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Seromolecular study of chicken infectious anemia in chickens, ostriches, and turkeys in Iran
©2013 Poultry Science Association, Inc.
Seromolecular study of chicken infectious anemia in chickens, ostriches, and turkeys in Iran Majid Gholami-Ahangaran,*1 Ezatollah Fathi-Hafshejani,* and Ravand Seyed-Hosseini† *Poultry Diseases Department, Veterinary Medicine Faculty, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran; and †Graduated from Diploma Degree in Poultry Health and Diseases, Shahrekord University, Shahrekord, Iran Primary Audience: Veterinarians, Researchers, Flock Supervisors SUMMARY Until recently, the chicken has been considered the only natural host and the main host for chicken anemia virus (CAV). However, CAV infection has now been reported in other avian species, including Japanese quail, jackdaws, rooks, and rare avian breeds. In this study, we examined chickens, ostriches, and turkeys for seromolecular detection of CAV in Iran. For the study, 375 serum and thymus samples from chickens, 84 serum and blood samples from ostriches, and 100 serum and blood samples from turkeys were collected from various locations in Iran. Serum samples were examined using ELISA. Deoxyribonucleic acid was extracted from the thymus, and blood samples were analyzed for detection of the VP2 genome of CAV by PCR. According to these results, 69.07 and 58.4% of chickens were positive for the antibody to CAV and the VP2 genome of CAV, respectively. In addition, in ostriches 27.38 and 34.52% were positive for the CAV antibody and the CAV VP2 genome, respectively. In turkey flocks, all turkeys (100%) were negative with respect to detection of the VP2 CAV genome and CAV antibody. It was concluded that CAV is distributed in broiler chickens and ostriches throughout Iran. Furthermore, ostriches may be considered a reservoir of CAV, and turkeys appear not to be susceptible to CAV infection. Key words: chicken, chicken anemia virus, Iran, ostrich, turkey 2013 J. Appl. Poult. Res. 22:404–409 http://dx.doi.org/10.3382/japr.2012-00567
DESCRIPTION OF PROBLEM The Circoviridae family contains 2 genera [1]. Chicken anemia virus (CAV) is the only member of the genus Gyrovirus, whereas the genus Circovirus currently comprises porcine circoviruses type 1 and 2, psittacine beak and feather disease virus, pigeon circovirus, canary circovirus, goose circovirus, and duck circovirus [1]. 1
Corresponding author: [email protected]
Members of the Circoviridae family are nonenveloped, regular icosahedrons and are the only bird viruses with a circular, single-stranded DNA genome [2]. These viruses share many epizootiological and pathological similarities (i.e., young age of affected birds, particular tropism for the lymphoid tissue and organs, related acquired immunosuppression and secondary infections) [3, 4].
Gholami-Ahangaran et al.: CHICKEN ANEMIA VIRUS Chicken anemia virus was first isolated and described in Japan by Yuasa et al. [5], but CAV antibodies have been detected in chicken sera worldwide [5, 6]. This virus has been found in most countries with poultry industries [7]. The virus spreads vertically from parental stock to progeny and horizontally by contact exposure with infected chickens or fomites. Chicken anemia virus infection induces either clinical or subclinical signs [8]. Clinical disease occurs when chicks are infected during the first 2 wk of life, but it can be avoided if hens transfer sufficient antibodies to their progeny. After 2 wk of age, chicks can be infected with the virus but do not develop clinical signs [9]. This virus can cause economically important losses with either the clinical or subclinical form of the disease in broiler chickens [10, 11]. Signs and lesions include stunting, increased mortality, anemia, bone marrow cell depletion, subcutaneous hemorrhage, and atrophy of secondary lymphoid organs [12]. This infection is often associated with opportunistic viral and bacterial infections and vaccination failures in chicken flocks [8, 13]. Chicken anemia virus was previously known as a much-conserved virus of one serotype [5] with several genetic groups [8]. However, an additional serotype has recently been reported [8, 14]. Until now, the chicken was considered to be the only natural host and the main host of CAV [4], but CAV infection has been reported in other avian species, including Japanese quail [15], fancy chicken breeds [16], jackdaws, rooks, and some rare avian breeds [17]. In contrast, the antibody to CAV was not found in some birds, such as the duck, pigeon, or pheasant [4]. In this study, chicken, ostrich, and turkey sera were tested by using a commercial ELISA for the presence of antibodies to CAV and for detection of the CAV genome by PCR in these birds in Iran.
