previous exposure to infectious agents, efficacy of vaccination, and duration of immunological response (Greene and Addie 2006; Greene and Schultz 2006). In addition, measurement of FPV serum antibodies has been recommended as a tool in disease outbreak management in animal shelters (Larson, Newbury et al. 2009). The objectives of this study were to determine the prevalence and factors associated with PAT against FPV, FHV, and FCV in cats entering animal shelters. It was hypothesized that the findings would provide objective data to support currently recommended vaccination protocols for shelter cats as well as identify factors that may be useful components of the risk assessment of individual animals during a disease outbreak. Materials and Methods Study Sites The study was conducted in 3 shelters in north Florida. Shelters 1 and 2 were open-admission municipal shelters and shelter 3 was a limited-admission privatelyfunded shelter located in the same county as shelter 2. The populations in the counties in which these shelters were located were 243,574 (shelter 1) and 857,040 (shelters 2 and 3) in The total number of cats admitted to each shelter in 2009 was 4,030, 12,509, and 1,411 for shelters 1, 2, and 3, respectively. Animals Cats entering the shelters between May 7, 2010 and August 15, 2010 were enrolled in the study. Information collected for each cat included date of intake, signalment (age, breed, sex, and neuter status), zip code of origin, source (stray, previously owned, confiscated, left at shelter night box), evidence of care-giving (relinquished, declawed, neutered, microchipped, collared/tagged, ear-tipped by a trap- 44

45 neuter-return program), health status at intake (healthy or not healthy), and outcome (adopted, transferred, euthanized, or reclaimed by owner). If the actual age was unknown, age was estimated as pediatric (<6 months), adolescent (6 to 11 months), young adult (1-5 years), or mature adult (>5 years). If no evidence of previous sterilization surgery was detected during physical examination, cats were classified as being reproductively intact. Address of origin was used to determine whether the community from which the cat originated was primarily urban or rural on the basis of population density, number of residents, housing density, income, and land use as recorded in an on-line directory (Advameg 2010). Prior vaccination history for ownerrelinquished cats was not known. Health status of cats was determined from examination by a veterinarian or trained technician. Body condition score (BCS) was evaluated using a 9-point scale with 1 being emaciated and 9 being obese (Nestle Purina 2010). For this study population, scores of 4, 5, or 6 were considered ideal. Cats were classified as not healthy if clinical signs of illness, injury, positive retroviral status, or any combination of these factors were identified. Cats having one of the following physical examination findings in the absence of other abnormalities were considered healthy: fleas, ear mites, mild dental disease, lactation, pregnancy, matted fur, and being under- or over-weight (BCS 2-3 or 7-8, respectively). The study protocol was approved by the University of Florida Institutional Animal Care and Use Committee. Serological Testing Blood (3 ml) was collected via jugular, cephalic, or femoral venipuncture into serum separator tubes within 1 day of admission to the shelter. According to shelter policy, cats are vaccinated as close to the time of admission as possible, therefore, in some cases, cats were vaccinated prior to sample collection. Serum was separated by 45

46 centrifugation and stored at -20ºC pending analysis. Serum antibody titers were determined by HI for FPV and by SN for FHV and FCV by a university-affiliated diagnostic laboratory. 1 PAT as defined by the laboratory in which assays were conducted and were 40, 8, and 32 for FPV, FHV, and FCV, respectively. Cats were tested for FeLV antigen and FIV antibodies using a commercially available point-of-care assay. 2 Statistical Analysis PAT prevalence was defined as the number of cats with PAT divided by the number of cats tested. Factors associated with PAT were determined by use of a χ 2 test; univariate logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI). A value of P <0.05 was considered significant. All potential factors were entered into a multivariate logistic regression model. Stepwise variable selection procedures were used to aid in model development. Interaction between selected explanatory variables was examined, and Hosmer Lemeshow tests were used to assess goodness of fit. All analyses were performed with statistical software. 3, 4 Results Cats Antibody titers were determined for 418 cats and kittens (Table 3-1). A total of 347 samples were collected from shelter 1, 63 samples from shelter 2, and 8 from shelter 3. Cats that were placed in the unhealthy category included 24 cats with signs of 1 Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY. 2 SNAP FIV/FeLV Combo test, IDEXX Laboratories, Westbrook, ME. 3 Epi Info 2008, Version 3.5.1, CDC, Atlanta, GA. 4 SAS SAS Institute Inc, Cary, NC. 46

47 upper respiratory infection, 4 cats with diarrhea, 13 cats with flea infestation in combination with other conditions, and 26 cats with other signs of illness or neglect. One of these 26 cats tested positive for FIV antibodies and 10 tested positive for FeLV antigen. Factors Associated with PAT Overall, 40.9% (171/418) of the cats admitted had PAT for FPV (Figure 3-1A). Cats greater than 1 year of age were more likely to have PAT against FPV than those <6 months of age (P<0.01), with the odds of PAT increasing with age. Cats that were neutered, relinquished by their owners, from an urban environment, or exhibiting signs of previous care-giving were more likely to have PAT against FPV than those that were not (P<0.01). Cats that were overweight (BCS 7-9) were more likely to have PAT against FPV than those that were underweight (BCS 1-3) (P<0.01). Cats vaccinated less than 24 hours prior to sample collection were more likely to have PAT than those that were not (P=0.03) and cats reclaimed by an owner were more likely to have PAT than those that were adopted (P<0.01) (Table 3-2). Overall, 10.5% (44/418) of cats had PAT for FHV (Figure 3-1B). Cats 6 months of age or older were more likely to have PAT against FHV than those <6 months of age (P<0.01), with the odds of PAT increasing with age. Being neutered (P<0.01), relinquished (P=0.05), from an urban environment (P=0.02), unhealthy (P=0.03), or exhibiting signs of previous care-giving (P<0.01) were also associated with PAT against FHV. Cats that had an ideal BCS (4-6) were less likely to have PAT than those that were underweight (BCS 1-3), and cats reclaimed by an owner had 5.7 times greater odds of PAT than those that were adopted (P<0.01) (Table 3-3). 47

