Bovine viral diarrhea virus outbreak in a beef cow herd in South Dakota Susan E. Kane, PhD; Larry D. Holler, DVM, PhD; Lyle J. Braun, MS; John D. Neill, PhD; Douglas B. Young, BA; Julia F. Ridpath, PhD; Christopher C. L. Chase, DVM, PhD
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Case Description—136 pregnant beef cows were purchased in the fall of 2003. The following spring, 128 cows calved as expected; 8 cows were believed to have aborted with the fetuses unavailable for evaluation. Of the 128 calves born, 8 died within 2 weeks after birth and 9 were born with congenital abnormalities. Clinical Findings—Cows and their calves were evaluated for bovine viral diarrhea virus (BVDV) infection. Forty-four of 120 calves, but 0 cows, tested positive for BVDV antigen by immunohistochemical staining of ear notch specimens. Treatment and Outcome—Five BVDV test–positive calves died shortly after weaning, and the remaining 39 BVDV test–positive calves were moved to an isolated feedlot and retested for BVDV at 5 to 6 months of age; 36 had positive results, which indicated that they were persistently infected (PI) with BVDV, whereas 3 had negative results, which indicated that they were transiently infected with BVDV at the time of the first test. All PI calves were infected with the same BVDV type 2a strain. As yearlings, 17 of the 36 PI calves died peracutely with lesions consistent with mucosal disease, 6 died without gross lesions, and 2 were euthanized because of chronic ill thrift. The remaining 11 PI calves appeared healthy and were sold for slaughter. Screening of the following year’s calf crop for BVDV by use of immunohistochemical staining of ear-notch specimens yielded negative results for all calves. Clinical Relevance—Introduction of BVDV into a naïve cow herd resulted in a loss of 44% of the calf crop subsequent to reproductive loss, poor thrift, and mucosal disease. (J Am Vet Med Assoc 2015;246:1358–1362)
O
ne hundred thirty-six 3-year-old pregnant Angus and Angus-cross beef cows with unknown vaccination histories were purchased by the South Dakota State University Antelope Range and Livestock Research Station from a producer in western South Dakota in the fall of 2003. The cows were bred to Angus bulls and were confirmed pregnant by transrectal palpation prior to purchase. Following purchase, the cows were transported to the research station in northwestern South Dakota where they were maintained on pasture and fed a ration that was formulated to meet or exceed National Research Council requirements.1 Water was available ad libitum at all times. One hundred twenty-eight of the 136 cows calved between March 25 and May 27, 2004, resulting in 128 live calves; 8 cows did not calve as expected and were assumed to have aborted, and the aborted fetuses were unavailable for evaluation. The cows and their calves From the Department of Veterinary and Biomedical Sciences, College of Agriculture and Biological Sciences, South Dakota State University, Brookings, SD 57006 (Kane, Holler, Braun, Chase); National Animal Disease Center, Agricultural Research Service, USDA, 1920 Dayton Ave, Ames, IA 50010 (Neill, Ridpath); and State of South Dakota Antelope Range and Livestock Research Station, South Dakota State University, 12988 Park Rd, Buffalo, SD 57720 (Young). Dr. Kane’s present address is Wenatchee Valley College, Wenatchee, WA 98801. Supported in part by Boehringer Ingelheim Vetmedica, Pfizer Animal Health, and Novartis Animal Health. Presented in abstract form at the 45th Annual Conference of the American Association of Bovine Practitioners, Montreal, September 2012. The authors thank Trey Patterson for technical assistance. Address correspondence to Dr. Chase ([email protected]). 1358
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ABBREVIATIONS BVDV PI TI
Bovine viral diarrhea virus Persistently infected Transiently infected
were maintained on pasture until the calves were weaned. Eight calves died within 2 weeks after birth and were not submitted for diagnostic testing. An additional 9 calves were born with congenital abnormalities such as corneal opacity, alopecia, and red hair, which raised concerns about a potential BVDV outbreak. In June, ear notch specimens were obtained from all calves and their dams and submitted for immunohistochemical staining for BVDV antigen as described.2 Bovine viral diarrhea virus antigen was not detected in any of the ear notch specimens obtained from the cows but was detected in the ear notch specimens of 44 of the 120 (37%) surviving calves, including 5 of the 9 calves with congenital abnormalities. Further genetic analysis of the BVDV antigen detected in positive earnotch specimens performed by the National Animal Disease Laboratory in Ames, Iowa, revealed that all calves were infected with the same BVDV type 2a strain. The calves were weaned in August, and 5 of the 44 BVDV test–positive calves died shortly thereafter. On October 6, 2004, the remaining 39 BVDV test– positive calves were transported to a university feedlot facility in Brookings, SD, where they were isolated as a group from other cattle and retested for BVDV. From each calf, a blood sample (approx 10 mL) was JAVMA, Vol 246, No. 12, June 15, 2015