MATERIALS AND METHODS
405
were collected from each flock. On average, all sampled flocks had a mortality higher than 1% per day for 10 d during the growing period. Eighty-four serum and blood samples were collected from 15 ostrich flocks with ages ranging from 8 to 15 mo of age. At least 5 serum samples were collected from each flock. These sera were collected from 5 processing facilities across Iran from 2009 to 2010. In addition, 100 serum and blood samples were collected from 10 healthy turkey flocks of different ages in various locations in Iran from 2009 to 2010. All commercial poultry flocks (chicken, ostrich, and turkey) examined in this study had not been vaccinated against CAV, and no clinical signs suggestive of CAV infection were observed in any of the flocks. All commercial poultry flocks were reared on litter, and feed and water were supplied ad libitum. ELISA Blood samples were collected via venipuncture of the wing vein. The blood was allowed to clot at room temperature for 30 min and centrifuged (1,500 × g, 15 min, room temperature). Serum was decanted into a microtube and inactivated by heating at 56°C for 30 min, and then stored at −20°C until tested. The sera were tested by indirect ELISA using a commercial competitive CAV ELISA Kit [18]. A serum dilution of 1:10 was used following the manufacturer’s instructions. The optical density value was read at a 650-nm wavelength on an ELISA reader [19] according to the manufacturer’s instructions. The presence or absence of the antibody to CAV was determined by sample-to-negative (S:N) ratios for each sample. Samples with an S:N value of less than 0.6 were defined as positive, and those with a value equal to or greater than 0.6 were interpreted as negative. A positive sample indicates the presence of CAV antibodies and previous exposure to CAV.
Flocks
PCR
Serum and thymus samples (375) were collected from 25 broiler chicken flocks in the Isfahan, Yazd, Tehran, and Chaharmahal-va-Bakhtiyari Provinces, in the central area of Iran, from 2009 to 2010. An average of 15 serum samples
Deoxyribonucleic acid extraction from tissue and blood samples was carried out using a High Pure Viral Nucleic Acid Kit [20] according to the manufacturer’s instructions. Polymerase chain reaction was carried out to amplify a fragment
JAPR: Research Report
406 of 713 bp from the viral protein 2 (VP2) gene of CAV. The sequence of the primers was as follows: forward primer: 5′-GCG CAC ATA CCG GTC GGC AGT; reverse primer: 5′-GGG GTT CGG CAG CCT CAC ACT AT [21]. Polymerase chain reaction amplification was performed in PCR buffer containing 1.5 mM MgCl2, 200 μM each deoxynucleotide 5′-triphosphate, 10 pM each primer, and 1.0 unit of Taq polymerase [22] in a 25-μL total reaction volume. The amplification was carried out in a thermal cycler [23] under the following conditions: initial denaturation of 94°C for 4 min, followed by 34 cycles of denaturation, annealing, and extension at 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, respectively, and a final extension at 72°C for 5 min. The PCR product was then analyzed by electrophoresis in 1% agarose gel and visualized under UV light after staining with ethidium bromide. In this study, Cuxhaven-1 strains of CAV [24] were provided and used as a positive control, and deoxyribonuclease-free water was used as a negative control.
RESULTS AND DISCUSSION ELISA All broiler chicken flocks were found to be positive at 7 to 8 wk of age. The rate of seropositive chickens among flocks ranged from 20 to 100%, and the prevalence was always more than 20%. The seroprevalence of CAV was 69.07% in chickens. The minimum and maximum of the mean S:N value were 0.18 and 0.65, respectively (Table 1). Eleven out of 15 ostrich flocks (73.33%) were positive for antibodies against CAV. The seroprevalence of CAV was 27.38% in ostriches. The mean CAV S:N value of the ostriches examined was 0.672, whereas the minimum and maximum S:N values were 0.20 and 0.98, respectively (Table 2). In this study, all turkey flocks (100%) were found to be negative for antibodies to CAV. The mean CAV S:N value of turkeys examined was 0.757, whereas he minimum and maximum S:N values were 0.62 and 0.98, respectively.
Table 1. Enzyme-linked immunosorbent assay results in the chicken flocks tested Flock no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1
CAV S:N ratio,1 mean ± SD
CV, %
Positive ELISA sample, %
Positive PCR sample, %
0.31 ± 0.18 0.59 ± 0.17 0.35 ± 0.18 0.20 ± 0.14 0.36 ± 0.08 0.37 ± 0.18 0.54 ± 0.27 0.29 ± 0.16 0.49 ± 0.17 0.57 ± 0.15 0.59 ± 0.19 0.40 ± 0.22 0.29 ± 0.17 0.31 ± 0.19 0.43 ± 0.17 0.49 ± 0.16 0.48 ± 0.12 0.18 ± 0.13 0.57 ± 0.21 0.32 ± 0.15 0.37 ± 0.23 0.65 ± 0.21 0.51 ± 0.26 0.49 ± 0.15 0.61 ± 0.23
58.3 30.6 51.2 69.2 22.3 48.5 50.3 54.7 35.2 26.5 32.1 55.8 59.8 61.9 40.2 34.1 25.4 73.4 37.0 47.9 61.9 32.8 51.2 31.1 37.2
100 46.7 86.7 100 100 80 53.3 100 66.8 60 20 73.3 86.7 86.7 73.3 60 66.7 100 26.7 100 80 20 46.7 66.7 26.7
86.6 40 73.3 100 100 80 40 80 46.6 40 13.3 73.3 80 73.3 66.6 46.6 53.3 93.3 20 86.6 60 6.6 26.6 46.6 26.7
CAV S:N ratio = chicken anemia virus sample-to-negative ratio.