48 Overall, 36.4% (152/418) of cats had PAT for FCV (Figure 3-1C). Cats 6 months of age or older were more likely to have PAT against FCV than those <6 months of age (P<0.01). Being neutered (P<0.01), relinquished (P=0.02), confiscated or anonymously left at the shelter (P=0.04), unhealthy (P<0.01), or showing signs of previous care-giving (P<0.01) were associated with PAT against FCV. Cats that had an ideal BCS (4-6) were less likely to have PAT than those that were underweight (BCS 1-3) (P<0.01), and cats that were euthanized were more likely to have PAT than those that were adopted (P=0.04) (Table 3-4). Several factors remained significantly associated with PAT for FPV, FHV or FCV when entered into a multivariate analysis model (Table 3-5). Cats that were neutered were more likely to have PAT against FPV than sexually intact cats. Some variables that were associated with PAT against FPV in the univariate analysis (source [P<0.01], environment of origin [P<0.01], signs of previous care-giving [P<0.01], and vaccination within 24 hours of sample collection [P=0.02]) were significantly associated with neuter status in the multivariate FPV model. The multivariate model for FHV PAT indicated that age (P<0.01), neuter status (P<0.01), and vaccination status (P=0.04) were each associated with PAT. The odds of having PAT against FHV increased with advancing age and cats that were neutered were more likely to have PAT than those that were sexually intact. Cats that were vaccinated less than 24 hours before sample collection were more likely to have PAT against FHV than those that were not. The multivariate model for FCV PAT indicated that age (P<0.01), source (P=0.01), and being vaccinated at intake (P=0.02) were each associated with PAT. The 48

49 odds of having PAT against FCV increased with advancing age and cats relinquished by their owners were more likely to have PAT against FCV than those that were strays or confiscated or left at the shelter at night. Cats that were vaccinated less than 24 hours before sample collection were more likely to have PAT against FCV than those that were not. The Hosmer Lemeshow goodness-of-fit test indicated a lack of fit for the multivariate model of FCV PAT, so factors with significant effects were further explored with interaction terms. A significant interaction between age and being vaccinated within 24 hours of sample collection was found (P<0.01) with cats 6-12 months of age and 1-5 years of age having increased odds of having PAT, and those >5 years of age having decreased odds as compared to cats <6 months of age. Factors that were not associated with PAT for FPV, FHV, and FCV in multivariate logistic regression were the environment of origin, health status at intake, and outcome. Discussion The majority of cats and kittens in the study population had insufficient antibody titers for FPV, FHV, and FCV. Increasing age, being neutered, being relinquished by an owner, and being vaccinated shortly after admission to the shelter were each associated with the presence of PAT. An animal shelter in Wisconsin has previously reported that 48% of cats had PAT against FPV (Tuzio 2009). The prevalence of PAT in predominantly adult, unvaccinated feral cats was 33%, 64%, and 21% for FPV, FHV, and FCV respectively (Fischer, Quest et al. 2007). Prevalence of PAT against FPV in the population of shelter cats in the study reported here was similar to previous reports. In contrast, PAT prevalence against FHV in feral cats was substantially higher, and that against FCV was lower than in the study population. 49

50 Evaluations of the risk factors for infection with FPV, FHV, and FCV specific for cats in animal shelters are scarce, but it has been shown that infection and death from FPV is more likely to occur in young kittens (Cave, Thompson et al. 2002). Increasing time in the shelter is correlated with disease from FHV and FCV, particularly for kittens (Dinnage, Scarlett et al. 2009). Cats with no record of previous vaccination and purebred cats are also at greater risk (Edinboro, Janowitz et al. 1999; Edwards, Coyne et al. 2008). The current data support the findings that cats <6 months of age are more vulnerable to infection with FPV, FHV and FCV. The increased odds of PAT among cats that were neutered or were known to have an owner suggests that these factors are likely surrogate markers for previous veterinary care which included vaccination. Despite these findings, it has been suggested that cat-related factors are not as important as facility-related factors (such as total feline intake, population density and the existence of designated isolation areas) in assessing risk of infectious disease in animal shelters (McCobb, Patronek et al. 2005; Edwards, Coyne et al. 2008). The potential effects of such factors on PAT were not assessed in the current study. One unexpected finding was the association of vaccination less than 24 hours prior to sample collection with the presence of PAT against FPV, FHV, and FCV. The minimum time from exposure to a virus to the development of a detectable primary or anamnestic antibody response in cats is currently unknown. However, in other species such as mice, woodchucks, and horses, primary adaptive immune response to live viral antigen exposure takes 5-7 days and a secondary anamnestic response takes 1-3 days as measured by cytotoxic T lymphocyte activity, virus-specific IgG and IgM levels, and response cell gene transcription (Tizard 2000; Goldsby, Kindt et al. 2003; Guy, 50

51 Mulrooney-Cousins et al. 2008; Miao, Hollenbaugh et al. 2010). The current data suggest that some cats in the current study were previously exposed to FHV and FCV naturally or via previous vaccination and subsequently developed an anamnestic response to vaccination in less than 24 hours. This supports the benefit of vaccination immediately on intake to an animal shelter. Additional studies may help determine the minimum time frame in which cats can be expected to develop PAT in a high-risk environment. Surprisingly, the type of community from which the cat originated, health status at intake, signs of previous care-giving other than being neutered, and outcome were not associated with PAT when evaluated in the multivariate model. These factors should not be used as the sole factor in determining an individual cat s need for vaccination at the time of admission, nor in risk assessment during a disease outbreak. This finding emphasizes the need for vaccination of all cats upon entry to an animal shelter regardless of their health status or evidence of previous veterinary care. 51

