A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry.

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

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.

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.

Causes of mortality in backyard poultry in eight states in the United States.

A comprehensive understanding of common diseases of backyard poultry flocks is important to providing poultry health information to flock owners, veterinarians, and animal health officials. We collected autopsy reports over a 3-y period (2015-2017) from diagnostic laboratories in 8 states in the United States; 2,509 reports were collected, involving autopsies of 2,687 birds. The primary cause of mortality was categorized as infectious, noninfectious, neoplasia or lymphoproliferative disease, or undetermined. Neoplasia or lymphoproliferative disease was the most common primary diagnosis and involved 42% of the total birds autopsied; 63% of these cases were diagnosed as Marek's disease or leukosis/sarcoma. Bacterial, parasitic, and viral organisms were commonly detected, involving 42%, 28%, and 7% of the birds autopsied, respectively, with 2 or more organisms detected in 69% of birds. Our findings demonstrate the importance of educating flock owners about disease prevention and biosecurity practices. The detection of zoonotic bacteria including paratyphoid salmonellae, Campylobacter spp., Listeria monocytogenes, and Mycobacterium avium, and the detection of lead and other heavy metals, indicate public health risks to flock owners and consumers of backyard flock egg and meat products.

Serotyping of Salmonella Enterica Isolated from Mice Caught on US Poultry Farms 1995 through 1998.

In this study, a collection of Salmonella enterica subspecies obtained from live mice caught on 32 poultry farms in the Northeast US between 1995 to 1998 was evaluated to provide a historical reference for serotype distribution during a time when egg contamination by serotype Enteritidis was at its peak. Of 821 mice cultured, 157 were positive (19.1%). Seven mice harbored two serotypes of Salmonella. Nine serotypes were detected, eight of which are often associated with foodborne illness. The three most prevalent serotypes were Enteritidis, Heidelberg, and Typhimurium. Enteritidis and Typhimurium were obtained from both spleens and intestines without preference according to type of sample. In contrast, Heidelberg was isolated most often from intestines and Schwarzengrund was most often obtained from spleens. These results support that the house mouse Mus musculus was a risk factor for introduction of multiple pathogenic Salmonella serotypes in poultry raised in the Northeast US during the mid-1990s. Isolates were submitted to the Food and Drug Administration and draft genomes for 64 isolates of Salmonella enterica serovar Enteritidis data have been released through the National Center for Biotechnology Information via the GenomeTrakr network.

Draft Genome Sequences of 64 Salmonella enterica Serotype Enteritidis Isolates Obtained from Wild Mice.

Salmonella enterica serotype Enteritidis is a foodborne pathogen of global concern, because it is frequently isolated from foods and patients. Draft genome sequences are reported here for 64 S Enteritidis strains isolated from the intestines and spleens of mice caught live on chicken farms in the U.S. Northeast. The availability of these genomes provides baseline information on the genomic diversity of S Enteritidis during the 1990s, when foodborne outbreaks traced to internal contamination of eggs were prevalent.

Development of Experimental Myopia in Chicks in a Natural Environment.

PURPOSE: The hypothesis that outdoor exposure might protect against myopia has generated much interest, although available data find only modest clinical efficacy. We tested the effect of outdoor rearing on form-deprivation myopia in chicks, a myopia model markedly inhibited by high-intensity indoor laboratory lighting. METHODS: Unilaterally goggled cohorts of White Leghorn chicks were maintained in a species-appropriate, outdoor rural setting during daylight hours to the extent permitted by weather. Control chicks were reared indoors with incandescent lighting. Besides ocular refraction and ultrasound, we determined dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content in retina and vitreous and measured mRNA expression levels of selected clock and circadian rhythm-related genes in the retina/RPE. RESULTS: Myopia developed in the goggled eyes of all cohorts. Whereas outdoor rearing lessened myopia by 44% at 4 days, a protective effect was no longer evident at 11 days. Outdoor rearing had no consistent effect on retinal or vitreous content of dopamine or DOPAC. Conforming to prior data on form-deprivation myopia, retina and vitreous levels of DOPAC were reduced in goggled eyes. Compared with contralateral eyes, the retinal expression of clock and circadian rhythm-related genes was modestly altered in myopic eyes of chicks reared indoors or outdoors. CONCLUSIONS: Outdoor rearing of chicks induces only a partial decrease of goggle-induced myopia that is not maintained, without evidence that retinal dopamine metabolism accounts for the partial myopia inhibition under these outdoor conditions. Although modest, alterations in retinal gene expression suggest that studying circadian signals might be informative for understanding refractive mechanisms.

