Seroprevalence and Risk Factors of Bovine Herpesvirus 1 Infection in Cattle Herds in the State of Paraná, Brazil

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1 Transboundary and Emerging Diseases ORIGINAL ARTICLE Seroprevalence and Risk Factors of Bovine Herpesvirus 1 Infection in Cattle Herds in the State of Paraná, Brazil J. A. Dias 1, A. A. Alfieri 2, J. S. Ferreira-Neto 3, V. S. P. Gonçalves 4 and E. E. Muller 5 1 Brazilian Agricultural Research Corporation, Embrapa-Rondônia, Porto Velho, Rondônia, Brazil 2 Laboratory of Animal Virology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Londrina, Paraná, Brazil 3 Laboratory of Epidemiology and Bioestatistics, Department of Veterinary Medicine and Zootechny, Universidade de São Paulo, São Paulo, São Paulo, Brazil 4 Laboratory of Epidemiology and Biostatistics, Faculty of Agronomy and Veterinary Medicine, Universidade de Brasília, Brasília, Distrito Federal, Brazil 5 Laboratory of Microbiology and Infectious Diseases, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Londrina, Paraná, Brazil Keywords: Cattle; BoHV-1; epidemiology; risk factors; serology; indirect ELISA Correspondence: A. A. Alfieri. Laboratory of Animal Virology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, PO box 6001, , Londrina, Paraná, Brazil. Tel.: ; Fax: ; alfieri@uel.br Received for publication October 15, 2011 doi: /j x Summary Epidemiological data describing bovine herpesvirus 1 (BoHV-1) infection in Brazilian cattle herds are scarce. A cross-sectional study was conducted in the state of Paraná between December 2001 and July 2002 with the objective of estimating the apparent prevalence of BoHV-1-seropositive herds and animals and identifying the potential risk factors for infection in farms with breeding animals in the state of Paraná in Southern Brazil. The state was divided into seven regions based on the livestock production dynamics of the different areas. Sampling was performed in two stages. Initially, herds were randomly selected, followed by a random selection of animals within the selected herds. Blood samples were collected from females, aged 24 months, from 2018 BoHV-1 non-vaccinated herds. Serum samples were tested for antibodies against BoHV-1 using an indirect enzyme-linked immunosorbent assay (ELISA). The apparent prevalence of seropositivity in the herds and animals in Paraná was 71.3% (95% CI: ) and 59.0% (95% CI: ), respectively. Multiple logistic regression analysis revealed that risk factors associated with the presence of the infection were as follows: beef herds [OR = 1.58 ( )], natural service [OR = 1.48 ( )], purchase of animals [OR = 1.90 ( )], pasture rental [OR = 2.24 ( )], existence of calving pens [OR = 1.56 ( )] and records of abortion in the last 12 months [OR = 1.45 ( )]. These results indicate that BoHV-1 infection is widespread in the state of Paraná. Introduction Bovine herpesvirus 1 (BoHV-1), which belongs to the Herpesviridae family in the Alphaherpesvirinae subfamily, is one of the main pathogens of cattle. Bovine herpesvirus 1 infection is responsible for considerable economic losses in beef and dairy cattle herds worldwide (Smith et al., 1995; Van Oirschot, 1998). Infection in adults and young cattle can be subclinical or can be accompanied by several clinical signs, such as supper respiratory disease [infectious bovine rhinotracheitis (IBR)], vulvovaginitis/balanoposthitis [infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB)], fever, conjunctivitis and reduction in milk production, and reproductive failures such as embryonic death and abortion (Wyler et al., 1989; Straub, 1990; Takiuchi et al., 2005). ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013)

2 Seroprevalence and Risk Factors of BoHV-1 J. A. Dias et al. The viruses of the Alphaherpesvirinae subfamily establish latent infections in the sensory ganglia and are periodically reactivated under stressful conditions or after treatment with corticoids (Ackermann et al., 1982). Infected cattle can present with intermittent episodes of viral excretion, which are not accompanied by any clinical signs, resulting in the dissemination and perpetuation of infection in the herds (Pastoret et al., 1982). Bovine herpesvirus 1 is widely distributed in beef and dairy cattle herds around the world (Lovato et al., 1995; Solis-Calderon et al., 2003; Boelaert et al., 2005; Guarino et al., 2008). However, some European countries with initially low infection rates have achieved the condition of being BoHV-1 free with the implementation of IBR eradication programmes (Ackermann and Engels, 2006). In South America, serological surveys have shown frequencies of seropositive cattle ranging from 37% to 67% (Obando et al., 1999; Guarino et al., 2008; Carbonero et al., 2011). In Brazil, most of the studies of BoHV-1 infection have been carried out in restricted geographical regions