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collected by jugular venipuncture into an evacuated anti-BVDV2 antibodies, all 6 had detectable titers blood collection tube containing EDTA. The buffy (range, 1:8 to 1:256) on December 8, 2004, whereas 2 coat fraction was recovered from each sample and had detectable titers (1:8) on February 15, 2005, and submitted for BVDV isolation, which was performed only 1 had a detectable titer (1:32) on March 31, 2005. by the tube method,3,4 and for a BVDV antigen capture During a 110-day period between May 5 and AuELISA.a Thirty-six calves had positive results for BVDV gust 22, 2005, 23 of the 36 PI calves (approx age, 13 to as determined by both virus isolation and the antigen 15 months; approx weight, 400 kg [880 lb]) died. Fifcapture ELISA and were considered PI with BVDV (PI teen of those calves died peracutely without any clinical calves), whereas the remaining 3 calves had negative signs of disease. The other 8 calves were found laterally results and were considered to have been TI with BVDV recumbent without any other clinical signs of disease (TI calves) at the time the ear notch specimen was and were unable to attain a sternal or standing position. obtained in June. Those calves failed to respond to palliative treatment From each of the 39 isolated calves, a blood sample (NSAID and oral and IV fluid administration) and died (approx 10 mL) was collected by jugular venipuncture or were euthanized within 6 to 12 hours. Many of those into evacuated blood collection tubes on December 8, calves died during or following days when the ambient 2004 (approx age, 9 months); February 15, 2005 (approx age, 11 months); and March 31, 2005 (approx age, 13 months) to obtain serum for determination of neutralizing antibody titers against BVDV types 1 (BVDV1) and 2 (BVDV2). The serum neutralization assays were performed in accordance with methods recommended by the World Organisation for Animal Health4 with a cytopathic Singer strain used as the reference strain for BVDV1 and a cytopathic A125 strain used as the reference strain for BVDV2. All 3 TI calves had antibodies against both BVDV1 and BVDV2 during the testing period. Of the 36 PI calves, only 1 had serum neutralizing antibody titers against both BVDV1 and BVDV2, whereas 3 calves had antibody titers against BVDV1 but not BVDV2 and 5 calves had antibody titers against BVDV2 but not BVDV1. The geometric mean antibody titers against both BVDV1 and BVDV2 for the TI calves were higher than those for the PI calves on each test date. For all 3 TI calves, the anti-BVDV1 antibody titer was < 1:2 and 1:512 on December 8, 2004, and February 15, 2005, respectively. On March 31, 2005, the anti-BVDV1 antibody titer was 1:256 for 1 calf, 1:512 for 1 calf, and 1:1,024 for the remaining calf. The anti-BVDV2 antibody titer was 1:4,096 for 2 of the TI calves and 1:8,192 for the remaining TI calf on December 8, 2004. On February 15, 2005, the anti-BVDV2 antibody titer was 1:512 for 1 TI calf and 1:1,024 for the remaining 2 TI calves. On March 31, 2005, the anti-BVDV2 antibody titer was 1:2,048 for all 3 TI calves. For the 4 PI calves that tested positive for anti-BVDV1 antibodies, 1 calf had a titer of 1:8 on December 8, 2004; 2 calves a titer of 1:8 and 2 others had a titer of 1:16 on February 15, 2005; and none of the calves had an anti-BVDV1 Figure 1—Flow chart of the clinical outcomes for the 2004 offspring of 136 pregnant Angus and Angus-cross cows that were purchased by the South Dakota State Uniantibody titer on March 31, 2005. For versity Antelope Range and Livestock Research Station in the fall of 2003 and were the 6 PI calves that tested positive for affected by a BVDV outbreak.
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temperature was greater than the seasonal mean temperature for Brookings, SD. Two other PI calves (approx weight, 160 kg [352 lb]) were euthanized because of poor weight gain and chronic ill thrift. The remaining 11 PI calves that appeared clinically normal with body weights ranging from 400 to 500 kg (880 to 1,100 lb) were sold for slaughter at the end of June 2005. The clinical outcomes for the calves born from the 136 purchased cows were summarized (Figure 1). Necropsy was performed on each of the 23 PI calves that died between May 5 and August 22, 2005. Results indicated that 17 of the 23 (74%) calves had lesions consistent with BVDV mucosal disease (extensive erosions and ulcerations throughout the gastrointestinal tract), whereas the remaining 6 (26%) calves did not have any gross lesions. Because calves were observed only 2 to 3 times/d and many died when the ambient temperature was abnormally high, some of the bodies had severe postmortem autolysis, which prevented histologic Figure 2—Distribution of the birth of 44 BVDV test–positive calves (gray bars) and 84 evaluation. However, when nonautolyzed BVDV test–negative calves (black bars) from the purchased cows of Figure 1. Calves born between March 25 and May 27, 2004. Eight cows did not calve as expected tissues were available for histologic evalu- were and were assumed to have aborted, and the fetuses were unavailable for evaluation. ation, the results invariably indicated Thus, 128 calves were born, of which 8 died < 1 month after birth and 9 were born extensive necrosis of lymphoid tissues, with congenital defects such as corneal opacity and alopecia. An ear notch specimen obtained from each of the 120 calves that survived > 1 month and was tested for especially the gut-associated lymphoid was the presence of BVDV antigen by use of an immunohistochemical staining technique. tissues. Noncytopathic and cytopathic Calves that died before they could be tested for BVDV are included in the BVDV test– BVDV were isolated from the tissues negative group. See Figure 1 for remainder of key. of 12 of the 17 calves with lesions and 2 of the 6 calves without lesions. Only Table 1—Mean birth weight and body weight and body weight noncytopathic BVDV was isolated from the tissues of 3 increase at 7 and 14 weeks old for 44 BVDV test–positive calves calves with lesions and 4 calves without lesions. Bovine and 76 BVDV test–negative calves. viral diarrhea virus was not isolated from the tissues of the remaining 2 calves with lesions; however, failure BVDV Mean weight Age test status Mean weight (kg) increase (kg [%]) to detect BVDV in those 2 calves was likely caused by severe postmortem autolysis. Birth Positive 35.0* — Negative 42.4 — Review of the calving records indicated that BVDV 7 weeks Positive 84.0* 47.6 (141.0) test–positive calves were born throughout the entire Negative 108.8 66.3 (159.1) calving season, rather than in separate clusters early 14 weeks Positive 127.9* 42.8 (50.7) or late in the season (Figure 2). Most of the BVDV Negative 167.5 58.8 (55.1) test–positive calves were born during the first half of Calves were tested for the presence of BVDV antigen by immuthe calving season, which corresponded to the period nohistochemical staining of ear notch