Diagnostic investigation of avian reovirus field variants circulating in broiler chickens in Pennsylvania of United States between 2017 and 2022.

Avian reovirus (ARV) has been continuously affecting the poultry industry in Pennsylvania (PA) in recent years. This report provides our diagnostic investigation on monitoring ARV field variants from broiler chickens in Pennsylvania. Genomic characterization findings of 72 ARV field isolates obtained from broiler cases during the last 6 years indicated that six distinct cluster variant strains (genotype I-VI), which were genetically diverse and distant from the vaccine and vaccine-related field strains, continuously circulated in PA poultry. Most of the variants clustered within genotype V (24/72, 33.3%), followed by genotype II (16/72, 22.2%), genotype IV (13/72, 18.1%), genotype III (13/72, 18.1%), genotype VI (05/72, 6.94%), and genotype I (1/72, 1.38%). The amino acid identity between 72 field variants and the vaccine strains (1133, 1733, 2408, 2177) varied from 45.3% to 99.7%, while the difference in amino acid counts ranged from 1-164. Among the field variants, the amino acid identity and count difference ranged from 43.3% to 100% and 0 to 170, respectively. Variants within genotype V had maximum amino acid identity (94.7-100%), whereas none of the variants within genotypes II and VI were alike. These findings indicate the continuing occurrence of multiple ARV genotypes in the environment.

Identification, 16S rRNA-based characterization, and antimicrobial profile of Gallibacterium isolates from broiler and layer chickens.

Gallibacterium spp., particularly G. anatis, have received much attention as poultry pathogens in recent years. We report here the presence and antimicrobial resistance profile of 69 Gallibacterium isolates obtained from 2,204 diagnostic submissions of broiler and layer chickens in 2019-2021. Gallibacterium-positive chickens had lesions primarily in the respiratory tract, reproductive tract, and related serosal surfaces. Gallibacterium spp. were initially identified based on their typical cultural characteristics on blood agar. The isolates were confirmed by a genus-specific PCR spanning 16S-23S rRNA and MALDI-TOF mass spectrometry. Phylogenetic analysis based on 16S rRNA gene sequence revealed distinct clades. Of the 69 isolates, 68 clustered with the reference strains of G. anatis and 1 with Gallibacterium genomospecies 1 and 2. Antimicrobial susceptibility testing of 58 of the 69 isolates by a MIC method showed variable responses to antimicrobials. The isolates were all susceptible to enrofloxacin, ceftiofur, florfenicol, and gentamicin. There was a high level of susceptibility to trimethoprim-sulfamethoxazole (98.0%), streptomycin (98.0%), amoxicillin (84.0%), sulfadimethoxine (71.0%), and neomycin (71.0%). All of the isolates were resistant to tylosin. There was resistance to penicillin (98.0%), erythromycin (95.0%), clindamycin (94.0%), novobiocin (90.0%), tetracycline (88.0%), oxytetracycline (76.0%), and sulfathiazole (53.0%). A high rate of intermediate susceptibility was observed for spectinomycin (67.0%) and sulfathiazole (40.0%). Our findings indicate a potential role of G. anatis as an important poultry pathogen and cause of subsequent disease, alone or in combination with other pathogens. Continuous monitoring and an antimicrobial susceptibility assay are recommended for effective treatment and disease control.

An unusual case of haemorrhagic bursa in a pullet flock associated with a genogroup 2 infectious bursal disease virus.