or were based on a limited number of samples without a standard sampling design. Such studies showed that seropositivity frequencies between 30% and 80% are frequently observed in non-vaccinated herds throughout the different geographical areas of Brazil (Lovato et al., 1995; Vidor et al., 1995; Richtzenhain et al., 1999; Takiuchi et al., 2001). In the state of Paraná, studies of BoHV-1 epidemiology have been conducted on farms with records of reproductive failures and have reported frequencies of seropositive animals ranging from 19.7% to 52.4% (Médici et al., 2000; Takiuchi et al., 2001). Because of the lack of unbiased epidemiological data, the aim of this study was to estimate the apparent prevalence of seropositivity in herds and individual animals and to investigate the risk factors associated with BoHV-1 infection in cattle herds in the state of Paraná. of Agriculture of Paraná (SEAB-PR) (Fig. 1). The census data used as the basis for the evaluation of samples and BoHV-1 apparent prevalence were the most updated available at the time of the study (Paraná, 2001). Table 1 presents the summary of the census data and also shows the samples studied in each of the bovine production regions. Study design The sampling design used in this study was similar to the study of bovine brucellosis in the state of Paraná by the National Program for Control and Eradication of Brucellosis and Tuberculosis, with modifications. The sampling design was developed by the Animal Defense Department, Ministry of Agriculture, Livestock, and Food Supply, and by the Epidemiology Division of SEAB-PR. To estimate the apparent prevalence of positive herds and animals, a sampling study was developed in two stages. First, a random selection of herds from a preestablished number of farms was collected, which represent the primary sampling units. Within these primary units, a pre-determined number of cows aged 24 months were randomly selected (secondary units) to determine the BoHV-1 infection status of the herd. The number of farms selected per region was estimated by the simple random sample formula proposed by Thrusfield (1995). The parameters adopted for the calculation were a confidence level of 0.95, estimated apparent prevalence of 50% and a standard error of The selection of herds in each region was random. In situations where herds have been substituted, the nearest herd with the similar characteristics was selected. The aim of the sampling plan for the secondary units was to estimate the minimal number of animals to be Materials and Methods Study population A cross-sectional study was conducted in the state of Paraná in Southern Brazil, which has a geographical area of km 2, with a total of cattle (IBGE, 2007) distributed across livestock farms (SEAB- PR, personal communication, 2007). To establish regional differences in the epidemiological parameters, the state was divided into seven different bovine production regions. The different production systems, management practices, types of breeding, average size of the herds and commercialization systems were evaluated for each region. Each region was integrated into a different regional administration office by the Secretary Fig. 1. Map of the state of Paraná, showing the division of regions used in this study. Detail shows the state of Paraná within Brazil. 40 ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013) 39 47

3 J. A. Dias et al. Seroprevalence and Risk Factors of BoHV-1 Table 1. Census data for the bovine population in the state of Parana, Brazil, in 2001, according to bovine production regions (Paraná, 2001) Productive circuits No Region Regional Administration Office Total herds with reproductive activities Total females aged 24 months Sampled herds Sampled females 1 North-west Umuarama and Paranavai Mid-western/north Campo Mourão, Maringa and Londrina Far north Cornélio Procópio, Ivaiporã and Jacarezinho Mid-south Laranjeiras do Sul, Guarapuava and Ponta Grossa West Cascavel and Toledo South-east Curitiba, União da Vitoria, Paranagua and Irati South-west Francisco Beltrão and Pato Branco State of Parana a a a Only herds that were not vaccinated against Bovine herpesvirus 1 were included in the study. examined within each farm to enable its classification as a focus or non-focus of BoHV-1 infection. Only unvaccinated females aged 24 months or older were sampled. For the calculations, the value of 50% was adopted as the intraherd apparent prevalence. In this process, Herdacc version 3 (University of Guelph) software was used, and the size of the sample was chosen based on desired sensitivity and specificity values of >90%. Therefore, in herds of up to 99 females, a total of 10 animals were sampled. In those made up of more than 99 females, 15 females were sampled. The selection of animals within the herd was made by using a systematic random sample procedure. A total