Reovirus Infections in Young Broiler Chickens Author(s): James F. Davis, Arun Kulkarni, and Oscar Fletcher Source: Avian Diseases, 57(2):321-325. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10515-021313-Case.1 URL: http://www.bioone.org/doi/full/10.1637/10515-021313-Case.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
AVIAN DISEASES 57:321–325, 2013
Case Report— Reovirus Infections in Young Broiler Chickens James F. Davis,AD Arun Kulkarni,B and Oscar FletcherC A
Department of Diagnostics, Georgia Poultry Laboratory Network, P.O. Box 20, Oakwood, GA 30566 B Department of Virology, Georgia Poultry Laboratory Network, P.O. Box 20, Oakwood, GA 30566 C College of Veterinary Medicine, North Carolina State University, Population Health and Pathobiology, 1060 William Moore Drive, Raleigh, NC 27607 Received 7 November 2012; Accepted 18 February 2013; Published ahead of print 27 February 2013 SUMMARY. Selected 10-to-21-day-old broiler chickens from flocks undergoing runting-stunting syndrome were found to have significant pancreatic damage, grossly and histologically. Six reoviruses, with sequences that varied, both from each other and from S1133 reovirus, were isolated from these pancreases and from pancreases of specific-pathogen-free leghorn sentinels placed on two of the broiler farms for 7 days. RESUMEN. Reporte de Caso—Infecciones por reovirus en pollos de engorde jo´venes. Se encontro´ dan˜o pancrea´tico significativo macrosco´pico y microsco´picamente en pollos de engorde de 10 a 21 de edad seleccionados por bajo crecimiento en parvadas con sı´ndrome de mala absorcio´n. Se aislaron seis reovirus, con secuencias que variaron tanto unos de otros como con respecto a la de cepa S1133, a partir del pa´ncreas de las aves afectadas y del pa´ncreas de aves Leghorn libres de pato´genos especı´ficos que fueron alojadas como centinelas en dos granjas de pollos de engorde por siete dı´as. Key words: reovirus, pancreas Abbreviations: aa 5 amino acid(s); CEK 5 chicken embryo kidney; H-SMS 5 hypoglycemia-spiking mortality syndrome; NBF 5 neutral buffered formalin; RSS 5 runting-stunting syndrome; RT 5 reverse transcriptase; SPF 5 specific pathogen free
Reoviruses are 75-nm RNA viruses (6,15) that have been reported to be vertically transmitted in chickens, resulting in tenosynovitis in the offspring (16). Reoviruses have been associated with degeneration and necrosis of pancreatic acinar cells in mice (7). In addition, two mouse Reovirus serotypes (1 and 3) have been shown to infect and injure the endocrine areas of the pancreas in mice, with a resulting drop in serum insulin levels that causes abnormalities in glucose tolerance (17). In poultry, reoviruses have been reported to infect and cause lymphoplasmacytic and fibroheterophilic tenosynovitis in tendons (6,8,10,11,13) and to also infect intestines (5,12), heart (1,3,14), and liver (9). This report describes pancreatic vacuolar change and atrophy in broiler chickens associated with reovirus infections. HISTORY
During early 2012, multiple groups of young broiler chicks, aged 10–21 days, were submitted to The Georgia Poultry Laboratory for necropsy and diagnoses. The primary complaints were sudden ‘‘spiking’’ mortality and poor growth rate. Runting-stunting syndrome (RSS) was suspected. CLINICAL FINDINGS AND PROCEDURES
Clinical signs included small runted chicks that were passing undigested feed. Some chicks were live and some were dead upon arrival at the laboratory. Live birds were euthanatized using CO2 gas and then heart-bled to obtain sera for antibody testing against D
Corresponding author. E-mail: [email protected]
reovirus by using ELISA (IDEXX Laboratories, Westbrook, ME). Blood from selected chicks also was submitted for blood glucose level determinations if the chicks seemed to be weak and ataxic. Upon necropsy, thin-walled intestines, consistent with RSS, were the primary lesions. In addition, a number of the 10–21-day-old chicks had what appeared to be pale, atrophic pancreases. Eleven severely runted broilers, from two of the affected farms, were submitted at 36 days of age for follow-up exams (five from one farm and six from the second farm). Blood was collected for ELISA testing. Pancreatic atrophy was found on necropsy in birds from both groups. All grossly abnormal pancreases were aseptically detached from the duodena by using sterile scissors and forceps, with extreme care being taken to ensure duodena were not cut. Each pancreas was cut into multiple sections, and half of the sections from each bird were submitted in sterile Whirl-PaksH (Nasco, Fort Atkinson, WI) for virus isolation attempts. The other sections were put into SurgipathH10% neutral buffered formalin ([NBF]; Leica Biosystems, Richmond, IL) for histopathology processing. We also collected 2.5-cm (1-inch)-long segments of duodena and jejuna in NBF for histopathology. Three groups of 12 21-day-old specific-pathogen-free (SPF) leghorn chickens were placed on two of the broiler farms where the affected chickens were grown, for 7 days, to serve as sentinels. Pancreases, duodena, and jejuna were all grossly normal, but they were collected and processed for virology and histopathology as described above. CLINICAL PATHOLOGY