Gholami-Ahangaran et al.: CHICKEN ANEMIA VIRUS
407
Table 2. Enzyme-linked immunosorbent assay results in the ostrich flocks tested Flock no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample size, no.
CAV S:N ratio,1 mean ± SD
CV, %
Positive ELISA sample, %
Positive PCR sample, %
8 6 6 7 5 6 7 5 8 4 3 7 5 4 3
0.66 ± 0.147 0.81 ± 0.121 0.73 ± 0.120 0.72 ± 0.126 0.77 ± 0.144 0.52 ± 0.144 0.68 ± 0.232 0.46 ± 0.241 0.74 ± 0.153 0.74 ± 0.127 0.82 ± 0.151 0.76 ± 0.168 0.72 ± 0.170 0.37 ± 0.156 0.52 ± 0.112
22.1 14.9 16.3 17.5 18.6 27.3 34 52.3 20.4 17 18.3 22 23.3 41.2 21.4
25 0 16.66 14.28 0 83.33 28.57 60 20 0 0 14.28 20 100 66.66
25 16.66 16.66 28.57 0 83.3 28.57 60 37.5 0 0 28.57 20 100 100
PCR In this study, all broiler chicken flocks (100%) were positive for CAV. On the basis of PCR results, 219 of 375 (58.4%) thymus samples from 25 broiler chicken flocks were positive for CAV (Table 1). The minimum and maximum values of positive chickens in all tested flocks were 6.6 and 100%, respectively. The CAV genome in this study was detected in 29 of the 84 (34.52%) blood samples collected from 15 ostrich flocks in Iran. The VP2 genome was detected in 80% of the examined flocks. The positivity rate ranged from 14.28 to 100% in all tested flocks (Table 2). In the turkey flocks tested, all turkeys (100%) were negative with respect to detection of the VP2 CAV genome in blood samples. Chicken anemia virus was first recognized in Japan [5]. Chicken anemia virus infection in chicken flocks has now been described in most countries and can result in economical losses from either clinical or subclinical infections [10, 11]. The CAV causes clinical disease in young chicks, and affected chicks experience increased mortality associated with anemia, aplasia of the bone marrow, and lymphoid depletion [25]. The clinical CAV infection is mainly noticed in young chicks with subcutaneous and muscular hemorrhages [26]. Older chickens are susceptible to this subclinical infection [27]. The clinical form of the disease is rare today because of the widespread practice of vaccinating breeders,
but the subclinical form of CAV is ubiquitous [28]. There are many reports of CAV infections in most chicken-producing countries in chicken flocks throughout the world [29], such as in Sudan [30], Japan [15], Turkey [31], and Jordan [32]. The clinical form of CAV infection was studied in chicken flocks in Iran [33–35]; however, the present study represents a seromolecular study for the prevalence of subclinical CAV infection in commercial broiler chickens in Iran. In this study, the seroprevalence of subclinical CAV infection was 69.07 and 100% in broiler chickens and broiler chicken farms, respectively. The PCR results confirm the serological results. This finding is accordance with and similar to previous reports in Iran of clinical CAV infection [35]. However, the infectivity of chicken flocks to CAV in flocks is expected because the individual viral prevalence of CAV in apparently healthy broiler chickens in Iran was reported as 24.58% and the rate of positive flocks reached 73.33% [36]. Therefore, CAV seems to be widespread in broiler chickens throughout Iran, and the results provide evidence of widespread distribution of the virus and a high incidence of infection among commercial broiler flocks in Iran, as has similarly been documented to occur worldwide in all major poultry-producing countries [29]. Until now, the chicken was thought to be the only host of CAV. However, in this study we found a high incidence of seropositivity to CAV in ostrich flocks, and these findings were con-
JAPR: Research Report
408 firmed with PCR. Whether CAV causes clinical disease in ostriches and whether the virus in these birds is CAV or another circovirus with cross-reacting properties need further investigation. Although a circovirus-like infection has been reported in ostriches [37], the distribution of this virus among ostriches is unknown. As far as is known, there are no reports showing that the circovirus of ostriches does cross-neutralize with CAV. In addition, circovirus has been observed by electron microscopy in the intestinal contents of an ostrich (Struthio camelus); however, it is uncertain whether the ostrich was actually infected with the virus or whether the virus was obtained from ingested plant matter [37]. Therefore, according to our findings, the ostrich can be considered a reservoir of CAV. We conclude, based on our review of the related literature, that although the chicken has been considered the natural host for CAV, antibodies to CAV have been detected in Japanese quail in Japan [15], in fancy chicken breeds in the Netherlands [16], and in jackdaws, rooks, and some rare avian breeds in Ireland [17]. In contrast, the antibody to CAV was not found in turkeys and ducks in the United Kingdom [38]; in pigeons, ducks, and pheasants in Ireland [17]; and in crows, pigeons, and ducks in Japan [15]. Furthermore, 1-d-old turkey poults inoculated with CAV did not show clinical signs of anemia and did not develop antibodies to the virus [38]. However, on the basis of negative results obtained with sera collected from a turkey flock, all flocks tested appeared not to have been infected with CAV. To our knowledge, this is the first report of the detection of serological and molecular CAV in species other than the chicken.
