Avian Encephalomyelitis in Layer Pullets Associated with Vaccination.

Avian encephalomyelitis (AE) was diagnosed in three flocks of leghorn layer pullets following AE vaccination. Ages of the birds were 11, 12, and 14 wk. The submissions came from three different companies located in two geographic areas of the Central Valley of California. The clinical signs included birds down on their legs, unilateral recumbency or sitting on their hocks, lethargy, reluctance to move, dehydration, unevenness in size, low weight, tremors of the head in a few birds, and mildly to moderately elevated mortality. The flocks had been vaccinated against fowl pox and AE with a combined product in the wing-web 2 wk prior to the onset of AE clinical signs. Histopathologic examination revealed lesions consistent with AE, including lymphocytic perivascular infiltration and neuronal central chromatolysis in the brain and spinal cord, as well as gliosis in the cerebellar molecular layer. The AE virus was detected by reverse-transcriptase PCR in the brain homogenate from three cases and peripheral nerves in one case. Additionally, the AE virus was isolated in specific-pathogen-free (SPF) embryonated eggs from brain tissue pool samples. Other avian viral infections capable of causing encephalitis, including avian paramyxoviruses, avian influenza virus (AIV), West Nile virus (WNV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), were ruled out by attempting virus isolation and molecular procedures.

Diagnosis and Control of a LPAI H5N8 Outbreak in a Japanese Quail (Coturnix coturnix japonica) Commercial Flock in the Central Valley of California.

In April 2014 an outbreak of low pathogenic avian influenza H5N8 North American genetic lineage was diagnosed in a commercial quail operation in Stanislaus County, California. Sudden increase in mortality prompted the submission of 20 Japanese quail hens (Coturnix c. japonica) to the California Animal Health and Food Safety Laboratory, Turlock Branch. Oropharyngeal and cloacal swabs tested positive for influenza A virus H5N8 by real-time reverse transcription-polymerase chain reaction. The virus was subsequently isolated. In vivo assay and sequencing of the hemagglutinin protein cleavage site classified the virus as a North American genetic lineage of low pathogenicity for chickens. Following the diagnosis, a rapid and coordinated response took place to contain the outbreak. The affected premise was depopulated, cleaned, and disinfected. Three areas from the affected premises-a 3 kilometer (km) radius (High Risk Zone), a 3-10 km area (Buffer Zone), and a 10-20 km (Surveillance Zone)-were established for avian influenza testing of commercial and noncommercial poultry operations. Surveillance testing and rapid control measures were successful in the control and eradication of the outbreak and revealed no area of spread of the virus from the index flock. This report describes the history, diagnosis, surveillance, and control measures applied to manage this outbreak.

Pathogenicity associated with coinfection with very virulent infectious bursal disease and Infectious bursal disease virus strains endemic in the United States.

The pathogenicity induced by co-challenge with the rB strain of very virulent Infectious bursal disease virus (vvIBDV) and IBDV pathotypes endemic in the United States was evaluated in specific pathogen-free chickens. Four- and 6-week-old birds were simultaneously challenged with a 10(5) 50% egg infectious dose (EID50) of rB mixed with a 10(5) EID50 of one of the following viruses: standard classic (STC), subclinical variant (Del-E), subclinical variant (T1), or avirulent serotype 2 (OH). Each challenge group consisted of 5 chickens. The severity of disease was assessed by comparing the 5-day mortality rates, bursal lesions (mean bursal lesion scores), and mean bursal-to-body weight ratios in each of the challenged groups. A mortality of 100% (10/10 and 5/5) was observed in birds inoculated with only the vvIBDV (rB) strain at 4 weeks and 6 weeks of age, respectively. Although the sample sizes were low, a significant reduction in mortality and severity of disease, based on mean bursal lesion scores, was observed in groups co-challenged with rB and the less virulent pathotypes Del-E, T1, or OH at 4 weeks of age. Co-challenge with rB and the antigenically similar STC strain did not result in a significant decrease in mortality compared to challenge with the pathogenic rB strain at 4 weeks of age, but a significant reduction in the mean bursa lesion score was observed. At 6 weeks of age, a significant decrease in mortality and mean bursa lesion score was observed in the rB groups co-challenged with STC, Del-E, or T1 but not OH.

Diversity of genome segment B from infectious bursal disease viruses in the United States.

