LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA 303

LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA 303 LEVEL AND LENGTH OF PERSISTENCE OF MATERNALLY DERIVED ANTIBODIES IN BROILER CHICKENS THAT ORIGINATE FROM BROILER BREEDER FLOCK INFECTED WITH CHICKEN ANEMIA VIRUS M. Kapetanov*, D. Orlic*, Maja Velhner* and Dragica Stojanovi** *Scientific Veterinary Institute Novi Sad Chicken anaemia (CV) is widespread all around the world in countries with intensive poultry production in heavy and light hybrids. This has been obser ved after semlogical examination maintained in many countries and where virus isolation was performed. At the beginning of investigation of pathogenesis and ethiology of this disease it was thought that the groups of chickens that originate from the parent flocks that are infected with the virus at the beginning of laying period are exposed to the highest risk of infection with CAV. Also, it is known that the disease will not occur if parent flocks have produced antibodies against CAV. There are nowdays data showing that protection of the progeny depends of level of transferred antibodies. Besides that knowing the level t,nd persistency of the protected values of maternally derived antibodies in progeny is very important. Key words: chicken anaemia virus, broiler breeder flock, broiler flock, maternal antibodies. Causative agent of chicken anaemia virus strain Gufu-1 was isolated for the first time in 1979 in Japan by Yuasa et al., (1979). The age related resistance after infection with this virus was determined at that time. On that occasion chicken anaemia could be successfully reproduced on 7 days old chickens while in older birds there were no clinical occurrence of the disease. In broiler breeder flocks specific antibodies for chicken anaemia could be detected after 6 weeks, so infected chickens produced antibodies which would unable vertical transmission of the disease (Buelow et al., 1986, Godwin et al.,1990, Dren et al., 1996, Kapetanov, 2002). If the broiler breeder flocks are infected before coming in to the lay, virus can easily be transmitted through eggs to the progeny. In that case symptoms of the disease can be seen during the first two weeks in chickens. Poor uniformity of the flock, as well as loss of appetite among chickens and somnolence, are common signs (Engstrom and Luthman, 1984, Vielitz and Langraf 1988, McNuly, 1991, Godwin et. al., 1991). According to the knowledge about the disease, only serologically negative flocks are dangerous for transmitting the disease to the progeny or for the appearance of the disease in typical form followed with the mortality. This danger is the result of the fact that hatched chickens are susceptible for CAV (Vielitz and Voss, 1994, Vielitz et al., 1991, Steeinhausen et. al., 1994). Besides that, chickens with maternally derived antibodies can be infected with the virus and shed the virus without showing any signs of the disease. It appears that critical moment for the disease is the lack of maternal antibodies in a day old chicken and they will succumb the disease if the infection occurs through vertical transmission of the virus (Yuasa etal., 1980).

304 LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA Materials and methods In this work we tested presence of specific antibodies at the end of production in broiler breeder flock. In broiler chickens, originating from this flock that was 54 weeks old, we determine level end length of persistence of maternal antibodies (MaAt). During research we collected 20 sera samples from broiler breeder flock age 54 weeks and 40 sera samples from their broiler chickens at one day of age, and at 1,2,3 and 4 weeks. Samples of blood sera were delivered to the Virology Laboratory of the Scientific Veterinary Institute where CAV antibodies were determined by ELISA method. For test to be valid optical density of negative control (650nm) must be higher or equal to 0.60 while positive control SN must be lower or equal to 0.50. Samples of SN ratio higher than 0.60 are considered negative in the range of test. Samples with SN ratio lower or equal with 0.60 are positive. Results The results of statistical analysis and parameters such as X, SD, CV and IV for determined value of SN that show concentration of antibodies are given in Tables. Table 1. The results of serological examination of blood sera from the broiler breeder flock on the presence of CAV antibodies at the age of 54 weeks. No. OG a ~SN C Results Neg. contr. 1,412 - - + Pos. contr. 0,403 1 0,274 0,194 + 2 0,661 0,468 + T 3 0,366 0,259 + h ' 4 0,562 0,398 + 5 0,525 0,371 + 6 0,274 0,194 + 7 0,669 0,400 * 8 0,419 0,297 + 9 0,268 0,190 + 10 0,320 0,226 + 11 0,655 0,464 + 12 0,338 0,239 + 13 0,361 0,255 + 14 0,669 0,400 + 15 0,295 0,209 + 16 0,270 0,191 + 17 0,350 0,247 + 18 0,300 0,212 + 19 0,574. 0,406 + 20 0,431 0,305 + x SD CV IV 0,296 0,097 0,485 0,190-0,468

LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA 305 a OD mean value of the optical density from sera tested in duplicates b SN quotient of the mean value of sample and negative control Broiler breeder flock at the end of production at the age of 54 weeks had antibodies for CAV in 20 (100%) of examined sera. The mean value of the extinction was 0.296 and IV ranged from 0.190 up to 0.468, Table No 1. In this experiment we examined level and length of persistence of MaAt in broiler chickens from the broiler breeder flock which acquired active protection for CAV. We sampled 10 blood sera of the broiler chickens of different age at day 1 and at 1,2,3 and 4 weeks. The results of examination are given in Tables and Figures. Table 2 The results of serology examination of blood sera of day old broiler chickens on presence of maternally derived antibodies for CAV with ELISA test. No. OG SN Results Neg. contr. 0,118 - - + Pos. contr. 0,06 1 0,058 0,49 + 2 0,062 0,52 + 3 0,065 0,55 + 4 0,060 0,50 + 5 0,061 0,51 + 6 0,060 0,50 + 7 0,064 0,54 + 8 0,058 0,49 + 9 0,064 0,54 + 10 0,62 0,52 + X Sd CV IV 0,516 0.022 0,22 0,49-0,55 In all 10 examined blood sera of day old chickens we have proved maternal antibodies. The mean values of the extinction of maternal antibodies was 0.516 and IV ranged from 0.49 to 0.55, (Table 2). The mean values of extinction of MaAt for CAV in day old chickens were lower for 174% from the mean values of the antibody extinction in broiler breeder flock age 54 weeks. In all 10 examined sera samples of broiler chickens age 1 week we could find maternal antibodies. The mean value of the extinction of antibodies was 0.532 and IV ranged from 0.49 to 0.58, Table 3. Presence of maternal antibodies for CAV was proved in all 10 (100%) of blood sera in broiler chickens at the age of 2 weeks. The mean value of the extinction of antibodies was 0.566 and IV ranged from 0.54 up to 0.59, Table 4. In further investigation of the presence of maternal antibodies for CAV in broiler chickens at the age of 3 and 4 weeks could n ot be proved.

306 LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA Table 3.The results of serological examination of blood sera of broiler chickens on the presence of maternally derived antibodies for CAV at the age of 1 week. No. OG SN Results f Neg. contr.0,118 - - + Pos. contr. 0,060 1 0,058 0,49 + 2 0,061 0,51 + 3 0,060 0,50 + 4 0,0600,50 +.'., 5 0,0620,52 + 6 0,065 0,55 + 7 0,065 0,55 + 8 9 0,066 0,069 0,55 0,58 + + 10 0,068 0,57 + x SD CV IV 0,532 0,032 0,32 0,49-0,58 Table 4.The results of serological examination of blood sera of broiler chickens on the presence of maternal antibodies for CAV at age of 2 weeks. No. OG SN Results Neg. contr. 0,118 - - + Pos. contr. 0,060 * 1 0,068 0,57 + '] 2 0,070 0,59 + 3 0,067 0,57 + 4 0,066 0,55 + 5 0,070 0,59 + 6 0,070 0,59 + 7 0,067 0,56 +,t 8 0,065 0,55 + 9 0,064 0,54 + 10 0,066 0,55 + x SD CV IV 0,566 0,019 0,19 0,54-0,59 Discussion At the beginning of our investigation we examined blood sera of the parent flock at the end of production (54 weeks old) which were infected during rearing with CAV, applying ELISA test. All the examined sera were positive on specific antibodies for CAV. The mean values of extinction of the examined samples were 0.296 ranging from 0.190 to 0.468. Pages-Mante et al., (1997) suggested that chickens should be vaccinated at 20 weeks of age regardless to the level of post infectious antibodies, because of

