ISRAEL JOURNAL OF VETERINARY MEDICINE Case Report CHICKEN INFECTIOUS ANEMIA IN YOUNG BROILER FLOCKS IN ISRAEL Davidson I.1*, Shkoda I.1, Elkin N.2, Ayali, G3., Hamzani E.3, Kass N.3, Smith B.4, Borochovitch H.4, Gilat G.5, Vol. 59 (4) 2003 Krispin H.6, Kedem M.7 and Perk S.1 1. Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel, 2. Or Yehuda, Neve Savion, Israel 3. Tzfat, Regional Poultry Disease Laboratory, Veterinary Services, Israel 4. Afula, Regional Poultry Disease Laboratory, Veterinary Services, Israel 5. Kiryat Tivon, Israel, 6. Yavne, Israel 7. Akko Regional Poultry Disease Laboratory, Veterinary Services, Israel Summary An outbreak of chicken infectious anemia virus-related clinical signs in broiler flocks is described. The flocks were derived from three broiler breeder flocks. The presence of CIAV sequences was determined by PCR in the progeny, broiler flocks, and CIAV-antibody levels in the parent broiler breeder flocks were determined by ELISA. These levels were low and inconsistent,. The progeny broiler flocks showed typical clinical signs of CIAV-infection and were PCR positive. The survey also included three control unaffected broiler flocks, that originated from breeder flocks of similar ages and genetic lines, and reared on other breeding farms. These differed in the maternal antibody levels transmitted to their progeny. Moreover the progeny flocks were negative clinically and by CIAV- PCR. The data illustrate the necessity of adequate levels of immunity of the parent flock to prevent the acute disease in their progeny. Introduction Chicken infectious anemia virus (CIAV) was first described in 1979 by Yuasa in Japan (1) as a circovirus with exceptional characteristics. Since then, CIAV has been detected by isolation or serology in many countries in both laying and broiler chickens (2). The disease prevalence in commercial flocks in Israel has recently been reported (5). Infections with CIAV are considered to be economically significant because clinical disease is associated with both vertical and horizontal transmission. CIAV infections are manifested by either clinical or subclinical signs. In young chicks of less than 3 weeks without CIAV maternal antibodies, infection results in stunting, runting, increased mortality, anemia, depletion of bone marrow cells and thymocytes, subcutaneous hemorrhages, and a decreased resistance to secondary bacterial diseases such as gangrenous dermatitis and campylobacter colonization. These effects are caused because CIAV infects stem cells of both the hematopoietic and lymphocyte cell lineages in the bone marrow and thymus. In older chickens the virus decreases the cell- mediated immune responses resulting in increased morbidity caused by various pathogens (3, 4). Most commercial flocks have been exposed to CIAV either by vaccination or natural exposure and develop antibodies which are transferred to their offspring. As a consequence, acute disease in young birds is rare. Once neutralizing antibodies develop, active viremia is curtailed and infectious CIAV is eliminated thereby preventing vertical transmission (6, 7). Several outbreaks of infectious anemia have been correlated with the absence of anti- CIAV antibodies in the respective parent flocks (3, 8, 9, 10). Recently, a number of young broiler flocks in Israel experienced outbreaks of clinical disease compatible with CIAV infection. In the present note we describe the epidemiology of the disease outbreaks, determine the presence of CIAV in the affected birds, and the relation to the level of immunity to CIAV in the parent broiler breeder flocks. Materials and Methods Chicken flocks Three broiler breeder flocks were the source of the broiler flocks included in the survey. The flock type, age and clinical signs were detailed in the results section for each flock mentioned. In each flock 3 to 5 birds were examined. DNA was obtained from the spleen, liver and feather tips of each bird (11). DNA amplification Genomic DNA was used to amplify a 480 bp fragment of CIAV using primers C1 (CCAAGAAGATACTCCACCCG) and C2 (TACGATACCGCTGTCTCCTC) as described (12). ELISA for CIAV antibodies CIAV antibodies were measured using the FlockChekR CAV (IDEXX, USA) according to the manufacturer recommendations at a 1:10 serum dilution. The ELISA results are expressed as the ratio between the samples assayed and the negative control (S/N). The quantity of antibodies is inversely proportional to the absorbance A(650 nm), and the S/N. Results and Discussion The broiler flocks detailed in Tables 2 and 3 with the progeny flocks of three breeder broiler flocks, A, B and C (Table 1). All the progeny broiler flocks were affected with typical clinical signs, such as stunting, runting, hemorrhages, increased mortality, gangrenous dermatitis and increased susceptibility to various diseases, possibly due to CIAV. Table 1 presents the CIAV-antibody levels of the parent flocks. The blood samples were obtained during 1 to 2 weeks preceding egg production of the broiler flocks described in Tables 2 and 3. The genetic line of the parent flocks B and C was the same, and the same grandparent flock produced flocks B and C at a 4 days interval. As shown in Table 1, the three parent flocks were of a similar age and their serological response to CIAV was low and inconsistent. From 22 to 23 sera from each flock were tested, the CIAV-antibody levels were low or negative, with a high variation in antibody titers. Flock A was re-examined two months afterwards and