Several phylogenetic lineages of the infectious bursal disease virus (IBDV) genome segment B have been identified. Although this genome segment has been shown to contribute to virulence, little is known about the genetic lineages that exist in the United States. The nucleotide genome segment B sequences of 67 IBDV strains collected from 2002 to 2011 in the United States were examined. Although they were from nine different states, a majority (47) of these viruses were from California. A 722-base pair region near the 5' end of genome segment B, starting at nucleotide 168 and ending at 889, was examined and compared to sequences available in GenBank. The nucleotide sequence alignment revealed that mutations were frequently observed and that they were uniformly spaced throughout the region. When the predicted amino acids were aligned, amino acids at positions 145, 146, and 147 were found to change frequently. Six different amino acid triplets were observed and the very virulent (vv) IBDV strains (based on presence of vvIBDV genome segment A sequence) all had the triplet T145, D146, and N147. None of the non-vvIBDV strains had this TDN triplet. Phylogenetic analysis of the 67 nucleotide sequences revealed four significant genome segment B lineages among the U.S. viruses. One of these included the genome segment B typically found in vvIBDV and three contained non-vvIBDV genome segment B sequences. When the available sequences in GenBank were added to the analysis, two additional lineages were observed that did not contain U.S. viruses; one included viruses from China and the other contained viruses from the Ivory Coast. Although the samples tested do not represent all poultry producing regions in the United States, serotype 1 viruses from states outside California all belonged to one genome segment B lineage. The other three lineages observed in the United States were populated with viruses exclusively found in California, except the serotype 2 lineage, where the type strain was a serotype 2 virus from Ohio. The data provide further evidence for the importance of genome segment B identification during routine molecular diagnosis of all IBDV strains.

Identification and pathogenicity of a natural reassortant between a very virulent serotype 1 infectious bursal disease virus (IBDV) and a serotype 2 IBDV.

Infectious bursal disease virus (IBDV) causes an economically important, immunosuppressive disease in chickens. There are two serotypes of the virus that contain a bi-segmented double-stranded RNA genome. In December 2008, the first very virulent (vv)IBDV was identified in California, USA and in 2009 we isolated reassortant viruses in two different locations. Genome segment A of these reassortants was typical of vvIBDV serotype 1 but genome segment B was most similar to IBDV serotype 2. The CA-K785 reassortant caused 20% mortality in chickens but no morbidity or mortality in commercial turkey poults despite being infectious. There have been previous reports of natural reassortants between vvIBDV and other serotype 1 strains, but a natural reassortant between IBDV serotypes 1 and 2 has not been described. The apparent reassorting of California vvIBDV with an endemic serotype 2 virus indicates a common host and suggests vvIBDV may have entered California earlier than originally thought.

Parasitic ventriculitis caused by Hadjelia truncata in California rock pigeons (Columba livia).

Severe ventriculitis and emaciation caused by the infestation of the nematode Hadjelia truncata occurred in meat-type breeder rock pigeons (Columba livia) in southern and central California. Hadjelia truncata can infest several species of birds, although it has only been reported as pathogenic in pigeons. The factors that contribute to H. truncata pathogenicity are not known. The gross and microscopic pathology caused by the infestation of H. truncata in the ventriculus of pigeons and its morphological identification are presented.

The diagnosis of very virulent infectious bursal disease in California pullets.

This report documents the occurrence of a very virulent infectious bursal disease virus (vvIBDV) in Northern California commercial brown pullets. Diagnosis was made from multiple accessions from two neighboring and epidemiologically related ranches submitted to the California Animal Health and Food Safety (CAHFS) laboratory. Pullets, 11 and 14 wk of age from ranch A (rA) and ranch B (rB) respectively, were submitted from infectious bursal disease virus vaccinated flocks experiencing a drastic increase in mortality. The December 2008 outbreak resulted in 26% and 34% mortality on rA and rB respectively. Gross and histologic lesions characteristic of acute vvIBDV were observed. Gross lesions included edematous bursas, hemorrhages at the junction of the proventriculus and gizzard as well as hemorrhages on skeletal muscles. Microscopic lesions included severe lymphoid necrosis and inflammation in edematous bursas, lymphoid necrosis in thymus, spleen, Peyer's patches and cecal tonsils. Diagnosis of vvIBDV was confirmed by molecular characterization of the IBDV from bursas as well as viral pathogenicity in specific-pathogen-free birds. RT-PCR and nucleotide sequencing of the hypervariable region of the VP2 (vVP2) gene segment of the IBDV genome was performed on rA, rB and embryo passaged rA virions.The amino acids compatible with vvIBDV isolates: 222(Ala), 242(Ile), 256(Ile), 294(Ile) and 299(Ser) were reported from both ranches. In addition, nucleotide sequencing of a fragment of the VP1 gene demonstrated the viruses have the segment B genotype associated with highly pathogenic vvIBDV. Inocula of 10(5.5) 50% egg infective dose of vvIBDV virus from rA and rB were introduced orally into two groups (g1 and g2 respectively) of 4 wk 2-day-old SPF leghorns. At 4 days postinoculation, there was 100% (22/22) morbidity in g1 and g2; 91% (20/22) mortality in g1; 100% (22/22) mortality for g2; 0% (0/20) morbidity and 0% (0/ 20) mortality was reported in the control group. This is the first occurrence of vvIBDV reported from birds in the United States.

