•Increase in viral lameness in commercial poultry caused by variant avian reoviruses.•Variant reoviruses are antigenically distinct from commercial vaccines.•Commercial vaccines do not provide ...adequate protection against disease.•New vaccines are needed to control disease.
Avian reoviruses are the causative agent of viral arthritis/tenosynovitis in chickens and turkeys. Clinical signs of disease include swelling of the hock joints accompanied by lesions in the gastrocnemius and digital flexor tendons causing lameness in addition to hydropericardium. The economic impact is significant as it results in poor weight gain, increased feed conversion ratios and condemnations at the processing plant. Vaccination with both live attenuated and inactivated oil emulsion vaccines have been used successfully for decades to control the disease. Current commercial vaccine strains belong to the same serotype and are antigenically and serologically distinct from circulating variant field viruses isolated from clinical cases of tenosynovitis. Since 2012, there has been a dramatic increase in the number of clinical cases of tenosynovitis in commercial poultry and commercial vaccines are unable to provide adequate levels of protection against disease. Producers have elected to use custom inactivated vaccines in the absence of any commercially available homologous vaccines. Identification and selection of field isolates for use in autogenous vaccines can be difficult especially when multiple reoviruses are co-circulating among flocks. In addition, field data suggests that in some cases the custom vaccines are providing adequate protection against disease but as new genetic variants emerge, new vaccines are needed.
Reoviral-induced tenosynovitis/viral arthritis is an economically significant disease of poultry. Affected birds present with lameness, unilateral or bilateral swollen hock joints or shanks, and/or ...reluctance to move. In severe cases, rupture of the gastrocnemius or digital flexor tendons may occur, and significant culling may be necessary. Historically, vaccination with a combination of modified live and inactivated vaccines has successfully controlled disease. Proper vaccination reduced vertical transmission and provided maternal-derived antibodies to progeny to protect against disease, at an age when they were most susceptible. Starting in 2011-2012, an increased incidence of tenosynovitis/viral arthritis was observed in chickens and turkeys. In chickens, progeny from reovirus-vaccinated breeders were affected, suggesting commercial vaccines did not provide adequate protection against disease. In turkeys, clinical disease was primarily in males, although females can also be affected. The most significant signs were observed around 14-16 wks of age and include reluctance to move, lameness, and limping on one or both legs. The incidence of tenosynovitis/viral arthritis presently remains high. Reoviruses isolated from clinical cases are genetically and antigenically characterized as variants, meaning they are different from vaccine strains. Characterization of the field isolates reveals multiple new genotypes and serotypes that are significantly different from commercial vaccines and each other. In 2012, a single prevalent virus was isolated from a majority of the cases submitted to the Poultry Diagnostic and Research Center at the University of Georgia. Genetic characterization of the σC protein revealed the early isolates belonged to genetic cluster (GC) 5. Soon after the initial identification of the GC5 variant reovirus, many broiler companies incorporated these isolates from their farms into their autogenous vaccines and continue to do so today. The incidence of GC5 field isolates has decreased significantly, likely because of the widespread use of the isolates in autogenous vaccines. Unfortunately, variant reoviruses belonging to multiple GCs have emerged, despite inclusion of these isolates in autogenous vaccines. In this review, an overview of nomenclature, sample collection, and diagnostic testing will be covered, and a summary of variant reoviruses isolated from clinical cases of tenosynovitis/viral arthritis over the past 10 yrs will be provided.
Prevention of tenosynovitis/viral arthritis caused by variant avian reoviruses within commercial broiler production has become increasingly more challenging because of the lack of protection afforded ...by the current commercially available vaccines. Avian reoviruses isolated from clinical cases of tenosynovitis/viral arthritis in recent years are antigenically distinct from nearly all of the commercially licensed modified live and inactivated biologics available in the United States. The emergence of new variants is likely shaped by a lack of homologous protection coupled with selection pressure influences and results in antigenically diverse populations of avian reoviruses. One tool available to the poultry industry is the use of autogenous (custom) vaccines. Although these can be effective, isolation, characterization, and screening of isolates from clinical cases is paramount for the selection of isolates to include in these vaccines. With no treatment options, control can only be attained via prevention of infection. To achieve this goal, commercially licensed products with antigenic applicability and broadly cross-protective vaccine strains are needed.
The
family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, ...one of the 15 recognized genera in the
family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus-single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5' and 3' ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses.
