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Coastal forested wetlands provide important ecosystem services along the southeastern region of the United States, but are threatened by anthropogenic and natural disturbances. Here, we examined the species composition, mortality, aboveground biomass, and carbon content of vegetation and soils in natural pine forests of the lower coastal plain in eastern North Carolina, USA. We compared a forest clearly in decline (termed “ghost forest”) adjacent to a roadside canal that had been installed as drainage for a road next to an adjacent forest subject to “natural” hydrology, unaltered by human modification (termed “healthy forest”). We also assessed how soil organic carbon (SOC) accumulation changed over time using 14C radiocarbon dating of wood sampled at different depths within the peat profile. Our results showed that the ghost forest had a higher tree density at 687 trees ha−1, and was dominated by swamp bays (Persea palustric), compared to the healthy forest, which had 265 trees ha−1 dominated by pond pine (Pinus serotina Michx). Overstory tree mortality of the ghost forest was nearly ten times greater than the healthy forest (p < 0.05), which actually contributed to higher total aboveground biomass (55.9 ± 12.6 Mg C ha−1 vs. 27.9 ± 8.7 Mg ha−1 in healthy forest), as the dead standing tree biomass (snags) added to that of an encroaching woody shrub layer during ecosystem transition. Therefore, the total aboveground C content of the ghost forest, 33.98 ± 14.8 Mg C ha−1, was higher than the healthy forest, 24.7 ± 5.2 Mg C ha−1 (p < 0.05). The total SOC stock down to a 2.3 m depth in the ghost forest was 824.1 ± 46.2 Mg C ha−1, while that of the healthy forest was 749.0 ± 170.5 Mg C ha−1 (p > 0.05). Carbon dating of organic sediments indicated that, as the sample age approaches modern times (surface layer year 2015), the organic soil accumulation rate (1.11 to 1.13 mm year−1) is unable to keep pace with the estimated rate of recent sea level rise (2.1 to 2.4 mm year−1), suggesting a causative relationship with the ecosystem transition occurring at the site. Increasing hydrologic stress over recent decades appears to have been a major driver of ecosystem transition, that is, ghost forest formation and woody shrub encroachment, as indicated by the far higher overstory tree mortality adjacent to the drainage ditch, which allows the inland propagation of hydrologic/salinity forcing due to SLR and extreme storms. Our study documents C accumulation in a coastal wetland over the past two millennia, which is now threatened due to the recent increase in the rate of SLR exceeding the natural peat accumulation rate, causing an ecosystem transition with unknown consequences for the stored C; however, much of it will eventually be returned to the atmosphere. More studies are needed to determine the causes and consequences of coastal ecosystem transition to inform the modeling of future coastal wetland responses to environmental change and the estimation of regional terrestrial C stocks and flux.

Abstract The fundamental methodological deficiency of megatrials is deliberate reduction of experimental control in order to maximize recruitment and compliance of subjects. Hence, typical megatrials recruit pathologically and prognostically heterogeneous subjects, and protocols typically fail to exclude significant confounders. Therefore, most megatrials do not test a scientific hypothesis, nor are they informative about individual patients. The proper function of a megatrial is precise measurement of effect size for a therapeutic intervention. Valid megatrials can be designed only when simplification can be achieved without significantly affecting experimental control. Megatrials should be conducted only at the end of a long process of therapeutic development, and must always be designed and interpreted in the context of relevant scientific and clinical information.

