EVALUATION OF AGREEMENT OF THE ELISA AND AGID TESTS IN THE DIAGNOSTICS OF CAPRINE ARTHRITIS-ENCEPHALITIS

Jarosław Kaba, Martin Ganter, Krzysztof Rypuła, Tadeusz Frymus

ABSTRACT

Caprine arthritis-encephalitis infection manifests itself as chronic inflammation of one or two carpal joints, neurological symptoms, shortened lactation period and decreased milk production. To the eradication of CAE infection, OIE recommended effective and recognized diagnostic tests like AGID and ELISA, which are based on detecting antibodies against glycoprotein gp135 or protein p28 of CAEV. Our study aimed at evaluating the agreement of these tests in order to diagnose CAE. In our investigations we used serum samples from 76 herds all over Poland. Our results confirm a high agreement of the AGID and ELISA tests in identifying CAE infection and show a high correlation between clinical symptoms and a positive result of diagnostic examination. Therefore, both these tests can be applied to projects of eradication of CAE infection in goat herds.

Key words: CAEV, AGID test, ELISA test, Kappa value..

INTRODUCTION

Clinical symptoms of caprine arthritis-encephalitis (CAE) were observed as early as in the 1960s [25]. The disease was first diagnosed in the USA in the 1970s [23]. The etiological factor - caprine arthritis-encephalitis virus (CAEV) belonging to the genus Lentivirus, the family Retroviridae, was isolated in 1980 [4, 33]. CAEV infection is the reason for a considerable loss of milk and meat herds of goats [6]. In adult animals, most frequently at the age of 2 years, the infection manifests itself as chronic inflammation of one or two carpal joints. Neurological symptoms are observed sporadically, usually in young kids aged 2-4 months [23]. In females, the infection results in worse milk quality, a shortened lactation period and decreased milk production - by 10-15 per cent [8, 14, 17, 19, 27]. Moreover, the infection affects also reproduction, and undermines the health of kids and curtails their growth [8, 31]. During the first lactation one can observe changes in the udder. I t becomes swollen and hard, milk production falls and the number of somatic cells increases [19]. Although the infection has a subclinical course, and the symptoms are manifest in only 20-30 per cent of the infected animals, yet CAE makes the farmers cull by 5-10 per cent more goats [17]. The above symptoms can occasionally be accompanied by chronic interstitial pneumonia [7].

At present CAE occurs worldwide. In Poland screening studies confirmed the appearance of specific CAEV antibodies in 26 per cent of herds. The infection in the herds was mostly due to the purchase of animals abroad [13]. CAE, owing to its high economical importance, has been researched in some countries and projects for its prophylaxis have already been introduced. They involve the cure of infected herds and their continuous screening as well [2, 20]. The lack of vaccine protecting the animals against this infection makes the issue of curing the herds even more urgent. In order to diagnose CAE in herds serological tests are made. The market offers effective and recognized diagnostic tests like AGID and ELISA recommended by OIE, which are based on detecting antibodies against glycoprotein gp135 or protein p28 of CAEV [1, 21]. Close antigen relationship between CAEV and maedi-visna virus as well as lowering of the tests’ manufacturing costs encouraged the application of the same antigens in d iagnosing both these diseases [24].

MATERIAL AND METHODS

Our study aimed at evaluating the agreement of the ELISA and immunodiffusion tests in agarose gel in order to diagnose CAE. Our material was serum collected from vena jugularis externa in goats at various ages. The total number of serum samples was 1074, collected from 76 herds all over Poland.

In order to evaluate the agreement of the ELISA and AGID tests, we used the collection of serum samples which included positive samples in both tests, negative samples in both tests and positive samples in one test and negative samples in the other, taken from both infected and non-infected herds. The owners of the herds from which the samples were taken, were also interviewed in details on epizootiology. Particular attention was given to the symptoms which might accompany or follow out of CAE virus infection (slimming and poor animal condition, udder diseases, laming, neurological symptoms). In the study we used ELISA Chekit CAEV/MVV (Dr. Bommeli AG, Bern, Switzerland). The test was performed in line with the manufacturer’s instructions. Optical density of a sample was determined with the ICN Flow Titertek Multiscan Plus Mk 11 reader (Labsystems, Espoo, Finland), the wave length being 405 nm. The final result of the examination was calculated with the formula in the test instruction:
a = [(OD _sample - OD_neg)/(OD _pos - OD _neg)] x 100% where:

a - result of the examined sample
OD_sample - optical density of the given sample
OD _neg - optical density of the negative control serum
OD _pos - optical density of the positive control serum

The obtained percentage value of the examined sample was estimated as follows: up to 30 per cent - negative result, 30-40 per cent - dubious result, over 40 per cent - positive result.