of females, aged 24 months, originating from 2018 herds that were not vaccinated against BoHV- 1, were evaluated. The potential risk factors for BoHV-1 in this study were assessed by the usage of an epidemiological questionnaire applied to all selected herds. For guidance to technicians, a handbook with detailed instructions for the completion of the questionnaire was distributed to each local office. The questionnaire was designed to assess the most important factors that might be associated with BoHV-1: type of management, method of breeding, predominant breeds, number of females, presence of wild animals, usage of artificial insemination (AI), purchase and sale of animals for reproduction, disposal method of abortion, rental grazing areas, common grazing areas, calving pens, veterinary assistance and history of abortion. For the detection of BoHV-1-specific antibodies, an indirect ELISA was carried out according to the method of Ferreira et al. (2005) with modifications. Briefly, Madin-Darby bovine kidney (MDBK) cells were used for the production of the antigen (BoHV-1). The cells were grown in Dulbecco s Modified Eagle Medium (DMEM; Invitrogen, Life Technologies, Carlsbad, CA, USA), supplemented with 7% mycoplasma- and virus-free foetal bovine serum (FBS; Invitrogen, USA), 55 mg/ml gentamicin, and 2.5 mg/ml amphotericin B (Sigma-Aldrich, Inc., St. Louis, MO, USA). The MDBK cells were confirmed to be free of bovine viral diarrhoea virus by a PCR assay (Pilz et al., 2007). The Los Angeles strain of BoHV-1 was amplified in DMEM supplemented with 1% FBS. The titre of the virus stock was calculated as the 50% tissue culture infective dose (TCID 50 ). To minimize the variation between the results obtained on each microplate, the optical density (OD) of each serum sample was expressed as an R (relation) value. The R value was calculated based on the OD values of 10 positive and 10 negative control sera, according to the method of Biondi et al. (1996). The highest R value in the negative serum was The cut-off was defined at 0.230, based on the highest R value observed in the negative serum, plus three standard deviations. Dias et al. (2008) reported that the sensitivity and specificity of the indirect ELISA were 98% and 93%, respectively, and that the results of the ELISA had a high correlation with the results of a virusneutralization test (j value of 0.954). Serological test for BoHV-1 Blood was collected in the period between December 2001 and July 2002 by puncturing the jugular vein using a sterile disposable needle and collecting the blood into a vacuum tube, which was previously labelled for identification. Serum samples were stored in plastic microtubes and were frozen at )20 C. Calculation of the apparent prevalence of seropositive animals The information from the questionnaires as well as the serology results and data regarding the condition of the herd was filled in a database using Microsoft Access Ò software. A herd was declared positive for the presence of BoHV-1 if it included at least one positive animal in ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013)

4 Seroprevalence and Risk Factors of BoHV-1 J. A. Dias et al. herds of up to seven females; two positive animals in herds of 8 99 females; and three positive animals in herds with more than 99 females. These criteria were established using the software Herdacc Ò version 3 (University of Guelph), based on the size of the population, an intraherd apparent prevalence of 50%, and the sensitivity and specificity of the diagnostic test used (indirect ELISA), with the goal of obtaining a herd sensitivity and specificity of >90%. The sampling design made it possible to determine the apparent prevalence of positive herds and of BoHV-1- seropositive adult females ( 24 months) in the state of Paraná and for each region of the state. Calculation of the estimated apparent prevalence values and the corresponding confidence intervals was made using the software EpiInfo 6.04d (Dean et al., 1994). Because the primary unit sample for each region was selected based on random systematic sampling (Cochran, 1977), the apparent prevalence of BoHV-1 foci was calculated using the number of positive herds and the number of herds sampled as parameters (Dean et al., 1994). For the total area of the state, because of the division of regions, the primary unit sample was considered to be a stratified random systematic sampling (Cochran, 1977). The parameters used for the calculation of the apparent foci prevalence were the status of the herd (BoHV-1 positive or negative), the region to which each herd belonged and the assigned weight of each herd sampled in the region. The assigned weight was calculated based on the ratio of the number of herds with reproductive activity to the total number of herds sampled in each region (Dean et al., 1994). The apparent prevalence of