Abstract Background Asthma can be difficult to diagnose, but bronchial provocation with methacholine, exercise or mannitol is helpful when used to identify bronchial hyperresponsiveness (BHR), a key feature of the disease. The utility of these tests in subjects with signs and symptoms of asthma but without a clear diagnosis has not been investigated. We investigated the sensitivity and specificity of mannitol to identify exercise-induced bronchoconstriction (EIB) as a manifestation of BHR; compared this with methacholine; and compared the sensitivity and specificity of mannitol and methacholine for a clinician diagnosis of asthma. Methods 509 people (6–50 yr) were enrolled, 78% were atopic, median FEV1 92.5% predicted, and a low NAEPPII asthma score of 1.2. Subjects with symptoms of seasonal allergy were excluded. BHR to exercise was defined as a ≥ 10% fall in FEV1 on at least one of two tests, to methacholine a PC20 ≤ 16 mg/ml and to mannitol a 15% fall in FEV1 at ≤ 635 mg or a 10% fall between doses. The clinician diagnosis of asthma was made on examination, history, skin tests, questionnaire and response to exercise but they were blind to the mannitol and methacholine results. Results Mannitol and methacholine were therapeutically equivalent to identify EIB, a clinician diagnosis of asthma, and prevalence of BHR. The sensitivity/specificity of mannitol to identify EIB was 59%/65% and for methacholine it was 56%/69%. The BHR was mild. Mean EIB % fall in FEV1 in subjects positive to exercise was 19%, (SD 9.2), mannitol PD15 158 (CI:129,193) mg, and methacholine PC20 2.1(CI:1.7, 2.6)mg/ml. The prevalence of BHR was the same: for exercise (43.5%), mannitol (44.8%), and methacholine (41.6%) with a test agreement between 62 & 69%. The sensitivity and specificity for a clinician diagnosis of asthma was 56%/73% for mannitol and 51%/75% for methacholine. The sensitivity increased to 73% and 72% for mannitol and methacholine when two exercise tests were positive. Conclusion In this group with normal FEV1, mild symptoms, and mild BHR, the sensitivity and specificity for both mannitol and methacholine to identify EIB and a clinician diagnosis of asthma were equivalent, but lower than previously documented in well-defined populations. Trial registration This was a multi-center trial comprising 25 sites across the United States of America. (NCT0025229).

Abstract Background Inhaled mannitol is a new bronchial provocation test (BPT) developed to improve portability and standardisation of osmotic challenge testing. Osmotic challenge tests have an advantage over the traditional methods of measuring airway hyperresponsiveness using methacholine as they demonstrate higher specificity to identify asthma and thus the need for treatment with inhaled corticosteroids (ICS). The safety and the efficacy of mannitol (M) as a BPT to measure airway hyperresponsiveness were compared to hypertonic (4.5%) saline (HS) in people both with and without signs and symptoms of asthma. Methods A phase III, multi-centre, open label, operator-blinded, crossover design, randomised trial, with follow-up. Asthmatics and non-asthmatics (6–83 yr) were recruited and 592 subjects completed the study. Mannitol was delivered using a low resistance dry powder inhaler and HS was delivered using an ultrasonic nebuliser. The FEV1 was measured 60 seconds after each dose of mannitol (5,10,20,40,80,160,160,160 mg) and after each exposure to HS (0.5,1.0,2.0,4.0,8.0 minutes). A 15% fall in FEV1 defined a positive test. Adverse events were monitored and diaries kept for 7 days following the tests. Results Mean pre-test FEV1 (mean ± SD) was 95.5 ± 14% predicted. 296 were positive to mannitol (M+) and 322 positive to HS (HS+). A post study physician conducted clinical assessment identified 82.3% asthmatic (44% classified mild) and 17.7% non-asthmatic. Of those M+, 70.1% were taking ICS and of those mannitol negative (M-), 81.1 % were taking ICS. The % fall in FEV1 for mannitol in asthmatics was 21.0% ± 5.7 and for the non-asthmatics, 5.5% ± 4.8. The median PD15 M was 148 mg and PD15 HS 6.2 ml. The sensitivity of M to identify HS+ was 80.7% and the specificity 86.7%. The sensitivity of M compared with the clinical assessment was 59.8% and specificity 95.2% and increased to 88.7% and 95.0% respectively when the M- subjects taking ICS were excluded. Cough was common during testing. There were no serious adverse events. The diarised events were similar for mannitol and HS, the most common being headache (17.2%M, 19%HS), pharyngolaryngeal pain (5.1%M, 3%HS), nausea (4.3%M, 3%HS), and cough (2.2%M, 2.4%HS). Conclusion The efficacy and safety of mannitol was demonstrated in non-asthmatic and clinically diagnosed asthmatic adults and children.

