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.

Characterization of Avian Pox in a Ruffed Grouse (Bonasa umbellus) from Pennsylvania.

A free-ranging, adult male ruffed grouse (Bonasa umbellus) was harvested by a hunter during November 2019 in Forest County, PA. The bird was submitted for necropsy due to a skin mass on its left leg. Upon necropsy, two proliferative skin masses were grossly visible, one on the left leg and one on the cere. An additional mass was present on the oropharyngeal mucosa covering the hard palate. These masses were diagnosed as avian pox based on histopathologic and cytologic findings, including marked epithelial hypertrophy, hyperplasia, vacuolar degeneration with eosinophilic stippling, and intracytoplasmic inclusion bodies. An avipoxvirus was detected using PCR and was identified as fowlpox virus through sequencing of the 4b core gene segment. The avipoxvirus from this case showed genetic similarity to isolates from Eastern wild turkeys (Meleagris gallopavo silvestris).

Caracterización de la viruela aviar en un grévol engolado (Bonasa umbellus) en el estado de Pensilvania. Un cazador recolectó un grévol engolado macho adulto silvestre (Bonasa umbellus) durante noviembre del 2019 en el condado de Forest, Pensilvania. El ave fue sometida a necropsia debido a una masa cutánea en su pata izquierda. Durante la necropsia, dos masas cutáneas proliferativas fueron claramente visibles, una en la pierna izquierda y otra en la cera. Había una masa adicional en la mucosa orofaríngea que cubría el paladar duro. Estas masas se diagnosticaron como viruela aviar con base en los hallazgos histopatológicos y citológicos, que incluyeron hipertrofia epitelial marcada, hiperplasia, degeneración vacuolar con punteado eosinofílico y cuerpos de inclusión intracitoplasmáticos. Se detectó un avipoxvirus mediante PCR y se identificó como virus de la viruela aviar mediante la secuenciación del segmento del gene 4b del centro viral. El avipoxvirus de este caso mostró similitud genética con aislamientos de pavos salvajes del este (Meleagris gallopavo silvestris).

Molecular epidemiology of Marek's disease virus in central Pennsylvania, USA.

The evolution of Marek's disease virus (MDV, Gallid herpesvirus 2) has threatened the sustainability of poultry farming in the past and its continued evolution remains a concern. Genetic diversity is key to understanding evolution, yet little is known about the diversity of MDV in the poultry industry. Here, we investigate the diversity of MDV on 19 Pennsylvanian poultry farms over a 3-year period. Using eight polymorphic markers, we found that at least twelve MDV haplotypes were co-circulating within a radius of 40 km. MDV diversity showed no obvious spatial clustering nor any apparent clustering by bird line: all of the virus haplotypes identified on the commercial farms could be found within a single, commonly reared bird line. On some farms, a single virus haplotype dominated for an extended period of time, while on other farms the observed haplotypes changed over time. In some instances, multiple haplotypes were found simultaneously on a farm, and even within a single dust sample. On one farm, co-occurring haplotypes clustered into phylogenetically distinct clades, putatively assigned as high and low virulence pathotypes. Although the vast majority of our samples came from commercial poultry farms, we found the most haplotype diversity on a noncommercial backyard farm experiencing an outbreak of clinical Marek's disease. Future work to explore the evolutionary potential of MDV might therefore direct efforts toward farms that harbor multiple virus haplotypes, including both backyard farms and farms experiencing clinical Marek's disease.

Modeling Marek's disease virus transmission: A framework for evaluating the impact of farming practices and evolution.

Marek's disease virus (MDV) is a pathogen of chickens whose control has twice been undermined by pathogen evolution. Disease ecology is believed to be the main driver of this evolution, yet mathematical models of MDV disease ecology have never been confronted with data to test their reliability. Here, we develop a suite of MDV models that differ in the ecological mechanisms they include. We fit these models with maximum likelihood using iterated filtering in 'pomp' to data on MDV concentration in dust collected from two commercial broiler farms. We find that virus dynamics are influenced by between-flock variation in host susceptibility to virus, shedding rate from infectious birds, and cleanout efficiency. We also find evidence that virus is reintroduced to farms approximately once per month, but we do not find evidence that virus sanitization rates vary between flocks. Of the models that survive model selection, we find agreement between parameter estimates and previous experimental data, as well as agreement between field data and the predictions of these models. Using the set of surviving models, we explore how changes to farming practices are predicted to influence MDV-associated condemnation risk (production losses at slaughter). By quantitatively capturing the mechanisms of disease ecology, we have laid the groundwork to explore the future trajectory of virus evolution.

Industry-Wide Surveillance of Marek's Disease Virus on Commercial Poultry Farms.

Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely. We conducted a 3-yr surveillance study to assess the prevalence of Marek's disease virus on commercial poultry farms, determine the effect of various factors on virus prevalence, and document virus dynamics in broiler chicken houses over short (weeks) and long (years) timescales. We extracted DNA from dust samples collected from commercial chicken and egg production facilities in Pennsylvania, USA. Quantitative PCR was used to assess wild-type virus detectability and concentration. Using data from 1018 dust samples with Bayesian generalized linear mixed effects models, we determined the factors that correlated with virus prevalence across farms. Maximum likelihood and autocorrelation function estimation on 3727 additional dust samples were used to document and characterize virus concentrations within houses over time. Overall, wild-type virus was detectable at least once on 36 of 104 farms at rates that varied substantially between farms. Virus was detected in one of three broiler-breeder operations (companies), four of five broiler operations, and three of five egg layer operations. Marek's disease virus detectability differed by production type, bird age, day of the year, operation (company), farm, house, flock, and sample. Operation (company) was the most important factor, accounting for between 12% and 63.4% of the variation in virus detectability. Within individual houses, virus concentration often dropped below detectable levels and reemerged later. These data characterize Marek's disease virus dynamics, which are potentially important to the evolution of the virus.

DNA from Dust: Comparative Genomics of Large DNA Viruses in Field Surveillance Samples.

The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek's disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately reflect virus in the field. The ability to directly sequence and compare field isolates of this virus is critical to understanding the genetic basis of rising virulence in the wild. Our approaches remove the prior requirement for cell culture and allow direct measurement of viral genomic variation within and between hosts, over time, and during adaptation to changing conditions.

Review of Nonfoodborne Zoonotic and Potentially Zoonotic Poultry Diseases.

Emerging and re-emerging diseases are continuously diagnosed in poultry species. A few of these diseases are known to cross the species barrier, thus posing a public health risk and an economic burden. We identified and synthesized global evidence for poultry nonfoodborne zoonoses to better understand these diseases in people who were exposed to different poultry-related characteristics (e.g., occupational or nonoccupational, operational types, poultry species, outbreak conditions, health status of flocks). This review builds on current knowledge on poultry zoonoses/potentially zoonotic agents transmitted via the nonfoodborne route. It also identifies research gaps and potential intervention points within the poultry industry to reduce zoonotic transmission by using various knowledge synthesis tools such as systematic review (SR) and qualitative (descriptive) and quantitative synthesis methods (i.e., meta-analysis). Overall, 1663 abstracts were screened and 156 relevant articles were selected for further review. Full articles (in English) were retrieved and critically appraised using routine SR methods. In total, eight known zoonotic diseases were reviewed: avian influenza (AI) virus (n = 85 articles), Newcastle disease virus (n = 8), West Nile virus (WNV, n = 2), avian Chlamydia (n = 24), Erysipelothrix rhusiopathiae (n = 3), methicillin-resistant Staphylococcus aureus (MRSA, n = 15), Ornithonyssus sylvarium (n = 4), and Microsporum gallinae (n = 3). In addition, articles on other viral poultry pathogens (n = 5) and poultry respiratory allergens derived from mites and fungi (n = 7) were reviewed. The level of investigations (e.g., exposure history, risk factor, clinical disease in epidemiologically linked poultry, molecular studies) to establish zoonotic linkages varied across disease agents and across studies. Based on the multiple outcome measures captured in this review, AI virus seems to be the poultry zoonotic pathogen that may have considerable and significant public health consequences; however, epidemiologic reports have only documented severe human cases clustered in Asia and not in North America. In contrast, avian Chlamydia and MRSA reports clustered mainly in Europe and less so in North America and other regions. Knowledge gaps in other zoonoses or other agents were identified, including potential direct (i.e., nonmosquito-borne) transmission of WNV from flocks to poultry workers, the public health and clinical significance of poultry-derived (livestock-associated) MRSA, the zoonotic significance of other viruses, and the role of poultry allergens in the pathophysiology of respiratory diseases of poultry workers. Across all pathogens reviewed, the use of personal protective equipment was commonly cited as the most important preventive measure to reduce the zoonotic spread of these diseases and the use of biosecurity measures to reduce horizontal transmission in flock populations. The studies also emphasized the need for flock monitoring and an integrated approach to prevention (i.e., veterinary-public health coordination with regard to diagnosis, and knowledge translation and education in the general population) to reduce zoonotic transmission.

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.

An observational study of the temporal and spatial patterns of Marek's-disease-associated leukosis condemnation of young chickens in the United States of America.

