Tamer A. Sharafeldin

Isolation and Characterization of a Turkey Arthritis Reovirus

SUMMARY. During the spring and summer of 2011, the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota received 14 submissions of 15-to-18-week-old tom turkeys that were recumbent with wing tip bruises (“wing walkers”) and uni- or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in specific-pathogen-free embryonated chicken eggs and QT-35 cells. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction, and gene sequence analysis. BLAST analysis on the basis of a 880 bp nucleotide sequence of the S4 gene confirmed all isolates as a reovirus. Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, -2, -3, and -5, and the other subgroup containing TARV-MN4. Isolates in subgroup I had a similarity of 97%–100% with each other, while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90%–93% similarity with turkey enteric reoviruses in the United States, while the other four isolates in subgroup I had 89%–97.6% similarity. These results indicate divergence within TARVs as well as from enteric viruses, which needs to be confirmed by complete genome sequence analysis. Further experimental studies are planned to determine the role of these isolates in turkey arthritis and to compare them with classical chicken reovirus. RESUMEN. Aislamiento y caracterización de un reovirus asociado con artritis en pavos. Durante la primavera y el verano del año 2011, el Laboratorio de Diagnóstico Veterinario el Estado de Minnesota en la Universidad de Minnesota recibió 14 casos de pavos machos de 15 a 18 semanas de edad, que mostraron recumbencia con moretones en la punta del ala (“aves que caminan con las alas”) e inflamación de la articulación del corvejón (articulación tibiotarsal) uni o bilateral. Ocasionalmente, los tendones del músculo gastrocnemio o flexores digitales se observaron rotos. Un total de cinco reovirus asociados con artritis en pavos (denominados TARV-MN1 al TARV-MN5) se aislaron en huevos embrionados de pollo libres de patógenos específico y en células QT-35. La identidad de los aislados se confirmó mediante microscopía electrónica, por transcripción reversa y reacción en cadena de la polimerasa, y por el análisis de secuencias. Mediante el análisis con la base de datos BLAST de las secuencias de nucleótidos de productos de 880 pares de bases que incluían al gene S4 confirmaron a todos los aislamientos como reovirus. El análisis filogenético dividió a los cinco aislamientos en dos subgrupos: subgrupo I que contenía a los virus TARV-MN1, TARV-MN2, TARV-MN3 y TARV-MN5 y el otro subgrupo que incluía al virus TARV-MN4. Los aislados en el subgrupo I mostraron una similitud de 97% -100% entre ellos, mientras que el subgrupo II (TARV-MN4) mostró una similitud de sólo 89.2% con los virus de subgrupo I. Este aislamiento mostró una similitud de 90%–93% con reovirus de pavo entéricos en los Estados Unidos, mientras que otros cuatro aislados en el subgrupo I mostraon una similitud de 89%–97.6%. Estos resultados indican divergencia dentro de los reovirus asociados con artritis en pavos, así como de los virus entéricos, lo cual debe ser confirmado por el análisis de las secuencias completas del genoma. Se han planeado otros estudios experimentales para determinar el papel de estos aislamientos en la artritis de los pavos y compararlas con reovirus de pollo clásicos.

The occurrence of enteric viruses in Light Turkey Syndrome

Two studies were conducted to determine the role of enteric viruses in Light Turkey Syndrome (LTS), which is characterized by lower weight in market age turkeys than their standard breed character. In the surveillance study, we selected four LTS and two non-LTS turkey flocks in Minnesota and collected faecal samples at 2, 3, 5 and 8-weeks of age. Astrovirus, rotavirus, and reovirus were detected alone or in various combinations in both LTS and non-LTS flocks. No coronavirus was detected in LTS flocks and no corona- or reovirus was detected in non-LTS flocks. In the second study, 2-week-old turkey poults were divided into two groups; Group A (challenged) was inoculated orally with 10% pooled faecal suspension from LTS flocks and group B (control) was inoculated with phosphate buffered saline (PBS). Clinical signs of depression, huddling, and lack of uniform size were observed in the challenged group but not in the control group. diarrhoea was observed in both groups but was more severe in the challenged group than in the control group. Birds in the challenged group shed astrovirus, rotavirus and reovirus, while the control group shed only astrovirus. Virus shedding in both groups was observed for up to nine weeks of age. Significantly lower body weights were seen in the challenged group starting at seven weeks of age and lasting until 20 weeks of age. These findings suggest that viral enteritis at an early age may set up conditions for the development of LTS in adult turkeys.