Whole blood glucose levels were evaluated from a small number of selected pale, runted, and apparently weak ataxic chicks by using a
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Table 1. Amino acid similarities of the sigma C gene of the GA/GPL reovirus isolates with reference strains.A % aa identity with the isolates from the present study Reference strainsB
Isolated fromC
12257D
12274E
12297F
12298G
12342H
12355I
DQ872797 DQ872798 DQ872800 FR694197 DQ996603 DQ996602 L139002
RSS RSS RSS RSS PEMS-turkey Healthy-turkey 1133 vaccine strain
55.3 46.6 47.9 56.6 45.0 45.0 45.6
51.4 87.3 89.5 51.1 70.5 70.2 56.5
87.3 49.5 51.4 87.6 48.9 49.5 47.3
51.5 43.5 44.5 52.5 44.5 45.5 43.5
86.7 48.7 51.6 88.3 48.4 50.0 48.7
49.5 67.9 68.3 48.3 81.9 83.8 53.0
A
Results expressed as the percentage of similarity. Bold indicates the highest aa identity of the isolate from the present study. GenBank accession numbers of the reference strains. C Isolation source of the reference strain. D–I GA/GPL/isolates with GenBank accession JX983603, JX983599, JX983600, JX983604, JX983601, and JX983602, respectively. B
hand-held digital glucose meter (FreeStyleH Blood Glucose Monitoring System; TheraSense, Inc., Alameda, CA). VIROLOGY
Virus isolation and identification. Pancreases were homogenized in phosphate-buffered saline supplemented with antibiotics. Crude virus suspension was clarified by centrifugation, filtered through a 0.2-mm membrane filter, and used for inoculation of primary chicken embryo kidney (CEK) cell monolayers. The first two serial passages produced isolated foci of detached cells within 72 hr postinoculation. After the third passage, there was marked rounding and detachment of cells. Virus isolates from this study were designated as GA/GPL/12257, GA/GPL/12274, GA/GPL/12297, GA/GPL/12298, GA/GPL/12342, and GA/GPL/12355. GA/GPL/ 12274 was isolated from the pancreases of the sentinel birds. The remaining isolates came from 14–17-day-old broilers. Viral RNA was extracted from the clarified supernatants of CEKinfected monolayers by using the RNeasy mini kit (QIAGEN, Valencia, CA) according to manufacturer’s instructions. The amplification of the sigma C gene was performed using the OneStep RT-PCR kit (QIAGEN) and previously described primers ScP1 and ScP4 (9). The reverse transcriptase (RT)-PCR products of the virus were directly sequenced using the same set of primers in both directions. Sequencing reaction was performed using 30 ng of template per reaction with the BigDye Terminator version 3.1 cycle
sequencing kit (Applied Biosystems, Foster City, CA) at the Georgia Genomics Facility, University of Georgia (Athens, GA). Sequence analysis. The nucleotide and deduced amino acid (aa) sequence data were aligned by MegAlign program version 8.0 (DNASTAR Inc., Madison, WI) using the ClustalW multiple sequence alignment algorithm. The phylogenetic tree was constructed by the neighbor-joining method with MegAlign package (DNASTAR Inc.) with 1000 replicates. The reovirus reference strains that were closely related to present isolates by basic local alignment search tool (BLAST) were selected for analysis. Nucleotide sequences from previously identified isolates from the United States and from the commonly used U.S. vaccine strain S1133 were selected for analysis to understand the extent of genetic identity of the isolates from the present study. The GenBank accession numbers of various isolates used for analysis are listed in Table 1 and Fig. 1. Strain S1133 was included in the analysis to determine the relatedness of the isolates to the vaccine. RESULTS
Virology. The primer pair ScP1 and ScP4 used for the amplification of the sigma C gene in this study consistently amplified approximately 1000-bp product as described previously (GenBank accession L39002). Three of the six isolates—GA/GPL/ 12274, GA/GPL/12297, and GA/GPL/12355—showed .86% aa identity with the previously identified reovirus isolated from RSS
Fig. 1. Neighbor-joining phylogenetic trees with 1000 bootstrap replicates showing the relationships among aa-deduced sequences when phylogenetic analyses are conducted on an approximately 1000-bp sequence of the sigma C region of reovirus isolates. The sequences were obtained from six field isolates and another eight reference strains indicated with GenBank accession number. The numbers at the fork indicate the bootstrap values (1000 replicates).
