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Tank milk, an extra tool for detection and eradication of IBR in cattle farms

Jean-Baptiste Hanon and Brigitte CaijCODA-CERVADirection opérationnelle des maladies viralesUnité des maladies enzootiques et (ré)émergentes.

Introduction

Infectious bovine rhinotracheitis (IBR) is a respiratory disease of cattle caused by bovine herpesvirus 1 (BoHV-1). Disease outbreaks can result in severe production losses, abortion and mortality. After infection with BHV-1 the animals seroconvert and become lifelong carriers of the virus that remains latent in the peripheral sensory ganglia. After reactivation caused by stress, the virus can be re-excreted and contaminate healthy animals. That is why several European countries, including Belgium, committed themselves to implement an eradication plan consisting in the detection and gradual eradication of all seropositive animals in order to obtain entirely IBR free herds. For detection of IBR positive animals, ELISA tests can be used on serum samples as well as on individual milk samples and on tank milk samples, as authorized by European legislation (Commission Decision 2004/558/EC amended by Decision 2010/433/EU).

Most European countries that implemented an IBR control plan use milk as a matrix for IBR detection in dairy herds for both acquiring and preserving the IBR free status of a herd. This is particularly so in the neighboring countries of Belgium (Germany, France, the Netherlands). In Belgium, the Royal decree on infectious bovine rhi-notracheitis (RD of 22 November 2006 amended by the RD of 16 February 2011) provides the possibility for a dairy herd to preserve the IBR status (status I3 or I4) on the basis of 6 tank milk analyses a year (plus an annual serology on other bovine animals than dairy cows). However, no IBR ELISA kits that may be used on milk have as yet been submitted for certification by the CODA-CERVA, the national reference laboratory, and can therefore not be used for official control purposes. As a result, IBR detection in herds is now done by means of analyses on the serum obtained from blood samples taken from individual adult bovine animals.

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Serological IBR statuses and detection tests used

When a herd has been infected, vaccination is recommended to limit virus circulation and contamination of naïve animals. The vaccines used for that purpose are made from a virus strain deleted in the glycoprotein gE. Based on the difference in the gE glycoprotein between a wild virus strain and the vaccine strain, animals infected with the wild virus have anti gE antibodies (gE+) and can thus be differentiated from healthy naïve or vaccinated animals that do not have anti-gE antibodies (gE -). However, vaccinated animals possess antibodies against other glyco-proteins of BHV-1 like antibodies against the glycoprotein gB (gB+). On the contrary, healthy animals that have not been infected don’t have any IBR antibodies. Based on the differences in immunological responses of naïve, vaccinated or infected animals, ELISA tests based on the detection of antibodies against gE or gB can determine the status of an animal. Different ELISA tests are available and can be used to establish the IBR status in accord-ance with the overview table below:

Status of the Animal gE ELISA gB ELISA Indirect ELISA

IBR infected + + +

Not infected and IBR vaccinated (deleted vaccine) - + +

Not infected and not vaccinated - - -

Evaluation of the use of ELISA tests for IBR detection in milk in view of using such tests in Belgium

In 2011, CODA-CERVA performed a study which resulted in a preliminary evaluation of several ELISA tests available on the European market that could be used for IBR detection in milk. The subject of that study was to estimate the features of those tests on individual milk and on tank milk samples (sensitivity, specificity, detectability) and their compliance with the official tests performed on serum. Several partners made a contribution to this study in which were involved 92 cattle farmers from all over Belgium, the regional veterinary laboratories (ARSIA and DGZ), the milk inspection bodies (Comité du lait and MCC) and 5 ELISA kits manufacturers.

Seven ELISA kits were evaluated in this study (2 gE ELISA kits, 1 gB ELISA kit and 4 indirect ELISA kits). The conclu-sions of the study were translated into recommendations that will be useful for a subsequent official certification procedure of IBR ELISA kits for milk in view for their use within the context of the IBR control program in Belgium.