Characterization of S class gene segments of a newly isolated turkey arthritis reovirus

We report on the complete characterization of S class gene segments of 12 newly isolated turkey arthritis reoviruses (TARVs) and compare it with that of a turkey enteric reovirus (TERV). Phylogenetic analysis of S2, S3 and S4 genome segments revealed grouping of all TARVs into two lineages while, on the basis of S1 genome segment, only one lineage was found. All TARVs had 95-100% nucleotide identity based on sigma C protein sequences (S1 segment) but varied from 90-100%, 88.9-100% and 88.7-100% on the basis of S2, S3, and S4 genome segments, respectively. Point mutations as well as possible re-assortments were observed in TARVs throughout the S class indicating the need for extensive epidemiological studies on these viruses in hatcheries and commercial farms, which would be useful in determining virus variation in the field.

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O’Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12–16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

One-Step Real-Time Reverse Transcription–PCR for the Detection of Turkey Reoviruses

SUMMARY During late 2010 and early 2011, an unusual problem of lameness and swollen hock joints in commercial turkeys was reported in the upper Midwest, which continues to this day. The disease caused substantial economic losses to turkey producers. Reovirus was isolated from tendons and joint fluids of lame turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory. This study was undertaken to develop a TaqMan real-time reverse transcription-PCR (rRT-PCR) assay for the early detection of turkey reoviruses (both enteric and lameness strains). A primer probe set was designed from the conserved region of the S4 segment of the turkey reovirus genome. The newly developed rRT-PCR was specific for the detection of turkey reoviruses. The detection limit of this assay was 10 genome copies per reaction. For the TARV-MN4 strain of turkey arthritis reovirus, one 50% tissue culture infectious dose was equivalent to 11.6 ± 0.2 genome copies. The highest coefficient of variation for intraexperimental and interexperimental variability was 0.08 and 0.06, respectively, indicating the reproducibility of the assay. This new test should be useful for the detection of turkey enteric and arthritis reoviruses. RESUMEN Método de transcripción reversa y PCR en un solo paso para la detección de reovirus de pavos. Durante finales del año 2010 y principios del 2011, se informó de un problema poco común de cojera e inflamación de corvejones en pavos comerciales en el Medio Oeste de los Estados Unidos, que continúa hasta este momento. La enfermedad ha causado substanciales pérdidas económicas a los productores de pavo. Se aisló un reovirus de los tendones y de los fluidos de las articulaciones de los pavos con cojera enviados al Laboratorio de Diagnóstico Veterinario de Minnesota. Este estudio se realizó para desarrollar un método de transcripción reversa y PCR en tiempo real (rRT-PCR) con sondas TaqMan para la detección temprana de los reovirus de pavo (cepas entéricas y cepas causantes de cojera). Se diseñó un conjunto de iniciadores para la región conservada del segmento S4 del genoma del reovirus de pavo. El método de rRT-PCR recientemente desarrollado era específico para la detección de reovirus de pavo. El límite de detección de este ensayo fue de 10 copias de genoma por reacción. Para la cepa TARV-MN4 de reovirus de pavo causante de artritis, una doses infectante 50% en cultivo de tejidos fue equivalente a 11.6 ± 0,2 copias del genoma. Los mayores coeficientes de variación para la variabilidad intraexperimental e interexperimental fueron de 0.08 y 0.06, respectivamente, indicando la reproducibilidad del ensayo. Esta nueva prueba puede ser útil para la detección de reovirus de pavos entéricos y causantes de artritis.

Etiology and pathology of epidemic outbreaks of avian influenza H5N1 infection in Egyptian chicken farms.