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Reovirus and pancreatic degeneration
Table 2. Whole blood glucose levels from selected ataxic chicks with pancreatic degeneration and atrophy (normal 5 150–300 mg/dl). Flock
Age (days)
Blood glucose (mg/dl)
A B C
14 14 14
286, 244, 199 73A, 214, 213, 185 77A, 164, 130A, 115A
A
Hypoglycemia.
cases from Georgia (Table 1). Amongst these three isolates, GA/ GPL/12297 and GA/GPL/12355 also shared .87% identity with the European isolate FR694197. Interestingly, isolate GA/GPL/ 12355, derived from 14-day-old broiler pancreases, was .81% identical to two turkey isolates from the United States (4). Finally, isolates GA/GPL/12257 and GA/GPL/12298 shared ,55% aa identity with all of the reference strains analyzed in this study. It is noteworthy that, like previously described reovirus isolates from RSS cases, (Table 1), none of the isolates from the present study shared .52% identity with commonly used vaccine strains S1133 or 1733. The GenBank designations for the six isolates are as follows: GA/ GPL/12257, JX983603; GA/GPL/12274, JX983599; GA/GPL/ 12297, JX983600; GA/GPL/12298, JX983604; GA/GPL/12342, JX983601; and GA/GPL/12355m, JX983602. Phylogenetic relationships among field isolates of reovirus. Based on the predicted aa sequence of the amplified region of the sigma C gene from six isolates and seven reference strains, a
Table 3. ELISA reovirus antibody titers of runted broilers with pancreatic degeneration and atrophy. Samples were collected 22 days after flock diagnosis of reovirus infection of pancreas. Farm
Age (days)
Individual bird ELISA titers against reovirus S-1133
Group geometric mean titer
A B
36 36
1166, 500, 75, 327, 176 88, 276, 2183, 0, 31, 190
306 83
phylogenetic tree was constructed (Fig. 1). All of the isolates from the present study fall into three distinct groups. In the first group (group 1), isolates 12274 and 12355 were grouped with reoviruses of turkey and chicken origin, with the latter from the cases of RSS. In the second group, isolates 12297 and 12342 were closely related to European and U.S. isolates FR694197 and DQ872797, respectively, both from chickens experiencing RSS (group 2). Isolates 12257 and 12298, although identical to each other up to 78%, did not belong to any of the reference strains used in the analysis (group 3). None of the isolates from the present study belong to the previously described U.S. isolates S1133, 1733, or ARV-136 (group 4). Serology. Whole blood glucose levels showed hypoglycemia in four of the 11 chicks tested (Table 2). ELISA testing on sera from the 11 follow-up broilers revealed very little antibody against reovirus on an ELISA kit with wells coated with S1133 antigen (IDEXX Laboratories; Table 3).
Fig. 2. (A) Section from jejunum shows normal villi and absence of crypt lesions. (B) Villous atrophy with no crypt lesions is in this section of jejunum. (C) Villous atrophy with multiple cystic and dilated crypts is in the jejunum. (D) Higher magnification shows villous atrophy and dilated, cystic crypts. Bar 5 250 mm. Age 5 17 days.
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Fig. 3. (A) Vacuolar degeneration and necrosis are in the pale-stained region of pancreas (left). Bar 5 250 mm. (B) Higher magnification of the pale region in panel A shows loss of granules in acinar cells. Bar 5 100 mm. (C) Higher magnification at margin of the pancreas shows vacuolar degeneration and necrosis of acinar cells. Bar 5 50 mm. (D) Higher magnification of lesion at the margin of the pancreas shows vacuolar degeneration and necrosis of acinar cells. Bar 5 25 mm. Age 5 17 days.
Histopathology. Sections from duodenum and jejunum from the 10–21-day-old broiler chickens had villous atrophy, sometimes with dilated and cystic crypts (Fig. 2). Lesions in pancreas included vacuolar degeneration, degeneration, and necrosis of acinar cells and also atrophy of acinar cells. Islets were not affected. Lesions were multifocal to regional (Figs. 3, 4). Similar pancreatic lesions of vacuolar degeneration and atrophy were found in the 36-day-old broilers. There were no lesions in the pancreas or intestines of the SPF leghorns.
