COMPARISON OF AGAR GEL IMMUNODIFFUSION TEST , ENZYME-LINKED IMMUNOSORBENT ASSAY AND PCR IN DIAGNOSTICS OF ENZOOTIC BOVINE LEUKOSIS

Bovine leukaemia virus (BLV) is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzymelinked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR) method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gp51) served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELISA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

Bovine leukaemia is a disease of cattle characterised by the development of malignant lymphomas (lymphosarcomas).In Europe and also in South Africa, the disease is better known as bovine leukosis.It is further divided into enzootic and sporadic bovine leukosis, which contributes to some confusion when people are not familiar with the different forms of the disease Š14¹.Enzootic bovine leukosis (EBL) is a disease of adult cattle caused by the retrovirus, the bovine leukaemia virus (BLV).Cattle may be infected at any age, including the embryonic stage Š8¹.Most infections are subclinical, but a proportion of cattle over 3 years of age develop persistent lymphocytosis, and a smaller proportion develop lymphosarcomas in various internal organs Š19¹.Natural infection has also been recorded in buffalos, sheep and capybaras.BLV-infected animals usually demonstrate a strong humoral response to BLV, which can be exploited for diagnostics by serological techniques Š1, 6¹.Eradication and control of the disease is based on early diagnostics and segregation of the carriers.The sensitivity of the testing strategy is a critical consideration, as false-negative test results may unnecessarily prolong the eradication efforts Š21¹.For a number of years, the AGID test has been the prescribed test for international trade Š22¹.In more recent years, the ELISA has replaced the AGID in eradication programmes Š7¹.Nowadays, sequence data of different BLV proviruses are available, enabling the development of PCR that is increasingly used for the diagnostics of the EBL and has advantages over serological tests Š2, 11, 12¹.The aim of this study was to evaluate the practical application of PCR in parallel with routine by used serological methods, for detection of BLV, considering conditions with very low incidence of BLV infection.

Samples / Uzorci
Serum samples were collected from dairy and beef herds from Slovenia.Only positive sera, as revealed with ELISA screening, were used in the further study.In BLV positive animals, samples were collected once more for sera and for DNA isolation.

AGID /AGID
AGID was performed in plastic Petri dishes (Ø 90 mm) filled with 15 ml of prewarmed liquid agar (Agar Mixture for Bovine Leucosis Immunodiffusion; Bommeli, Switzerland) to get a layer 2.5 mm thick.Cooled agar was punched to get a ring of wells.The centre well was filled with 25 ml of antigen (Bovine Leucosis Antigen for Immunodiffusion; Bommeli, Switzerland), and brim wells were filled with 50 ml of positive control serum (Bovine Leucosis Control Serum Positive, for

Materials and methods / Materijal i metode rada
Immunodiffusion; Bommeli, Switzerland) or sample serum.We evaluated results after 72 hours of incubation at room temperature in a humid atmosphere.

ELISA / ELISA test
The commercially available ELISA kits were used according to the manufacturer's instructions.The microtiter plates were coated with antigen p24 in screening tests (LEUCOTEST; Bommeli, Switzerland) or with gP51 protein in confirmatory test (ELISA Bovine Leukosis Serum Verification; Pourquier, France).Positive and negative controls provided with the kits were run with each assay.After all the prescribed incubation and rinse steps, the plates were measured on a spectrophotometer (TECAN, Austria) and the results were interpreted as recommended by the manufacturer.