Epidemic outbreaks of avian influenza (AI) virus H5N1 have been frequently reported in Egypt during the last nine years. Here we investigate the involvement of AI H5N1 in outbreaks of acute respiratory disease that occurred in several commercial chicken farms in Egypt in 2011, and we describe to the pathology caused by the virus in the course of the outbreak. Twenty-one chicken farms with history of acute respiratory symptoms and high mortalities were screened for AI H5N1. Virus identification was based on hemagglutination inhibition test, and PCR detection and sequencing of the hemagglutinin and neuraminidase genes. Virus distribution was determined by immunohistochemical staining of AI antigens in organs of infected birds. Standard H&E staining was performed for histological examination of affected organs. Eighty-one % of the examined birds, representing 100% of the screened farms, were positive for AI H5N1 virus. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of the isolated virus reveals its affiliation to clade 2.2.1. Viral antigens were localized in the endothelial cells of the heart, liver, lungs and skin, where pathological lesions including congestion, hemorrhages, multifocal inflammation and necrosis were concurrently observed. According to the pattern of the viral antigen and lesion distribution in the visceral organs, we suggest cardiovascular and circulatory failures as the probable cause of death during these outbreaks. In conclusion, the present study further confirms the epidemic status of AI H5N1 virus in Egypt and reveals the highly pathogenic nature of the local isolates.

A Newly Emergent Turkey Arthritis Reovirus Shows Dominant Enteric Tropism and Induces Significantly Elevated Innate Antiviral and T Helper-1 Cytokine Responses

Newly emergent turkey arthritis reoviruses (TARV) were isolated from tendons of lame 15-week-old tom turkeys that occasionally had ruptured leg tendons. Experimentally, these TARVs induced remarkable tenosynovitis in gastrocnemius tendons of turkey poults. The current study aimed to characterize the location and the extent of virus replication as well as the cytokine response induced by TARV during the first two weeks of infection. One-week-old male turkeys were inoculated orally with TARV (O’Neil strain). Copy numbers of viral genes were estimated in intestines, internal organs and tendons at ½, 1, 2, 3, 4, 7, 14 days Post inoculation (dpi). Cytokine profile was measured in intestines, spleen and leg tendons at 0, 4, 7 and 14 dpi. Viral copy number peaked in jejunum, cecum and bursa of Fabricius at 4 dpi. Copy numbers increased dramatically in leg tendons at 7 and 14 dpi while minimal copies were detected in internal organs and blood during the same period. Virus was detected in cloacal swabs at 1–2 dpi, and peaked at 14 dpi indicating enterotropism of the virus and its early shedding in feces. Elevation of IFN-α and IFN-β was observed in intestines at 7 dpi as well as a prominent T helper-1 response (IFN-γ) at 7 and 14 dpi. IFN-γ and IL-6 were elevated in gastrocnemius tendons at 14 dpi. Elevation of antiviral cytokines in intestines occurred at 7dpi when a significant decline of viral replication in intestines was observed. T helper-1 response in intestines and leg tendons was the dominant T-helper response. These results suggest the possible correlation between viral replication and cytokine response in early infection of TARV in turkeys. Our findings provide novel insights which help elucidate viral pathogenesis in turkey tendons infected with TARV.

Molecular characterization of L class genome segments of a newly isolated turkey arthritis reovirus

Seven strains of turkey arthritis reovirus (TARV) isolated from cases of turkey arthritis were characterized on the basis of their L class genome segment sequences, which were then compared with those of turkey enteric reovirus (TERV) and chicken reovirus (CRV). All three L class gene segments of TARVs and TERVs and their encoded proteins λA, λB, and λC were similar in size to those of CRV reference strain S1133. The conserved motifs such as C2H2 zinc-binding motif and conserved polymerase region were present in λA and λB, respectively. A conserved motif for ATP/GTP-binding site and an S-adenosyl-l-methionine (SAM)-binding pocket for methyltransferase were observed in λC protein of TARVs and TERVs with only one substitution as compared to that in CRV. We propose a new genotype classification system for avian reoviruses (ARVs) based on the nt identity cut-off value for each of the L class. Based on this new genotype classification, all ARVs were divided into six, seven and eight genotypes in L1, L2 and L3 genes, respectively. Interestingly TARVs and TERVs grouped with three CRVs (two arthritic strains from Taiwan and one enteritic strain from Japan) in genotype L1-I and formed a different genotypes (L2-I, L3-I) from CRVs in L2 and L3 genes. The maximum nucleotide divergence was observed in genotypes of L1 and L2 genes but less at amino acid level indicates mostly changes were synonymous type. Compared to L1 and L2 genes, the nonsynonymous changes were more in L3 gene. Point mutations and possible reassortments among TARVs, TERVs and CRVs were also observed.