showed that a seroconversion had occurred about the same time that the eggs for the progeny broiler flocks were laid. The major clinical signs were recorded in young broilers at the age when the flocks were sampled (Tables 2 and 3) included a generalized weakness, depression, stunting, growth retardation, ruffled feathers, gangrenous dermatitis and increased mortality. At necropsy the carcasses, bone marrow and liver were pale and internal and external hemorrhages were observed. Another part of the CIAV-infection related losses were indirect, and consisted of aggravation of other, unrelated diseases that affected these flocks at the same time. These included the increased expenses of medications and decreased profitability. Because the variety of losses observed in the affected flocks, and the difficulties in their quantitative assessment, they were categorized by three parameters in Tables 2 and 3; gangrenous dermatitis when it appeared to any extent was indicative, morbidity refers to all the above findings, while mortality refers to increased mortality, as high as 40-50%. Table 2 summarizes the findings in the progeny broiler flocks derived from the parent broiler flock A from the eggs laid within 1 to 2 weeks preceding the first blood sample (Table 1). The present survey also includes three unrelated broiler flocks, (D-F), as controls for the affected broiler flocks. Flocks D-F originated from other breeder flocks of similar ages and genetic origin, which were reared on other breeding farms. While all broiler flocks that originated from breeder flock A showed typical clinical signs of CIAV and were CIAV-PCR positive, the three control flocks (D, E, and F) were negative clinically and by CIAV-PCR. Tables 3a and 3b summarize the broiler flocks derived from breeder flocks B and C, whose CIAV-antibody status was detailed in Table 1. On several farms, such as farms I, VI and VII (Table 2) and farms I, II and III (Table 3a) the broiler flocks were the progeny of other breeder flocks of the same genetic line. The disease did not affect these three broiler flocks, and the PCR assays were negative for CIAV. Altogether, we demonstrate now the close association between the clinical signs and the positive CIAV-PCR in young broiler flocks. In summary, the CIAV-related conditions were investigated in view of the acute outbreaks reported in broilers and an awareness of the CIAV impact on poultry productivity. The present communication presents data that strengthen the necessity for providing adequate immunity of breeder flocks as a means of avoiding the acute disease in their progeny. Table 1. CIAV antibody status of the three broiler breeder flocks Flock Genetic Age No. at Arithmetic Positive/ Standard CV* strain bleeding (days) Total tested mean (Amn) Deviation A I 169 3/23 0.895 0.323 36.1 230 21/22 0.318 0.156 49.2 257 22/22 0.252 0.2 79.4 B II 182 24/24 0.911 0.279 30.6 244 24/24 0.082 0.016 19.5 C II 189 21/23 0.319 0.166 52.0 256 23/23 0.084 0.033 39.2 * Coefficient of variation = Standard deviationX100/Amn Table 2. CIAV presence in the progeny flocks of broiler breeder flock A hatched from eggs laid within 1-2 weeks after bleeding at 169 days. Farm Line Broiler flock Clinical signs CIAV-PCR age at sampling (days) No. Morbidity Mortality birds tested Organ Gangrenous dermatitis PCR I A 16 + + + 3 liver + 3 spleen + 3 feathers + 19 + + + 3 liver + 3 spleen + 3 feathers + I D 18 - - - 3 liver - 3 spleen - 3 feathers - II A 19 + + + 3 liver + 3 spleen + 3 feathers + III A 42 + + + 4 liver + 4 spleen + 4 feathers + IV A 22 + + + 3 liver + 3 spleen + 3 feathers + V A 18 + + + 4 liver + 4 spleen + 4 feathers + VI E 18 - - - 5 liver - 5 spleen - 5 feathers - VII F 30 - - - 4 liver - 4 spleen - 4 feathers - Table 3a: CIAV presence in progeny flocks of broiler breeder flock B hatched from eggs laid within 1 week after bleeding at 182 days Farm Line Broiler flock age at Clinical signs CIAV-PCR sampling (days) Gangrenous Morbidity Mortality No. birds Organ PCR dermatitis tested I B 16 + + + 3 liver + 3 spleen + 3 feathers + D 16 - - - 4 liver - 4 spleen - 4 feathers - II B 16 + + + 3 liver + 3 spleen + 3 feathers + E 13 - - - 3 liver - 3 spleen - 3 feathers - III B 17 + + + 4 liver + 4 spleen + 4 feathers + 22 + + + 4 liver + 4 spleen + 4 feathers + F 22 - - - 5 liver - 5 spleen - Table 3b: CIAV presence in progeny flocks of broiler breeder flock C, hatched from eggs laid 4 weeks after bleeding at 189 days. FarmLine Eggs laid at no. Broiler flockClinical signs CIAV-PCR weeks post bleeding age at event GangrenousMorbidity Mortality No. dermatitis Organ PCR birds tested I C 1 16 + + + 6 liver + 6 spleen + 6 feathers + II C 1 17 + + + 5 liver + 5 spleen + 5 feathers + III C 2 15 + + + 3 liver + 3 spleen + 3 feathers + IV C 3 38 + + + 5 liver + 5 spleen + 5 feathers + V C 4 3 - - - 10 liver - 10 spleen - Acknowledgement This study was supported by Grant US-3535-04R from the USA-Israel Agricultural Research and Development Fund (BARD). LINKS TO OTHER ARTICLES IN THIS ISSUE References 1. Yuasa, N., Taniguchi, T. & Yoshida I. (1979). Isolation and some characteristics of an agent inducing anemia in chicks. Avian Dis. 23, 366-385. 2. Schat, K.A. (2003). Circovirus infections, Chicken infectious anemia. In: Y.M. Saif, H.J. Barnes, A.M. Fadly, J. R. Glisson, L.R. McDougald, and D.E. Swayne (eds) Diseases of Poultry, 11th edition, Iowa State Press, Ames, Iowa, pp182-202. 3. Fehler, F., & Winter, C. 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