An unusual case of infectious bursal disease (IBD) was observed in eight-week-old commercial caged pullets. This flock (House 1) exhibited a one-day spike in mortality. On gross necropsy examination, enlarged, diffusely haemorrhagic bursas were observed. This lesion has been frequently described in cases of very virulent infectious bursal disease virus (vvIBDV). A five-week-old caged pullet flock (House 2) in an adjacent building did not display haemorrhagic bursa lesions. Microscopic examination of bursas from the eight-week-old pullets in House 1 showed marked diffuse haemorrhages and extensive lymphoid necrosis. Histopathology of bursas from the five-week-old pullets in House 2 showed severe, diffuse lymphoid depletion without haemorrhages. IBD ELISA results from birds in House 1 at 9 weeks had a GMT of 6395 and birds in House 2 had a GMT of 82 in the same timeframe. Diagnostic testing for avian influenza virus, Mycoplasma gallisepticum, Mycoplasma synoviae, virulent Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, chicken anaemia virus and fowl pox virus were negative. The predicted amino acid sequence of the hypervariable region of VP2 indicated the IBDV observed in both flocks (1/chicken/USA/1300OH/19 from House 1 and 1/chicken/USA/1301OH/19 from House 2) was identical and was not a vvIBDV. Their sequences were similar to a genogroup 2 IBDV from Ontario, Canada (EF138967). No mortality was observed when the 1/chicken/USA/1300OH/19 virus was inoculated into specific-pathogen-free (SPF), four-week-old pullets. Gross and microscopic lesions were observed in bursa tissue, but the bursal haemorrhages observed in the original field case were not reproduced in challenged SPF pullets.

Complete Genome Sequences of Seven Avibacterium paragallinarum Isolates from Poultry Farms in Pennsylvania, USA.

Avibacterium paragallinarum, the causative agent of infectious coryza, causes significant economic losses to the poultry industry due to increased culling rates in growing chickens and decreased egg production in layers. We present the complete genome sequences of seven strains of Avibacterium paragallinarum isolated from poultry farms in Pennsylvania during 2019.

Surveillance for Heterakis spp. in Game Birds and Cage-Free, Floor-Raised Poultry in Pennsylvania.

Histomoniasis is a significant disease of gallinaceous birds caused by Histomonas meleagridis. Transmission of this parasite is dependent on use of the cecal nematode Heterakis gallinarum. To define the host range of this nematode, cecal contents from 399 game birds and poultry, representing eight species, were examined for Heterakis spp. The majority of these species (five of eight) were infected with Heterakis nematodes. Heterakis gallinarum was detected in free-ranging wild turkeys (Meleagridis gallopovo), captive-raised ring-necked pheasants (Phasianus colchicus), chukars (Alectoris chukar), and domestic chickens (Gallus gallus domesticus), whereas H. isolonche was found in ruffed grouse (Bonasa umbellus). No Heterakis species were identified in the domestic turkey (Meleagridis gallopovo), American woodcock (Scolopax minor), and dabbling duck (Anas spp.) samples. Genetic characterization indicated that nematodes identified as H. gallinarum were present in two distinct clades. One clade of H. gallinarum sequenced from this study grouped with chicken-derived sequences from other countries. The other group of sequences consisted of a sister clade to a group of parasites morphologically identified as H. isolonche. Currently it is unknown if this group represents a genetic variant of H. gallinarum, a variant of H. isolonche, or a novel species. These results indicate Heterakis infection varies among poultry and game bird species but is common among select gallinaceous species in Pennsylvania.