Detection and Characterization of Newcastle Disease Virus in Formalin-Fixed, Paraffin-Embedded Tissues from Commercial Broilers in Egypt

SUMMARY Newcastle disease (ND) is highly contagious and causes severe economic losses to the poultry industry due to high morbidity and mortality. In this report, we describe the detection of Newcastle disease virus (NDV) in formalin-fixed tissues from an outbreak of ND on broiler farms in Egypt. The affected birds experienced respiratory and/or nervous signs and a 75% mortality rate. Tissue samples were collected and placed in 10% neutral buffered formalin followed by embedding in paraffin. RNA was extracted from 80-µm formalin-fixed paraffin-embedded tissue blocks and recovered in 60 µl of elution buffer. All samples were negative for influenza virus by real-time reverse-transcription (RT)-PCR but positive for NDV. These flocks were known to have been vaccinated with a live NDV vaccine (LaSota strain). The nucleic acid sequences of the virus detected in this study were similar to those of a velogenic virus at its cleavage site 111GRRQKR*F117 and clustered with class II genogroup VII lineage of NDV, with a nucleotide sequence identity of 94%–99%. Although extraction and amplification of NDV from paraffin-embedded tissues from experimentally infected birds has been reported previously, this study reports on the use of RT-PCR on formalin-fixed tissues from actual field samples. RESEMEN Detección y caracterización del virus de la enfermedad de Newcastle en tejidos fijados en formalina e incluidos en parafina y provenientes de pollos de engorde de Egipto. La enfermedad de Newcastle (ND) es altamente contagiosa y causa graves pérdidas económicas a la industria avícola debido a la alta morbilidad y mortalidad. En este reporte se describe la detección del virus de la enfermedad de Newcastle (NDV) en tejidos fijados en formalina recolectados durante un brote de la enfermedad de Newcastle en las granjas de engorde en Egipto. Las aves afectadas experimentaron signos respiratorios y/o nerviosos y una tasa de mortalidad del 75%. Se recolectaron muestras de tejidos y se colocaron en formalina neutra amortiguada al 10% y finalmente las muestras se incluyeron en parafina. Se extrajo el ARN a partir de bloques de tejido fijado con formalina e incluido en parafina de 80 micras y se recuperó en 60 µl del buffer de elución. Todas las muestras fueron negativas para el virus de la influenza aviar mediante un método de transcripción reversa y PCR en tiempo real, pero resultaron positivas para la presencia del virus de Newcastle. Se conocía que estas parvadas habían sido vacunadas con una vacuna viva de Newcastle (cepa LaSota). Las secuencias de los ácidos nucleicos del virus detectado en este estudio fueron similares a las secuencias de un virus velogénico en el sitio de disociación 111GRRQKR*F117 y se agruparon en el genogrupo clase II, linaje VII del virus de Newcastle, con una identidad en la secuencia de nucleótidos del 94% al 99%. Aunque la extracción y la amplificación del virus de Newcastle a partir de tejidos en parafina procedentes de aves infectadas experimentalmente se ha reportado anteriormente, este estudio informa sobre el uso de un método de RT-PCR en tiempo real con tejidos fijados en formalina provenientes de muestras reales de campo.

Prevalence of parvovirus in Minnesota turkeys

&NA; Poult enteritis syndrome (PES) is characterized by enteritis and decreased body weight gain in growing turkey poults between one d and 7 wk of age. Another syndrome called light turkey syndrome (LTS) causes a decrease in body weight of adult tom turkeys in Minnesota leading to huge economic losses. Reovirus, rotavirus, and astrovirus have been found in LTS and PES flocks in Minnesota. We tested 80 fecal pools collected from four LTS flocks and 35 fecal pools from non‐LTS flocks for the presence of parvovirus. In addition, 116 fecal and meconium samples from turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) also were tested. The samples were tested by PCR using primers for the non‐structural 1 (NS1) gene of parvovirus. Of the 80 samples from LTS flocks, 41 were positive for parvovirus while 20 of 35 samples from non‐LTS flocks were positive. The prevalence of parvovirus in fecal samples submitted to MVDL was relatively low; only five of the 116 pools were positive. The partial NS1 gene sequences from LTS and non‐LTS samples showed 98 to 100% nt identity except for one divergent turkey parvovirus (TuPV) strain that revealed 90% identity and clustered with chicken‐like parvoviruses. The presence of this divergent strain suggests circulation of a recombinant strain of TuPV in Minnesota turkeys. Our results indicate that TuPVs are circulating in both LTS and non‐LTS flocks of turkeys in Minnesota, and further experimental studies are indicated to study the role of TuPV in LTS.