seropositive adult females was calculated for each region and also for the whole state. In this case, secondary units were chosen by means of a grouping sample that was stratified by region (Cochran, 1977). Parameters used in this calculation were status of the animal (positive or negative), region to which the sampled herd belonged, identification of the herd in the study and assigned weight of each sampled animal, which is calculated by the following formula (Dean et al., 1994): females >24 months in region weight ¼ females>24 months in region females >24 months in sampled herd females>24 months sampled in herd The first factor in this formula represents the level to which each selected animal represents the region, and the second represents the level to which each animal represents the herd. Study of risk factors For the combined data for the state, a cross-sectional study was conducted. Two groups of herds (positive and negative) were formed. These groups were compared with regard to the variables evaluated in the epidemiological questionnaire, which enabled the measurement of the strength of the association of these variables with the presence of BoHV-1 infection. In each herd, 15 variables were analysed, which were linked to the breeding type and management practices. The variable categories were organized to present a growing risk scale, based on information obtained in the literature (Kahrs, 1977; Wyler et al., 1989; Wentink et al., 1993; Van Schaik et al., 1998, 2002). These variables were re-categorized as necessary. The lowest risk category was used as a basis of comparison for the other categories. Quantitative variables were also categorized into quartiles. Univariate analysis was conducted to verify the association between risk variables and the status of a herd, and BoHV-1 infection was coded 0 (absence of positive animals) and 1 (presence of one, two or three seropositive animals, according to the previously described criteria, dependent variable) using the chi-squared (v 2 ) test and Fisher s exact test. Variables of invariable analysis having a P-value of 0.20 were subjected to additional analyses. A multiple logistic regression analysis was performed (Hosmer and Lemeshow, 1989). The model was adopted with herd seropositivity (yes/no) as the outcome and was built by using the forward selection procedure. The analyses were performed by using the software SPSS version 9.0 (SPSS Inc., 1999). Results Characterization of the study population In the state of Paraná, the number of cattle in each herd varied from 1 to 3527, with a median of 19 animals (first quartile = 9; third quartile = 51). The median number of animals was higher in regions 1 and 2, in beef, dairy and mixed herds (Fig. 2). Prevalence Of 2018 analysed herds, 1481 tested positive for BoHV-1. Table 2 presents the results of BoHV-1 herd apparent prevalence in the seven regions and also in total for the state of Paraná. The values of herd apparent prevalence stratified by production purpose are presented in Table 3. The apparent prevalence of animals with serum antibodies against BoHV-1 is shown in Table 4. Of evaluated animals, 7125 were seropositive, yielding an apparent prevalence of 59.0% (95% CI: ). 42 ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013) 39 47

5 J. A. Dias et al. Seroprevalence and Risk Factors of BoHV-1 Table 4. Apparent prevalence of bovine herpesvirus 1 (BoHV-1)-seropositive animals in the regions of Parana State, Brazil Animals Regions Sampled Positive Prevalence (%) 95% CI Total Fig. 2. Box plot showing the distribution of cattle herd size according to the breeding type in regions of the state of Paraná, Brazil. Table 2. Apparent prevalence of bovine herpesvirus 1 (BoHV-1)-seropositive herds in the regions of Parana State, Brazil Region Herds Sampled Positive Prevalence (%) 95% CI Total Design effect = Table 3. Apparent prevalence of bovine herpesvirus 1 (BoHV-1) infection stratified by breeding type (beef, dairy and mixed) in regions of Parana State, Brazil Regions BoHV-1 herd prevalence Beef Dairy Mixed % 95% CI % 95% CI % 95% CI Total Table 5 presents the results of the univariate analysis of potential risk factors for BoHV-1 infection in the state of Paraná. Those variables with values of P < 0.2 were included in the multiple logistic regression analysis. The variables associated with BoHV-1 in the multiple logistic regression were as follows: beef herds, natural service, purchased animals, pasture rental, presence of calving pens and records of abortion in the last 12 months (Table 6). Discussion The seroprevalence of BoHV-1 was high in all of the geographical areas, both at the herd and at the animal levels. The apparent prevalence (71.3%) of BoHV-1 infection in cattle herds was lower than previous findings (Médici et al., 2000), during which 92% of the herds evaluated were BoHV-1 positive and