CONCLUSIONS AND APPLICATIONS
1. Chicken anemia virus was found to be widespread in broiler chickens and ostriches throughout Iran. 2. In this study, seropositivity to CAV was found in ostrich flocks, and these findings were confirmed by PCR. Therefore, the ostrich can be considered a reservoir of CAV. 3. In this study, all turkey flocks (100%) were found to be negative with regard
to antibodies to CAV and detection of the VP2 CAV genome in blood samples. Therefore, it seems turkeys are not susceptible to CAV infection.
REFERENCES AND NOTES 1. Todd, D., A. N. J. Scott, E. Fringuelli, H. L. Shivraprasad, D. Gavier-Widen, and J. A. Smyth. 2007. Molecular characterization of novel circoviruses from finch and gull. Avian Pathol. 36:75–81. 2. Twentyman, C. M., M. R. Alley, J. Meers, M. M. Cooke, and P. J. Duignan. 1999. Circovirus-like infection in a southern black-backed gull (Larus dominicanus). Avian Pathol. 28:513–516. 3. Bassami, M. R., D. Berryman, G. E. Wilcox, and S. R. Raidal. 1998. Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus. Virology 249:453–459. 4. Schat, K. A. 2003. Chicken infectious anaemia. Pages 182–202 in Diseases of Poultry. 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. Swayne, ed. Iowa State Press, Ames. 5. Yuasa, N., T. Taniguchi, and I. Yoshida. 1979. Isolation and some characteristics of an agent-inducing anaemia in chicks. Avian Dis. 23:366–385. 6. McNulty, M. S., T. J. Cormor, and F. McNeilly. 1989. A survey of specific pathogen-free chicken flocks for antibodies to chicken anemia agent, avian nephritis virus and group A rotavirus. Avian Pathol. 18:215–220. 7. Ducatez, M. F., A. A. Owoade, J. O. Abiola, and C. P. Muller. 2006. Molecular epidemiology of chicken anemia virus in Nigeria. Arch. Virol. 151:97–111. 8. Simionatto, S., C. A. V. Lima-Rosa, E. Binneck, A. P. Ravazzolo, and C. W. Canal. 2006. Characterization and phylogenetic analysis of Brazilian chicken anaemia virus. Virus Genes 33:5–10. 9. Canal, C. W., D. J. Ferreira, M. Macagnan, L. C. B. Fallavena, H. L. S. Moraes, and V. B. Wald. 2004. Prevalence of antibodies against CAV in broiler breeder in southern Brazil. Pesqui. Vet. Bras. 24:89–92. 10. McNulty, M. S., S. G. McIlroy, D. W. Bruce, and D. Todd. 1991. Economic effects of subclinical chicken anemia agent infection in broiler chickens. Avian Dis. 35:263–268. 11. Yuasa, N., and K. Imai. 1986. Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (eAA). Avian Pathol. 15:639–645. 12. Adair, B. M. 2000. Immunopathogenesis of chicken anemia virus infection. Dev. Comp. Immunol. 24:247–255. 13. Cloud, S. S., J. K. Rosenberger, and H. S. Lillehoj. 1992. Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. II. Alterations of in vitro lymphoproliferation and in vivo immune responses. Vet. Immunol. Immunopathol. 34:353–366. 14. Spackman, E., S. S. Cloud, C. R. Pope, and J. K. Rosenberger. 2002. Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate I. Pathogenesis. Avian Dis. 46:945–955. 15. Farkas, T., M. Maeda, H. Sugiura, K. Kai, K. Hirai, K. Ostuki, and T. Hayashi. 1998. A serological survey of chickens, Japanese quail, pigeons, ducks and crows for an-
