Abstract: Background: The reticuloendotheliosis virus (REV) in chicken induces a variety of deleterious effects, tumors and increased mortalities causing considerable economic losses. In this study the reticuloendotheliosis infection status were performed through antibody determination and histopathological findings in chickens in delta Egypt. Materials and Methods: Fourteen cross-bred chicken flocks varied from 12-25 weeks age and reared in two provinces in delta Egypt were investigated. About 691 birds were submitted for pathological investigations. Two hundred and seventy one serum samples were collected for serology. Further 20 samples from seropositive and seronegative birds were used for hematological screening. Results: In total of 691 birds 278 birds (40.2%) unusual tumor formation in the head and neck regions and enlarged liver, spleen and kidneys. The features of common pathologic lesions were shown as multiple neoplastic nodules of homogeneous lymphocytes in the livers and spleens. Histopathology of the head region revealed hypercellularity with intercellular transudate, focal aggregation of inflammatory cells around the diluted blood vessels and hemorrhages in tumor mass. Visceral lymphomas occurred in a 25 weeks old chicken flock. The serological prevalence for the REV antibody ranged from 25.0-100.0% at 12th and 25th weeks of age, respectively. The total WBCs, heterophils and monocytes count were significant higher in birds with tumor. Conclusion: The present study describes the first report for prevalence of REV in cross-bred chicken in delta region in Egypt. The serological results suggested circulation of REV within chicken in delta Egypt. Further study is paramount for improving the control measures for REV in chickens.
INTRODUCTION
Reticuloendotheliosis viruses (REV) are a group of avian retroviruses that infect turkeys, ducks, quails and chickens causing marked immunosuppression and disease which is sporadically neoplastic1,2. The virus is widespread and the infection can be transmitted by vertical or horizontal means. The horizontal transmission takes place by direct and indirect contact. The virus could be detected in feces, cloacal swabs and litter and could be disseminated by needles used for administration of antibiotics and vitamins in hatcheries as well as by inoculation with contaminated biologics3. The introduction of the virus into chicken flocks via commercial vaccines contaminated with this virus has been documented4,5. The REV was probably transmitted to the commercial chickens through congenital transmission6.
The REV infections can cause various pathologic lesions1. The most common clinical diseases induced by REV are including runting-stunting disease syndrome, characterized by growth retardation and poor feathering with acute reticular cell neoplasia and chronic B-cell and T-cell lymphomas. The losses and mortalities in affected flocks with REV can be significant due to tumor and or immunosuppression7. Although REV is wide spread, REV-induced clinical disease is infrequently diagnosed in commercial flocks8. Reticuloendotheliosis cases have been rarely diagnosed in commercial chicken flocks, even though REV infection has been identified in a high proportion of chicken farms by serology and virus isolation8. Serological studies have however, indicated that REV infection may be common in commercial chicken flocks9, although isolation of REV from seropositive chicken flocks has been either unsuccessful or difficult2,9. An enzyme-linked immunosorbent assay (ELISA) was successful tool for the detection of avian reticuloendotheliosis10. The ELISA has advantages over other tests for the routine detection of avian leukosis virus infection in chickens11,12. A microplate indirect enzyme-linked immunosorbent assay (ELISA) for antibodies against reticuloendotheliosis virus (REV) was consistently more sensitive than indirect immunofluorescent-antibody tests and suitable for screening chicken flocks12.
There are no commercial vaccines are available for control of REV infection and no method has been routinely used by industry to control REV infection in commercial turkey and chicken flocks13-15. This study characterized an outbreak of a visceral lymphoma in a commercial cross-bred chicken flock in delta region of Egypt.
MATERIALS AND METHODS
Flock history: Between 2011 and 2012, in 14 cross-bred chicken flocks aged between 12-25 weeks, showed runting, paleness of the face, swelling in the head region, lameness and abnormal feathering as well as tumor-like lesions in the skin, liver and spleen were observed and REV infection was suspected. The farms are located in two provinces in delta Egypt, Kafrelsheikh and Elgharbia provinces. The birds were reared on floor. The birds were vaccinated against Marek’s disease, newcastle disease, infectious bronchitis, infectious bursal disease, fowl pox, infectious laryngeotracheitis and avian influenza. Apparent healthy, diseased and dead birds showed above mentioned lesions were submitted to the Laboratory of Poultry Diseases, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, Egypt for final diagnosis. Age and number of submitted birds are shown in (Table 1).