52 Table 3-1. Demographics of 418 cats and kittens admitted to 3 animal shelters in north Florida. Factor Category No. Percent Stray Owned Source Other 21 5 Rural Environment Urban None Microchip 19 5 Ear tipped 7 2 Identification* Collar 6 1 < 6 mos mos yrs Age >5 yrs MI MN 36 9 FI Sex FS DSH DLH Breed Other 18 4 Healthy Health status Not healthy Body condition score Vaccinated at intake Outcome Yes No Adopted Transferred Reclaimed 7 2 Euthanized Present 17 4 Signs of previous Yes care No *One cat had an ear tip as well as a microchip and was counted in each category. Body condition score was not recorded for 4 cats. 52

53 Table 3-2. Results of univariate logistic regression analysis of factors associated with PAT against FPV in 418 cats and kittens admitted to animal shelters. Factor Category No. tested No. with PAT Prevalenc e (%) OR 95% CI P-value < 6 mos REF NA NA NA 6-<12 mos yrs <0.01 Age >5 yrs <0.01 Neuter Intact REF NA NA NA status Neutered <0.01 Stray REF NA NA NA Owned <0.01 Source Other Rural REF NA NA NA Environment Urban <0.01 Intake health Healthy REF NA NA NA status Not healthy Signs of No REF NA NA NA care-giving Yes <0.01 Body REF NA NA NA condition score <0.01 Vaccinated at intake Outcome No REF NA NA NA Yes Adopted REF NA NA NA Transferred Reclaimed <0.01 Euthanized Present

54 Table 3-3. Results of univariate logistic regression analysis of factors associated with PAT against FHV in 418 cats and kittens admitted to animal shelters. Factor Category No. tested No. with PAT Prevalence (%) OR 95% CI P-value < 6 mos REF NA NA NA 6-<12 mos < yrs <0.01 Age >5 yrs <0.01 Neuter Intact REF NA NA NA status Neutered <0.01 Stray REF NA NA NA Owned Source Other Rural REF NA NA NA Environment Urban Intake health Healthy REF NA NA NA status Not healthy Signs of No REF NA NA NA care-giving Yes <0.01 Body REF NA NA NA condition <0.01 score Vaccinated at intake Outcome No REF NA NA NA Yes Adopted REF NA NA NA Transferred Reclaimed Euthanized Present

55 Table 3-4. Results of univariate logistic regression analysis of factors associated with PAT against FCV in 418 cats and kittens admitted to animal shelters. Factor Category No. tested No. with PAT Prevalence (%) OR 95% CI P-value < 6 mos REF NA NA NA 6-<12 mos < yrs <0.01 Age >5 yrs <0.01 Neuter Intact REF NA NA NA status Neutered <0.01 Stray REF NA NA NA Owned Source Other Rural REF NA NA NA Environment Urban Intake health Healthy REF NA NA NA status Not healthy <0.01 Signs of No REF NA NA NA care-giving Yes <0.01 Body REF NA NA NA condition <0.01 score Vaccinated at intake Outcome No REF NA NA NA Yes Adopted REF NA NA NA Transferred Reclaimed Euthanized Present

56 Table 3-5. Results of multivariate analysis of factors associated with PAT against FPV, FCV, and FHV in 418 cats and kittens admitted to animal shelters. Disease Factor Category OR 95% CI Feline panleukopenia Feline herpesvirus-1 Feline calicivirus Neuter status Age Neuter status Vaccinated at intake Age Origin Vaccinated at intake Neutered REF NA NA Intact < 6 mos. REF NA NA 6-<12 mos yrs >5 yrs Neutered REF NA NA Intact No REF NA NA Yes < 6 mos. REF NA NA 6-<12 mos yrs >5 yrs Stray REF NA NA Owned Other No REF NA NA Yes

57 No. of cats No. of cats No. of cats FPV titer A FHV titer B >6144 FCV Titer C Figure 3-1. Distribution of antibody titers for FPV (A), FHV (B), and FCV (C) in 418 cats and kittens at entry into animal shelters. Titers to the right of the dotted line were considered protective. 57

58 CHAPTER 4 DETECTION OF PROTECTIVE ANTIBODY TITERS AGAINST FELINE PANLEUKOPENIA, FELINE HERPESVIRUS-1, AND FELINE CALICIVIRUS IN CATS USING A POINT-OF-CARE ELISA Background In the general pet cat population, illness caused by FPV is relatively uncommon and FHV and FCV infections usually result in mild illness. In contrast, these diseases are a constant and serious threat to the health and welfare of cats residing in animal shelters. Outbreaks of FPV are frequently managed by depopulation of both clinically diseased and healthy exposed cats (Pappas 2005; Patterson, Reese et al. 2007; King 2010). Upper respiratory disease caused by FHV and FCV is associated with increases in length of shelter stay, animal care costs, and euthanasia (Edinboro, Janowitz et al. 1999; Dinnage, Scarlett et al. 2009). Because of these threats, rapid recognition of cats at risk for infection, particularly FPV, is critical in the high-risk environment of an animal shelter. Serum antibody titers have been shown to be a useful measurement of protection against infection (FPV) or clinical disease (FHV and FCV), and their determination has been recommended as part of disease outbreak management in animal shelters (Scott and Geissinger 1999; Lappin, Andrews et al. 2002; Larson, Newbury et al. 2009). However, titer determination at a reference laboratory is technical, time-consuming, and often cost-prohibitive for animal shelters. The availability of a rapid semi-quantitative point-of-care assay for PAT would be a valuable tool for disease outbreak management. A commercially available point-of-care ELISA 1 previously found to be an accurate method of identifying PAT against canine parvovirus (TiterCHEK; 1 TiterCHEK CDV/CPV. Synbiotics Corp, San Diego, CA. 58