Gholami-Ahangaran et al.: CHICKEN ANEMIA VIRUS tibodies to chicken anaemia virus (CAV) in Japan. Avian Pathol. 27:316–320. 16. de Wit, J. J., J. H. Van-Eck, R. P. Crooijmans, and A. Pijpers. 2004. A serological survey for pathogens in old fancy chicken breeds in central and eastern part of The Netherlands. Tijdschr. Diergeneeskd. 129:324–327. 17. Campbell, G. 2001. Investigation into evidence of exposure to infectious bursal disease virus (IBDV) and chick infectious anaemia virus (CIAV) in wild birds in Ireland. Pages 230–233 in Proc. 2nd Int. Symp. Infectious Bursal Disease and Chicken Infectious Anaemia, Rauischholzhausen, Germany. Institut für Geflügelkrankheiten, Justus Liebig University, Giessen, Germany. 18. Flockchek, CIAV, IDEXX, Westbrook, ME. 19. Bio-Rad, Hercules, CA. 20. High Pure Viral Nucleic Acid Kit, Roche, Basel, Switzerland. 21. Natesan, S., J. M. Kataria, K. Dhama, S. Rahul, and N. Bhardwaj. 2006. Biological and molecular characterization of chicken anaemia virus isolates of Indian origin. Virus Res. 118:78–86. 22. Fermentas, Glen Burnie, MD. 23. Mastercycler Gradient, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany. 24. Thymovac vaccine, Lohmann Animal Health, Cuxhaven, Germany. 25. McNulty, M. S. 1991. Chicken anaemia agent: A review. Avian Pathol. 20:187–203. 26. Yuasa, N., K. Imai, K. Watanabe, F. Saito, M. Abe, and K. Komi. 1987. A etiological examination of an outbreak of hemorrhagic syndrome in a broiler flock in Japan. Avian Pathol. 16:521–526. 27. Von Bülow, V. 1991. Avian infectious anemia and related syndromes caused by chicken anemia virus. Crit. Rev. Poult. Biol. 3:1–17. 28. Sommer, F., and C. Cardona. 2003. Chicken anemia virus in broilers: Dynamics of infection in two commercial broiler flocks. Avian Dis. 47:1466–1473. 29. Cardona, C., B. Lucio, P. Oconnell, J. Jagne, and K. Schat. 2000. Humoral immune responses to chicken infec-
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tious anaemia virus in three strains of chickens in a closed flock. Avian Dis. 44:661–667. 30. Ballal, A., A. M. Elhussein, and I. S. A. Abdelrahim. 2005. Serological survey of chicken infectious anemia in commercial chicken flocks in Khartoum state . Sudan. J. Anim. Vet. Adv. 4:666–667. 31. Hadimli, H. H., O. Ergains, L. Guler, and U. S. Uan. 2008. Investigation of chicken infectious anemia virus infection by PCR and ELISA in chicken flocks. Turk. J. Vet. Anim. Sci. 32:79–84. 32. Rouassan, D. A. 2006. Serological survey on the prevalence of CIVA in commercial broiler chicken flock in Northern Jordan. Int. J. Poult. Sci. 5:544–546. 33. Toroghi, R., A. H. Shoushtari, S. Charkhkar, and M. Niazi. 2003. The first report of incidence of chicken infectious anaemia disease in broiler flocks of Iran. Pages 240– 243 in Proc. 13th Iranian Vet. Assoc. Congr., Tehran, Iran. Iranian Vet. Assoc. Publishing Co., Tehran, Iran. 34. Mahzounieh, M., I. Karimi, and T. Zahraei Salehi. 2005. Serological evidence of chicken infectious anaemia in commercial chicken flocks in Shahrekord, Iran. Int. J. Poult. Sci. 4:500–503. 35. Farhoodi, M., R. Toroghi, M. R. Basami, M. Kianzadeh, and S. Charkhkar. 2007. Infection of chicken infectious anaemia virus in broiler flocks of Iran. Arch. Razi Inst. 62:1–6. 36. Gholami-Ahangaran, M., H. Momtaz, N. Zia-Jahromi, and M. Momeni. 2011. Genomic detection of the chicken anaemia virus from apparently healthy commercial broiler chickens in Iran. Rev. Méd. Vét. (Toulouse) 162:604–606. 37. Els, H. J., and D. Josling. 1998. Viruses and virus-like particles identified in ostrich gut contents. J. S. Afr. Vet. Assoc. 69:74–80. 38. McNulty, M. S., T. J. Connor, F. M. C. Neilly, K. S. Kilrkapatrick, and J. B. McFerran. 1988. A serological survey of domestic poultry in the United Kingdom for antibody to chicken anaemia agent. Avian Pathol. 17:315–324.
Acknowledgments
This study was supported financially by the Islamic Azad University, Shahrekord Branch, Iran.