specimens. At each age, the during which most of the cows calved. weight of each calf was obtained and the mean was calculated for each group. Similarly, the amount and percentage of weight gained The mean birth weight for the 44 calves that tested between measurements was determined for each calf, and the repositive for BVDV antigen by immunohistochemical spective mean weight increases were calculated. staining of ear notch specimens (35 kg [77 lb]) was — = Not applicable. *Value is significantly (P < 0.05) less than the corresponding value for the BVDV test–negative calves. significantly less than the mean birth weight for the 76 BVDV test–negative calves (42.4 kg [93.3 lb]). The mean body weight for the BVDV test–positive calves In the spring of 2005, ear notch specimens were remained less than that for the BVDV test–negative obtained from all calves born at the research station calves at 7 and 14 weeks of age; however, the mean where the PI calves were born in 2004. Results of impercentage of body weight increase from birth, which munohistochemical staining for BVDV antigen were accounted for differences in birth weight among calves, negative for all calves. did not differ significantly between the BVDV test– positive and BVDV test–negative calves at either 7 or 14 Discussion weeks old (Table 1). This indicated that the growth rate of the BVDV test–positive calves was similar to that of Bovine viral diarrhea virus continues to be a major the BVDV–test negative calves through 14 weeks of age. cause of reproductive disease in cattle.5–7 Currently, 1360
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Other reports21,22 of BVDV mucosal disease outbreaks in beef herds were retrospectively evaluated. In the present report, we described the effect of BVDV on the growth and health of a group of PI calves and the subsequent development of mucosal disease in those calves in a prospective manner. Infection with BVDV alters the growth of affected cattle.23 Although the mean birth weight and body weights at 7 and 14 weeks of age for the BVDV test–positive calves of the present report were less than those for their BVDV test– negative herd mates, the actual amount and percentage of weight increase between measurements did not differ significantly between the 2 groups of calves. This finding indicated that the growth rate for the BVDV test–positive calves was similar to that of the BVDV test–negative calves. It is generally believed that most PI calves die within the first few months after birth, but that notion was not upheld in the present outbreak because all 36 PI calves were clinically normal for > 1 year until the first calf succumbed to mucosal disease. Moreover, 11 of those 36 (31%) calves remained healthy, achieved finished market weight, and were sold for slaughter. The serum neutralizing anti-BVDV antibody titers for the BVDV test–positive calves are difficult to interpret. The consistently high anti-BVDV2 antibody titers of the 3 TI calves were likely the result of an active infection and continuous exposure to calves PI with a BVDV2a strain. The presence of anti-BVDV1 antibody titers in all 3 TI calves in the absence of vaccination against or natural exposure to BVDV1 was likely the result of a cross-reaction between the BVDV1 reference strain and the BVDV2 strain to which the calves were exposed.20 Only 9 of the 36 PI calves had antibodies against BVDV, and those titers were typically low and tended to decrease over the 6-month observation period. It is unlikely that the antibodies were indicative of an acquired immunity against the persistent strain of BVDV because immunotolerance of PI calves to the persistent strain is documented.9 It is worth noting, however, that PI calves are able to respond to BVDV strains other than the persistent strain, and BVDV is an RNA virus that can generate variants during replication.24,25 It is possible that the anti-BVDV antibodies present in the PI calves were the result of passive immunity. Anecdotally, maternal antibodies have been reported to persist in calves for 6 to 8 months after weaning. Admittedly, this would be atypical and has not yet been established under experimental settings; maternal antibody titers are typically very high during the neonatal period and then gradually decline over time. The decay of maternal antibodies could have accounted for the decline in antiBVDV antibody titers observed in the PI calves over the 6-month observation period. Unfortunately, we cannot confirm that the anti-BVDV antibody titers in the PI calves were the result of maternal antibodies because the titers were very low and were not determined for the calves prior to 9 months of age. It also possible that the kinetics of maternal antibody clearance in PI calves is not the same as those in non–PI calves, and this has not been evaluated. Seventeen of the 23 PI calves that died had lesions characteristic of mucosal disease. The other 6 calves died Scientific Reports
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BVDV strains are classified into 1 of 2 genotypes (BVDV1 or BVDV2), each of which is further subdivided into 2 biotypes (cytopathic and noncytopathic) on the basis of whether a given strain causes lytic changes in cultured cells.8,9 Infection of a pregnant cow with a noncytopathic strain of BVDV prior to 40 days of gestation can result in early embryonic death, whereas infection between 40 and 150 days of gestation can result in a fetus that is PI with BVDV, and infection during the latter stages of the second and third trimesters can cause abortion or congenital abnormalities in the fetus or less severe outcomes such as the birth of calves with poor vigor or no clinically apparent abnormalities.6,10 Cattle PI with BVDV (PI cattle) are the major reservoir for the virus,11,12 and current recommendations for BVDV control focus on the detection and removal of PI cattle.11–17 Persistently infected cattle that survive the first year of life often die from mucosal disease, which results when an animal becomes infected with a cytopathic strain of BVDV that is substantially similar to the noncytopathic