BVD: effective testing for control or eradication

Andrew MacPherson, Medical Affairs Veterinarian at IDEXX Laboratories, reports on research into using earnotch samples to test calves for bovine viral diarrhoea.

INTRODUCTION

Bovine viral diarrhoea (BVD) is a disease affecting cattle that is caused by the BVD virus. The virus is endemic in most cattlefarming countries and causes significant economic losses worldwide (Yarnall and Thrusfield, 2017).

BVD manifests on dairy and beef farms. As it is immunosuppressive it can present in many ways, including as ill-thrift, reproductive losses and/or decreased milk production, with a predisposition to concurrent infectious disease. These variations in manifestation often impede management efforts and rapid diagnosis. In addition, and despite its name, the absence of diarrhoea does not rule out the presence of the BVD virus on farm, as acute infection can be subclinical and cause mild or undetected disease.

When a cow first becomes infected with the virus, no matter what their age, an acute transient infection (TI) occurs. These animals do not have a significant role in the ongoing transmission of BVD on farm, as they excrete only small amounts of the virus in a period of a few days (Evans et al., 2019).

When a pregnant cow who has never been infected with BVD (and therefore hasn’t developed antibodies against the virus) is infected during gestation, the fetus will also become infected. If the dam is infected between day 30 and 125 of gestation, the fetus will recognise the virus as part of itself, and be born as a ‘BVD carrier’ or a persistently infected (PI) calf.

Unlike TI cattle, PI cattle never develop immunity to the virus. They go on to excrete enormous amounts of BVD virus during their lifetimes, which makes them the most important sources of new infections for naïve animals on dairy and beef farms. The early detection and immediate removal of PI animals is therefore an essential step in any BVD control programme.

THE BVD ANTIGEN-ELISA EARNOTCH TRIAL

Maternal antibodies against BVD have been reported to have a half-life of 21 days and to persist for up to 30 weeks after birth. In New Zealand this has been thought to result in a ‘diagnostic gap’, where PI calves test negative via serum soon after birth. As a result, testing using antigen-enzyme-linked immunosorbent assay (ELISA) has not been recommended before calves are 35 days old. However, other studies have found that the use of tissue samples (eg, by ear notching) reduces the amount of maternal antibody interference (Kuhne et al., 2005; Hill et al., 2007).

In response to these studies, Cognosco conducted a trial in spring 2019 to assess the sensitivity and specificity of calves’ earnotch samples from day 38 postpartum back to as close to their birth as possible.

TRIAL METHODS

The study enrolled 1,030 calves from a total 11 dairy herds where the BVD virus had been detected in bulk tank milk and/ or where individual animals had been confirmed as BVD-virus antigen-positive before the 2019 spring calving season.

All herds had evidence of multiple years of exposure to the virus associated with high or very high antibody titres in bulk tank milk.

Ear-notch samples were collected from all replacement calves at approximately 3, 10, 24 and 38 days of age. The day 38 ear-notch samples were submitted for antigen-ELISA (BVDV Ag/Serum Plus ELISA, IDEXX Laboratories) and realtime polymerase chain reaction (PCR) testing (IDEXX RealPCR BVDV, IDEXX Laboratories). If they were positive, all previous samples (ie, days 3, 10 and 24) were also tested using antigen-ELISA and real-time PCR.

Blood samples were collected from calves who tested positive by antigenELISA or real-time PCR at day 38, at an average of 102 (standard deviation 13, range 76–127) days of age (day 100), to determine the antibodies to the BVD virus and the presence, or not, of BVD virus antigen. The results were reported as test positive or negative based on the manufacturers’ cut-off points for real-time PCR (cycle threshold) and antigen-ELISA (sample to negative control values).

Calves were defined as PI if they were positive to the antigen-ELISA or real-time PCR test at day 38 and positive to the antigen-ELISA in serum at day 100. Calves were defined as TI if they were positive to the antigen-ELISA at Day 38 and negative to the antigen-ELISA in serum at day 100. Animals who tested negative at day 38 using both antigen-ELISA and real-time PCR assay were defined as not infected.

Calves with serum immunoglobulin concentrations of <10g/L (based on samples from 10–15 calves in the herd) were defined as having not received adequate amounts of antibodies via colostrum from the dams (failure of passive transfer [FPT]).

RESULTS

Of the calves with test results on day 38, 26 (2.5%) were positive for the BVD virus by real-time PCR. However, only five of the 26 were defined as PI.

For the PI calves, all ear-notch samples at all time points tested positive on both antigen-ELISA and real-time PCR. One calf was subject to euthanasia on farm before the final sampling, having shown symptoms consistent with BVD. The other four PI calves tested positive for serum antigen-ELISA on day 100, and three of the four also tested positive for antibody by antibody-ELISA at this time (likely due to the persistence of maternal antibodies in circulation).

Results showed that PI calves tested positive with the antigen-ELISA and the real-time PCR at all time points, while TI calves tested negative at all time points with the antigen-ELISA in contrast to the real-time PCR, which did not differentiate between PI and TI calves.