59 Gray, Crawford et al. 2011) has been recommended for evaluation of FPV PAT in cats (Day, Horzinek et al. 2010), even though it is only labeled and approved for use in dogs. A feline-specific, point-of-care ELISA 2 has been developed for the determination of PAT against FPV, FHV and FCV. No information has been published about the accuracy of these two assays in cats compared to gold standard laboratory analysis. The objective of this study was to determine the sensitivity, specificity, and interobserver and inter-assay agreement of these 2 semi-quantitative point-of-care assays for the detection of PAT against FPV, FHV and FCV and to determine if the assays would be useful in the management of a FPV outbreak in a shelter. Materials and Methods Samples A total of 356 serum samples from cats and kittens entering 3 shelters in north Florida between May 7, 2010 and August 15, 2010 were collected for analysis. A total of 56 archived serum samples collected from feral cats undergoing elective ovariohysterectomy or orchiectomy through a trap-neuter-return program 3 were also used. Blood (3 to 5 ml) was collected via jugular, cephalic or femoral venipuncture into serum separator tubes within one day of admission to the shelter. Serum was separated by centrifugation, aliquoted into duplicate cryovials and stored at -20ºC pending analysis. The study protocol was approved by the University of Florida Institutional Animal Care and Use Committee. 2 Immunocomb Feline VacciCheck. Biogal Galed Laboratories, Kibbutz Galed, Israel. 3 Operation Catnip, Gainesville, FL. 59

60 Serological Testing Serum antibody titers were measured by use of a HI assay for FPV and by a SN assay for FHV and FCV at a university-affiliated diagnostic laboratory. 4 PAT as defined by the laboratory in which assays were conducted was 40, 8, and 32 for FPV, FHV, and FCV, respectively. The canine assay utilizes color-coded plastic wells coated with purified canine parvovirus antigen. One microliter of serum or plasma is placed into a well and incubated with polyclonal rabbit anti-dog IgG conjugated to horseradish peroxidase. A chromogenic substrate is added and the subsequent color reaction is compared visually to positive and negative control wells on the plate. Samples with color reactions of equal or greater intensity than the positive control are considered to have PAT against canine parvovirus (equivalent to HI titers 80 in dogs). Those with reactions of less intensity than the positive control well are considered negative for PAT (TiterCHEK 2010). The assay also includes canine distemper virus antigen-coated wells and can simultaneously determine PAT against canine distemper virus when used with canine samples. The feline assay utilizes a plastic, comb-shaped card with FPV, FHV, and FCV antigen-coated test spots (in addition to a positive reference spot) attached to each of 12 teeth and a reagent-filled, 6-row, multi-compartment developing plate. Using capillary tubes and a piston (provided in the kit) or a calibrated pipetter, a serum, plasma (5 µl) or whole blood (10 µl) sample is deposited in the first row of the developing plate. The comb is then inserted into the rows allowing for binding of antibodies present in the 4 Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY. 60

61 sample to the antigen spots on the comb device. At timed intervals, the comb is transferred to the remaining wells for washing, binding of labeled secondary antibodies (anti-cat IgG), and color development, resulting in the production of a gray color tone. The intensity of the gray tone is visually compared to the positive reference spot and to a gray scale provided with the kit to determine whether PAT is present. The kit instructions define PAT as HI titers 40 for FPV and SN titers 16 and 32 for FHV and FCV, respectively. Test spots equal to or darker than the positive reference spot are considered indicative of PAT. Those lighter than the positive reference are considered negative for PAT (Figure 4-1). Alternatively, a software program developed for the assay can be used to measure the relative density of spots on scanned images and to calculate a quantitative titer equivalent (Immunocomb 2007). Once developed, test spots are permanent allowing for archiving and review of results. A set of feral (n=56) and shelter (n=20) cat serum samples were thawed and analyzed to evaluate both point-of-care assays according to manufacturer s instructions during pilot testing. Due to poor sensitivity of the canine assay for FPV antibodies in the pilot study, only the feline assay was subsequently used in the definitive study. In the definitive study, 356 serum samples from shelter cats were thawed and tested for FPV, FHV, and FCV PAT using the feline point-of-care assay according to manufacturer s instructions. Results derived from the first run in the software program were used for calculation of sensitivity and specificity. Test combs were also evaluated visually by observers blinded to the software results. To assess inter-observer variability, assay results were evaluated by visual assessment of the color intensity using the scale provided in the kit 61

62 by 3 different observers blinded to the previous results. The numeric scale score was translated into a positive or negative result according to manufacturer s instructions and compared to those reported by the assay software. To assess inter-assay variability, a subset of 60 serum samples was re-tested on a different day. To assess repeatability of scanned results, 476 samples (416 unique samples from pilot and definitive testing plus 60 samples from inter-assay testing) were scanned and analyzed by the software program a total of 3 times. Statistical Analysis Sensitivity for each assay was calculated as the number of cats with positive results for PAT divided by the total number of cats with positive results on the gold standard assay (true positives) plus the total number of cats with positive results on the gold standard assay but negative results on the test assay (false negatives). Specificity was calculated as the number of cats with negative results divided by the total number of cats with negative results on the gold standard assay (true negatives) plus the total number of cats with negative results on the gold standard assay but positive results on the test assay (false positives). PAT prevalence was defined as the number of cats with PAT as determined in the gold-standard assays divided by the number of cats tested. Positive predictive value (PPV) was calculated as the number of true-positives divided by the total number of positive results. Negative predictive value (NPV) was calculated as the number of true-negatives divided by the total number of negative results. Overall accuracy was calculated as the number of true positives plus true negatives divided by the total number of results. Because PPV and NPV vary depending on actual prevalence in the specific population being tested, PPV and NPV were also calculated for theoretical PAT population prevalences of 25%, 50%, and 75% to mimic conditions 62