strain with which it is PI (the cytopathic strain frequently emerges from a mutation of the noncytopathic strain).18,19 Information regarding the mortality rate of PI calves within 1 year after birth and the percentage of PI cattle that die as a result of mucosal disease or appear clinically normal is lacking. The present report provided additional information about the clinical outcomes associated with the apparent introduction of a single strain of BVDV2 into a herd of 3-year-old pregnant beef cows. The BVDV outbreak described in the present report was particularly notable because of the substantial impact that BVDV infection had on the 2004 calf crop. We assumed that the 8 suspected abortions and 8 neonatal deaths were related to BVDV infection, and 44 calves subsequently tested positive for BVDV at least once. Thus, 44% (60/136) of the 2004 calf crop for this herd was lost or otherwise affected by BVDV. The source of most BVDV outbreaks is generally traced back to the introduction of a PI animal into a BVDV-naïve herd. However, immunohistochemical staining of ear notch specimens for BVDV antigen yielded negative results for all 136 purchased cows; therefore, it is unlikely that there was a PI cow among the purchased cows, and the exact point source of BVDV entry into this herd is unknown. The present report highlighted the risk producers assume when they purchase pregnant cattle, a common practice in the beef and dairy industries. Currently, there is no affordable or safe method to determine whether the fetus a pregnant cow is carrying is PI with BVDV. The detection of maternal viremia is not a viable option for identification of dams carrying PI calves because viremia associated with BVDV infection has a short duration (generally < 10 days) and must occur during a particular time (40 to 150 days) of gestation to result in a PI calf.6 Screening pregnant cows for the presence of anti-BVDV antibody titers is also not a useful method for identification of dams carrying PI calves because currently available neutralization assays cannot differentiate between anti-BVDV antibodies that resulted from vaccination and those that resulted from natural infection, and cattle are commonly vaccinated against BVDV.20
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without any gross lesions, and BVDV infection would not have been suspected without knowledge that the calves were PI. Thus, it is recommended that BVDV diagnostic tests be routinely included in the necropsy workup for cattle that die suddenly, even those without gross lesions. Outbreaks of mucosal disease have been previously reported in 2 beef herds in Saskatchewan, Canada.21,22 In 1 report, 28 of 105 (27%) yearling cattle were PI with BVDV, and 20 (71%) of those cattle died from mucosal disease. Interestingly, 1 PI calf was born in that herd the year following the birth of the cattle that died from mucosal disease.21 Five PI calves were born the second year following the birth of the cattle that died from mucosal disease, and 3 of those 5 PI calves were born to cohorts of the PI cattle that died from mucosal disease.21 Hence, it is important to screen all cows and heifers for BVDV for at least 2 years following an outbreak to identify and remove PI cattle. Similar to the herd of the present report, most of the PI cattle in that study21 were > 1 year old when they died of mucosal disease. In the other report,22 71 of 560 (13%) calves were suspected to be PI with BVDV; 20 calves died before weaning and were likely PI, and the remaining 51 calves were confirmed to be PI with BVDV on the basis of virus isolation or necropsy results. Of the 51 confirmed PI calves, 47 died at < 1 year old, and 12 (26%) of those had no or only mild lesions associated with mucosal disease.22 The presence of PI cattle is a threat to the health and overall profit of any cattle operation.5,12,13 An increase in producer awareness of BVDV and the use of appropriate vaccination regimens, surveillance, and eradication efforts have resulted in a decrease in the incidence of PI cattle.10,12,13,16 However, until an affordable and practical test to screen fetuses for BVDV is developed, the risk of introducing BVDV into a herd should be considered whenever pregnant cows are purchased, and biosecurity practices (quarantine of purchased cows and the calves from those cows) should be implemented until appropriate testing can be performed.12,13 Additionally, all cattle that die suddenly with or without gross lesions should be tested for BVDV. The present report is a reminder that diligence in BVDV prevention is vital for beef operations because a BVDV outbreak can have devastating effects on productivity. Also, although it is often recommended that any animal with a positive result for BVDV antigen by immunohistochemical staining of an ear notch specimen be culled,2,26 1 positive result is insufficient to definitively identify an animal as PI with BVDV. For the herd of the present report, 3 of the 44 (6.8%) calves that tested positive for BVDV antigen by immunohistochemical staining of an ear notch specimen subsequently tested negative for BVDV on virus isolation and by antigen capture ELISA performed on buffy coat samples and were determined to be only TI with BVDV at the time of the positive test. This finding emphasizes the importance of performing follow-up testing of BVDV test– positive cattle to definitively determine whether they are truly PI with BVDV. Proper biosecurity and vaccination programs and the rapid identification and removal of PI cattle are strongly recommended to minimize BVDVassociated losses.