FPT was found in 30.3% of the calves, but the within-herd prevalence varied from 13% to 53%. This figure correlates to the estimated percentage of FPT in other trials in New Zealand.

DISCUSSION

Contrary to previous recommendations about the age of testing, this trial concluded that using ear-notch samples on calves aged up to and including 38 days provided no evidence that the age of the calves at testing affected the test results for either the antigen-ELISA or the realtime PCR assay.

The diagnostic gap may occur in calves of this age when serum, rather than ear-notch samples, is used or where an antigen-ELISA targets NS3 rather than Erns antigens (Fux and Wolf, 2012).

This study confirms a method for identifying and removing PI calves while avoiding the removal or unnecessary retesting of TI calves.

Thirty percent of calves sampled had FPT, conversely suggesting that 70% of calves did in fact receive effective amounts of colostrum. This indicates that the effect of maternal antibodies will likely vary both within and between herds.

However, for the small number of PI calves detected in the current study, there was no evidence of a consistent change in the negative control or cycle threshold values with increasing calf age at sampling. The five PI calves were first sampled at two, two, three and seven days old (one calf did not have a calving date but was less than a week old when sampled).

CONCLUSION

The control or eradication of BVD from New Zealand bovine herds can only be made possible through a combination of effectively testing all animals and using appropriate diagnostic tests for a specific, predetermined objective.

At the farm level BVD can be controlled by testing for and eliminating PI animals combined with imposing strict biosecurity measures to prevent reintroduction. From an epidemiological point of view, slaughtering TI animals has little benefit. Therefore, it is important to consider the test method carefully.

When the goal of BVD testing is to identify PI calves rather than TI calves, the antigen-ELISA (using ear-notch tissue) has been shown as superior as it achieved 100% sensitivity and specificity for the detection of PI calves. The PCR test could not differentiate between PI and TI; doing so would require an antigen test 28 days later.

REFERENCES: Evans CA, Pinior B, Larska M, Graham D, Schweizer M, Guidarini C, Decaro N, Ridpath

J, Gates MC. Global knowledge gaps in the prevention and control of bovine viral diarrhoea (BVD) virus. Transboundary and Emerging Diseases 66, 640–52, doi:10.1111/tbed.13068, 2019

Fux R, Wolf G. Transient elimination of circulating bovine viral diarrhoea virus by colostral antibodies in persistently infected calves: A pitfall for BVDV-eradication programs? Veterinary Microbiology 161, 13–9, 2012

Hill FI, Reichel MP, McCoy RJ, Tisdall DJ.

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle. New Zealand Veterinary Journal 55, 45–8, 2007

Kuhne S, Schroeder C, Holmquist G, Wolf

G, Horner S, Brem G, Ballagi A. Detection of bovine viral diarrhoea virus infected cattle: Testing tissue samples derived from ear tagging using an Erns capture ELISA. Journal of Veterinary Medicine, Series B 52, 272–7, 2005

Yarnall MJ, Thrusfield MV. Engaging veterinarians and farmers in eradicating bovine viral diarrhoea: A systematic review of economic impact. Veterinary Record 181(13), 347, 2017

Infectious canine hepatitis –

it’s still out there

Michael Hardcastle, Veterinary Anatomic Pathologist at Gribbles Veterinary, Auckland, discusses the prevalence and diagnosis of ICH, and its persistence despite the availability of vaccination.

FIGURE 1:Intestinal serosal haemorrhage and a peritoneal effusion.

DOGS AND CATS in New Zealand are fortunate to be relatively free of contagious diseases, with many viral diseases preventable or minimised by vaccination. Disease due to canine parvovirus-2 is probably the most significant of these. However, our laboratories occasionally diagnose other viral diseases, and it is worth highlighting that these organisms are still in circulation despite the availability of vaccination.

A one-month-old, cross-bred puppy from north Auckland presented collapsed, with cold extremities and a temperature of 35.6°C. They had reportedly been clinically normal the previous evening. An in-house haemogram showed anaemia with a haematocrit of 21.1% (reference interval 37.3–61.7) and possible thrombocytopenia (26K/µL, reference interval 148–484 with a caution to check the blood film). Biochemistry showed increased alanine transaminase (357U/L, reference interval 8–75). The puppy died despite supportive care. At postmortem examination, there were areas of intestinal serosal haemorrhage and a serous peritoneal effusion (Figure 1), a few strands of fibrin adherent to the liver and pancreatic oedema.

Two other puppies from the same litter died suddenly. In-house laboratory testing of the three surviving litter mates showed variable haematocrits (28.6%, 30.8% and 22.8%) but no thrombocytopenia. Their mother was a rescue dog with neonatal puppies when adopted; she had no known vaccination history.

A range of fixed tissues submitted from the postmortem of the first puppy was processed for histopathology. It showed a range of lesions; the most significant were prominent intranuclear inclusion bodies in most hepatocytes and some endothelial cells, often filling the nucleus (Figure 2, white arrow), along with multifocal necrosis of hepatocytes (Figure 2, blue arrow). Intranuclear inclusion bodies were also seen in macrophages or endothelial cells of the bone marrow, spleen, lymph node, lung, kidney, heart and small intestine, where they were associated with mesenteric vasculitis and haemorrhage. Other findings included lymphoid necrosis/apoptosis in the spleen and lymph nodes consistent with a viral infection.