Pathological and histopathological examination: Postmortem examination was performed on diseased and freshly dead birds collected from all investigated flocks. In total of 102 suspected tissues as livers, spleens, gizzard and proventriculus were examined for gross and microscopic lesions. The tissue specimens were fixed in 10% neutral buffered formalin solution, embedded in paraffin, sectioned at approximately 5-6 micrometers and stained with Hematoxylin and Eosin (H and E) for microscopical examination16.
REV antibody detection: In total 12-22 serum samples were collected from each flock for detection of antibodies against REV. Serologic testing was performed by IDEXX REV Ab test using a commercial ELISA kit (IDEXX, Westbrook, ME, USA), according to the manufacturer’s instructions. In a flock, if the flock seropositive prevalence lay above 10% was considered to be infected with REV12.
Hematological studies: Based on the results of serological examination 20 heparinized blood samples from each seropositive and seronegative birds were submitted to hematological examinations.
RESULTS
Clinical and necropsy findings: Affected chickens showed runting, paleness of the face, swelling in the head region and abnormal feathering. Out of submitted 691 birds 278 birds showed tumors (Table 1). The percent of dead birds showing tumor lesions ranged 6.7-51 and 50%.
| Table 1: | Investigated farms in this study, total number in each farm, number and age of investigated bird and percentage of birds with tumors |
| Fig. 1: | Unusual tumor formation in the head region gross lesions of head region of cross-bred chicken 25 weeks of age with tumors formation |
Percentage of birds with tumor lesions started at 12th weeks of age and gradually increased in other flocks to reach 28.6% at 18th weeks of age, 44.7% at 22nd weeks of age and 51.5% at 25th weeks of age (Table 1).
The postmortem examination of dead and sacrificed birds revealed moderately to markedly emaciated carcasses, unusual tumor formation in the head region (Fig. 1), congested and enlarged liver, spleen, proventriculus and gizzard with whitish nodular infiltrations in livers and spleens were commonly observed in submitted chickens. In addition, neoplastic nodules in the intestine, pancreas and mesentery could be detected (Fig. 2).
Histopathological pictures: Multiple nodules were observed in the liver, spleen and intestine consisted of homogeneous
immature lymphocytes. The head region showed presence of collagen fibers in circular and in parallel or interlake arrangement, hypercellularity, clusters of lipoblasts of varying size, intercellular transudate, dilated blood vessels and aggregation of inflammatory cells surround the blood vessel (Fig. 3a-h).
Some areas were more cellular than others, these cells mainly fibroblasts, which separated from each other by intercellular transudate and focal localized aggregation of inflammatory cells mainly lympho-reticular cells, lymphocytes and histocytes. Aggregation of inflammatory cells surrounded congested blood vessels (Fig. 3). The dilated blood vessels sometimes contain transudate and inflammatory cells (Fig. 3). Mitotic figures of fibroblast were observed (Fig. 4).
| Fig. 2: | Congested and enlarged liver, proventriculus and gizzered with whitish nodular infiltrations organ obtained from chicken with 25 weeks age |
| Table 2: | Reticuloendotheliosis antibody positive percentage in 14 chicken flocks with age ranged from 12-25 weeks using ELISA |
Clusters of lipoblasts of varying size were distributed throughout the tumor mass (Fig. 4). Several nests of tumor giant cell insinuated throughout the tumor mass and hemorrhages were detected (Fig. 5).
Serological investigation: All 14 serologically tested flocks had antibodies to REV. The seropositive prevalence of the REV antibody in cross-bred chicken flocks were 25% (5 out of 20) at 12 weeks of age flock, 65% (13 out of 20) at 14 weeks of age, 83% (10 out of 12) at 15 weeks, 95% (19 out of 20) at 21 weeks, 82% (14 out of 17) at 22 weeks and chicken flock at 25 weeks of age represented significant high ELISA titers against REV 100% (22 out of 22) (Table 2).
Hematological results: The results of blood picture and differential leucocytic count of investigated birds are summarized in Table 3. The total WBCs count revealed significant higher in birds with tumor compared to the birds without lesions. In addition, there were significant differences in heterophils and monocytes count in examined chicken flock as compared with control normal birds.
DISCUSSION
Reticuloendotheliosisis considered as one of the most prevalent pathogens that cause neoplasms in various avian species1. The REV has been frequently detected in chickens by serology and virology surveys, few RE field cases have been reported2,8,9. Several reports have described RE outbreaks related to vaccination with contaminated FPV17,18.