Nota de investigación- Vigilancia de Heterakis spp. en aves de caza y aves comerciales criadas en piso sin jaulas en Pennsylvania. La histomoniasis es una enfermedad importante de las aves gallináceas causada por Histomonas meleagridis. La transmisión de este parásito depende de la interacción con el nematodo cecal Heterakis gallinarum. Para definir el rango de hospedadores de este nematodo, se examinaron los contenidos cecales de 399 aves de caza y aves domésticas, que representaron a ocho especies, para detectar Heterakis spp. La mayoría de estas especies (cinco de ocho) estaban infectadas con nematodos Heterakis. Heterakis gallinarum se detectó en pavos silvestres (Meleagridis gallopavo), faisanes comunes criados en cautiverio (Phasianus colchicus), perdiz chukar (Alectoris chukar) y pollos domésticos (Gallus gallus domesticus), mientras que H. isolonche se encontró en el grévol engolado (Bonasa umbellus). No se identificaron especies de Heterakis en las muestras de pavo doméstico (Meleagridis gallopavo), chocha americana (Scolopax minor) y pato chapuceadores (Anas spp.). La caracterización genética indicó que los nematodos identificados como H. gallinarum estaban presentes en dos clados distintos. Un clado de H. gallinarum secuenciado de este estudio agrupado con secuencias derivadas de pollos de otros países. El otro grupo de secuencias consistió en un clado hermano de un grupo de parásitos identificados morfológicamente como H. isolonche. Actualmente se desconoce si este grupo representa una variante genética de H. gallinarum, una variante de H. isolonche o una especie nueva. Estos resultados indican que la infección por Heterakis varía entre las aves domésticas y las especies de aves de caza, pero es común entre las especies de gallináceas seleccionadas en Pennsylvania.

Detection of CTX-M-1 extended-spectrum beta-lactamase among ceftiofur-resistant Salmonella enterica clinical isolates of poultry.

Salmonella enterica resistance to extended-spectrum cephalosporins (ESC) conferred by cefotaximases (blaCTX-M) is a growing concern in the United States. Among food-producing animals, poultry are a major reservoir of ESC-resistant Salmonella. A retrospective study was carried out to further characterize 38 ceftiofur-resistant clinical Salmonella enterica isolates obtained from poultry during 2007-2018. Of the isolates tested, 31 displayed resistance to ceftriaxone and harbored blaCMY-2, whereas 7 isolates demonstrated resistance or reduced susceptibility to cefepime in addition to ceftriaxone resistance. These 7 isolates displayed extended-spectrum ß-lactamase activity, harbored blaCTX-M-1, and were recovered only from recent poultry diagnostic submissions made in 2011-2018 as opposed to the 31 isolates that were recovered in 2007-2018. Further characterization of the blaCTX-M-1 gene determined that it was located on conjugative IncN/ST1 and IncI1/ST87 plasmids in the isolates from commercial turkeys and broilers, respectively. These plasmids have been responsible for extensive spread of blaCTX-M-1 in livestock, poultry, and humans in Europe. Potential transfer of IncN and IncI1 plasmids and/or nontyphoidal Salmonella carrying these plasmids through the food chain, or by other means to humans, may result in treatment failures. Our study demonstrates the importance of further characterization of ceftiofur-resistant S. enterica isolates detected by veterinary diagnostic laboratories to identify the sources of blaCTX-M-1 and to mitigate the spread of ESC-resistant Salmonella in the poultry production pyramid.

A Retrospective Study of Salmonella Enteritidis Isolated from Commercial Layer Flocks.

Consumption of shell eggs has been associated with Salmonella Enteritidis (SE) infections in humans in the United States. Because of this, the Pennsylvania Egg Quality Assurance Program (PEQAP) was developed and implemented in 1994. The PEQAP involves periodic flock testing and management practices to minimize SE contamination of shell eggs. Subsequently, the U.S. Food and Drug Administration (FDA) introduced a mandatory federal program in 2010 and 2012 for shell egg producers modeled closely after PEQAP to reduce the incidence and prevalence of SE during production, storage, and transport nationwide. In this study, a retrospective epidemiologic analysis was conducted by characterizing SE isolated from commercial layer environment samples and shell eggs submitted to the Animal Diagnostic Laboratory at The Pennsylvania State University using phage typing and pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the relatedness of SE isolates from hen house environments and shell eggs and to optimize the existing protocols of egg quality assurance programs by identifying the best layer-house environmental sampling time points in order to minimize SE contamination of shell eggs. A total of 94 SE isolates from 65 hen flocks on 35 premises in Pennsylvania recovered during 2007 to 2015 were used in this study. The SE phage type 8 and PFGE fingerprint type JEGX01.0004 most commonly associated with human SE infection was also the predominant type present in layer-house environments and shell eggs. This reconfirms hen house environmental monitoring is an effective method to identify SE-infected flocks. Further, the PEQAP program allowed SE detection of infected flocks earlier than the FDA program as it included an additional environmental test at 29-31 wk of age, enabling the earlier prevention of SE-contaminated shell eggs going to the market. Therefore, it is recommended to refine the sampling time points of the current FDA Egg Rule by adding hen house environmental testing at 29-31 wk of age.