LUCRARI stiinwice MEDICINA VETERINARA, VOL. xxxvi, 2003, TIMISOARA 307 continuous exposure to CAV. The same author stated that the highest mean values of antibodies were determined at 10 weeks after vaccination, at 30. weeks of age. In order to test persistence and level of MaAt in broiler chickens that originate from the tested broiler breeder flock (age 54 weeks), we examined their blood sera when 1 day old and at the end of 1,2,3 and 4 weeks of age. During examination we determined presence of MaAt for CAV in 100% of examined day old chickens or chickens at the end of first and second week. In older broilers, at 3 and 4 weeks, we could not determine presence of MaAt for CAV. This data are in accordance to the results of Pages-Mante et al., (1997) that j show the highest values of MaAt in a day old chickens originated from the broiler breeder flock at the age of 30 weeks that was vaccinated, while the lowest antibody values were in chickens 30 days old coming from vaccinated broiler breeder flock 60 weeks old. They also observed that MaAt decreases linearly with half-life of 10 days. Yuasa et al., (1980) and Otaki et al., (1992) emphasise that the level of antibodies for CAV in broiler breeder flock, which could transfer MaAt to the progeny, are protective during the first two weeks of life, i.e. until age resistance develop. Speed and successes rate of acquiring resistance is slow in the case of immunosupression what is the cause with simultaneous infection with GB virus or in bursectomised chickens (Yuasa et al., 1980, Yuasa et. al., 1988). According to the examinations currently described about the transfer of infection and time when the disease occurs only serologically negative flocks are dangerous for transfer of the disease to their progeny, or appearance of the disease in typical form with mortality. Although chickens with maternal antibodies can be infected with the virus and shed the virus, they do not show any symptoms of the disease (Yuasa et al., 1980). References 1. BUlow V V, 1988, Unsatisfactory sensitivity and specificity of indirect immunofluorescence testes for the presence or absence of antibodies to chicken anaemia agent (CAA) in sera of SPF and broiler breeder chickens, J Vet Med, B 35 : 594-600. 2. Dren Csaba, Tiber Farkas, Istvan Nemeth. 1996, Serological survey an tha prevolence of chicken anaemia virus infection in Hungarian chucken flocks, Veterinary Microbiology 50 (1996) 7-16 3. Goodwin M.A., Steffens W.L., Davis J.F., Brown J., Latimer K.S., Dickson T.G. 1991. Diagnoses of infections by the socalled chick anemia agent. Anemia and direct transmission electron microscopic detection of virus. Avian Dis. 35, 869-871. 4. Goryo, M., H. Sugimura, S. Matsumoto, T. Umemura, and C. Itakura. 1985. Isolation of agent inducing chicken anaemia. Avian Pathol 14:483-496. 5. Kapetanov M, Ispitivanje zastitne vrednosti nasle*enih antitela protiv virusa zarazne anemije pili*a (VZAP), Doktorska disertacija, Beograd, 2002.

308 LUCRARI STIINTIFICE MEDICINA VETERINARA, VOL. XXXVI, 2003, TIMISOARA 6. McNeilly F., Allan G.M., Moffett D.A., McNulty M.S. 1991. Detection of chicken anaeima agent in chickens by imunofluorescence and imunoperoxidase staining. Avian Pathol. 20,125-132 7. Otaki, Y., Saito, K., Tajima, M. Nomura, Y. 1992. Persistence of maternal antibody to chicken anaemia agent, and its effect on the susceptibility of youang chickens. Avian Pathology, 21, 147-151. 8. Pages A, N Saut,:, C Artigas S E, 1997, Experimental evaluation of an inactivated vaccine against chicken anaemia virus, Avian Pathology, 26: 721-729. 9. Steinhuisenm W H, Jagt HJM, Schrier CC, 1994, The use of a live attenuated CAV vaccine in breder flock in the Netherlands. Inter. Symp, In IBD and CAV, Germany, 482-497. 10. Vielitz E., Conrad C., Voss M., BUlow V.v., Dorn P., Bachmeier J., Lohren U. 1991. Immunization against chicken anaemia agent (CAA) a field trial. Dtsch. Tierarztl.Wschr. 98, 144-147. 11. Vielitz E, Voss M, 1994, Experiences with a comercial CAV vaccine. International simp. Inf. bursal disease and chicken inf. anemia,rauschholyhausen, Germany, 465-497. 12. Yuasa, N. and K. Imai. 1988. Efficacy of Mareks disease vaccine, herpesvirus of turkeys, in chickens infected with chicken anemia agent. In S. Kato, T. Horiuchi, T. Mikami, and K. Hirai (eds.), Advances in Mareks Disease Research, pp. 358-363. Japanese Association on Mareks Disease, Osaka. 13. Yuasa, N., T. Taniguchi, and I. Yoshida.1979. Isolation and some characteristics of an agent inducing anemia in chicks. Avian Dis 23:366-385. 14. Yuasa N, T Noguchi, K Furata, I Yoshida, 1980, Maternal antibody and its effect on the susceptibility of chicks to chicken anaemia agent, Avian Dis, 24:197-201. Or Milos Kapetanov, Scientific Veterinary Institute, Novi Sad, Rumena*ki put 6, 21000 Novi Sad