DISCUSSION
Protection of day-old chicks against reovirus by maternal antibodies is achieved by vaccination of breeding stock. This vaccination is mostly performed in the United States using strains S1133 and 1733 or from a naturally apathogenic strain 2177. The observed sequence discrepancy between the isolates reported in the present study and the vaccine strain S1133 warrants further investigation. The reoviruses isolated from the pancreases and then sequenced differed significantly from previously reported reoviruses. These viruses seem to be closely associated with the increase in mortality in these broiler flocks. Severe hypoglycemia, such as that found with
hypoglycemia-spiking mortality syndrome (H-SMS) of broilers does not seem to be the cause of the flock mortality because pancreases examined in studies of H-SMS consistently were histologically normal (2), whereas the pancreases examined in our study were not. The hypoglycemia observed in birds from our study was likely due to the significant pancreatic damage found in these birds. Further investigations need to be done, including bird inoculations, to determine the pathogenicity and pathogenesis of these reoviruses. In addition, studies on the effect of these reovirus infections on pancreatic hormone and enzyme levels need to be conducted. Vaccination trials also need to be conducted to determine whether any currently available vaccines will protect against these reoviruses. The ELISA titers measured in the follow-up birds and the sequencing results suggest that the reoviruses infecting these birds were not closely related to S1133. Therefore, it seems likely that S1133-based vaccines will not protect against these reoviruses. In addition, the fact that the sequences of all six of these viruses varied with each other may produce additional problems with production of a vaccine that will protect against the pancreatic damage. None of the isolates from the present study belong to the previously described five genotyping clusters of reoviruses (9). We are not concluding that the reoviruses that we isolated from these diseased pancreases are the etiologic agents responsible for inducing the lesions. Further experimental investigations, including SPF bird inoculations, need to be conducted to determine whether this is the case.
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Fig. 4. (A) Low power view shows atrophy of a large area of pancreas. The lobe at the top is relatively unaffected. Bar 5 500 mm. (B) Higher magnification shows loss of granules in acinar cells. Tip of unaffected pancreas is in upper right. Bar 5 100 mm. (C) Higher magnification shows the atrophy of acinar cells. An uninvolved islet is in the lower left. Bar 5 50 mm. (D) Higher magnification shows atrophy of acinar cells. Note the absence of granules. Bar 5 25 mm. Age 5 17 days. REFERENCES 1. Bagust, T. J., and H. A. Westbury. Isolation of reoviruses associated with diseases of chickens in Victoria. Aust. Vet. J. 51:406–407. 1975. 2. Davis, J. F. Hypoglycemia-spiking mortality syndrome. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 1269–1272. 2008. 3. Davis, J. F., A. Kulkarni, and O. Fletcher. Myocarditis in 9- and 11day-old broiler breeder chicks associated with a reovirus infection. Avian Dis. 56:786–790. 2012. 4. Day, J. M., M. J. Pantin-Jackwood, and E. Spackman. Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses. Virus Genes 35:235–242. 2007. 5. Dees, T. A., R. E. Wooley, and J. B. Gratzek. Pathogenicity of bacteria-free filtrates and a viral agent isolated from turkeys with infectious enteritis. Am. J. Vet. Res. 33:165–170. 1972. 6. Glass, S. E., S. A. Naqi, C. F. Hall, and K. M. Kerr. Isolation and characterization of a virus associated with arthritis in chickens. Avian Dis. 17:415–424. 1973. 7. Jacoby, R. O., J. G. Fox, and M. Davison. Biology and diseases of mice, 2nd ed. In: Laboratory animal medicine. J. G. Fox, ed. Academic Press, San Diego, CA. p. 72. 2002. 8. Jones, R. C. Other reovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 310–328. 2008.