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.

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.

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.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

An unusual cutaneous fowl pox outbreak occurred in 8-wk-old broilers in California. Rounded and longitudinal, proliferative scratch-associated lesions were found only in feathered areas of the body. Both sides of the hip, the lower abdomen, pericloacal area, and lateral lower neck area were involved. The head, legs, feet, and toes did not have lesions. Birds in only one section of one of five houses were affected. Fifteen percent condemnations occurred in birds from the affected house due to the skin lesions. A diagnosis of fowl pox was achieved by histopathology, viral isolation, and direct electron microscopy. The unusual distribution of pox lesions was assumed to be associated with skin scratches. There was no evidence that mosquitoes or other types of insects were involved in this outbreak. To the knowledge of the authors, this is the first report of this kind of unusual fowl pox in the United States.

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.

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.

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.

Breeder squab candidates between the ages of 6 and 16 wk were submitted to the California Animal Health and Food Safety Laboratory, Turlock branch, as a result of respiratory distress and increased mortality. These cases were submitted from one Northern California commercial squab operation on three separate occasions occurring between December 2007 and March 2008. Severe trichomoniasis was identified, primarily in the tracheal epithelium and lung of squabs, with few or no lesions in the oral cavity, crop, esophagus, and livers, where the organism commonly infiltrates. Infiltration of the trachea and lung sections with trichomonads was associated with a severe inflammatory response in the surrounding tissue. Diagnosis was confirmed with the use of histopathology and an immunoperoxidase special stain. Oxytetracycline supportive antibiotic therapy to prevent secondary bacterial infections was administered to remaining squabs on the farm, but no specific treatment regimen was instituted. This novel respiratory presentation of trichomoniasis continued over a period of 3 mo, until mortality gradually returned to normal.

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.

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.

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.

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.

An outbreak of infectious catarrhal enteritis, associated with the flagellated protozoan Spironucleus meleagridis (syn. Hexamita meleagridis), is reported from a commercial flock of chukar partridges in California. The disease affected birds between the ages of 4 and 6 wk and resulted in diarrhea, listlessness, depression, and high mortality. Concurrent infection with other intestinal pathogens, including Cryptosporidia, group E Salmonella, long-segmented filamentous microorganisms (LSFMOs), and Rotavirus-like virus particles, was found in some but not all affected birds. Dermatitis of the face, shanks, and feet, suggestive of B-complex vitamin deficiency, was present in most affected birds as well. Flagellated protozoan parasites could be found in the lumen of the duodenum and jejunum and in the intestinal crypts. In some cases the flagellates were wedged between epithelial cells or were located intracellularly within cells of the mucosal epithelium and the intestinal lamina propria.

A disease characterized by paresis and paralysis was seen in 7-9-day-old broiler chicks after vaccination in the neck area at day-of-age with a live virus vaccine containing viruses of Marek's disease, fowl pox, and infectious bursal disease. Affected birds presented with variable signs of ataxia, lateral recumbency, leg paralysis, and twisting or S-shaped flexure of the neck. Gross lesions noted at necropsy included swelling and edema of the subcutaneous tissues and muscles of the neck at the injection site area. A heavy mononuclear inflammatory cell infiltration was seen in the subcutaneous tissues, connective tissues, and muscles of the neck at the injection site. In some cases, the inflammatory process extended along fascial planes to involve the epidural spaces surrounding the spinal cord. Fatty changes with possible demyelination of nerve fibers were noted in some sections of the spinal cord adjacent to the inflammatory lesions. Clusters of poxviruses were found within some inflammatory lesions on transmission electron photomicrographs.