DNA isolation and PCR / Izolovanje DNK i PCR
Viral DNA was isolated from whole blood using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and/or from serum using QIAamp UltraSens Virus Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.The env-nested PCR was performed with the external primers ENV1 (5'-TCT GTG CCA AGT CTC CCA GAT A-3') and ENV4 (5'-AAC AAC AAC CTC TGG GAA GGG T-3') and internal primers ENV2 (5'-CCC ACA AGG GCG GCG CCG GTT T-3') and ENV3 (5'-GCG AGG CCG GGT CCA GAG CTG G-3') Š2¹.The reaction mix for first amplification was performed with a 50 ml volume containing 20 ml template DNA, 0.5 ml of each primer, 4 ml dNTPs (10 mM each), 5 ml 10 x PCR buffer, 3 ml MgCl 2 (1.5 mM) and 0.25 ml Platinum Polymerase (Invitrogen, 5 U/ml).3 ml of PCR product of the first amplification was used as a template for the second amplification.The amplification protocols previously described by Beier et al were used.PCR products were electrophoresed in a 2% agarose gel and visualised with ethidium bromide staining.
To assess the ability of the AGID, confirmatory ELISA and PCR for the correct discrimination of positive and negative samples, the 30 positive sera as revealed by the screening ELISA were compared as well as 10 negative sera.The results demonstrate that all AGID-positive sera showed reactivity in confirmatory ELISA.Moreover, 9 of the AGID-negative sera showed reactivity in confirmatory ELISA.On the other hand, comparison of confirmatory ELISA and PCR resulted in 100% matching, with the exception of 2 samples where AGID and PCR were negative (Table 1).Other PCR negative samples were negative also in serological tests.In view of the increasing economic impact of BLV infection on the cattle industry, the availability of a highly sensitive and specific assay for the identification of BLV-infected cattle is of critical importance.Ideally, the assay should be practical, inexpensive, and able to be adapted for large-scale use Š16¹.According to our results, AGID, ELISA and PCR methods are quite adequate for routine diagnostics.The AGID test has been the prescribed test for international trade for a number of years due to its high level of sensitivity and specificity.In recent years, many authors reported that the prevalence of BLV infection in the examined herds might have been underestimated, because the prevalence of the disease was based on data obtained by AGID, which unlike ELISA, is significantly less sensitive Š23¹.From this point of view, our results obtained from AGID showed as lower sensitivity in comparison with confirmatory ELISA, and the reading of the AGID test is more subjective Š5, 9¹.Nowadays, the detection of antibodies by ELISA has been widely used in veterinary diagnostic laboratories for serologic diagnosis of BLV infection.In general, ELISA is highly practical and relatively inexpensive, so it can be easily implemented for large-scale testing, which is usually needed in serological surveys and in control-and-eradication programmes Š13¹.Screening ELISA is applicable for the first step in making a diagnosis due to its good sensitivity and reduced specificity; it gives, as expected, more positive results.Using confirmatory ELISA, which is more specific, we retested these positive samples and reduced the level of positive results.Serologically confirmed positive results should be confirmed also with direct virus detection using molecular methods Š17, 20¹.The detection of viral sequences by PCR provides a precise and suitable method for the direct diagnosis of BLV infection, particularly as a confirmation test after serological testing in case of serologically doubtful results and when maternal antibodies still persist in the serum of the calves Š18¹.The reliability of the PCR method was demonstrated as well in our study where two different serological methods in comparison gave the same result as PCR.In view of this, when choosing a diagnostic test for BLV, it is essential to analyse the aim pursued.In high seroprevalence herds, high specific tests such as AGID and ELISA should be used, to ensure that animals considered as positive are truly positive, even at the risk of obtaining false-negative results Š10, 15¹.When running a large number of samples, direct virological methods would be better, but the price and the simplicity of serological methods make them more advisable Š10¹.On the other hand, in lowseroprevalence herds it is advisable to choose methods with high sensitivity, such as PCR, to ensure that all animals considered as negative are truly negative, even to asume the risk of encountering false-positive results Š2¹.This reference is derived from the fact that PCR methods are able to detect animals, which might test seronegative Š4¹.In this respect, serological tests and tests for direct viral detection must complement each other.

Table 1 .
Distribution of AGID values, confirmatory ELISA values and PCR within 30 positive sera and 10 negative sera tested by screening ELISA /