The immunodiffusion test in agarose gel Capriclear^Ž (Central Veterinary Laboratory, Weybridge, Great Britain) was performed according to the manufacturer’s instructions with our own modification. The examined serum was placed into outer wells twice at 24-hour interval. The control positive serum was placed once into the wells at 6 and 12 h, and the central well was with the antigen. The result was read after 48 hours and it was considered positive when distinct precipitation lines with antigen gp135 or p28 occurred. The evaluation of the test agreement was performed calculating the Kappa value using the programme Win Episcope 2.0 (free sherware) [32]. Only the results which were positive and negative in both tests were considered for the evaluation and calculation of the Kappa value:

Kappa = 2x (a x d - b x c) / [(a + c) x (c + d) + (a + b) x (b + d)]. )]. The obtained results are given below [32].

RESULTS AND DISCUSSION

The screening examinations were carried out with the ELISA test and we received 187 positive results coming from animals from 25 farms. Among these, 161 samples proved positive also in the AGID test. In 24 cases no positive result was confirmed. Two positive sera in the ELISA examination were dubious in the AGID examination and consequently they were excluded from calculating the Kappa value. Therefore the Kappa value was calculated from 185 positive samples. Among 155 negative samples in the ELISA test, 149 cases turned out to be negative also in the AGID test, and six samples were positive (Tab. 1). To evaluate the agreement of the ELISA and AGID tests, negative sera collected from 3 randomly selected negative herds were used (A, AY, BD) - total 95 samples, and from 5 randomly selected positive herds (H, AA, AB, AF, AZ) - 60 negative samples, which made up the collection of sera used for the evaluation of the agreement of both tests. The Kappa value of test agreement calculated on this collection was 0.82 (Tab. 2). High test agreement was confirmed by the results from other authors [11]. The same tests were used to diagnose maedi-visna disease in sheep and it occurred that more positive results were obtained with the ELISA test than with the other one, which testifies to a higher sensitivity of this method rather than of the AGID test [10, 16, 29]. It is usually due to the lower specificity of a test [32]. These relationships can be vital while interpreting serological results in single animals.

In this study we adopted two most frequent techniques for diagnosing CAE [10, 29]. In the herds diagnosed with the ELISA test, two of them were found positive (herds L and N), while their diagnosing with the AGID test proved they were negative (Tab. 1). In these herds examined with the ELISA test, we received one positive result for each of them, although the animals did not manifest any clinical symptoms which might suggest CAE infection. The remaining results obtained in the 20 herds diagnosed with the ELISA test are in line with the results obtained with the AGID test (Tab. 1). In the herds B, C, D, Z, AA, AB, AW, AZ, BE, BH, BL, BO, BW the owners observed arthritis in their animals which is a frequent symptom of CAE infection [31]. In the remaining seven herds, in which the owners did not observe clinical symptoms, the percentage of serologically positive animals was below 40 per cent. Poor condition of the animals in herds B, C, AA, AB, AF, A W, BH, BJ, BW could also be connected with CAE infection. The reports on udder diseases do not univocally explain whether the symptoms observed in herds B, D, H, AA, AB, AW, AZ, BH, BI, BL, BO, BW, BX could be connected with CAEV infection. Neurological symptoms which occurred in three herds (B, Z, BO), should not be directly associated with CAEV, either. These symptoms are observed mostly in young kids, and in the interview it was impossible to estimate the kids’ age. [23]. We also lack information on the character of the observed symptoms, because neurological symptoms may often accompany other diseases [31].

The data in the literature show that serological tests applied to diagnosing CAE are of various sensitivity and specificity. The immunodiffusion test in the agarose gel with the antigen gp 135 is more sensitive than the test with the antigen p28 [1]. Similarly, the sensitivity of the test with the CAE antigen is higher than that with maedi-visna virus in sheep, eg. 91.0 and 56.0 per cent, respectively [15]. The AGID test is usually less sensitive than the ELISA, therefore it is recommended for screening of goat herds [9, 10, 29]. In the projects for CAE eradication, the ELISA test with the CAEV antigen or with the maedi-visna virus antigen is more useful [18, 29, 30]. However, the test with the CAEV antigen is highly sensitive (100.0%) and specific (95.6%) [28]. The application of the recombined CAE virus as an antigen reveals a higher specificity, but simultaneously a lower test sensitivity [3]. Nevertheless, the ELISA test based on protein antigen of the CAEV recombinant and glycoprotein gp 46 in sheep manifested a very high sensitivity and specificity, i.e. 99.4 per cent and 99.3 per cent, respectively.