Seromolecular study of chicken infectious anemia in chickens, ostriches, and turkeys in Iran Majid Gholami-Ahangaran,*1 Ezatollah Fathi-Hafshejani,* and Ravand Seyed-Hosseini† *Poultry Diseases Department, Veterinary Medicine Faculty, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran; and †Graduated from Diploma Degree in Poultry Health and Diseases, Shahrekord University, Shahrekord, Iran Primary Audience: Veterinarians, Researchers, Flock Supervisors SUMMARY Until recently, the chicken has been considered the only natural host and the main host for chicken anemia virus (CAV). However, CAV infection has now been reported in other avian species, including Japanese quail, jackdaws, rooks, and rare avian breeds. In this study, we examined chickens, ostriches, and turkeys for seromolecular detection of CAV in Iran. For the study, 375 serum and thymus samples from chickens, 84 serum and blood samples from ostriches, and 100 serum and blood samples from turkeys were collected from various locations in Iran. Serum samples were examined using ELISA. Deoxyribonucleic acid was extracted from the thymus, and blood samples were analyzed for detection of the VP2 genome of CAV by PCR. According to these results, 69.07 and 58.4% of chickens were positive for the antibody to CAV and the VP2 genome of CAV, respectively. In addition, in ostriches 27.38 and 34.52% were positive for the CAV antibody and the CAV VP2 genome, respectively. In turkey flocks, all turkeys (100%) were negative with respect to detection of the VP2 CAV genome and CAV antibody. It was concluded that CAV is distributed in broiler chickens and ostriches throughout Iran. Furthermore, ostriches may be considered a reservoir of CAV, and turkeys appear not to be susceptible to CAV infection. Key words: chicken, chicken anemia virus, Iran, ostrich, turkey 2013 J. Appl. Poult. Res. 22:404–409 http://dx.doi.org/10.3382/japr.2012-00567
DESCRIPTION OF PROBLEM The Circoviridae family contains 2 genera [1]. Chicken anemia virus (CAV) is the only member of the genus Gyrovirus, whereas the genus Circovirus currently comprises porcine circoviruses type 1 and 2, psittacine beak and feather disease virus, pigeon circovirus, canary circovirus, goose circovirus, and duck circovirus [1]. 1
Corresponding author: [email protected]
Members of the Circoviridae family are nonenveloped, regular icosahedrons and are the only bird viruses with a circular, single-stranded DNA genome [2]. These viruses share many epizootiological and pathological similarities (i.e., young age of affected birds, particular tropism for the lymphoid tissue and organs, related acquired immunosuppression and secondary infections) [3, 4].
Gholami-Ahangaran et al.: CHICKEN ANEMIA VIRUS Chicken anemia virus was first isolated and described in Japan by Yuasa et al. [5], but CAV antibodies have been detected in chicken sera worldwide [5, 6]. This virus has been found in most countries with poultry industries [7]. The virus spreads vertically from parental stock to progeny and horizontally by contact exposure with infected chickens or fomites. Chicken anemia virus infection induces either clinical or subclinical signs [8]. Clinical disease occurs when chicks are infected during the first 2 wk of life, but it can be avoided if hens transfer sufficient antibodies to their progeny. After 2 wk of age, chicks can be infected with the virus but do not develop clinical signs [9]. This virus can cause economically important losses with either the clinical or subclinical form of the disease in broiler chickens [10, 11]. Signs and lesions include stunting, increased mortality, anemia, bone marrow cell depletion, subcutaneous hemorrhage, and atrophy of secondary lymphoid organs [12]. This infection is often associated with opportunistic viral and bacterial infections and vaccination failures in chicken flocks [8, 13]. Chicken anemia virus was previously known as a much-conserved virus of one serotype [5] with several genetic groups [8]. However, an additional serotype has recently been reported [8, 14]. Until now, the chicken was considered to be the only natural host and the main host of CAV [4], but CAV infection has been reported in other avian species, including Japanese quail [15], fancy chicken breeds [16], jackdaws, rooks, and some rare avian breeds [17]. In contrast, the antibody to CAV was not found in some birds, such as the duck, pigeon, or pheasant [4]. In this study, chicken, ostrich, and turkey sera were tested by using a commercial ELISA for the presence of antibodies to CAV and for detection of the CAV genome by PCR in these birds in Iran.
MATERIALS AND METHODS
405
were collected from each flock. On average, all sampled flocks had a mortality higher than 1% per day for 10 d during the growing period. Eighty-four serum and blood samples were collected from 15 ostrich flocks with ages ranging from 8 to 15 mo of age. At least 5 serum samples were collected from each flock. These sera were collected from 5 processing facilities across Iran from 2009 to 2010. In addition, 100 serum and blood samples were collected from 10 healthy turkey flocks of different ages in various locations in Iran from 2009 to 2010. All commercial poultry flocks (chicken, ostrich, and turkey) examined in this study had not been vaccinated against CAV, and no clinical signs suggestive of CAV infection were observed in any of the flocks. All commercial poultry flocks were reared on litter, and feed and water were supplied ad libitum. ELISA Blood samples were collected via venipuncture of the wing vein. The blood was allowed to clot at room temperature for 30 min and centrifuged (1,500 × g, 15 min, room temperature). Serum was decanted into a microtube and inactivated by heating at 56°C for 30 min, and then stored at −20°C until tested. The sera were tested by indirect ELISA using a commercial competitive CAV ELISA Kit [18]. A serum dilution of 1:10 was used following the manufacturer’s instructions. The optical density value was read at a 650-nm wavelength on an ELISA reader [19] according to the manufacturer’s instructions. The presence or absence of the antibody to CAV was determined by sample-to-negative (S:N) ratios for each sample. Samples with an S:N value of less than 0.6 were defined as positive, and those with a value equal to or greater than 0.6 were interpreted as negative. A positive sample indicates the presence of CAV antibodies and previous exposure to CAV.