presented history of reproductive problems. Bovine production regions 1 and 2, which have a predominance of beef cattle breeding, had the highest apparent prevalence levels (94.1% and 91.9%, respectively). At the time of this study, these regions contained 40% of all cattle in the state and presented the highest median number of cattle in each herd (Fig. 2). The lowest herd apparent prevalence (51.5%) within the state of Paraná occurred in region 4 and might be related to the fact that cattle within this region are used predominantly for dairy production. The dairy farms in this region are characterized by smaller herds, with a median of 11 cattle per herd (Fig. 2). Additionally, the intensive dairy herds presented elevated milk production per animal and good management practices, which included the replacement of animals that presented factors that interfere with milk production and consequently increase production costs. With this management system, the individual animals are removed in cases of low milk production, reproduction problems, mastitis, foot problems and others. While studying the variability of BoHV-1 prevalence in three districts of Kenya, McDermott et al. (1997) concluded ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013)

6 Seroprevalence and Risk Factors of BoHV-1 J. A. Dias et al. Table 5. Distribution of the variables analysed as possible risk factors for bovine herpesvirus 1 (BoHV-1) in cattle herds from Parana State, Brazil Variables a Categories Cases/Exposures Frequency (%) Management type 1: extensive b 1159/ : semi-confined/confined 312/ Breeding type c 1: dairy b 585/ : beef 290/ : mixed 603/ Predominant breeds 1: pure 496/ : others 940/ Number of females c 0: <23 females b 1032/ : 23 females 449/ Number of females 24 months c 0: <13 females b 1002/ : 13 females 479/ Presence of wild animals 0: no b 1075/ : yes 406/ Use of artificial insemination (AI) c 1: only A. I. b 105/ : A. I./natural service 153/ : only natural service 1189/ Buying animals for reproduction c 0: no b 708/ : yes 763/ Selling animals for reproduction c 0: no b 1095/ : yes 372/ Disposal method of abortion 0: bury/disposes/burns b 564/ : leave in the field 515/ Rental grazing areas c 0: no b 1239/ : yes 232/ Common grazing areas c 0: no b 1269/ : yes 204/ Calving pens c 0: no b 1027/ : yes 427/ Veterinary assistance 0: yes b 433/ : no 1017/ History of abortion c 0: no b 1156/ : yes 299/ a Some questions were not answered by the owners. b Baseline category. c P < 0.20 in the univariate analysis and is included in the multiple logistic regression. Table 6. Results of the multiple logistic regression analysis for risk factors associated with bovine herpesvirus 1 (BoHV-1) infection in cattle herds with reproductive activity in the state of Parana, Brazil Variables b SE OR 95% CI P Breeding Beef herds Buying of breeders Natural service Rental of grazing areas Presence of calving pen History of abortion Log likelihood statistic = , Nagelkerke R 2 = 8.7. that variations in the prevalence between regions may be partially explained by factors such as the size of the herd, measures to control diseases, the type of breeding and the age of the animals. The herd apparent prevalence of BoHV-1 infection in the entire state, stratified by the type of breeding, was greater in beef cattle herds (83.3%) than in dairy (71.3%) and mixed herds (71.4%) (Table 3). Studies conducted in beef cattle herds with reproductive failures in the states of Rio Grande do Sul (Vidor et al., 1995) and Paraná (Médici et al., 2000) found frequencies of positive herds of 71% and 100%, respectively. A characteristic of management in this type of breeding may be a lower rate of cow replacement as well as a lower level of sanitary control of reproductive diseases compared with dairy farms. The apparent prevalence of BoHV-1-seropositive animals in the state of Paraná was 59.0% (95% CI: ). In a previous study carried out in some of the same geographical areas, the rate of BoHV-1-seropositive animals was 52% (Takiuchi et al., 2001). The north-western and mid-western/northern regions of Paraná presented the 44 ª 2012 Blackwell Verlag GmbH Transboundary and Emerging Diseases. 60 (2013) 39 47