Despite descriptions of runting-stunting syndrome (RSS) in broiler chickens dating back over 40 years, the aetiology has not yet been described. A novel chicken astrovirus (CkAstV) was isolated in an ...LMH liver cell line from the intestines of chickens affected with RSS. Clinical RSS is characterized by retarded growth and cystic crypt lesions in the small intestine. In 1-day-old broiler chickens infected with the CkAstV isolate, virus was only detected in the intestinal epithelial cells during the first few days after infection. Notably, the preferred host cells are the crypt epithelial cells following initial replication in the villous epithelial cells, thus implying viral preference for immature intestinal cells. Nevertheless, the CkAstV isolate did not induce remarkable pathological changes, despite the presence of the virus in situ. Serial chicken-to-chicken passages of the virus induced increased virulence, as displayed by decreased weight gain and the presence of cystic lesions in the small intestine reproducing clinical RSS in chickens. The analysis of the full-length genome sequences from the isolated CkAstV and the CkAstV from the bird-to-bird passages showed >99 % similarity. The data obtained in this study suggest that the CkAstV isolate is capable of inducing RSS following serial bird-to-bird passages in broilers and is as an aetiological agent of the disease.
Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies ...and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.
During a series of pathology surveys in four production complexes of a U.S. broiler integrator, the technical services veterinarians of an animal health company noted a high incidence of severe ...gizzard erosions and ulcerations (GEU), prompting further clinical investigation and a battery trial. No growth-promoting antibiotics or ionophore coccidiostats were used during the period of these surveys. All used tribasic copper chloride (TBCC) at ≤120 ppm added copper in broiler rations.
was isolated from 83% and 67% of gizzard lesions cultured in two complexes, and cecal
most probable number determinations were higher in severely affected than in mildly affected or unaffected birds. Histopathology revealed both acellular koilin fusion defects characteristic of copper toxicity, as well as inflammatory cell infiltrates. Intralesional bacilli suggestive of
were noted in 78% of affected flocks examined. Species E
was isolated from one bird in one complex, and that bird had a single intranuclear inclusion body; no other flocks had Adenoviruses isolated or detected on PCR, nor any inclusion bodies. Other viruses detected were thought to be incidental. A pilot study using feed with supplemental copper from TBCC or copper sulfate and challenge with one of the isolated
strains reproduced the lesions. A battery study was conducted with an unchallenged negative control group fed a diet with 16 ppm added copper, a group fed the control diet and orally challenged with 10
organisms of a field strain of
at 21 and 22 days, and a group treated with the same diet containing 250 ppm added copper from TBCC and orally challenged with
. Birds were necropsied at 23 and 28 days. All challenged groups developed lesions, with those receiving both TBCC and
having significantly higher gross and histopathological lesion scores than the unchallenged negative controls. Lesions were qualitatively similar to those in the field and contained suspected
bacilli. Because the levels of TBCC used in the commercial birds and in the battery trial generally have been considered safe, and because
is usually regarded as a pathogen of the lower GI tract, the possible association of these two agents with GEU is a novel observation and warrants further investigation.
RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability ...to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.
Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human ...illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry.
Multiple oncogenic viruses, including lymphoproliferative disease virus (LPDV) and reticuloendotheliosis virus (REV), have been detected in wild turkeys (Meleagris gallopavo). The prevalence of ...infection with these viruses appears to be more common than overt disease; thus, data on the manifestation of associated disease in wild turkeys are scarce. Diagnostic records from wild turkeys submitted to the Southeastern Cooperative Wildlife Disease Study from 1980 to 2017 were reviewed to identify cases of neoplasia. Neoplasia was reported in 59 of 851 (6.9%) wild turkeys submitted. Of the cases of neoplasia tested by polymerase chain reaction, LPDV was detected in 34 of 58 (59%), REV in 10 of 39 (26%), both viruses in 3 of 39 (8%), and no retroviruses detected in 5 of 39 (13%) turkeys. The most common gross lesions observed among turkeys with neoplasms were emaciation (30/40; 75%); nodules in the skin (26/59; 44%), liver (17/59; 29%), or spleen (9/59; 15%); and splenomegaly (14/59; 24%). Microscopically, nodules were composed of pleomorphic round cells with large eccentric nuclei and prominent nucleoli resembling lymphocytes or lymphoblasts (57/59; 97%) except for 2 cases, one of myeloid cell origin and the other with primarily spindloid cells. This study indicates the need to characterize the pathogenesis and potential health threat posed by REV and LPDV to wild turkeys. Experimental infection studies and the development of additional diagnostic tests to confirm the role of retroviruses in lymphoproliferative disease are warranted.
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