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National Research Council. Nutrient requirements of beef cattle. 7th ed. Washington, DC: National Academy Press, 2000;102–112. Njaa BL, Clark EG, Janzen E, et al. Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalinfixed skin biopsy specimens. J Vet Diagn Invest 2000;12:393–399. South Dakota State University Animal Disease Research and Diagnostic Laboratory. BVD virus isolation and outgrowth ELISA. VSD.ARDRL. SOP. VIR. 4100.04. Brookings, SD: South Dakota State University, 2007. OIE. Bovine viral diarrhoea. 2.10.6. In: Manual of diagnostic tests and vaccines for terrestrial animals. Paris: OIE, 2004;1051–1063. Grooms DL. Reproductive consequences of infection with bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2004;20:5–19 Brock KV, Grooms DL, Givens MD. Reproductive disease and persistent infections. In: Goyal SM, Ridpath J, eds. Bovine viral diarrhea virus: diagnosis, management and control. Ames, Iowa: Blackwell Publishing, 2005;145–156. González Altamiranda EA, Kaiser GG, et al. Effect of bovine viral diarrhea virus on the ovarian functionality and in vitro reproductive performance of persistently infected heifers. Vet Microbiol 2013;165:326–332. Ridpath JF, Bolin SR, Dubovi EJ. Segregation of bovine viral diarrhea virus into genotypes. Virology 1994;205:66–74. Gillespie JH, Baker JA, McEntee K. A cytopathogenic strain of virus diarrhoea virus. Cornell Vet 1960;50:73–79. Baker JC. Clinical aspects of bovine virus diarrhoea virus infection. Rev Sci Tech 1990;9:25–41. Fulton RW, Whitley EM, Johnson BJ, et al. Prevalence of bovine viral diarrhea virus (BVDV) in persistently infected cattle and BVDV subtypes in affected cattle in beef herds in south central United States. Can J Vet Res 2009;73:283–291. Van Campen H. Epidemiology and control of BVD in the US. Vet Microbiol 2010;142:94–98. Hans H. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet Microbiol 1999;64:89–107. Ridpath J. Preventive strategy for BVDV infection in North America. Jpn J Vet Res 2012;60(suppl):S41–S49. Houe H. Risk assessment. In: Goyal SM, Ridpath JF, eds. Bovine viral diarrhea virus: diagnosis, management, and control. Oxford, England: Blackwell Publishing Ltd, 2008;35–64. Rossmanith W, Wilhelm J. Control of BVDV-infection on common grassland—the key for successful BVDV-eradication in Lower Austria. Prev Vet Med 2005;72:133–137. Brock K. Strategies for the control and prevention of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2004:20:171–180. Bolin SR. The pathogenesis of mucosal disease. Vet Clin North Am Food Anim Pract 1995;11:489–500. Brownlie J, Clarke MC, Howard CJ. Experimental production of fatal mucosal disease in cattle. Vet Rec 1984;114:535–536. Fairbanks K, Schnackel J, Chase C. Evaluation of a modified live virus type-1a bovine viral diarrhea virus vaccine (Singer strain) against a type-2 (strain 890) challenge. Vet Ther 2003;4:24–34. Taylor LF, Janzen ED, Ellis JA, et al. Performance, survival, necropsy, and virological findings from calves persistently infected with the bovine viral diarrhea virus originating from a single Saskatchewan beef herd. Can Vet J 1997;38:29–37. Taylor LF, Van Donkersgoed J, Radostits OM, et al. Investigation of an outbreak of mucosal disease in a beef cattle herd in southwestern Saskatchewan. Can Vet J 1994;35:425–432. Webb BT, Norrdin RW, Smirnova NP, et al. Bovine viral diarrhea virus cyclically impairs long bone trabecular modeling in experimental persistently infected fetuses. Vet Pathol 2012;49:930–940. Jones LR, Zandomeni R, Weber EL. Quasispecies in the 5′ untranslated genomic region of bovine viral diarrhoea virus from a single individual. J Gen Virol 2002;83:2161–2168 Chase CC. The impact of BVDV infection on adaptive immunity. Biologicals 2013 41:52–60 Thür B, Zlinszky K, Ehrensperger F. Immunohistology as a reliable and efficient method for the diagnosis of BVDV infections. Schweiz Arch Tierheilkd 1996;138:476–482. JAVMA, Vol 246, No. 12, June 15, 2015
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Case Description—136 pregnant beef cows were purchased in the fall of 2003. The following spring, 128 cows calved as expected; 8 cows were believed to have aborted with the fetuses unavailable for evaluation. Of the 128 calves born, 8 died within 2 weeks after birth and 9 were born with congenital abnormalities. Clinical Findings—Cows and their calves were evaluated for bovine viral diarrhea virus (BVDV) infection. Forty-four of 120 calves, but 0 cows, tested positive for BVDV antigen by immunohistochemical staining of ear notch specimens. Treatment and Outcome—Five BVDV test–positive calves died shortly after weaning, and the remaining 39 BVDV test–positive calves were moved to an isolated feedlot and retested for BVDV at 5 to 6 months of age; 36 had positive results, which indicated that they were persistently infected (PI) with BVDV, whereas 3 had negative results, which indicated that they were transiently infected with BVDV at the time of the first test. All PI calves were infected with the same BVDV type 2a strain. As yearlings, 17 of the 36 PI calves died peracutely with lesions consistent with mucosal disease, 6 died without gross lesions, and 2 were euthanized because of chronic ill thrift. The remaining 11 PI calves appeared healthy and were sold for slaughter. Screening of the following year’s calf crop for BVDV by use of immunohistochemical staining of ear-notch specimens yielded negative results for all calves. Clinical Relevance—Introduction of BVDV into a naïve cow herd resulted in a loss of 44% of the calf crop subsequent to reproductive loss, poor thrift, and mucosal disease. (J Am Vet Med Assoc 2015;246:1358–1362)
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ne hundred thirty-six 3-year-old pregnant Angus and Angus-cross beef cows with unknown vaccination histories were purchased by the South Dakota State University Antelope Range and Livestock Research Station from a producer in western South Dakota in the fall of 2003. The cows were bred to Angus bulls and were confirmed pregnant by transrectal palpation prior to purchase. Following purchase, the cows were transported to the research station in northwestern South Dakota where they were maintained on pasture and fed a ration that was formulated to meet or exceed National Research Council requirements.1 Water was available ad libitum at all times. One hundred twenty-eight of the 136 cows calved between March 25 and May 27, 2004, resulting in 128 live calves; 8 cows did not calve as expected and were assumed to have aborted, and the aborted fetuses were unavailable for