63 found in various cat populations. Inter-observer and inter-assay agreement were assessed by calculating the kappa coefficient. 5 Observations with kappa values <0.6 were considered to have poor agreement, those 0.6 and 0.8 to have good agreement, and those >0.8 to have very good agreement (Newman, Browner et al. 2007). Results Pilot Testing of Canine and Feline Assays PAT prevalence for FPV in the 76 samples tested was 93% as determined by HI. The sensitivity of the canine assay for the study population was 28%, resulting in a NPV of 9% and overall accuracy of 33% (Table 4-1). The specificity and PPV of the assay were each 100%. The assay never reported a positive PAT result for samples with titers <640 (PAT is 40). The feline assay reported valid results for 73 of the 76 samples, demonstrating a sensitivity of 72%, a NPV of 21%, and overall accuracy of 74%. The specificity and PPV of the assay were each 100%. Three invalid results reported by the software program did not appear grossly abnormal and could be scored visually (these results were not included in calculations of sensitivity and specificity). Because of the poor sensitivity of the canine assay for feline samples, no further testing was performed with it. Definitive Testing of Feline Assay Of the 356 samples tested, 145 (41%) were identified by HI to have PAT for FPV. As identified by SN, 35 (10%) and 127 (36%) had PAT for FHV and FCV, respectively. Performance of the feline ELISA is reported in Table 4-2. Valid results were obtained for 5 GraphPad Software, Inc. Quantify agreement with kappa. Available online at: 63

64 344 of 356 samples. The software program reported invalid results for a total of 12 samples. One of these 12 samples was also not able to be evaluated visually due to lack of development of any of the test spots on that tooth of the comb, including the positive control. The remaining invalid results did not appear grossly abnormal and could be read visually. Ten of these 11 invalid results occurred on the second tooth of the comb, suggesting a possible manufacturing or software defect for this tooth. A large proportion of erroneous results was due to false-negative results reported for samples with FPV HI titers of 40 (58 out of 89 [65%] discordant FPV results) but false-negative results were reported for titers as high as 1:2560. False-negative results for FHV and FCV represented a variety of SN titers (FHV range 8-24; FCV range ). The predictive value of a positive or negative result was calculated using the actual prevalence of PAT in the samples from the study population (Table 4-2). Because predictive values change when prevalence changes, PPV and NPV were also calculated for theoretical PAT prevalences of 25%, 50%, and 75% (Table 4-3). The strength of agreement between the software program and visual interpretation by 3 observers is reported in Table 4-4. In general, agreement was good for FPV (κ= ) and FHV (κ= ), and very good for FCV (κ= ). A total of 60 samples was assayed a second time to evaluate inter-assay variability. The interassay agreement was very good for FPV (κ=0.84, CI= ), good for FHV (κ=0.69, CI= ), and very good for FCV (κ=0.93, CI= ). Among the 476 samples scanned 3 times on different days using the software program, valid results were available for 450 samples. Invalid results were reported for 19 samples during the first scan, 13 samples during the second scan, and 15 samples 64

65 during the third scan, representing a total of 25 different serum samples. Invalid results were consistently reported across all 3 scans for 8 samples. Excluding samples with invalid results during any scan, there were 57 samples (12%) with variable results between the three scans for FPV, 44 (9%) for FHV, and 10 (2%) for FCV. Four samples tested for FPV resulted in a different titer value reported for each of the 3 scans; the remainder of the discordant results for FCV, FHV, and FPV resulted in 2 like titers and 1 different titer among the 3 scans. Four of the 57 (7%) variable FPV analyses resulted in discordant PAT results (ie, present vs. absent). Two of the 44 (5%) variable FHV analyses and 1 of the 10 (10%) FCV analyses resulted in discordant PAT results. These errors would have resulted in an error in clinical diagnosis. Discussion Low sensitivity for FPV antibodies rendered the canine point-of-care assay inappropriate for use in cats. This is not surprising given that the assay relies on the detection of anti-dog IgG and the manufacturer makes no claims about its use in other species. The feline point-of-care assay also exhibited a high number of false-negative results for FPV PAT, resulting in low sensitivity and low negative predictive value, but was highly accurate in the assessment of FHV and FCV PAT. When a subset of samples was analyzed a second time, and when developed combs were scanned multiple times visually or by computer, discordant results for PAT occurred in 5%-10% of samples. Unfortunately the utility of PAT determination in the control of respiratory infection transmission in shelters is unknown. The assays evaluated in this study were designed to assess the need for individual cats and dogs to receive booster vaccines rather than reliance on a predetermined immunization schedule. These semi-quantitative assays are designed to provide a 65

66 positive result when serum antibody levels consistent with protection against infection are present. Although cats with PAT against FPV are considered to be immune to infection, those with PAT against FHV and FCV are protected only against severe clinical disease (Scott and Geissinger 1999; Lappin, Andrews et al. 2002; Lappin, Sebring et al. 2006; Lappin, Veir et al. 2009). It is not possible to use antibody titers to determine the converse, if cats are susceptible to infection, because the innate and cellmediated arms of the immune system also contribute to host defense but are more difficult to measure. While sensitivity and specificity are fixed characteristics of diagnostic tests, variations in actual PAT prevalence influence the predictive value of positive or negative results. That is, PPV decreases as prevalence decreases and NPV decreases as prevalence increases. Recent work by our laboratory indicates a PAT prevalence of 41% for FPV, 11% for FHV, and 36% for FCV in the population of shelter cats that provided samples for this study. Another author has reported similar protective titers against FPV (48%) in shelter cats in Wisconsin (Tuzio 2009). Confirmatory testing of questionable results using gold standard assays may be indicated in some cases. The first step in the management of a disease outbreak in an animal shelter is the identification and isolation of cats with active disease on the basis of clinical signs or diagnostic testing (Hurley 2009; Tuzio 2009). Then, measurement of antibody titers can be used to assign infection risk categories to exposed, asymptomatic cats. Exposed, asymptomatic cats with PAT are at lower risk for infection and may be placed for adoption. Those without PAT may be in a subclinical or incubation phase of infection and are kept in quarantine for the duration of the disease incubation period or otherwise 66