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Typical features of detection in tank milk

Tank milk consists of milk obtained from one or more milking sessions of all lactating cows of a dairy farm. Hence, the analysis of a tank milk sample for the detection of antibodies should, in principle, make it possible to detect the presence of infected adult animals in that herd, except for dry cows or cows from which the milk of which has not been collected in the tank (e.g. due to mammitis). It is therefore important to perform the milk tank analysis several times until all animals of the herd have been tested in the course of one year. The concentration of anti-bodies or immunoglobulins in milk is about 10 % of that in blood serum. Moreover, this concentration varies in the course of lactation: it is lower when milk production is at its highest level, due to a diluting effect. In addition, when IBR prevalence in a herd is low, the milk of infected animals is diluted with that of the healthy cows. It is therefore important to have sufficiently sensitive ELISA tests that allow the detection of low amounts of antibod-ies.When compared to individual serological tests, IBR detection on tank milk has the following advantages:- sampling is easy and may be done through the milk inspection services- costs are low due to the smaller number of samples to be tested - early detection of a possible contamination of an IBR free herd due to repetitive testing at regular times over the year (as opposed to the annual serological screening)

The most important disadvantage of the method is the poor stability of fresh milk samples. Appropriate logistic facilities are required for the prompt transport of the samples to the lab. Adding a preservative to the sample may possibly help to increase the sample’s lifetime. On the other hand, samples can be preserved by freezing without any problem once the milk has been skimmed.

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Conclusions

Thanks to the study performed by CODA-CERVA the different types of ELISA kits for the detection of IBR from milk samples could be characterized. The overall characteristics of the 3 categories of ELISA tests may be listed as follows:

gE ELISA gB ELISA Indirect ELISA

Individual milk Sensitivity +/- ++ +++

Specificity + to +++ +++ ++

Tank milk Sensitivity - - +++

Detectability -(>10 to 15%)

+/-(>10%)

+++(<2%)

Specificity + +++ +++

Legend: +++ excellent / ++ very good / + good / +/- mediocre / - bad

It appears that indirect ELISA kits are very efficient for detecting IBR in tank milk in non vaccinated herds and should therefore be recommended in view of the preservation of I4 IBR free statuses. The sensitivity and the de-tectability of indirect ELISA in tank milk are significantly superior to those obtained with annual serological detec-tion on a random sample of animals, as required by the Royal decree on IBR: the size of the sample for serological testing is appropriate for detecting a 15 % IBR prevalence whereas with indirect ELISA on tank milk it is possible to detect an infection level of less than 2% in cows. In addition, the repetitive testing of tank milk (every 2 months) allows earlier detection of possible re-infections of a herd when compared to a serological test that is performed only once a year.

For I3 status IBR free animals (vaccination authorized), indirect ELISA is not an appropriate method since it does not allow to distinguish vaccinated animals from infected animals. The gE ELISA kits have low sensitivity in tank milk and do not allow the detection of infected animals within a herd with low prevalence (detectability >10 to 15%). This lack of sensitivity is partly compensated for by the fact that tests on tank milk are repeated every two months. It can indeed be expected that when an IBR free herd is infected, the rate of infected cows will soon exceed 15%. Moreover, there are certain milk treating methods that make it possible to increase the immunoglob-ulin concentration of tank milk samples and improve the sensitivity and the detectability of gE ELISA kits. Thanks to these methods, higher sensitivity might be achieved than with the annual serological detection on a random sample of animals, i.e. the procedure that is currently applied in Belgium.

Under these circumstances, it is recommended to adopt as soon as possible a procedure for the official certifica-tion of indirect ELISA kits for the detection of IBR in tank milk, within the context of IBR control in Belgium. In the meantime there have already been contacts with neighboring countries that have included the use of controlled IBR ELISA kits for milk in their official control program and proposals for mutual recognition of controlled batches from other countries were formulated.

Contact: brigitte.cay@coda-cerva.be