Systemic erysipelas infection was seen in 2-to-4-day-old poults from three separate ranches owned by the same company. The affected poults were all from the same breeder source; poults from other breeder sources were seemingly unaffected. Mortality increased on days 2 and 3, ranging from 2% to 8.5%. Birds submitted were dehydrated and very weak, with one half of the poults submitted having died during transport to the lab. Gross lesions included swollen, congested livers and spleens, as well as hemorrhagic breast muscle in one case. Toes were swollen and reddish-purple in color. The poults had been toe-trimmed during hatchery processing using a commercial microwave. Histologically, periportal inflammation with heterophilic infiltration in the liver was noted. Spleens showed hyalinization of arteries, lymphoid depletion, and necrosis. Toe joints showed purulent synovitis and cellulitis. Gram stains done on impression smears of liver and spleen showed rare to moderate numbers of small gram-positive rods. Erysipelothrix rhusiopathiae was isolated from 18 of 22 livers cultured, five of six toe joints cultured, and from the yolk sac in two birds.

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the molecular epidemiology of 26 Mycoplasma gallisepticum (MG) isolates obtained from turkeys located in the central valley of California. The MG isolates were recovered from 5 different companies and 13 ranches. Each company had unique MG strains. No evidence of spread of MG between companies was detected. RAPD analysis of MG isolates within a ranch during an outbreak revealed only a single strain involved in each outbreak. RAPD analysis identified an isolate from 1 ranch with a banding pattern identical to that of the 6/85 vaccine strain, which had been used on that particular ranch. Similar RAPD banding patterns of isolates from different ranches within the same company suggested horizontal spread of MG between ranches. The use of 2 primer sets in RAPD analysis was critical to prevent misinterpretation of relationships between different isolates.

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate 7 strains of Mycoplasma gallisepticum. Six commercially available primers or primer combinations were screened for their ability to differentiate vaccine and type strains. Although major and minor bands were produced with each primer, many of the primers were unsuitable for strain differentiation. The use of primer 6 and combined primers 3 and 4 resulted in complementary RAPD banding patterns for each M. gallisepticum strain. Eleven different isolates representing 7 different strains were segregated into 7 different patterns, corresponding to the 7 strains.

Seven 18-wk-old pullets from a commercial layer flock experiencing increased mortality associated with neurologic and respiratory symptoms were submitted to the California Veterinary Diagnostic Laboratory System at the Turlock Branch for necropsy. Clinical signs included depression, torticollis, swollen eyelids, conjunctivitis, and sinusitis. Meningoencephalitis and suppurative inflammation of the cranial air spaces were found on histopathology. The brain, sinuses, and air spaces of the cranium were infected with Pasteurella multocida. Complicating the condition was Mycoplasma gallisepticum infecting the sinus and paramyxovirus-I affecting the trachea.

A mixed breed rooster, from a backyard flock of 13 chickens, was received at California Veterinary Diagnostic Laboratory System-Turlock Branch for postmortem examination. The bird presented with thickened, featherless, scab-encrusted skin around the head region. Numerous sticktight fleas were found attached to the encrusted skin. Microscopic evaluation of the skin revealed a lymphoplasmacytic reaction in the dermis with visible embedded flea mouthparts. Also noted histologically in this region were epidermal hyperplasia and ballooned epidermal cells containing intracytoplasmic inclusions indicative of fowl poxvirus.

A meat turkey ranch had a history of producing flocks with low market weights. Live bird samples and environmental samples from the ranch were collected over a period of eight months and analyzed at the California Veterinary Diagnostic Laboratory System-Turlock Branch. The disease condition consistently present was enteritis associated with Ascaridia dissimilis infection in birds from growout houses. After aggressive control measures for A. dissimilis were implemented, subsequent flocks reached expected market weights.

Esophageal trichomoniasis has been rarely reported in chickens. At the California Veterinary Diagnostic Laboratory System-Turlock Branch, this disease was recently diagnosed in two cases submitted from backyard chicken flocks. The esophageal lesions observed were similar to those seen in several other important diseases of chickens. The causative trichomonad organisms were readily demonstrated on wet smears and by histologic studies. In both cases, the investigated flocks were afflicted with several concurrent diseases. California has experienced an increase in the number of small nontraditional chicken production operations. These facilities are sometimes in close proximity to commercial poultry operations and biosecurity barriers occasionally fail. The poor husbandry practices often used in these small flocks make them a potential reservoir for rare diseases such as trichomoniasis and also for disease organisms that are devastating to commercial poultry.