Flocks
PCR
Serum and thymus samples (375) were collected from 25 broiler chicken flocks in the Isfahan, Yazd, Tehran, and Chaharmahal-va-Bakhtiyari Provinces, in the central area of Iran, from 2009 to 2010. An average of 15 serum samples
Deoxyribonucleic acid extraction from tissue and blood samples was carried out using a High Pure Viral Nucleic Acid Kit [20] according to the manufacturer’s instructions. Polymerase chain reaction was carried out to amplify a fragment
JAPR: Research Report
406 of 713 bp from the viral protein 2 (VP2) gene of CAV. The sequence of the primers was as follows: forward primer: 5′-GCG CAC ATA CCG GTC GGC AGT; reverse primer: 5′-GGG GTT CGG CAG CCT CAC ACT AT [21]. Polymerase chain reaction amplification was performed in PCR buffer containing 1.5 mM MgCl2, 200 μM each deoxynucleotide 5′-triphosphate, 10 pM each primer, and 1.0 unit of Taq polymerase [22] in a 25-μL total reaction volume. The amplification was carried out in a thermal cycler [23] under the following conditions: initial denaturation of 94°C for 4 min, followed by 34 cycles of denaturation, annealing, and extension at 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, respectively, and a final extension at 72°C for 5 min. The PCR product was then analyzed by electrophoresis in 1% agarose gel and visualized under UV light after staining with ethidium bromide. In this study, Cuxhaven-1 strains of CAV [24] were provided and used as a positive control, and deoxyribonuclease-free water was used as a negative control.
RESULTS AND DISCUSSION ELISA All broiler chicken flocks were found to be positive at 7 to 8 wk of age. The rate of seropositive chickens among flocks ranged from 20 to 100%, and the prevalence was always more than 20%. The seroprevalence of CAV was 69.07% in chickens. The minimum and maximum of the mean S:N value were 0.18 and 0.65, respectively (Table 1). Eleven out of 15 ostrich flocks (73.33%) were positive for antibodies against CAV. The seroprevalence of CAV was 27.38% in ostriches. The mean CAV S:N value of the ostriches examined was 0.672, whereas the minimum and maximum S:N values were 0.20 and 0.98, respectively (Table 2). In this study, all turkey flocks (100%) were found to be negative for antibodies to CAV. The mean CAV S:N value of turkeys examined was 0.757, whereas he minimum and maximum S:N values were 0.62 and 0.98, respectively.
Table 1. Enzyme-linked immunosorbent assay results in the chicken flocks tested Flock no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1
CAV S:N ratio,1 mean ± SD
CV, %
Positive ELISA sample, %
Positive PCR sample, %
0.31 ± 0.18 0.59 ± 0.17 0.35 ± 0.18 0.20 ± 0.14 0.36 ± 0.08 0.37 ± 0.18 0.54 ± 0.27 0.29 ± 0.16 0.49 ± 0.17 0.57 ± 0.15 0.59 ± 0.19 0.40 ± 0.22 0.29 ± 0.17 0.31 ± 0.19 0.43 ± 0.17 0.49 ± 0.16 0.48 ± 0.12 0.18 ± 0.13 0.57 ± 0.21 0.32 ± 0.15 0.37 ± 0.23 0.65 ± 0.21 0.51 ± 0.26 0.49 ± 0.15 0.61 ± 0.23
58.3 30.6 51.2 69.2 22.3 48.5 50.3 54.7 35.2 26.5 32.1 55.8 59.8 61.9 40.2 34.1 25.4 73.4 37.0 47.9 61.9 32.8 51.2 31.1 37.2
100 46.7 86.7 100 100 80 53.3 100 66.8 60 20 73.3 86.7 86.7 73.3 60 66.7 100 26.7 100 80 20 46.7 66.7 26.7
86.6 40 73.3 100 100 80 40 80 46.6 40 13.3 73.3 80 73.3 66.6 46.6 53.3 93.3 20 86.6 60 6.6 26.6 46.6 26.7
CAV S:N ratio = chicken anemia virus sample-to-negative ratio.
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Table 2. Enzyme-linked immunosorbent assay results in the ostrich flocks tested Flock no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample size, no.