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

Surveillance for Pasteurella multocida in Ring-Necked Pheasants (Phasianus colchicus) After an Outbreak of Avian Cholera and Apparently Successful Antibiotic Treatment.

Avian cholera is a significant disease of domestic and wild birds caused by the bacterium Pasteurella multocida (PM). In poultry, a major source of PM infection is chronic carriers, domestic birds that have become infected and recovered or had subclinical infections. Although outbreaks of avian cholera in ring-necked pheasants (Phasianus colchicus) have been reported, the potential for chronic carriers is unknown. To address this, we conducted surveillance for PM in a flock of captive ring-necked pheasants after an outbreak of avian cholera that responded positively to antibiotic treatment based on resolution of morbidity and mortality. At approximately 1 mo after antibiotic treatment, oropharyngeal swabs were collected from 300 pheasants (out of a total population of ~2300) in a single winter holding pen. All samples were tested for PM through routine aerobic bacterial culture, but none of the samples were positive. In addition, there were no additional outbreaks within this infected pen over the subsequent months. These data provide preliminary evidence to suggest that pheasants that respond to antibiotic therapy may be less likely to become chronic carriers of PM than other poultry species, such as chickens (Gallus domesticus). However, due to marked phenotypic and biologic differences between PM strains, additional studies are needed to further support or refute these findings and better understand avian cholera in this species.

Risk Factors Associated With Salmonella in Laying Hen Farms: Systematic Review of Observational Studies.

Salmonella contamination of laying hen flocks and shell eggs is associated with various management and environmental factors. Foodborne outbreaks of human salmonellosis have been traced back to consumption of Salmonella-contaminated shell eggs. In the present study, a systematic literature review was conducted to identify and provide an evidence-based overview of potential risk factors of Salmonella contamination of laying hens, layer premises, and shell eggs. This systematic literature search was conducted using AGRICOLA, CAB Abstracts, and PubMed databases. Observational studies that identified risk factors for Salmonella contamination of layer flocks and shell eggs were selected, and best evidence was synthesized to summarize the results. Altogether, 13 cross-sectional studies and four longitudinal studies published in English were included in the review. Evidence scores were assigned based on the study design and quality of the study to grade the evidence level. The strength of association of a risk factor was determined according to the odds ratios. In this systematic review, the presence of previous Salmonella infection, absence of cleaning and disinfection, presence of rodents, induced molting, larger flock size (>30,000 hens), multiage management, cage housing systems, in-line egg processing, rearing pullets on the floor, pests with access to feed prior to movement to the feed trough, visitors allowed in the layer houses, and trucks near farms and air inlets were identified as the risk factors associated with Salmonella contamination of laying hen premises, whereas high level of manure contamination, middle and late phase of production, high degree of egg-handling equipment contamination, flock size of >30,000, and egg production rate of >96% were identified as the risk factors associated with Salmonella contamination of shell eggs. These risk factors demonstrated strong to moderate evidence of association with Salmonella contamination of laying hens and shell eggs. Eggshells testing positive for Salmonella were 59 times higher when fecal samples were positive and nine times higher when floor dust samples were positive. Risk factors associated with Salmonella Enteritidis infection in laying hens were flock size, housing system, and farms with hens of different ages. As a summary, this systematic review demonstrated that Salmonella contamination of laying hen flocks and shell eggs in layer production systems is multifactorial. This study provides a knowledge base for the implementation of targeted intervention strategies to control Salmonella contamination of laying hen flocks and shell eggs.