evaluation. The cows and their calves From the Department of Veterinary and Biomedical Sciences, College of Agriculture and Biological Sciences, South Dakota State University, Brookings, SD 57006 (Kane, Holler, Braun, Chase); National Animal Disease Center, Agricultural Research Service, USDA, 1920 Dayton Ave, Ames, IA 50010 (Neill, Ridpath); and State of South Dakota Antelope Range and Livestock Research Station, South Dakota State University, 12988 Park Rd, Buffalo, SD 57720 (Young). Dr. Kane’s present address is Wenatchee Valley College, Wenatchee, WA 98801. Supported in part by Boehringer Ingelheim Vetmedica, Pfizer Animal Health, and Novartis Animal Health. Presented in abstract form at the 45th Annual Conference of the American Association of Bovine Practitioners, Montreal, September 2012. The authors thank Trey Patterson for technical assistance. Address correspondence to Dr. Chase ([email protected]). 1358
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ABBREVIATIONS BVDV PI TI
Bovine viral diarrhea virus Persistently infected Transiently infected
were maintained on pasture until the calves were weaned. Eight calves died within 2 weeks after birth and were not submitted for diagnostic testing. An additional 9 calves were born with congenital abnormalities such as corneal opacity, alopecia, and red hair, which raised concerns about a potential BVDV outbreak. In June, ear notch specimens were obtained from all calves and their dams and submitted for immunohistochemical staining for BVDV antigen as described.2 Bovine viral diarrhea virus antigen was not detected in any of the ear notch specimens obtained from the cows but was detected in the ear notch specimens of 44 of the 120 (37%) surviving calves, including 5 of the 9 calves with congenital abnormalities. Further genetic analysis of the BVDV antigen detected in positive earnotch specimens performed by the National Animal Disease Laboratory in Ames, Iowa, revealed that all calves were infected with the same BVDV type 2a strain. The calves were weaned in August, and 5 of the 44 BVDV test–positive calves died shortly thereafter. On October 6, 2004, the remaining 39 BVDV test– positive calves were transported to a university feedlot facility in Brookings, SD, where they were isolated as a group from other cattle and retested for BVDV. From each calf, a blood sample (approx 10 mL) was JAVMA, Vol 246, No. 12, June 15, 2015
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collected by jugular venipuncture into an evacuated anti-BVDV2 antibodies, all 6 had detectable titers blood collection tube containing EDTA. The buffy (range, 1:8 to 1:256) on December 8, 2004, whereas 2 coat fraction was recovered from each sample and had detectable titers (1:8) on February 15, 2005, and submitted for BVDV isolation, which was performed only 1 had a detectable titer (1:32) on March 31, 2005. by the tube method,3,4 and for a BVDV antigen capture During a 110-day period between May 5 and AuELISA.a Thirty-six calves had positive results for BVDV gust 22, 2005, 23 of the 36 PI calves (approx age, 13 to as determined by both virus isolation and the antigen 15 months; approx weight, 400 kg [880 lb]) died. Fifcapture ELISA and were considered PI with BVDV (PI teen of those calves died peracutely without any clinical calves), whereas the remaining 3 calves had negative signs of disease. The other 8 calves were found laterally results and were considered to have been TI with BVDV recumbent without any other clinical signs of disease (TI calves) at the time the ear notch specimen was and were unable to attain a sternal or standing position. obtained in June. Those calves failed to respond to palliative treatment From each of the 39 isolated calves, a blood sample (NSAID and oral and IV fluid administration) and died (approx 10 mL) was collected by jugular venipuncture or were euthanized within 6 to 12 hours. Many of those into evacuated blood collection tubes on December 8, calves died during or following days when the ambient 2004 (approx age, 9 months); February 15, 2005 (approx age, 11 months); and March 31, 2005 (approx age, 13 months) to obtain serum for determination of neutralizing antibody titers against BVDV types 1 (BVDV1) and 2 (BVDV2). The serum neutralization assays were performed in accordance with methods recommended by the World Organisation for Animal Health4 with a cytopathic Singer strain used as the reference strain for BVDV1 and a cytopathic A125 strain used as the reference strain for BVDV2. All 3 TI calves had antibodies against both BVDV1 and BVDV2 during the testing period. Of the 36 PI calves, only 1 had serum neutralizing antibody titers against both BVDV1 and BVDV2, whereas 3 calves had antibody titers against BVDV1 but not BVDV2 and 5 calves had antibody titers against BVDV2 but not BVDV1. The geometric mean antibody titers against both BVDV1 and BVDV2 for the TI calves were higher than those for the PI calves on each test date. For all 3 TI calves, the anti-BVDV1 antibody titer was < 1:2 and 1:512 on December 8, 2004, and February 15, 2005, respectively. On March 31, 2005, the anti-BVDV1 antibody titer was 1:256 for 1 calf, 1:512 for 1 calf, and 1:1,024 for the remaining calf. The anti-BVDV2 antibody titer was 1:4,096 for 2 of the TI calves and 1:8,192 for the remaining TI calf on December 8, 2004. On February 15, 2005, the anti-BVDV2 antibody titer was 1:512 for 1 TI calf and 1:1,024 for the remaining 2 TI calves. On March 31, 2005, the anti-BVDV2 antibody titer was 1:2,048 for all 3 TI calves. For the 4 PI calves that tested positive for anti-BVDV1 antibodies, 1 calf had a titer of 1:8 on December 8, 2004; 2 calves a titer of 1:8 and 2 others had a titer of 1:16 on February 15, 2005; and none of the calves had an anti-BVDV1 Figure 1—Flow chart of the clinical outcomes for the 2004 offspring of 136 pregnant Angus and Angus-cross cows that were purchased by the South Dakota State Uniantibody titer on March 31, 2005. For versity Antelope Range and Livestock Research Station in the fall of 2003 and were the 6 PI calves that tested positive for affected by a BVDV outbreak.