7 J. A. Dias et al. Seroprevalence and Risk Factors of BoHV-1 highest apparent prevalence levels of BoHV-1-seropositive animals, 71.1% and 66.2%, respectively. As these areas of Paraná form a border with the states of São Paulo and Mato Grosso do Sul, there is an intense movement of animals, mainly from beef herds, between these three states. The epidemiological data describing BoHV-1 infection in herds and animals in these areas of the state of Paraná are very similar to what have been described in the states of São Paulo and Mato Grosso do Sul (Richtzenhain et al., 1999; Takiuchi et al., 2001). The multiple logistic regression analysis of the risk factors for BoHV-1 infection in cattle herds in the state of Paraná showed that beef cattle herds have a greater probability of being positive than dairy or mixed herds (Table 5). The beef cattle herds sampled in this study comprised the highest median cattle numbers in relation to the other types of breeding for all of the regions included in this study (Fig. 2). The association between herd size and BoHV-1 infection may be attributed to other variables that are strongly related to herd size, such as the frequency of the purchase of animals, participation in cattle shows, professional visits and the presence of regular and temporary workers on the farms (Van Schaik et al., 1998). Acquisition of animals during incubation periods, or during acute or latent phases of BoHV-1 infection, is described as the main factor contributing to the introduction of the infection into a herd (Msolla et al., 1981; Pastoret et al., 1982). In this study, the purchase of animals for breeding purposes was identified as a risk factor for BoHV-1 infection in the multiple logistic regression analysis (Table 6). While studying the risk factors for the introduction of BoHV-1 in the Netherlands, Van Schaik et al. (1998) showed that farms that purchased animals had a 3.5-fold greater chance of being positive than those farms that were closed, based on an average purchase of 4.5 animals per year. Several authors have reported that semen is an important source of BoHV-1 shedding (Straub, 1990; Wentink et al., 1993; Van Oirschot, 1995; Rocha et al., 1999). In this study, farms using natural service as the reproduction method had a greater probability of being positive than those using artificial insemination (AI) (Table 5). Studies have shown that the shedding of BoHV-1 in semen may be intermittent and that the virus can be isolated from seronegative bulls, making the identification of these animals in the herd more difficult (Huck et al., 1971; Xia et al., 1995; Gee et al., 1996). In the AI centres, although the storage conditions of semen favour the preservation of the virus, strict control standards have been established with the aim of ensuring the absence of BoHV-1 in the semen. The farms that use calving pens had a 1.56-fold greater chance of being BoHV-1 positive than those that did not using calving pens (Table 5). Use of a calving pen is a good management practice that facilitates birth assistance and calf management. However, when this practice is carried out in an uncontrolled way, it can facilitate the transmission of microorganisms that are shed during birth. Considering the physiological immunosuppression that occurs during pregnancy and parturition, animals with latent infections may undergo virus reactivation and may excrete the virus into the environment. The grouping of animals in the calving pen should be considered a management failure that facilitates intraherd BoHV-1 transmission. One of the weaknesses of this study was the possible classification error introduced by imperfect serological tests. However, classification was improved by using a test with elevated sensitivity (indirect ELISA), while specificity would be improved by interpreting the results in series. Considering the population under investigation, it should be possible to reduce the current BoHV-1 seroprevalence if tests with high sensibility and specificity (virusneutralization) were used; this however would have been unfeasible because of the large sample number. The high apparent prevalence of animals and herds with BoHV-1 infection during the study period shows that the virus is widely distributed in all regions of the state, mainly within beef cattle herds. The results of the analysis of risk factors indicate that characteristics of each farm as well as characteristics of the management practices used at each farm are associated with BoHV-1 infection. In Brazil, there is not an official programme for IBR control. The BoHV-1 infection frequency in dairy and beef cattle herds in the state of Paraná and the economic losses caused by this viral infection for Brazilian agribusiness show the urgent need for the adoption of BoHV-1 prevention and control measures. This study provides a basis for planning such measures in the state of Paraná. Acknowledgements The authors would like to thank to the Secretary of Agriculture of Paraná State and the Ministry of Agriculture, Livestock, and Food Supply for help during the sampling design and sample collection. This study was financially supported by CAPES, CNPq, FINEP and Fundação Araucária (FAP/PR). Alfieri, A.A. is a recipient of a CNPq fellowship. References Ackermann, M., and M. Engels, 2006: Pro and contra IBReradication. Vet. Microbiol. 113, Ackermann, M., E. Peterhans, and R. Wyler, 1982: DNA of bovine herpesvirus 1 in trigeminal ganglia of latently infected calves. Am. J. Vet. 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