Infectious laryngotracheitis (ILT) was diagnosed as the cause of an outbreak of respiratory disease in broiler chickens in California. The classical form of ILT is characterized by dyspnea, gasping, coughing, and expectoration of bloody exudate. Most of the broilers submitted to the diagnostic laboratory showed a non-classical presentation of ILT, in which mucoid tracheitis and conjunctivitis were the most consistent lesions. Historically, most of the ILT cases diagnosed in our laboratory have consisted of layers with classical signs and lesions. It is not known whether this non classical presentation of ILT in broilers is due to differences in the way broilers respond to ILT infection or to the nature of the ILT virus isolate.

Severe cephalic swelling and facial cellulitis in turkeys associated with fowl cholera were present in seven accessions submitted to two laboratories in a 2-year period. Flocks ranged in age from 6 to 18 weeks and included both toms and hens. Interestingly, turkeys with facial cellulitis had no gross internal lesions of fowl cholera, whereas birds with gross lung, liver, and air-sac lesions did not have swollen heads. Histologically, the facial cellulitis was characterized by extensive fibrinonecrotic inflammation of the deep dermis with heterophilic perivasculitis and thrombosis. Additional characterization of Pasteurella multocida isolates from these cases was conducted retrospectively from lyophilized cultures. Serogrouping, serotyping, and an enzyme-linked immunosorbent assay (ELISA) for dermonecrotic factor were performed. All isolates were serogroup A or unencapsulated. Serotype 1 was the most prevalent serotype isolated in association with facial cellulitis. ELISA results for dermonecrotic toxin were inconclusive.

Serotypes of Salmonella from turkeys and their environment identified at the California Veterinary Diagnostic Laboratory System (CVDLS) between 1984 and 1989 are reported. Between 1988 and 1989, Salmonella serotypes from turkeys were compared from two sources: from routine submissions to the CVDLS (primarily associated with the National Poultry Improvement Plan), and from a random sample of turkey farms conducted by the National Animal Health Monitoring System (NAHMS). Serotype isolation frequencies were ver similar under these two systems. Serotypes identified most frequently under both systems were (listed from most to least frequent): Salmonella kentucky, S. anatum, S. heidelberg, S. reading, and S. senftenberg. This isolation pattern was different from that found in humans in California; only S. heidelberg was relatively common in both humans and turkeys during this period.

Between April of 1990 and March of 1992, calves on a Holstein calf ranch experienced subcutaneous decubital abscesses involving the brisket region, dorsal aspect of the carpus, and lateral aspect of the stifle joints. Fifty out of 2,500 (2%) Holstein calves between the ages of 3 and 12 weeks were affected. Needle aspirates of brisket abscesses from 8 calves and 6 live or dead calves with 1 or more decubital abscesses were submitted for examination. Two of the 6 calves in addition had bronchopneumonia. Mycoplasma bovis was isolated from all abscesses and 1 lung. Formalin fixed tissues taken from the affected areas also revealed M. bovis by immunoperoxidase staining. No evidence of joint involvement was apparent, and no mycoplasma was isolated from the joints adjacent to affected areas. Attempts to isolate mycoplasma from milk and environmental samples were unsuccessful.

An unidentified, pleomorphic, gram-negative rod (PGNR) bacterium has been isolated from domestic fowl with respiratory disease. The PGNR was isolated in 5% of turkey accessions and 3% of chicken accessions, primarily from the respiratory tract. Preliminary characterization of this organism included reviewing accession records, conducting cultural and biochemical tests, and analyzing cellular fatty acids. The PGNR was also compared with other bacteria capable of inhabiting the avian respiratory system. Biochemical and cellular fatty acid analysis failed to identify the organism, however all 14 isolates were similar.