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temperature was greater than the seasonal mean temperature for Brookings, SD. Two other PI calves (approx weight, 160 kg [352 lb]) were euthanized because of poor weight gain and chronic ill thrift. The remaining 11 PI calves that appeared clinically normal with body weights ranging from 400 to 500 kg (880 to 1,100 lb) were sold for slaughter at the end of June 2005. The clinical outcomes for the calves born from the 136 purchased cows were summarized (Figure 1). Necropsy was performed on each of the 23 PI calves that died between May 5 and August 22, 2005. Results indicated that 17 of the 23 (74%) calves had lesions consistent with BVDV mucosal disease (extensive erosions and ulcerations throughout the gastrointestinal tract), whereas the remaining 6 (26%) calves did not have any gross lesions. Because calves were observed only 2 to 3 times/d and many died when the ambient temperature was abnormally high, some of the bodies had severe postmortem autolysis, which prevented histologic Figure 2—Distribution of the birth of 44 BVDV test–positive calves (gray bars) and 84 evaluation. However, when nonautolyzed BVDV test–negative calves (black bars) from the purchased cows of Figure 1. Calves born between March 25 and May 27, 2004. Eight cows did not calve as expected tissues were available for histologic evalu- were and were assumed to have aborted, and the fetuses were unavailable for evaluation. ation, the results invariably indicated Thus, 128 calves were born, of which 8 died < 1 month after birth and 9 were born extensive necrosis of lymphoid tissues, with congenital defects such as corneal opacity and alopecia. An ear notch specimen obtained from each of the 120 calves that survived > 1 month and was tested for especially the gut-associated lymphoid was the presence of BVDV antigen by use of an immunohistochemical staining technique. tissues. Noncytopathic and cytopathic Calves that died before they could be tested for BVDV are included in the BVDV test– BVDV were isolated from the tissues negative group. See Figure 1 for remainder of key. of 12 of the 17 calves with lesions and 2 of the 6 calves without lesions. Only Table 1—Mean birth weight and body weight and body weight noncytopathic BVDV was isolated from the tissues of 3 increase at 7 and 14 weeks old for 44 BVDV test–positive calves calves with lesions and 4 calves without lesions. Bovine and 76 BVDV test–negative calves. viral diarrhea virus was not isolated from the tissues of the remaining 2 calves with lesions; however, failure BVDV Mean weight Age test status Mean weight (kg) increase (kg [%]) to detect BVDV in those 2 calves was likely caused by severe postmortem autolysis. Birth Positive 35.0* — Negative 42.4 — Review of the calving records indicated that BVDV 7 weeks Positive 84.0* 47.6 (141.0) test–positive calves were born throughout the entire Negative 108.8 66.3 (159.1) calving season, rather than in separate clusters early 14 weeks Positive 127.9* 42.8 (50.7) or late in the season (Figure 2). Most of the BVDV Negative 167.5 58.8 (55.1) test–positive calves were born during the first half of Calves were tested for the presence of BVDV antigen by immuthe calving season, which corresponded to the period nohistochemical staining of ear notch specimens. At each age, the during which most of the cows calved. weight of each calf was obtained and the mean was calculated for each group. Similarly, the amount and percentage of weight gained The mean birth weight for the 44 calves that tested between measurements was determined for each calf, and the repositive for BVDV antigen by immunohistochemical spective mean weight increases were calculated. staining of ear notch specimens (35 kg [77 lb]) was — = Not applicable. *Value is significantly (P < 0.05) less than the corresponding value for the BVDV test–negative calves. significantly less than the mean birth weight for the 76 BVDV test–negative calves (42.4 kg [93.3 lb]). The mean body weight for the BVDV test–positive calves In the spring of 2005, ear notch specimens were remained less than that for the BVDV test–negative obtained from all calves born at the research station calves at 7 and 14 weeks of age; however, the mean where the PI calves were born in 2004. Results of impercentage of body weight increase from birth, which munohistochemical staining for BVDV antigen were accounted for differences in birth weight among calves, negative for all calves. did not differ significantly between the BVDV test– positive and BVDV test–negative calves at either 7 or 14 Discussion weeks old (Table 1). This indicated that the growth rate of the BVDV test–positive calves was similar to that of Bovine viral diarrhea virus continues to be a major the BVDV–test negative calves through 14 weeks of age. cause of reproductive disease in cattle.5–7 Currently, 1360
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Other reports21,22 of BVDV mucosal disease outbreaks in beef herds were retrospectively evaluated. In the present report, we described the effect of BVDV on the growth and health of a group of PI calves and the subsequent development of mucosal disease in those calves in a prospective manner. Infection with BVDV alters the growth of affected cattle.23 Although the mean birth weight and body weights at 7 and 14 weeks of age for the BVDV test–positive calves of the present report were less than those for their BVDV test– negative herd mates, the actual amount and percentage of weight increase between measurements did not differ significantly between the 2 groups of calves. This finding indicated that the growth rate for the BVDV test–positive calves was similar to that of the BVDV test–negative calves. It is generally believed that most PI calves die within the first few months after birth, but that notion was not upheld in the present outbreak because all 36 PI calves were clinically normal for > 1 year until the first calf succumbed to mucosal disease. Moreover, 11 of those 36 (31%) calves remained healthy, achieved finished market weight, and were sold for slaughter. The serum neutralizing anti-BVDV antibody titers for the BVDV test–positive calves are difficult to interpret. The consistently high anti-BVDV2 antibody titers of the 3 TI calves were likely the result of an active infection and continuous exposure to calves PI with a BVDV2a strain. The presence of anti-BVDV1 antibody titers in all 3 TI calves in the absence of vaccination against or natural exposure to BVDV1 was likely the result of a cross-reaction between the BVDV1 reference strain and the BVDV2 strain to which the calves were exposed.20 Only 9 of the 36 PI calves had antibodies against BVDV, and those titers were typically low and tended to decrease over the 6-month observation period. It is unlikely that the antibodies were indicative of an acquired immunity against the persistent strain of BVDV because immunotolerance of PI calves to the persistent strain is documented.9 It is worth noting, however, that PI calves are able to respond to BVDV strains other than the persistent strain, and BVDV is an RNA virus that can generate variants during replication.24,25 It is possible that the anti-BVDV antibodies present in the PI calves were the result of passive immunity. Anecdotally, maternal antibodies have been reported to persist in calves for 6 to 8 months after weaning. Admittedly, this would be atypical and has not yet been established under experimental settings; maternal antibody titers are typically very high during the neonatal period and then gradually decline over time. The decay of maternal antibodies could have accounted for the decline in antiBVDV antibody titers observed in the PI calves over the 6-month observation period. Unfortunately, we cannot confirm that the anti-BVDV antibody titers in the PI calves were the result of maternal antibodies because the titers were very low and were not determined for the calves prior to 9 months of age. It also possible that the kinetics of maternal antibody clearance in PI calves is not the same as those in non–PI calves, and this has not been evaluated. Seventeen of the 23 PI calves that died had lesions characteristic of mucosal disease. The other 6 calves died Scientific Reports