Marek's disease, a disease primarily affecting immature chickens, is a worldwide problem that has on at least three occasions threatened the poultry industry in the United States. A rich dataset to study the epidemiology of this disease is available because the United States Department of Agriculture has required mandatory inspections of all commercially sold poultry of significant scale since the mid-20th century with over 99% of all chickens inspected. This dataset includes monthly totals aggregated by state since 1961 of the number of "young chickens" inspected and the number with "leukosis", a condemnation category that is almost always associated with Marek's disease in this category of birds. The objective of this study was to analyze temporal and spatial patterns in this condemnation data to gain insight into the ecology and epidemiology of the causative virus. We extracted visual patterns in the data using seasonal trend decomposition, and we tested for statistical significance using extended linear modeling techniques. The analysis confirmed previous findings that there are differences in leukosis condemnation rates between states, across years, and within years. The analysis also revealed several patterns not previously highlighted, including spatial and temporal autocorrelations in leukosis condemnation, changes to the amplitude of seasonality over time, and increasing within-year variation in condemnation rate over time. These patterns suggest that locally shared farm practices, virus transmission between farms, or viral persistence may be important to understanding the dynamics of the disease. We also discuss the plausibility of other potential explanations for these patterns.

Brachyspira intermedia and Brachyspira pilosicoli are commonly found in older laying flocks in Pennsylvania.

Anaerobic intestinal spirochetes (genus Brachyspira) include several species that are recognized as pathogens of poultry. Surveys undertaken in Europe and Australia have shown that layer and breeder flocks are often colonized by the pathogenic species Brachyspira intermedia and Brachyspira pilosicoli, but similar surveys have not been conducted in the United States. In the current study, DNA was extracted from fecal samples (n=50) collected from each of 21 flocks of laying hens >40 wk of age in Pennsylvania, and this material was tested for B. intermedia and B. pilosicoli using a duplex PCR. Negative samples also were tested using a Brachyspira genus-specific PCR. The consistency of the feces was observed, and manure handling systems and medication histories were recorded. Brachyspira intermedia was detected in 662 (63.1%) samples from 17 (81%) flocks, with a within-flock prevalence of 10%-100%. Brachyspira pilosicoli was detected in 112 (10.7%) samples from 5 flocks (23.8%), with a within-flock prevalence of 8%-82%. Four of the flocks had both pathogenic species present, three had no pathogenic species detected, and two had no Brachyspira species detected. Nine flocks had many fecal samples with a wet appearance and/or a caramel color, and all of these were colonized with one or the other of the two pathogenic species. Nine of 12 flocks with manure that was mainly dry also were colonized. Differences in colonization rates between flocks with or without wet manure were not significant. Colonization with pathogenic Brachyspira species, and particularly B. intermedia, occurs very commonly in layer flocks >40 wk of age in Pennsylvania. The significance of this high rate of colonization requires further investigation.

Isolation and molecular characterization of a new Muscovy duck parvovirus from Muscovy ducks in the USA.

Between 1997 and 1999 several cases of a new disease in Muscovy ducks were reported in Pennsylvania, USA. The cases were characterized by locomotor dysfunction, weakness, recumbency, 40 to 60% morbidity and 10 to 40% mortality. The most characteristic microscopic lesions were moderate to severe degenerative rhabodomyopathy. In order to characterize the aetiological agent, virus isolation was attempted from the spleen, liver, heart, skeletal muscle and intestine by inoculation of 14-day-old Muscovy duck embryos with tissue homogenates. Deaths occurred on the second egg passage and parvoviruses were isolated by serial passage of allantoic fluid from dead embryos and then in Muscovy duck embryo fibroblast (MDEF) cultures. Parvovirus particles were observed in allantoic fluids and supernatants of MDEF cultures by transmission electron microscopy. Two genomic fragments, comprising 1108 nucleotides of the right open reading frame that codes for the structural viral proteins 1, 2 and 3, were amplified by polymerase chain reaction from one of the isolates, Muscovy duck parvovirus (MDPV)/PSU-31010. Comparison of this fragment with available sequences of other MDPV and related goose parvovirus (GPV) isolates showed that it had only 84.5% sequence identity with other MDPV isolates and 84.6% identity with the GPV isolates. This region shares over 99% identity among previously sequenced MDPV isolates and 95% identity among the related GPV isolates. This suggests that MDPV/PSU-31010 is divergent from all other sequenced MDPV and GPV isolates, and may represent a new group of avian parvoviruses.

Perinatal litigation and related nursing issues.

Obstetric and neonatal nurses are expected to provide an abundance of guidance, support, monitoring, and education to women and their babies during and after delivery. Nurses should adhere to standards of professional nursing practice. This will ensure that optimal and safe care is provided for the mother and fetus or neonate. Perinatal nurses are vulnerable to litigation should complications occur. Perinatal nurses are responsible for providing routine assessments as well as initiating and performing emergency interventions. This includes recognition of the symptoms of complications in the mother and the neonate, resuscitation, and activation of the emergency system. Occasionally, nurses are obliged to question the practice of other health care providers. Although perinatal nurses continue to be at risk for malpractice vulnerability, risk reduction techniques are available to them. This article provides the nurse with knowledge of legal proceedings and strategies to reduce liability when caring for pregnant women and newborns.

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.