Mycotic pododermatitis and mycotic pneumonia in commercial turkey poults in northern California.

Seven 5-week-old broad-breasted white commercial meat turkeys were submitted to the California Animal Health and Food Safety laboratory in Turlock with a history of respiratory illness. The primary diagnostic findings were mycotic pododermatitis and mycotic pneumonia. The unique feature of this case was the colonization of footpad epidermis and subcutis by fungal hyphae in commercial turkey species. No fungal cultures were undertaken at the time of the necropsy; therefore, paraffin-embedded tissue sections of lung and footpads were used to extract, amplify, and sequence mycotic DNA. A mixed population of fungi was identified in both lung and footpads by polymerase chain reaction amplification of part of the large subunit ribosomal RNA gene using broad-range fungal primers and DNA sequencing. In footpads, sequences matching Cryptococcus saitoi and Cladosporium and Cudoniella species were identified. It is believed that these fungi were opportunistic pathogens originating from the litter. The fungi identified from lungs were Aspergillus species, most closely matching Aspergillus flavus and Arxiozyma telluris (most likely a contaminant). Mycotic pododermatitis in avian species is considered a rare pathologic finding, and few documented reports are available. The on-farm prevalence of footpad lesions was estimated at 3%, and there was no associated increase in the incidence of lameness or weight depression in affected birds. Microscopically, a granulomatous inflammatory reaction associated with fungal hyphae was observed in lung parenchyma. Disruption of keratinized epidermis, encrustations, and acute inflammation were also noted in footpads invaded with fungal hyphae.

Characteristics of a very virulent infectious bursal disease virus from California.

An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvwIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (105.5 50% egg infective dose [EID50]) and a low dose (102.0 EID50) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 10(2) EID50 and 10(5) EID50 of the STC virus. When challenged with 10(2) EID50 of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription-polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 10(5) EID50 of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Persistence of immunity in commercial egg-laying hens following vaccination with a killed H6N2 avian influenza vaccine.

The California poultry industry experienced an outbreak of H6N2 avian influenza beginning in February 2000. The initial infections were detected in three commercial egg-laying flocks and a single noncommercial backyard flock but later spread to new premises. The vaccination of pullet flocks with a commercially prepared, killed autogenous vaccine prior to their placements on farms with infected or previously infected flocks was used as a part of the eradication programs for some multiage, commercial egg production farms. The purpose of this study was to follow three vaccinated flocks on two commercial farms to track the immune responses to vaccination. The antibody-mediated responses of the three flocks followed in this study were markedly different. One flock achieved 100% seroconversion at 12.5 wk of age, but by 32 wk of age, all of the hens were seronegative by agar gel immunodiffusion (AGID). In contrast, at 32 wk of age, flocks from the other farm (flocks 2A and 2B) were 95% and 72% seropositive by AGID, respectively. Of the differences that were identified between the vaccination protocols on the two farms, the distinction that could explain the level of disparity between responses is the delivery of the second dose of vaccine with a bacterin on the first farm, which may have interfered with the persistence of immunity in this flock. Hens from flocks 2A and 2B were experimentally challenged at 25 wk of age with H6N2 avian influenza virus. Hens from flock 2A did not transmit virus to naive contact-exposed hens, but hens from flock 2B did. At 34 wk of age, hens from flock 2A were again challenged and naive contact-exposed hens were infected in this second trial. These challenge experiments served to demonstrate that despite detectable antibody responses in flocks 2A and 2B, the birds were protected from infection for less than 21 wk after the second vaccination.

Comparison of a Salmonella enteritidis-specific polymerase chain reaction assay to delayed secondary enrichment culture for the detection of Salmonella enteritidis in environmental drag swab samples.