Two unrelated flocks of chicks experienced elevated mortality from 4 to 10 days of age. Clinical signs in affected birds included a full range of neurological disorders. Livers and spleens were grossly enlarged at necropsy. Enterococcus durans was isolated from multiple organs, including brains. Histologically, there were multifocal coagulative necrosis in the liver, areas of malacia in brain stem, and cerebellar white matter. Inoculation of 1-day-old chicks with a suspension of E. durans resulted in bacteremia but did not produce the lesions observed in field cases.

Monensin was given via gavage to 5- and 6-week-old broad-breasted white turkeys. The birds were allotted into four groups--three groups given monensin and one group of controls--in two experimental trials. Treated turkeys in Trial 1 received one dose of monensin per day for 4 days at 4.7 mg monensin/kg body weight, 8.8 mg/kg, or 17.6 mg/kg. Treated turkeys in Trial 2 received one dose of monensin per day for 5 days at 1.93 mg/kg, 4.7 mg/kg, or 8.8 mg/kg. Turkeys receiving the lowest dose showed no clinical signs of myopathy. Birds receiving 4.7 mg monensin/kg developed ataxia after the third dose and rear limb paresis and paralysis after the fifth dose. Turkeys receiving 8.8 mg/kg were ataxic after the second dose and paretic or paralyzed after the fourth dose. Turkeys receiving 17.6 mg/kg were ataxic 3 hours after the first dose and paretic or paralyzed 8 hours after the first dose. Histologically, a necrotizing skeletal myopathy was present in the muscles of the rear limbs. A dose-related response was observed in the percentage of myofibers damaged in birds that survived until the end of the trials. Intrafiber edema and vacuolation were observed in histologic sections from myocardium of turkeys from the two highest dose groups.

A necrotizing skeletal myopathy of rear limbs was diagnosed in 17 flocks of commercial turkeys. The mean mortality attributed to the myopathy was 2.29% (range = 0.13-9.7%) over a mean period of 9.6 days (range = 6-14 days). The mean age of the birds at the time of onset was 7.4 weeks (range = 4-10.5 weeks). Clinically, birds experienced an episode of watery droppings and high-pitched crying, followed by rear-limb paresis or paralysis. Creatine kinase and aspartate aminotransferase were markedly elevated in birds with the myopathy. Grossly, a few birds had pale streaking in the muscles of the thighs and legs. Histologically, acute and subacute degeneration was present in myofibers of the legs, abdomen, thighs, back, and tail. The subacute lesion was characterized by marked sarcolemmal cell proliferation. Feed analyses ruled out selenium deficiency and the presence of mycotoxins as etiologies. Monensin was present in approved usage or only slightly elevated levels. A known potentiating antibiotic was being used concurrently with monensin in only one flock.

Nine previously vaccinated turkeys were inoculated intravenously with Pasteurella anatipestifer, and blood samples were taken periodically to evaluate the potential of chronically infected turkeys to serve as reservoirs of infection for blood-feeding arthropod vectors. Vertebral osteomyelitis (spondylitis), as yet unreported in the literature in association with infection with the organism, was found in the thoracic vertebrae of five out of nine inoculated turkeys, and P. anatipestifer was isolated from the thoracic vertebrae of three of the five. The organism was isolated from the peripheral blood of six turkeys 24 hours postinoculation and from the peripheral blood of one turkey 7 days postinoculation. The organism was also isolated from the heart blood of two birds at necropsy--from one at 21 days and, following an intramuscular injection of dexamethasone, from the other turkey at 38 days postinoculation.

An outbreak of diarrhea and neurological disease in California racing pigeons caused by avian paramyxovirus type 1 (PMV-1) is documented. Predominant clinical signs were polydipsia, ataxia, poor balance, torticollis, head tremors, inability to fly, and diarrhea that was unresponsive to therapy. Gross pathologic findings were often unremarkable or non-specific. The predominant histologic lesions were interstitial nephritis, chronic tubular necrosis, lymphoplasmacytic infiltration within the kidney, liver, and pancreas, and focal non-suppurative encephalitis. Pigeons from 20 submissions demonstrated characteristic clinical signs of PMV-1 infection. Pigeons from 17 submissions exhibited typical histopathology. Serologic evidence of PMV-1 infection was present in pigeons from 13 submissions, and PMV-1 was isolated from pigeons received in six submissions. None of these pigeons had been vaccinated against PMV-1.