67 removed from the population. This is an especially useful tool in the face of a FPV outbreak because the incubation period is short, there is no carrier state, and PAT is highly predictive of immunity. This contrasts with infection with FHV or FCV which often results in chronic carrier states and the development of partial immunity, making titer interpretation more difficult. Inaccurate antibody test results can have a substantial impact on management outcomes in any environment and their implications must be considered. During a shelter disease outbreak, failure to detect PAT may result in an exposed low-risk cat being kept unnecessarily in shelter quarantine or being euthanized. Failure to detect the absence of PAT may cause a high-risk exposed cat to be kept in the shelter population or to be adopted, potentially facilitating the spread of infection. A screening test with a high specificity for PAT, such as the feline point-of-care ELISA in this study, can help limit disease transmission, but the low sensitivity of this assay for FPV may result in inefficient management decisions such as increasing numbers of cats being held in quarantine and unnecessary euthanasia. However, given the high PPV of the feline ELISA, it may be a useful tool in the context of a shelter where large numbers of animals are routinely euthanized. Despite its lack of sensitivity, it will correctly identify a portion of animals at low risk for disease, ensuring that those selected to remain in the population are likely to be protected. Cost, efficiency, and ease of use play an important role when choosing a diagnostic test in an animal shelter. The feline ELISA can be performed at the shelter on an as-needed basis using only 5 microliters of serum or plasma or 10 microliters of whole blood for all three titers. The assay takes approximately 30 minutes to perform 67

68 (not including sample collection and preparation), but is a multi-step process requiring a proficient technician and can be time consuming for shelter staff if there are many cats to evaluate. In this study, efficiency of sample handling was substantially improved through use of a calibrated pipetter rather than the capillary and piston provided with the test kit, and ease of test spot interpretation was improved through use of the scanning software. In comparison, titer measurement at most reference laboratories requires 1 milliliter of serum or plasma and less shelter staff time because the samples are analyzed off-site. Availability of test results takes longer than with the ELISA, which may be problematic during disease outbreaks when prompt triage of exposed cats is essential. The feline point-of-care screening ELISA, which had high diagnostic accuracy and can be performed in less than an hour, can be a useful tool for evaluation of PAT for FHV or FCV, but only identified about half of cats with FPV PAT. Improvements in accuracy and repeatability of FPV PAT determination could make this tool a valuable component of a disease outbreak response, assisting shelters in saving as many cats as possible while preventing the spread of infection to other cats, inadvertently adopting infected cats to the public, or resorting to depopulation in the event of an infectious disease outbreak. 68

69 Table 4-1. Results of pilot testing of 2 semi-quantitative point-of-care assays for the detection of PAT when used to detect FPV antibody titers in a population of shelter and feral cats. Canine assay Feline assay Invalid results 0 3 HI titer No. of samples Negative Positive Negative Positive < Point-of-care assay results reported as positive or negative (indicating the presence or absence of PAT) were compared to the gold-standard HI assay. HI titers and feline assay titers 40 are considered positive for PAT. Canine assay titers 80 are considered positive for PAT against canine parvovirus. Table 4-2. Performance of a feline point-of-care assay for the detection of PAT against FPV, FHV, and FCV in 344 shelter cats. Overall accuracy (%) PPV (%) NPV (%) [CI] [CI] [CI] Disease Feline panleukopenia Feline herpesvirus-1 Feline calicivirus Sensitivity (%) [CI] 49 [ ] 91 [ ] 90 [ ] Specificity (%) [CI] 99 [98-100] 97 [ ] 91 [ ] 76 [ ] 93 [ ] 88 [ ] 96 [ ] 78 [ ] 85 [ ] 74 [ ] 99 [ ] 94 [ ] 69

70 Table 4-3. Calculated positive and negative predictive values of a feline point-of-care ELISA for 3 different prevalence levels of FPV, FHV, and FCV. 25% Prevalence 50% Prevalence 75% Prevalence Disease PPV (%) NPV (%) PPV (%) NPV (%) PPV (%) NPV (%) Feline panleukopenia Feline herpesvirus Feline calicivirus Table 4-4. Percent of concordant results and strength of agreement between scanned images interpreted by a software program and visual interpretation by 3 observers when using a point-of-care assay for the detection of PAT against FPV, FHV, and FCV in 356 shelter cats. Disease Agreement (%) Kappa CI Feline panleukopenia Feline herpesvirus Feline calicivirus

71 A B Figure 4-1. A feline point-of-care ELISA kit for the determination of PAT against FPV. FHV, and FCV. A) A 12-toothed comb with antigen test spots is moved through wells of the developing plate at timed intervals. B) The results are displayed as gray color tones and are compared to a Comb Scale for interpretation. 71