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BVDV strains are classified into 1 of 2 genotypes (BVDV1 or BVDV2), each of which is further subdivided into 2 biotypes (cytopathic and noncytopathic) on the basis of whether a given strain causes lytic changes in cultured cells.8,9 Infection of a pregnant cow with a noncytopathic strain of BVDV prior to 40 days of gestation can result in early embryonic death, whereas infection between 40 and 150 days of gestation can result in a fetus that is PI with BVDV, and infection during the latter stages of the second and third trimesters can cause abortion or congenital abnormalities in the fetus or less severe outcomes such as the birth of calves with poor vigor or no clinically apparent abnormalities.6,10 Cattle PI with BVDV (PI cattle) are the major reservoir for the virus,11,12 and current recommendations for BVDV control focus on the detection and removal of PI cattle.11–17 Persistently infected cattle that survive the first year of life often die from mucosal disease, which results when an animal becomes infected with a cytopathic strain of BVDV that is substantially similar to the noncytopathic strain with which it is PI (the cytopathic strain frequently emerges from a mutation of the noncytopathic strain).18,19 Information regarding the mortality rate of PI calves within 1 year after birth and the percentage of PI cattle that die as a result of mucosal disease or appear clinically normal is lacking. The present report provided additional information about the clinical outcomes associated with the apparent introduction of a single strain of BVDV2 into a herd of 3-year-old pregnant beef cows. The BVDV outbreak described in the present report was particularly notable because of the substantial impact that BVDV infection had on the 2004 calf crop. We assumed that the 8 suspected abortions and 8 neonatal deaths were related to BVDV infection, and 44 calves subsequently tested positive for BVDV at least once. Thus, 44% (60/136) of the 2004 calf crop for this herd was lost or otherwise affected by BVDV. The source of most BVDV outbreaks is generally traced back to the introduction of a PI animal into a BVDV-naïve herd. However, immunohistochemical staining of ear notch specimens for BVDV antigen yielded negative results for all 136 purchased cows; therefore, it is unlikely that there was a PI cow among the purchased cows, and the exact point source of BVDV entry into this herd is unknown. The present report highlighted the risk producers assume when they purchase pregnant cattle, a common practice in the beef and dairy industries. Currently, there is no affordable or safe method to determine whether the fetus a pregnant cow is carrying is PI with BVDV. The detection of maternal viremia is not a viable option for identification of dams carrying PI calves because viremia associated with BVDV infection has a short duration (generally < 10 days) and must occur during a particular time (40 to 150 days) of gestation to result in a PI calf.6 Screening pregnant cows for the presence of anti-BVDV antibody titers is also not a useful method for identification of dams carrying PI calves because currently available neutralization assays cannot differentiate between anti-BVDV antibodies that resulted from vaccination and those that resulted from natural infection, and cattle are commonly vaccinated against BVDV.20
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without any gross lesions, and BVDV infection would not have been suspected without knowledge that the calves were PI. Thus, it is recommended that BVDV diagnostic tests be routinely included in the necropsy workup for cattle that die suddenly, even those without gross lesions. Outbreaks of mucosal disease have been previously reported in 2 beef herds in Saskatchewan, Canada.21,22 In 1 report, 28 of 105 (27%) yearling cattle were PI with BVDV, and 20 (71%) of those cattle died from mucosal disease. Interestingly, 1 PI calf was born in that herd the year following the birth of the cattle that died from mucosal disease.21 Five PI calves were born the second year following the birth of the cattle that died from mucosal disease, and 3 of those 5 PI calves were born to cohorts of the PI cattle that died from mucosal disease.21 Hence, it is important to screen all cows and heifers for BVDV for at least 2 years following an outbreak to identify and remove PI cattle. Similar to the herd of the present report, most of the PI cattle in that study21 were > 1 year old when they died of mucosal disease. In the other report,22 71 of 560 (13%) calves were suspected to be PI with BVDV; 20 calves died before weaning and were likely PI, and the remaining 51 calves were confirmed to be PI with BVDV on the basis of virus isolation or necropsy results. Of the 51 confirmed PI calves, 47 died at < 1 year old, and 12 (26%) of those had no or only mild lesions associated with mucosal disease.22 The presence of PI cattle is a threat to the health and overall profit of any cattle operation.5,12,13 An increase in producer awareness of BVDV and the use of appropriate vaccination regimens, surveillance, and eradication efforts have resulted in a decrease in the incidence of PI cattle.10,12,13,16 However, until an affordable and practical test to screen fetuses for BVDV is developed, the risk of introducing BVDV into a herd should be considered whenever pregnant cows are purchased, and biosecurity practices (quarantine of purchased cows and the calves from those cows) should be implemented until appropriate testing can be performed.12,13 Additionally, all cattle that die suddenly with or without gross lesions should be tested for BVDV. The present report is a reminder that diligence in BVDV prevention is vital for beef operations because a BVDV outbreak can have devastating effects on productivity. Also, although it is often recommended that any animal with a positive result for BVDV antigen by immunohistochemical staining of an ear notch specimen be culled,2,26 1 positive result is insufficient to definitively identify an animal as PI with BVDV. For the herd of the present report, 3 of the 44 (6.8%) calves that tested positive for BVDV antigen by immunohistochemical staining of an ear notch specimen subsequently tested negative for BVDV on virus isolation and by antigen capture ELISA performed on buffy coat samples and were determined to be only TI with BVDV at the time of the positive test. This finding emphasizes the importance of performing follow-up testing of BVDV test– positive cattle to definitively determine whether they are truly PI with BVDV. Proper biosecurity and vaccination programs and the rapid identification and removal of PI cattle are strongly recommended to minimize BVDVassociated losses.
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