9. Kant, A., F. Black, L. Born, D. Van-Roozelaar, J. Heijmans, A. Gielkns, and A. T. Huurne. Classification of Dutch and German avian reoviruses by sequencing of the sC protein. Vet. Res. 34:203–212. 2003. 10. Levisohn, S., A. Gur-Lavi, and J. Weisman. Infectious synovitis in turkeys: isolation of a tenosynovitis virus-like agent. Avian Pathol. 9:1–4. 1980. 11. Page, R. K., O. J. Fletcher, and P. Villegas. Infectious synovitis in young turkeys. Avian Dis. 26:924–927. 1982. 12. Pantin-Jackwood, M. J., J. M. Day, M. W. Jackwood, and E. Spackman. Enteric viruses detected by molecular methods in commercial chickens and turkey flocks in the United States between 2005 and 2006. Avian Dis. 52:235–244. 2008. 13. Rosenberger, J. K. Reovirus infections. 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, eds. Iowa State University Press, Ames, IA. pp. 283–293. 2003. 14. Shivaprasad, H. L., M. Franca, P. R. Woolcock, R. Nordhausen, J. M. Day, and M. Pantin-Jackwood. Myocarditis associated with reovirus in turkey poults. Avian Dis. 53:523–532. 2009. 15. Spandios, D. A., and A. F. Graham. Physical and chemical characterization of an avian reovirus. J. Virol. 19:968–976. 1976. 16. Van der Heide, L., and M. Kalbac. Infectious tenosynovitis (viral arthritis): characterization of a Connecticut viral isolant as a reovirus and evidence of viral egg transmission by reovirus-infected broiler breeders. Avian Dis. 19:683–688. 1975. 17. Ward, R., M. M. McNeal, M. B. Farone, and A. L. Farone. Reoviridae. In: The mouse in biomedical research, vol. 2. Fox, J. G., ed. Academic Press, Burlington, MA. p. 256. 2007.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
AVIAN DISEASES 57:321–325, 2013
Case Report— Reovirus Infections in Young Broiler Chickens James F. Davis,AD Arun Kulkarni,B and Oscar FletcherC A
Department of Diagnostics, Georgia Poultry Laboratory Network, P.O. Box 20, Oakwood, GA 30566 B Department of Virology, Georgia Poultry Laboratory Network, P.O. Box 20, Oakwood, GA 30566 C College of Veterinary Medicine, North Carolina State University, Population Health and Pathobiology, 1060 William Moore Drive, Raleigh, NC 27607 Received 7 November 2012; Accepted 18 February 2013; Published ahead of print 27 February 2013 SUMMARY. Selected 10-to-21-day-old broiler chickens from flocks undergoing runting-stunting syndrome were found to have significant pancreatic damage, grossly and histologically. Six reoviruses, with sequences that varied, both from each other and from S1133 reovirus, were isolated from these pancreases and from pancreases of specific-pathogen-free leghorn sentinels placed on two of the broiler farms for 7 days. RESUMEN. Reporte de Caso—Infecciones por reovirus en pollos de engorde jo´venes. Se encontro´ dan˜o pancrea´tico significativo macrosco´pico y microsco´picamente en pollos de engorde de 10 a 21 de edad seleccionados por bajo crecimiento en parvadas con sı´ndrome de mala absorcio´n. Se aislaron seis reovirus, con secuencias que variaron tanto unos de otros como con respecto a la de cepa S1133, a partir del pa´ncreas de las aves afectadas y del pa´ncreas de aves Leghorn libres de pato´genos especı´ficos que fueron alojadas como centinelas en dos granjas de pollos de engorde por siete dı´as. Key words: reovirus, pancreas Abbreviations: aa 5 amino acid(s); CEK 5 chicken embryo kidney; H-SMS 5 hypoglycemia-spiking mortality syndrome; NBF 5 neutral buffered formalin; RSS 5 runting-stunting syndrome; RT 5 reverse transcriptase; SPF 5 specific pathogen free
Reoviruses are 75-nm RNA viruses (6,15) that have been reported to be vertically transmitted in chickens, resulting in tenosynovitis in the offspring (16). Reoviruses have been associated with degeneration and necrosis of pancreatic acinar cells in mice (7). In addition, two mouse Reovirus serotypes (1 and 3) have been shown to infect and injure the endocrine areas of the pancreas in mice, with a resulting drop in serum insulin levels that causes abnormalities in glucose tolerance (17). In poultry, reoviruses have been reported to infect and cause lymphoplasmacytic and fibroheterophilic tenosynovitis in tendons (6,8,10,11,13) and to also infect intestines (5,12), heart (1,3,14), and liver (9). This report describes pancreatic vacuolar change and atrophy in broiler chickens associated with reovirus infections. HISTORY
During early 2012, multiple groups of young broiler chicks, aged 10–21 days, were submitted to The Georgia Poultry Laboratory for necropsy and diagnoses. The primary complaints were sudden ‘‘spiking’’ mortality and poor growth rate. Runting-stunting syndrome (RSS) was suspected. CLINICAL FINDINGS AND PROCEDURES
Clinical signs included small runted chicks that were passing undigested feed. Some chicks were live and some were dead upon arrival at the laboratory. Live birds were euthanatized using CO2 gas and then heart-bled to obtain sera for antibody testing against D
Corresponding author. E-mail: [email protected]