The first puppy was diagnosed as a case of infectious canine hepatitis (ICH), caused by canine adenovirus-1 (CAV-1). In a puppy with haemorrhagic lesions, canine herpesvirus-1 could also have been considered; however, it tends to affect younger puppies than this one, mainly presents with renal haemorrhages, has fewer and smaller inclusions and does not target hepatocytes.

Clinically, diagnostic options for ICH in New Zealand are limited as serology, polymerase chain reaction or other tests are not readily available through commercial diagnostic laboratories or the Ministry for Primary Industries’ Animal Health Laboratory, according to Laboratory Technical Officer for Diagnostic and Surveillance Services Danni Thornton. Therefore the diagnosis is typically based on a consistent history, clinical signs, antemortem clinical

FIGURE 2:Histopathology showing prominent intranuclear inclusion bodies in most hepatocytes and in some some endothelial cells (white arrow); multifocal necrosis of hepatocytes (blue arrow).

pathology and postmortem examination with histopathology.

ICH is spread by direct or indirect contact with infected urine, faeces, saliva and respiratory secretions (Hornsey et al., 2019). It causes viraemia after oronasal exposure and initial localisation in the tonsils. It mainly affects young dogs and tends to be seen sporadically or as small outbreaks in kennels. The severity of disease seems to be dependent on neutralising antibody titre (Greene, 2012). It can present as sudden death in peracute infections. In less acute diseases, clinical signs may range from vomiting, melaena, fever, tachypnoea, tachycardia, abdominal pain, hepatomegaly, non-specific nervous signs, mucosal petechiae, pallor and mild icterus to mild pharyngitis, tonsillitis, coughing (pneumonia), cervical lymphadenomegaly and dependent oedema, or inapparent infection. Convalescing dogs may

develop corneal oedema (‘blue eye’), considered a hypersensitivity reaction to immune-complex deposition. In animals who survive, it seems that the liver can regenerate rapidly (Cullen and Stalker, 2016), although some sources suggest that partially immune dogs may develop chronic hepatitis (Greene, 2012).

Clinicopathologic findings include leukopaenia with lymphopaenia and neutropaenia, progressing to neutrophilia and lymphocytosis during recovery. Thrombocytopenia is common and clotting times are variably prolonged. Liver enzymes (alanine transaminase, alkaline phosphatase and aspartate transaminase) increase proportionate to the degree of hepatic necrosis, but hyperbilirubinaemia is uncommon. Proteinuria and hypoglycaemia may be identified. Hepatocellular intranuclear inclusions might be found antemortem on cytology or in liver biopsies (Greene, 2012).

At postmortem there may be lymph node oedema or haemorrhage and blotchy/‘paint brush’ haemorrhages on the gastrointestinal tract or serosa, or haemorrhage in other organs such as the kidney, lung, bone and brain. There may be slight icterus. The liver may be large and friable; there may be ascites with fibrin strands; and the gall bladder may also be enlarged and oedematous. Haemorrhages are considered to be largely due to a consumptive coagulopathy, since endothelial damage initiates the clotting cascade (Cullen and Stalker, 2016).

Treatment options are supportive and include fluid therapy (crystalloids, plasma or whole blood), glucose infusions and strategies to reduce ammonia production (eg, enemas) (Greene, 2012).

ICH is seen sporadically in New Zealand, with cases reported anecdotally every few years and occasionally mentioned in Surveillance magazine (Anonymous, 2010). It is interesting that it persists in New Zealand despite our lack of wild carnivores (overseas, reservoirs for ICH and other infectious diseases such as canine distemper). According to Massey University Associate Professor Nick Cave, possible reservoir species in New Zealand include mustelids (Nick Cave, personal communication, July 2020). However, ICH frequently causes subclinical infections, is shed for months in urine and is robust in the environment, resisting disinfectants (Greene, 2012). This makes its persistence in the New Zealand canine population, despite the availability of vaccination, probably not surprising – especially since the prevalence of vaccination is unknown but certainly less than 100%.

REFERENCES:

Anonymous. Surveillance 37(4), 19–29, 2010

Cullen JM, Stalker MJ. Liver and biliary system. In: Maxie GM (ed.) Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. 6th Edtn. Pp. 310–2. Elsevier, St. Louis, Missouri, USA, 2016

Greene CE. Infectious canine hepatitis and canine acidophil cell hepatitis. In: Greene CE (ed). Infectious Diseases of the Dog and Cat. 4th Edtn. Pp. 42–8. Elsevier, St. Louis, Missouri, USA, 2012

Hornsey SJ, Philibert H, Godson DL, Snead ECR.

Canine adenovirus type 1 causing neurological signs in a 5-week-old puppy. BMC Veterinary Research 15, 418, 2019 https://doi.org/10.1186/s12917-019-2173-5

ACKNOWLEDGEMENTS The author wishes to thank Mark Anderson of Vets North, Helensville, for providing the clinical information and gross images relating to this case.