72 CHAPTER 5 CONCLUSIONS AND SUMMARY This investigation sought to fill in some of the gaps in the existing literature regarding the response of cats to vaccination against FPV, FHV, and FCV as well as to provide objective data upon which an individual animal risk assessment for infection with FPV, FHV, and FCV can be made. In combination with knowledge of the accuracy of available point-of-care tests for the detection of PAT against FPV, FHV, and FCV, this information can be used by shelter managers and veterinarians to develop an evidence-based response to the control of these important infectious diseases in animal shelters. Effect of MDA on Serological Response to Vaccination Data from a colony of specific-pathogen-free cats was used to describe the effects of MDA on vaccination and the influence of the type of vaccination on the development of PAT. In this study, kittens were grouped according to the level of MDA present as determined by pre-vaccination titers at 8 weeks of age and were vaccinated against FPV, FHV, and FCV 3 times using either a MLV or IA vaccine. In the overall study population, 15% of kittens never developed PAT against FPV by 17 weeks of age. Forty-four percent of kittens did not develop PAT against FHV and 4% did not develop PAT against FCV during the study period. These findings suggest MDA can interfere with vaccination response at 14 weeks of age with lack of PAT development evident at least up to 17 weeks of age, which is longer than previous reports (Gaskell and Povey 1982; Johnson and Povey 1983; Johnson and Povey 1985; Greene and Addie 2006; Richards, Elston et al. 2006). 72

73 Regarding the type of vaccine, this study found that use of a MLV product induced PAT against FPV more quickly than an IA vaccine. In addition, the IA vaccine protocol was only effective at inducing PAT by 17 weeks of age in kittens with low levels of MDA. Against FHV, vaccination with an IA product was only effective in kittens with low levels of MDA. In kittens with high MDA, the type of vaccine product did not have a significant effect on the development of PAT against FHV both products were equally effective. Concerning FCV, no differences were found in kittens with high MDA between the MLV and IA vaccine. Among kittens with low MDA, however, the MLV vaccine was more effective at inducing PAT against FCV than the IA vaccine. In the absence of individual antibody titer assessment, kitten vaccination protocols should continue through at least 16 weeks of age and should incorporate the use of MLV products to increase the odds of effective immunization against FPV, FHV, and FCV. These data offer additional support for currently accepted professional veterinary medical guidelines, which recommend vaccination of naïve kittens through at least 16 weeks of age with a MLV product (Richards, Elston et al. 2006; Griffin 2009). This contrasts with historical recommendations to vaccinate kittens up to 12 weeks. These historical recommendations, which were not based on empirical titer information in kittens with PAT, are still widely used in veterinary practices. Cats that are not vaccinated through 16 weeks may not develop PAT and may be at greater risk for infection and disease from FPV, FHV, and FCV than those that receive additional vaccinations. The exact age at which MDA will no longer interfere with vaccination response remains unknown. Given our current understanding, it may be prudent to extend vaccination even longer (e.g., to 20 weeks of age) in the face of potential 73

74 exposure during an outbreak of FPV. Future studies should be aimed at more precisely defining this period. Prevalence and Risk Factors for PAT The prevalence of FPV, FHV, and FCV PAT in cats in animal shelters was evaluated. Serum samples from more than 400 cats and kittens collected within 24 hours of admission to 3 different shelters in northern Florida revealed a PAT prevalence against FPV of 40.9%, 10.5% for FHV, and 36.4% for FCV. These data indicate that the vast majority of cats entering animal shelters do not have PAT against these common infections, confirming that these animals are at high risk for infection with FPV, FHV, and FCV (Richards, Elston et al. 2006; Griffin 2009). After multivariate analysis, cats in the study population that were neutered were significantly more likely to have PAT against FPV than those that were un-neutered. The presence of PAT against FHV was significantly associated with increasing age, being neutered, and having received a vaccination against FHV within 24 hours of shelter admission. The presence of PAT against FCV was significantly associated with increasing age, vaccination within 24 hours of shelter admission and being relinquished to the shelter by an owner. However, a portion of cats in each risk group lacked PAT, so risk factor analysis alone is insufficient to definitively determine individual cat risk. These data identify animal factors that may be useful in the assessment of an individual animal s risk of acquiring disease from FPV, FHV, or FCV in an animal shelter. They also support the notion that a protective immune response may be induced less than 24 hours after vaccination in some cats, affirming recommendations for vaccination of cats immediately at or before admission to animal shelters or other high risk environments (Griffin 2009). 74

75 For FPV, FHV, and FCV, factors not significantly associated with PAT in multivariate analysis included the health status of the cat, the environment of origin (rural or urban), and the final outcome for the cat (adoption, euthanasia, transfer, return to owner). The lack of universal association between PAT and factors suggestive of previous veterinary care (i.e., relinquished by an owner, declawed, neutered, microchipped, collared/tagged, ear-tipped by a trap-neuter-return program) support current professional guidelines calling for vaccination of all cats, regardless of their source or physical condition (Griffin 2009). Detection of PAT A feline-specific, point-of-care enzyme-linked immunosorbent assay (ELISA) 18 with the potential to overcome the technical and financial challenges of diagnostic laboratory testing has been manufactured for the determination of PAT against FPV, FHV and FCV. The accuracy of this point-of-care titer assessment tool in a population of shelter cats was evaluated. The sensitivity of this test for FPV PAT detection (49%) is too low for optimal use during an outbreak of FPV. The large number of false-negative results could undermine an FPV outbreak response by increasing the numbers of cats held in quarantine and/or euthanized. However, given the high PPV of the feline ELISA (96%), it may be a useful tool in the context of a shelter where large numbers of animals are routinely euthanized, in which case approximately half of animals at low risk for disease will be correctly identified, ensuring that those selected to remain in the population will be protected. 18 ImmunoComb Feline VacciCheck. Biogal Galed Laboratories, Kibbutz Galed, Israel. 75