Isolation and molecular characterization of newly emerging avian reovirus variants and novel strains in Pennsylvania, USA, 2011-2014.

Avian reovirus (ARV) infections of broiler and turkey flocks have caused significant clinical disease and economic losses in Pennsylvania (PA) since 2011. Most of the ARV-infected birds suffered from severe arthritis, tenosynovitis, pericarditis and depressed growth or runting-stunting syndrome (RSS). A high morbidity (up to 20% to 40%) was observed in ARV-affected flocks, and the flock mortality was occasionally as high as 10%. ARV infections in turkeys were diagnosed for the first time in PA in 2011. From 2011 to 2014, a total of 301 ARV isolations were made from affected PA poultry. The molecular characterization of the Sigma C gene of 114 field isolates, representing most ARV outbreaks, revealed that only 21.93% of the 114 sequenced ARV isolates were in the same genotyping cluster (cluster 1) as the ARV vaccine strains (S1133, 1733, and 2048), whereas 78.07% of the sequenced isolates were in genotyping clusters 2, 3, 4, 5, and 6 (which were distinct from the vaccine strains) and represented newly emerging ARV variants. In particular, genotyping cluster 6 was a new ARV genotype that was identified for the first time in 10 novel PA ARV variants of field isolates.

Acute septicemia caused by Streptococcus gallolyticus subsp. pasteurianus in turkey poults.

Streptococcus gallolyticus, previously known as Streptococcus bovis biotypes I and II/2, is a well-known cause of sepsis and meningitis in humans and birds. The present case report describes an outbreak of fatal septicemia associated with S. gallolyticus subsp. pasteurianus (S. bovis biotype II/2) in 11 turkey flocks in Pennsylvania between 2010 and 2013. Affected poults were 2-3 wk of age. Major clinical observation was sudden increase in mortality among turkey poults without any premonitory clinical signs. Postmortem examination findings revealed acute septicemia with lesions such as fibrinous pericarditis, meningitis, splenic multifocal fibrinoid necrosis, hepatitis, osteochondritis, myositis, and airsacculitis. Gram-positive cocci were isolated from several organs by routine bacterial culture. Biotyping identified bacteria as streptococci, whereas 16S ribosomal RNA gene sequencing identified them as S. gallolyticus subsp. pasteurianus. Antibiotic susceptibility profiles revealed that all the strains isolated were sensitive to penicillin and erythromycin with different sensitivity profiles for other antibacterial agents tested. The present study reports the first confirmed case of acute septicemia in turkey poults caused by S. gallolyticus subsp. pasteurianus.

Fingerprinting of poultry isolates of Enterococcus cecorum using three molecular typing methods.

Enterococcus cecorum is an emerging challenge to the broiler industry. The organism has been implicated in septicemia, spondylitis, arthritis, and osteomyelitis in commercial broilers and broiler breeders, which lead to economic losses attributed to increased mortality and culling rates, decreased average processing weights, and increased feed conversion ratios. The current study evaluated the genetic variability of 30 clinical isolates of E. cecorum from outbreaks in Pennsylvania, using 3 molecular typing methods, namely, pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA analysis, and enterobacterial repetitive intergenic consensus-PCR (polymerase chain reaction), in order to understand their genetic relatedness and to identify possible pathogenic clones. The study revealed the existence of genotypic polymorphism among E. cecorum associated with clinical disease. Of the 3 typing methods used, PFGE analysis demonstrated higher genetic variability of E. cecorum isolates compared to PCR-based methods. Also, each molecular typing method was evaluated in terms of typeability, discriminatory power, and reproducibility for application of these typing methods in fingerprinting of E. cecorum in future reference. Pulsed-field gel electrophoresis provided the most reliable results with greater discriminatory power and higher reproducibility compared to the 2 PCR-based methods.

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02.

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.