reovirus by using ELISA (IDEXX Laboratories, Westbrook, ME). Blood from selected chicks also was submitted for blood glucose level determinations if the chicks seemed to be weak and ataxic. Upon necropsy, thin-walled intestines, consistent with RSS, were the primary lesions. In addition, a number of the 10–21-day-old chicks had what appeared to be pale, atrophic pancreases. Eleven severely runted broilers, from two of the affected farms, were submitted at 36 days of age for follow-up exams (five from one farm and six from the second farm). Blood was collected for ELISA testing. Pancreatic atrophy was found on necropsy in birds from both groups. All grossly abnormal pancreases were aseptically detached from the duodena by using sterile scissors and forceps, with extreme care being taken to ensure duodena were not cut. Each pancreas was cut into multiple sections, and half of the sections from each bird were submitted in sterile Whirl-PaksH (Nasco, Fort Atkinson, WI) for virus isolation attempts. The other sections were put into SurgipathH10% neutral buffered formalin ([NBF]; Leica Biosystems, Richmond, IL) for histopathology processing. We also collected 2.5-cm (1-inch)-long segments of duodena and jejuna in NBF for histopathology. Three groups of 12 21-day-old specific-pathogen-free (SPF) leghorn chickens were placed on two of the broiler farms where the affected chickens were grown, for 7 days, to serve as sentinels. Pancreases, duodena, and jejuna were all grossly normal, but they were collected and processed for virology and histopathology as described above. CLINICAL PATHOLOGY
Whole blood glucose levels were evaluated from a small number of selected pale, runted, and apparently weak ataxic chicks by using a
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Table 1. Amino acid similarities of the sigma C gene of the GA/GPL reovirus isolates with reference strains.A % aa identity with the isolates from the present study Reference strainsB
Isolated fromC
12257D
12274E
12297F
12298G
12342H
12355I
DQ872797 DQ872798 DQ872800 FR694197 DQ996603 DQ996602 L139002
RSS RSS RSS RSS PEMS-turkey Healthy-turkey 1133 vaccine strain
55.3 46.6 47.9 56.6 45.0 45.0 45.6
51.4 87.3 89.5 51.1 70.5 70.2 56.5
87.3 49.5 51.4 87.6 48.9 49.5 47.3
51.5 43.5 44.5 52.5 44.5 45.5 43.5
86.7 48.7 51.6 88.3 48.4 50.0 48.7
49.5 67.9 68.3 48.3 81.9 83.8 53.0
A
Results expressed as the percentage of similarity. Bold indicates the highest aa identity of the isolate from the present study. GenBank accession numbers of the reference strains. C Isolation source of the reference strain. D–I GA/GPL/isolates with GenBank accession JX983603, JX983599, JX983600, JX983604, JX983601, and JX983602, respectively. B
hand-held digital glucose meter (FreeStyleH Blood Glucose Monitoring System; TheraSense, Inc., Alameda, CA). VIROLOGY
Virus isolation and identification. Pancreases were homogenized in phosphate-buffered saline supplemented with antibiotics. Crude virus suspension was clarified by centrifugation, filtered through a 0.2-mm membrane filter, and used for inoculation of primary chicken embryo kidney (CEK) cell monolayers. The first two serial passages produced isolated foci of detached cells within 72 hr postinoculation. After the third passage, there was marked rounding and detachment of cells. Virus isolates from this study were designated as GA/GPL/12257, GA/GPL/12274, GA/GPL/12297, GA/GPL/12298, GA/GPL/12342, and GA/GPL/12355. GA/GPL/ 12274 was isolated from the pancreases of the sentinel birds. The remaining isolates came from 14–17-day-old broilers. Viral RNA was extracted from the clarified supernatants of CEKinfected monolayers by using the RNeasy mini kit (QIAGEN, Valencia, CA) according to manufacturer’s instructions. The amplification of the sigma C gene was performed using the OneStep RT-PCR kit (QIAGEN) and previously described primers ScP1 and ScP4 (9). The reverse transcriptase (RT)-PCR products of the virus were directly sequenced using the same set of primers in both directions. Sequencing reaction was performed using 30 ng of template per reaction with the BigDye Terminator version 3.1 cycle
sequencing kit (Applied Biosystems, Foster City, CA) at the Georgia Genomics Facility, University of Georgia (Athens, GA). Sequence analysis. The nucleotide and deduced amino acid (aa) sequence data were aligned by MegAlign program version 8.0 (DNASTAR Inc., Madison, WI) using the ClustalW multiple sequence alignment algorithm. The phylogenetic tree was constructed by the neighbor-joining method with MegAlign package (DNASTAR Inc.) with 1000 replicates. The reovirus reference strains that were closely related to present isolates by basic local alignment search tool (BLAST) were selected for analysis. Nucleotide sequences from previously identified isolates from the United States and from the commonly used U.S. vaccine strain S1133 were selected for analysis to understand the extent of genetic identity of the isolates from the present study. The GenBank accession numbers of various isolates used for analysis are listed in Table 1 and Fig. 1. Strain S1133 was included in the analysis to determine the relatedness of the isolates to the vaccine. RESULTS
Virology. The primer pair ScP1 and ScP4 used for the amplification of the sigma C gene in this study consistently amplified approximately 1000-bp product as described previously (GenBank accession L39002). Three of the six isolates—GA/GPL/ 12274, GA/GPL/12297, and GA/GPL/12355—showed .86% aa identity with the previously identified reovirus isolated from RSS
Fig. 1. Neighbor-joining phylogenetic trees with 1000 bootstrap replicates showing the relationships among aa-deduced sequences when phylogenetic analyses are conducted on an approximately 1000-bp sequence of the sigma C region of reovirus isolates. The sequences were obtained from six field isolates and another eight reference strains indicated with GenBank accession number. The numbers at the fork indicate the bootstrap values (1000 replicates).