76 Although test sensitivity for the detection of PAT against FHV (91%) and FCV (90%) were acceptable, PAT detection is not as useful against these disease agents as it does not correspond to immunity as strongly as for FPV. The development of chronic carrier states and partial immunity associated with FHV and FCV infection confound titer interpretation. PAT determination is an important means of individual animal risk assessment for use in animal shelters during an outbreak of FPV. Practical means of accurately assessing titers remains a challenge for animal shelters with limited resources. Creating an Evidence-Based Plan for Disease Control Disease Prevention The information gleaned from these and previous studies can be utilized to formulate an evidence-based plan for the control of FPV, FHV and FCV in shelter cats. First and foremost, all cats should be vaccinated at or before the time of entry with a MLV product. Kittens should receive additional vaccinations at 2-week intervals until at least 16 weeks of age. In an outbreak of FPV or in a shelter with episodic or endemic FPV cases, vaccination to 20 weeks of age may be warranted. Risk factor analysis was used to define factors indicative of a high likelihood of PAT against FPV, FHV and FCV at the time of shelter admission as well as risk factors for infection (Tables 5-1 and 5-2). In some populations, the number of risk factors found may indicate a greater likelihood of protection. This was the case for the population of cats studied here with 32% of cats with 0-2 factors, 47% of cats with 3-4 factors, and 75% of cats with 5-7 factors having PAT. This information can be used to assist shelter practitioners in making sound clinical judgments regarding which animals are most likely to acquire disease and guide care in the shelter setting. For example, practitioners may 76

77 allocate the most bio-secure housing to those animals deemed to be the most vulnerable or at greatest risk for disease. Outbreak Response Regarding management of a FPV outbreak, intake diversion and complete separation between animals with clinical signs of disease, healthy exposed animals, and healthy unexposed animals are the keys to stopping an outbreak. Individual animal risk assessment is crucial to limiting disease transmission among healthy exposed animals already in the population as well as those that must enter a population in the midst of an outbreak in the event that intake diversion is not possible. Individual animal risk assessment should be based on the presence or absence of clinical signs, known risk factors for infection, the odds of the presence of PAT and, when feasible, the actual results of PAT determination. When diversion of intake is not possible, and after vaccination against FPV with a MLV product, cats deemed to have a high likelihood of possessing PAT at admission (e.g., neutered cats), could be selectively admitted to the shelter and directed toward an adoption pathway even in the midst of a disease outbreak. Such cats must be kept separate from cats already in the shelter in order to limit inadvertent disease transmission, but would theoretically be at little risk of acquiring FPV infection themselves. Even in these low risk cats, definitive PAT determination is ideal, given that no factor was universally associated with PAT and the consequences of placing a cat infected with FPV into the community could be devastating to a sheltering organization. In contrast, cats deemed unlikely to have PAT at admission should be vaccinated and diverted into foster care or their intake postponed until the threat of disease was minimized. If admission to the shelter were absolutely necessary, they should be 77

78 vaccinated and separated from cats already in the shelter to prevent exposure and allow for further assessment of their individual risk (Table 5-2). An example of how various factors predictive of the presence of PAT might be utilized to create an algorithm for response is presented in Figure 5-1. The plan presented here is based on the odds of the presence of PAT at intake to the shelter and makes the assumption that factors found to be significant in multivariate analyses (i.e., being neutered) indicate a greater chance of its presence. In addition, although parallels can be drawn, since the risk factors are based on the measurement of PAT at intake to the shelter, this model may only be applicable to the decision-making process for cats that must enter the shelter during a disease outbreak, not those already in the shelter. In the latter case, other factors including the likelihood of exposure, age at vaccination, timing of vaccination relative to exposure, and length of stay in the shelter all play an important role in individual animal risk assessment. Although definitive PAT determination for every cat is ideal, in shelters with limited resources such a plan can be utilized to prioritize cats for PAT determination. If definitive PAT testing is pursued, cats in high or medium risk categories could be selected for testing and moved to the low risk category if found to have PAT. When PAT is determined, the PPV and NPV of the test used should be considered in order to understand how false results will influence individual animal outcome. Future studies should be directed at identifying risk factors for PAT for cats already present in the shelter, identifying risk factors for the absence of PAT, and assessing the success of various evidence-based disease control plans in the shelter. 78

79 Table 5-1. Factors associated with PAT against FPV, FHV, and FCV for cats entering animal shelters based on univariate analysis. FPV FHV FCV 1 year of age 6 months of age a 6 months of age a Neutered a Neutered a Neutered Owner-relinquished Owner-relinquished Owner-relinquished a Urban origin Urban origin Not healthy d Signs of care-giving b Not healthy d Signs of care-giving b BCS c >7 Signs of care-giving b BCS c 4-6 Vaccinated at intake BCS c 4-6 Vaccinated at intake e Vaccinated at intake e a Factors retained significance in multivariate analysis b Signs of care-giving include: being relinquished by an owner, declawed, neutered, microchipped, collared/tagged, or ear-tipped by a trap-neuter-return program c Body condition score measured on a 9-point scale d Cats that were not healthy at intake were significantly more likely to have PAT, although these titer values likely represent response to active infection rather than protection e Factors were significant in multivariate analysis only Table 5-2. Presumed risk factors for infection with FPV, FHV, and FCV for cats in animal shelters based on analysis of factors not significantly associated with PAT. FPV FHV FCV < 1 year of age < 6 months of age < 6 months of age Un-neutered Un-neutered Un-neutered Stray Rural origin Stray Confiscated or abandoned Healthy at intake Healthy at intake Rural origin No signs of care-giving a No signs of care-giving a No signs of care-giving a BCS b <4 or >6 BCS b <4 or >6 BCS b <7 Not vaccinated at intake a Signs of care-giving include: being relinquished by an owner, declawed, neutered, microchipped, collared/tagged, or ear-tipped by a trap-neuter-return program b Body condition score measured on a 9-point scale 79

80 Figure 5-1. Evidence-based individual risk assessment protocol for cats entering animal shelters during an outbreak of FPV. Note: See Table 5-1 for factors predictive of PAT. 80