CONCLUSIONS

In the examinations evaluating the agreement of the ELISA and AGID tests, by means of the immunoperoxidase test and Western blot technique, the Kappa value was 0.71 to 0.73 and 0.72 [11]. The examination detecting protein p28 by means of the Western blot technique serves usually as a reference test [6, 11, 26]. As a test complementary to serological examinations the PCR technique is used, which reveals the virus or its structures in the tissues and shows latent infection as well. [10, 12]. However, the most certain way is to isolate the virus on the culture of cells of the synovial membrane of the articular capsule of healthy goats, which can be conducted by taking synovial membranes of articular capsules, lymph nodes and monocytes, milk and peripheral blood [5, 10].

Our results confirm a high agreement of the AGID and ELISA tests in identifying CAE infection and show a high correlation between clinical symptoms and a positive result of diagnostic examination. Therefore, both these tests can be applied to projects of eradication of CAE infection in goat herds.

REFERENCES

1. Adams D.S., Gorham J.R., 1986. The gp135 of caprine arthritis encephalitis virus affords greater sensitivity than the p28 in immunodiffusion serology. Res. Vet. Sci. 40, 157-160.

2. Braddock A., 1997. Caprine arthritis encephalitis accreditation scheme. Goat Vet. Soc. J. 17, 33.

3. Celer V., Zanoni R.G., Peterhans E., 1993. Porovnání různých antigenů pro diagnostiku CAEV testem ELISA. [Comparison of Different Antigens for CAEV Diagnosis by Elisa].Vet. Med. - Czech. 38: 237-244.

4. Crawford T. B., Adams D.S., Cheevers W.P., Cork L.C., 1980. Chronic arthritis in goats caused by a retrovirus. Science, 207, 997-999.

5. DaSilvaTeixeira M.F., Lambert V., Mselli-Lakhal L., Chettab A., Chebloune Y., Mornex J.F., 1997. Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses. Am. J. Vet. Res. 58, 579-584.

6. Ding E.Y., Xiang W.H., 1997. Immune responses in goats to caprine arthritis-encephalitis virus. Viral Immunol. 10, 111-115.

7. Ellis T.M., Robinson W.F., Wilcox G.E., 1988. The pathology and etiology of lung lesions in goats infected with caprine arthritis-encephalitis virus. Aust. Vet. J. 65, 69-73.

8. Greenwood P.L., North R.N., Kirkland P.D., 1995. Prevalence, spread and control of caprine arthritis-encephalitis virus in dairy goat herds in New South Wales. Aust. Vet. J. 1995, 72, 341-345.

9. Hanson J., Hydbring E., Olsson K.A., 1996. long term study of goats naturally infected with caprine arthritis-encephalitis virus. Acta Vet. Scand. 37, 31-39.

10. Harkiss G.D., Watt N.J., 1990. Lentivirus infections and their detection. Goat Vet. Assoc. J. 11, 19-25.

11. Heckert R.A., McNab W.B., Richardson S.M., Briscoe M.R., 1991. Evaluation of an enzyme-linked immunosorbent assay for the detection of antibodies to caprine arthritis-encephalitis virus in goat serum. Can. J. Vet. Res. 56, 237-241.

12. Johnson L.K., Meyer A.L., Zink M.C., 1992. Detection of ovine lentivirus in seronegative sheep by in situ hybrydization, PCR and cocultivation with susceptible cells. Clin. Immunol. Immunopathol. 65, 254-261.

13. Kaba J., Ganter M., Bickhardt K., Prandota J., 1997. Prevalence of antibodies to caprine arthritis-encephalitis virus in breeding goats in Poland. Epidemiologie et Sante Animale, 11.C16, 31-32.

14. Kaba J., Ryniewicz Z., Ganter M., 2000. Changes in goat milk productivity caused by caprine arthritis encephalitis virus infection. Preliminary data. Seventh International Conference on Goats. Tours, France, 815-816.

15. Knowles D., Cheevers W., McGuire T., Stem T., Gorham J., 1990. Severity of arthritis is predicated by antibody response to gp135 in chronic infection with caprine arthritis-encephalitis virus. J. Virol. 64, 2396-2398.