During a 3-year period, 4 cases of bovine abortion attributable to Listeria ivanovii were diagnosed from 243 bovine fetuses submitted for diagnostic evaluation. Listeria monocytogenes was isolated only once from a bovine fetus during this same time period. Pathologic findings were similar to those seen in abortions attributable to L monocytogenes. Consistent management factors were not recognized and breed susceptibility was not apparent. Listeria ivanovii is most often associated with abortions from sheep and is rarely reported from cattle. On the basis of findings in this study, L ivanovii must be included as a potential cause of bovine abortions.

Six hundred fourteen meat turkeys were submitted for necropsy from 24 California ranches as part of the National Animal Health Monitoring System (NAHMS). Enteritis was the most frequent pathologic diagnosis in birds 18 days old or younger and the second most frequent diagnosis in birds 19-70 days old. Hemorrhagic enteritis was the most frequent diagnosis in birds aged 19-70 days. Tibial dyschondroplasia, bronchopneumonia, and ascaridiasis were ranked one through three in frequency of diagnoses in birds over 70 days of age. Salmonella was isolated from 71% of flocks tested, and Mycoplasma meleagridis was isolated from 33% of tested flocks over 70 days of age. Antibodies to several disease agents were detected, including hemorrhagic enteritis (100% of flocks over 70 days old) and Newcastle disease (63% of flocks over 70 days old).

Systemic trichomoniasis was responsible for severe morbidity and mortality in a commercial squab operation. Squabs of 1 to 3 weeks of age were primarily affected. Extensive caseous necrosis of the liver, heart, lung, and intestinal mesentery was observed grossly. Histologically, extensive heterophilic inflammation surrounded the necrotic areas and was observed perivascularly in areas that were uninvolved grossly. Numerous trichomonads were demonstrated in affected tissues with Bodian's silver stain.

Two cases of infectious coryza in meat chickens are reported. The first case involved 6-week-old broiler chickens in which only Haemophilus paragallinarum was isolated. The second case involved 11-week-old roaster chickens in which H. paragallinarum and Mycoplasma synoviae were isolated. Both farms were in close proximity to layer-chicken farms where infectious coryza had been previously diagnosed. In both cases, only certain houses on the farm were affected, and mortality in these houses increased slightly. At processing, the condemnation rates for affected houses were considerably higher than rates for unaffected houses. Condemnations for affected houses were mostly due to airsacculitis. A dissecting fibronopurulent cellulitis was a prominent lesion in the second case. This lesion could lead to confusion with chronic fowl cholera and swollen-head syndrome.

Herpesvirus infection was diagnosed in a toucan. The herpesvirus was isolated from the liver and identified by electron microscopy in the liver and in cell culture. A negative immunofluorescent reaction was obtained when virus-infected cell cultures were reacted with a conjugate to the herpesvirus of Pacheco's disease. The main pathologic finding in the toucan consisted of a severe necrotizing hepatitis with intranuclear inclusions in the liver and spleen. A presumptive diagnosis of chlamydiosis was also made, based on a positive direct fluorescent monoclonal antibody reaction to chlamydial antigens in impression smears of liver and spleen. Chlamydial isolation attempts were unsuccessful. The toucan had been in contact with two macaws that had died 5 days before the toucan died and were diagnosed by histology as having herpesvirus hepatitis.

Case submissions and a field investigation indicated that oil-adjuvanted killed vaccines may produce long-lived residual lesions in chickens. The lesions typically are yellow opaque cysts along the fascial planes separating the superficial and deep pectoral muscles. Microscopic evaluation shows that most lesions are cysts with thin fibrous capsules, sometimes associated with lymphocytic aggregates and, more rarely, a granulomatous reaction.

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.