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Table 2. Whole blood glucose levels from selected ataxic chicks with pancreatic degeneration and atrophy (normal 5 150–300 mg/dl). Flock
Age (days)
Blood glucose (mg/dl)
A B C
14 14 14
286, 244, 199 73A, 214, 213, 185 77A, 164, 130A, 115A
A
Hypoglycemia.
cases from Georgia (Table 1). Amongst these three isolates, GA/ GPL/12297 and GA/GPL/12355 also shared .87% identity with the European isolate FR694197. Interestingly, isolate GA/GPL/ 12355, derived from 14-day-old broiler pancreases, was .81% identical to two turkey isolates from the United States (4). Finally, isolates GA/GPL/12257 and GA/GPL/12298 shared ,55% aa identity with all of the reference strains analyzed in this study. It is noteworthy that, like previously described reovirus isolates from RSS cases, (Table 1), none of the isolates from the present study shared .52% identity with commonly used vaccine strains S1133 or 1733. The GenBank designations for the six isolates are as follows: GA/ GPL/12257, JX983603; GA/GPL/12274, JX983599; GA/GPL/ 12297, JX983600; GA/GPL/12298, JX983604; GA/GPL/12342, JX983601; and GA/GPL/12355m, JX983602. Phylogenetic relationships among field isolates of reovirus. Based on the predicted aa sequence of the amplified region of the sigma C gene from six isolates and seven reference strains, a
Table 3. ELISA reovirus antibody titers of runted broilers with pancreatic degeneration and atrophy. Samples were collected 22 days after flock diagnosis of reovirus infection of pancreas. Farm
Age (days)
Individual bird ELISA titers against reovirus S-1133
Group geometric mean titer
A B
36 36
1166, 500, 75, 327, 176 88, 276, 2183, 0, 31, 190
306 83
phylogenetic tree was constructed (Fig. 1). All of the isolates from the present study fall into three distinct groups. In the first group (group 1), isolates 12274 and 12355 were grouped with reoviruses of turkey and chicken origin, with the latter from the cases of RSS. In the second group, isolates 12297 and 12342 were closely related to European and U.S. isolates FR694197 and DQ872797, respectively, both from chickens experiencing RSS (group 2). Isolates 12257 and 12298, although identical to each other up to 78%, did not belong to any of the reference strains used in the analysis (group 3). None of the isolates from the present study belong to the previously described U.S. isolates S1133, 1733, or ARV-136 (group 4). Serology. Whole blood glucose levels showed hypoglycemia in four of the 11 chicks tested (Table 2). ELISA testing on sera from the 11 follow-up broilers revealed very little antibody against reovirus on an ELISA kit with wells coated with S1133 antigen (IDEXX Laboratories; Table 3).
Fig. 2. (A) Section from jejunum shows normal villi and absence of crypt lesions. (B) Villous atrophy with no crypt lesions is in this section of jejunum. (C) Villous atrophy with multiple cystic and dilated crypts is in the jejunum. (D) Higher magnification shows villous atrophy and dilated, cystic crypts. Bar 5 250 mm. Age 5 17 days.