The California Egg Quality Assurance Program uses the delayed secondary enrichment culture method for detecting Salmonella Enteritidis in environmental drag swabs obtained from commercial layer complexes. The turnaround time for this method is variable and is dependent on the prevalence of Salmonella, level of the Salmonella identification, and capabilities of the performing laboratory. On a sample basis, a range of 4 to 8 days is required to identify a Salmonella sp. to the serogroup level. Additional time is required to serotype group D Salmonella isolates. A Salmonella Enteritidis-specific polymerase chain reaction (PCR) assay was developed for use on drag swabs and chick box papers, and had a turnaround time of 3-4 days. The delayed secondary enrichment culture method and the Salmonella Enteritidis-specific PCR assay were compared on 942 drag swab and 85 chick box paper samples submitted from 217 and 22 premises, respectively, as part of the California Egg Quality Assurance Program. The PCR assay identified 43 positive Salmonella Enteritidis samples from 22 premises, whereas the culture method identified 24 group D Salmonella-positive samples from 16 premises. There was a significant difference (P = 0.001) in the proportion of positive samples as determined by the two assays. Complete serotyping of the group D Salmonella-positive cultures confirmed Salmonella Enteritidis in all but one sample that was identified as Salmonella Jamaica and was negative by the PCR assay.

Diagnosis of zinc phosphide poisoning in chickens using a new analytical approach.

Approximately 200 chickens were found dead after the flooring of a slat-and-litter house was breached. No clinical signs of illness were observed in the surviving birds. During necropsy, rolled oats were found in the chickens' crops and gizzards, and the contents had a petroleum-like odor. Histopathologic examination revealed severe pulmonary edema and congestion of the chickens' lungs, hearts, livers, and kidneys. Based on the history and necropsy findings, zinc phosphide exposure was suspected. Diagnosis of zinc phosphide poisoning has previously been based on history of exposure, identification of the bait material in the gastrointestinal tract, and chemical detection of phosphine gas. However, currently available diagnostic methods are nonconfirmatory, and may produce false positive results. The objective of this case report was to determine whether the sudden death described in these chickens was caused by the ingestion of zinc phosphide, by developing a sensitive and highly specific gas chromatography/mass spectrometry (GC/MS) methodology for analysis of the gastrointestinal samples submitted to the laboratory. It was also found that the determination of zinc concentrations in liver or kidney tissue or stomach contents is not a reliable indicator of zinc phosphide poisoning.

Acute depigmentation of fertile brown eggs in a commercial layer operation.

Rapid depigmentation of brown eggs is an infrequent but startling event in the commercial egg industry that can result in significant economic losses. Loss of shell pigment in brown-shelled eggs is caused by various factors. In many cases, the exact cause of flock-wide pigment loss remains undetermined. A rapid decline in shell pigmentation was observed in 2 flocks of Hyline brown layers. The lack of evidence of an infectious disease process suggested a feed or management problem. On the basis of a small-scale, "in-house" feeding trial, the feed was identified as the cause of depigmentation. Feed analysis by liquid chromatography with mass spectrometry confirmed the presence of 4,4'-dinitrocarbanilide, a major component of nicarbazin (NCZ). There was no evidence of increased mortality, and only a slight but transient drop in the egg production was observed. Depigmentation effects were rapidly reversed after replacing the feed with NCZ-free feed.

Environmental Survey for Listeria Species in California Milk Processing Plants.

This survey was undertaken to determine the prevalence of Listeria monocytogenes and Listeria spp. in environmental samples obtained from milk processing plants located throughout California. Identification of Listeria spp. other than L. monocytogenes was not made. Milk processing plants were categorized as to type of product produced and subdivided into areas based on manufacturing activity occurring in that area. A total of 597 environmental samples from 156 plants were analyzed in the six month period of January through July 1987. Listeria spp. were isolated from 75 (12.6%) of the samples. Thirty-eight of the Listeria spp. (50.7%) were identified as L. monocytogenes . Forty-six plants (29.5%) yielded positive results for Listeria spp., of which, 31 plants (19.9%) were positive for L. monocytogenes . Listeria was recovered more frequently from fluid milk product plants and frozen milk product plants. Likewise, Listeria was isolated most frequently from the packaging-filling room location within a plant and least frequently from the raw milk receiving room. Although Listeria spp. was isolated most frequently from the conveyor site, the drain site within a plant location gave a Listeria isolation rate similar to the total number of Listeria positive sites within that plant location.