CAV S:N ratio,1 mean ± SD
CV, %
Positive ELISA sample, %
Positive PCR sample, %
8 6 6 7 5 6 7 5 8 4 3 7 5 4 3
0.66 ± 0.147 0.81 ± 0.121 0.73 ± 0.120 0.72 ± 0.126 0.77 ± 0.144 0.52 ± 0.144 0.68 ± 0.232 0.46 ± 0.241 0.74 ± 0.153 0.74 ± 0.127 0.82 ± 0.151 0.76 ± 0.168 0.72 ± 0.170 0.37 ± 0.156 0.52 ± 0.112
22.1 14.9 16.3 17.5 18.6 27.3 34 52.3 20.4 17 18.3 22 23.3 41.2 21.4
25 0 16.66 14.28 0 83.33 28.57 60 20 0 0 14.28 20 100 66.66
25 16.66 16.66 28.57 0 83.3 28.57 60 37.5 0 0 28.57 20 100 100
PCR In this study, all broiler chicken flocks (100%) were positive for CAV. On the basis of PCR results, 219 of 375 (58.4%) thymus samples from 25 broiler chicken flocks were positive for CAV (Table 1). The minimum and maximum values of positive chickens in all tested flocks were 6.6 and 100%, respectively. The CAV genome in this study was detected in 29 of the 84 (34.52%) blood samples collected from 15 ostrich flocks in Iran. The VP2 genome was detected in 80% of the examined flocks. The positivity rate ranged from 14.28 to 100% in all tested flocks (Table 2). In the turkey flocks tested, all turkeys (100%) were negative with respect to detection of the VP2 CAV genome in blood samples. Chicken anemia virus was first recognized in Japan [5]. Chicken anemia virus infection in chicken flocks has now been described in most countries and can result in economical losses from either clinical or subclinical infections [10, 11]. The CAV causes clinical disease in young chicks, and affected chicks experience increased mortality associated with anemia, aplasia of the bone marrow, and lymphoid depletion [25]. The clinical CAV infection is mainly noticed in young chicks with subcutaneous and muscular hemorrhages [26]. Older chickens are susceptible to this subclinical infection [27]. The clinical form of the disease is rare today because of the widespread practice of vaccinating breeders,
but the subclinical form of CAV is ubiquitous [28]. There are many reports of CAV infections in most chicken-producing countries in chicken flocks throughout the world [29], such as in Sudan [30], Japan [15], Turkey [31], and Jordan [32]. The clinical form of CAV infection was studied in chicken flocks in Iran [33–35]; however, the present study represents a seromolecular study for the prevalence of subclinical CAV infection in commercial broiler chickens in Iran. In this study, the seroprevalence of subclinical CAV infection was 69.07 and 100% in broiler chickens and broiler chicken farms, respectively. The PCR results confirm the serological results. This finding is accordance with and similar to previous reports in Iran of clinical CAV infection [35]. However, the infectivity of chicken flocks to CAV in flocks is expected because the individual viral prevalence of CAV in apparently healthy broiler chickens in Iran was reported as 24.58% and the rate of positive flocks reached 73.33% [36]. Therefore, CAV seems to be widespread in broiler chickens throughout Iran, and the results provide evidence of widespread distribution of the virus and a high incidence of infection among commercial broiler flocks in Iran, as has similarly been documented to occur worldwide in all major poultry-producing countries [29]. Until now, the chicken was thought to be the only host of CAV. However, in this study we found a high incidence of seropositivity to CAV in ostrich flocks, and these findings were con-
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408 firmed with PCR. Whether CAV causes clinical disease in ostriches and whether the virus in these birds is CAV or another circovirus with cross-reacting properties need further investigation. Although a circovirus-like infection has been reported in ostriches [37], the distribution of this virus among ostriches is unknown. As far as is known, there are no reports showing that the circovirus of ostriches does cross-neutralize with CAV. In addition, circovirus has been observed by electron microscopy in the intestinal contents of an ostrich (Struthio camelus); however, it is uncertain whether the ostrich was actually infected with the virus or whether the virus was obtained from ingested plant matter [37]. Therefore, according to our findings, the ostrich can be considered a reservoir of CAV. We conclude, based on our review of the related literature, that although the chicken has been considered the natural host for CAV, antibodies to CAV have been detected in Japanese quail in Japan [15], in fancy chicken breeds in the Netherlands [16], and in jackdaws, rooks, and some rare avian breeds in Ireland [17]. In contrast, the antibody to CAV was not found in turkeys and ducks in the United Kingdom [38]; in pigeons, ducks, and pheasants in Ireland [17]; and in crows, pigeons, and ducks in Japan [15]. Furthermore, 1-d-old turkey poults inoculated with CAV did not show clinical signs of anemia and did not develop antibodies to the virus [38]. However, on the basis of negative results obtained with sera collected from a turkey flock, all flocks tested appeared not to have been infected with CAV. To our knowledge, this is the first report of the detection of serological and molecular CAV in species other than the chicken.
CONCLUSIONS AND APPLICATIONS
1. Chicken anemia virus was found to be widespread in broiler chickens and ostriches throughout Iran. 2. In this study, seropositivity to CAV was found in ostrich flocks, and these findings were confirmed by PCR. Therefore, the ostrich can be considered a reservoir of CAV. 3. In this study, all turkey flocks (100%) were found to be negative with regard
to antibodies to CAV and detection of the VP2 CAV genome in blood samples. Therefore, it seems turkeys are not susceptible to CAV infection.
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Acknowledgments
This study was supported financially by the Islamic Azad University, Shahrekord Branch, Iran.