Serum biochemistry values, plasma mineral levels, and whole blood heavy metal measurements in wild northern goshawks (Accipiter gentilis).

Serum chemistry panels and complete mineral and heavy metal screens were performed on blood samples from eight adult northern goshawks (Accipiter gentilis) breeding in Pennsylvania. Serum chemistry panels were performed to determine the health status of each bird. Biochemical values measured included serum glucose, sodium, potassium, chloride, magnesium, uric acid, creatine kinase, and aspartate transaminase. Glucose, creatine kinase, and aspartate transaminase values were elevated when compared with published values for northern goshawks and other species of raptors. Complete mineral screens were performed to better document the blood mineral content of northern goshawks. Plasma calcium, copper, iron, magnesium, total phosphorus, potassium, sodium, and zinc levels were determined. Whole blood heavy metal screens were done to evaluate the northern goshawk's exposure to environmental arsenic, cadmium, lead, thallium, and selenium. Arsenic, cadmium, lead, and thallium whole blood levels of less than 0.05 ppm in all birds indicated that the northern goshawks were not being exposed to significant levels of heavy metals in their environment. Whole blood selenium levels of the northern goshawks were above the minimum dietary requirement for avian species (0.130-0.200 ppm) and below published toxic selenium concentrations.

Evaluation of the efficacy of a live fowlpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral challenge.

Infectious laryngotracheitis (ILT) is caused by an alphaherpesvirus, and latency can be produced by previous exposure to vaccine virus. The main sites of latency for the ILT virus have been shown to be the trigeminal ganglion and the trachea. Reactivation of latent virus is one factor related to the production of clinical signs. The development of a genetically engineered ILT vaccine has been suggested for many years as a tool to eliminate viral latency. Several approaches have been suggested. Included among them is the development of a thymidine kinase-deficient mutant or the insertion of ILT viral glycoproteins into a viral vector such as a poxvirus. A commercially available, live, fowlpox-vectored infectious laryngotracheitis + avian encephalomyelitis (FP-LT+AE) vaccine was used in field trials in leghorn pullet flocks and evaluated by tracheal challenge in a laboratory setting with the use of the National Veterinary Services Laboratory (Ames, IA) ILT challenge virus. Interference of the pigeon pox vaccine, which is often administered concurrently with fowlpox vaccine, was also evaluated when given in conjunction with the FP-LT+AE vaccine. Overall, the results indicate that the FP-LT+AE vaccine provides adequate protection against ILT viral challenge. Proper administration is essential. In one flock, inadequate protection was most likely a result of either poor vaccine administration or previous exposure to pox virus. In addition, the simultaneous administration of pigeon pox vaccine did not appear to interfere with protection against ILT viral challenge.

Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States.

The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.

The role of disinfectant resistance of Salmonella enterica serotype enteritidis in recurring infections in Pennsylvania egg quality assurance program monitored flocks.

The Pennsylvania egg quality assurance program (PEQAP) has made major gains in the reduction of Salmonella enterica serotype enteritidis (S. enteritidis). However, S. enteritidis continues to be a major food safety concern for the commercial egg laying industry. Despite intensive control efforts through PEQAP, some commercial egg layer houses still remain positive for S. enteritidis. The primary objective of this study was to determine whether S. enteritidis isolates obtained from historically environmentally S. enteritidis-positive houses were resistant to commonly used disinfectants. Archived S. enteritidis isolates (environmental, rodent, or egg) were compared with recently obtained isolates from the environment, rodents, or eggs from the same S. enteritidis-positive house. In addition, the isolates were compared with archived isolates from those premises that appeared to have eliminated S. enteritidis from their layer facilities. The official methods of the use-dilution analysis of the Association of Official Analytical Chemists were used to evaluate each disinfectant product. Two phenolic, one quaternary ammonium, and one combination product containing quaternary ammonium and formaldehyde were evaluated, in addition to one sodium hypochlorite detergent. All products diluted according to the manufacturers' recommendations killed the S. enteritidis isolates in this test system. There was no difference in susceptibility or resistance to the disinfectants used between the isolates from those facilities that remained S. enteritidis-positive and those that appeared to have eliminated S. enteritidis from their facility.