The present study demonstrated that REV infection in the field is quite common in poultry flocks in Egypt. Affected chickens suffered from illness, sudden death, abnormal swelling in the head region, runting, mild proventriculitis and feathering abnormality.
| Fig. 3(a-h): | Pathological changes in the head showing the (a) Collagen fibers in circular, (b) In parallel or interlake in tumor mass arrangement in tumor mass, (c-d) Hypercellularity in tumor mass, (e) Clusters of lipoblasts of varying size in tumor mass, (f) Intercellular transudate in tumor mass, (g) Dilated blood vessels and (h) Aggregation of inflammatory cells surround the blood vessel |
| Table 3: | Hematological changes in cross-bred chickens during REV outbreak |
| *Significant different from control at p<0.001 and **Control is cross-bred chicken was serologically negative for REV and clinically healthy birds | |
| Fig. 4: | Section from head region with tumor formation showing mitotic figure of tumor mass (H and E x1000) |
| Fig. 5(a-c): | Section from head region showing nest of tumor giant cells in tumor mass H and E, (a) x100, (b) x250 and (c) x1000 |
Similar findings were also observed in breeder chicken flocks infected with REV at 13th, 39th and 63rd weeks of age19. Mortality due to tumor lesions were demonstrated in sporadic cases at 12th weeks of age and gradually increased in other flocks with advanced age. Percentage of examined birds with tumor reach 28.6% at 18th weeks of age, 42.7% at 22nd weeks of age and 51.5% at 25th weeks of age. These findings are in accordance with previous study conducted in broiler breeder showed bone and visceral tumor20.
The gross lesions found on autopsy in commercial cross-bred chickens at 12-25 weeks of age, which were creamy and friable tumor in the head, congested and enlarged liver, spleen, proventriculus and gizzard with nodular tumor infiltration. These findings in agreement with previous studies20,21 conducted in commercial broiler breeder flock, the breeder suffered from visceral and bone tumor due to infection with avian leukosis virus associated with REV and Marek’s disease virus infections.
Histopathological examination results revealed that the tumor in the head region consists of solid mass of collagen fibers, fibroblasts and aggregation of inflammatory cells mainly large mononuclear lymphoreticular cells, lymphocytes and histocytes. Mitotic figures of fibroblasts with lipoblasts of varying size with several nests of tumor giant cells insinuated throughout the tumor mass and hemorrhages were detected Similar findings were reported in previous studies8,21. Macroscopically, neoplastic nodules were observed in the livers, spleens and intestines of investigated chickens and tissue samples. These findings were similar to those described previously in chickens infected with REV4,16,22,23. Lymphoma outbreak of REV infection is closely related to virus dosage and host age4,24,25. Based on these results, the pathognomonic lesions were related to REV infection. Finally, a lymphoma outbreak seemed to be caused by REV infection.
The ELISA is more effective at evaluating the presence of REV antibodies than other serologic tests, including agar gel precipitation test and indirect immunofluorescent antibody test12. Concerning the serological results, ELISA titers against REV were detected in positive result with a low titer obtained in 12 weeks old positive chicken flock (25%) and then significantly increased to the highest value in a positive flock (100%) at the age 25th weeks. Similar results were demonstrated in study conducted in Taiwan for serological and virological surveys of reticuloendotheliosis in chickens19. Moreover, study in USA showed that all tested 6 broiler breeder flocks were infected with reticuloendotheliosis2. In contrast to the higher frequency in this study, previous study showed that 14 of 44 broiler breeder flocks in USA were seropositive on a single sampling9. Moreover, REV seropositive rates can increase when chickens are infected with a high dose of REV18,26,27.
Hematological examination revealed significant increase in the total WBCs count and heterophils. Similar results were described in chicken infected with reticuloendothrliosis virus and in broiler breeder chicken infected with avian leucosis virus2,20, but different from this results in that there was significant decrease in the number of monocytes in REV-infected chickens as compared with normal control chickens. The significant increase in total WBCs count and the significant difference in heterophils and monocytic count may refer to multi-causal agents of tumor formation.
The findings of this study indicate the wide spread REV infection in commercial cross-bred chickens in Egypt. The serological results suggested circulation of REV within chicken in delta Egypt. Further study is paramount for improving the control measures for REV in chickens.
ACKNOWLEDGEMENTS
We thank the co-workers in the Institute of Poultry Diseases, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt for their cooperation and technical assistance.