16. Kołodziej P., Klimentowski S., Rypuła K., Marzec G., Kubica B., 1996. Badania porównawcze metod AGID i ELISA w rozpoznawaniu zakażeń lentiwirusowych owiec. Życie Wet. 71, 376-378.

17. Krieg A., Peterhans E., 1990. Die caprine arthritis-encephalitis in der Schweiz, epidemiologische und klinische Untersuchungen. Schweiz. Arch. Tierheilk. 132, 345-352.

18. Kwang J., Keen J., Cutlip R.C., Kim H.S., DeLaConcha-Bermejillo A., 1995. Serological diagnosis of caprine lentivirus infection by recombinant immunoassays. Small Rumin. Res. 16, 171-177.

19. Larondelle C., Richard Y., Issartial J., 1992. Factors affecting somatic cell counts in goat milk. Small Rumin. Res. 8, 129-139.

20. MacDiarmid S.C., 1984. A voluntary scheme to accredit flocks and herds free from caprine arthritis encephalitis infection (CAE). N. Z. Vet. J. 32, 166-171.

21. Manual of standards for diagnostic tests and vaccines. Office International des Epizooties, Paris, 1996.

22. Nord K., Ådnøy T., 1997. Effects of infection by caprine arthritis-encephalitis virus on milk production of goats. J. Dairy Sci. 80, 2391-2397.

23. Norman S., Smith M.C., 1983. Caprine arthritis-encephalitis: review of the neurologic form in 30 cases. J. Am. Vet. Med. Assoc. 182, 1342-1345.

24. Pasick J., 1998. Use of recombinant meadi-visna virus protein ELISA for the serologic diagnosis of lentivirus infections in small ruminants. Can. J. Vet. Res. 62, 307-310.

25. Rajya B.S., Singh C.M., 1964. The pathology of pneumonia and associated respiratory disease of sheep and goats. First occurrence of Jagskie and Maedi in sheep and goats in India. J. Am. Vet. Med. Assoc. 25, 61-67.

26. Rimstad E., East N.E., Torten M., Higgins J., DeRock E., Pedersen N.C., 1993. Delayed seroconvertion following naturally acquired caprine arthritis-encephalitis virus infection in goats. Am. J. Vet. Res. 54, 1858-1862.

27. Ryan D.P., Greenwood P.L., Nicholls P.J., 1993. Effect of caprine arthritis-encephalitis virus infection on milk cell count and N-acetyl-b-glucosaminidase activity in dairy goats. J. Dairy Res. 60, 299-306.

28. Saman E., VanEynde G., Luljan L., Extramiana B., Harkiss G., Tolari F., Gonzalez L., Amorena B., Watt N., Badiola J., 1999. A new sensitive serological assay for detection of lentivirus infections in small ruminants. Clin. Diagn. Lab. Immunol. 6, 734-740.

29. Schalie J.V., Bradway D.S., Besser T.E., Evermann J.F., 1994. Evaluation of a kinetic enzyme-linked immunosorbent assay for detection of caprine arthritis-encephalitis virus-specific antibodies. J. Vet. Diagn. Invest. 6, 30-33.

30. Schroeder B.A., Oliver R.E., Cathcart A., 1985. The development and evaluation of an ELISA for the detection of antibodies to caprine arthritis-encephalitis virus in goat sera. N. Z. Vet. J. 33, 213-215.

31. Smith M.C., Cutlip R., 1988. Effects of infection with caprine arthritis-encephalitis virus on milk production in goats. J. Am. Vet. Med. Assoc. 193, 63-67.

32. Thrusfield M. Veterinary Epidemiology. Blackwell Science, Oxford 1999.

33. Zanoni R., Nauta I.M., Pauli U., Peterhans E., 1991. Expression in Escherichia coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna virus strain ZZV 1055, application of recombinat protein in enzyme-linked immunosorbent assay for detection of caprine and ovine lentiviruses. J. Clin. Microbiol. 29, 1290-1294.

34. 6th Report of ICTV, Oxford University Press, 1997.

Martin Ganter
Clinic for pigs, small ruminants, forensic medicine and ambulatory service
School of Veterinary Medicine
Bischofsholer Damm 15, D-30173 Hannover, Germany

Krzysztof Rypuła
Department of Epizootiology and Veterinary Administration with Clinic
Agricultural University of Wrocław
Pl. Grunwaldzki 45, 50-366 Wrocław, Poland

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed ‘Discussions’ in each series and hyperlinked to the article.