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Fig. 3. (A) Vacuolar degeneration and necrosis are in the pale-stained region of pancreas (left). Bar 5 250 mm. (B) Higher magnification of the pale region in panel A shows loss of granules in acinar cells. Bar 5 100 mm. (C) Higher magnification at margin of the pancreas shows vacuolar degeneration and necrosis of acinar cells. Bar 5 50 mm. (D) Higher magnification of lesion at the margin of the pancreas shows vacuolar degeneration and necrosis of acinar cells. Bar 5 25 mm. Age 5 17 days.
Histopathology. Sections from duodenum and jejunum from the 10–21-day-old broiler chickens had villous atrophy, sometimes with dilated and cystic crypts (Fig. 2). Lesions in pancreas included vacuolar degeneration, degeneration, and necrosis of acinar cells and also atrophy of acinar cells. Islets were not affected. Lesions were multifocal to regional (Figs. 3, 4). Similar pancreatic lesions of vacuolar degeneration and atrophy were found in the 36-day-old broilers. There were no lesions in the pancreas or intestines of the SPF leghorns.
DISCUSSION
Protection of day-old chicks against reovirus by maternal antibodies is achieved by vaccination of breeding stock. This vaccination is mostly performed in the United States using strains S1133 and 1733 or from a naturally apathogenic strain 2177. The observed sequence discrepancy between the isolates reported in the present study and the vaccine strain S1133 warrants further investigation. The reoviruses isolated from the pancreases and then sequenced differed significantly from previously reported reoviruses. These viruses seem to be closely associated with the increase in mortality in these broiler flocks. Severe hypoglycemia, such as that found with
hypoglycemia-spiking mortality syndrome (H-SMS) of broilers does not seem to be the cause of the flock mortality because pancreases examined in studies of H-SMS consistently were histologically normal (2), whereas the pancreases examined in our study were not. The hypoglycemia observed in birds from our study was likely due to the significant pancreatic damage found in these birds. Further investigations need to be done, including bird inoculations, to determine the pathogenicity and pathogenesis of these reoviruses. In addition, studies on the effect of these reovirus infections on pancreatic hormone and enzyme levels need to be conducted. Vaccination trials also need to be conducted to determine whether any currently available vaccines will protect against these reoviruses. The ELISA titers measured in the follow-up birds and the sequencing results suggest that the reoviruses infecting these birds were not closely related to S1133. Therefore, it seems likely that S1133-based vaccines will not protect against these reoviruses. In addition, the fact that the sequences of all six of these viruses varied with each other may produce additional problems with production of a vaccine that will protect against the pancreatic damage. None of the isolates from the present study belong to the previously described five genotyping clusters of reoviruses (9). We are not concluding that the reoviruses that we isolated from these diseased pancreases are the etiologic agents responsible for inducing the lesions. Further experimental investigations, including SPF bird inoculations, need to be conducted to determine whether this is the case.
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Fig. 4. (A) Low power view shows atrophy of a large area of pancreas. The lobe at the top is relatively unaffected. Bar 5 500 mm. (B) Higher magnification shows loss of granules in acinar cells. Tip of unaffected pancreas is in upper right. Bar 5 100 mm. (C) Higher magnification shows the atrophy of acinar cells. An uninvolved islet is in the lower left. Bar 5 50 mm. (D) Higher magnification shows atrophy of acinar cells. Note the absence of granules. Bar 5 25 mm. Age 5 17 days. REFERENCES 1. Bagust, T. J., and H. A. Westbury. Isolation of reoviruses associated with diseases of chickens in Victoria. Aust. Vet. J. 51:406–407. 1975. 2. Davis, J. F. Hypoglycemia-spiking mortality syndrome. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 1269–1272. 2008. 3. Davis, J. F., A. Kulkarni, and O. Fletcher. Myocarditis in 9- and 11day-old broiler breeder chicks associated with a reovirus infection. Avian Dis. 56:786–790. 2012. 4. Day, J. M., M. J. Pantin-Jackwood, and E. Spackman. Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses. Virus Genes 35:235–242. 2007. 5. Dees, T. A., R. E. Wooley, and J. B. Gratzek. Pathogenicity of bacteria-free filtrates and a viral agent isolated from turkeys with infectious enteritis. Am. J. Vet. Res. 33:165–170. 1972. 6. Glass, S. E., S. A. Naqi, C. F. Hall, and K. M. Kerr. Isolation and characterization of a virus associated with arthritis in chickens. Avian Dis. 17:415–424. 1973. 7. Jacoby, R. O., J. G. Fox, and M. Davison. Biology and diseases of mice, 2nd ed. In: Laboratory animal medicine. J. G. Fox, ed. Academic Press, San Diego, CA. p. 72. 2002. 8. Jones, R. C. Other reovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 310–328. 2008.
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