The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hc1, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J–induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J–infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.

Myeloblastosis-associated virus type 1 (MAV-1) is an exogenous avian retrovirus with oncogenic potential. MAV-1 was detected in young chicks hatching from eggs produced by an experimental genetic line of egg-type chickens. Transmissibility of MAV-1 had not been documented previously. This investigation was intended to partially characterize the virus involved and to study its transmissibility and oncogenicity in naturally and contact-infected chickens. Commercially produced white and brown layer pullets free of exogenous avian leukosis viruses were commingled at hatch with naturally MAV-1–infected chickens. The original MAV-1–infected chickens were discarded after approximately 8 wk, and the contact-exposed chickens were maintained in isolation for 36 wk. Young specific-pathogen-free (SPF) single comb white leghorn chickens were added to the group to study possible horizontal transmission of MAV-1 in young chickens. Upon weekly virus isolation attempts, MAV-1 was readily isolated from the contact-exposed white layers but not from the brown layers between 36 and 53 wk of age (18 wk in total). Three-week-old SPF chickens were readily infected with MAV-1 by contact as early as 1 wk postexposure. Throughout 22 hatches derived from the white and brown MAV-1–contact-exposed layers (between 36 and 53 wk of age), MAV-1 was frequently detected in the white layer progeny, whereas the virus was seldom isolated from the progeny produced by the brown layers during the same 18-wk period. MAV-1 induced a persistent infection in some of the SPF chickens that were exposed by contact at 3 wk of age. Gross tumors were not detected in any of the originally infected experimental chickens at 8 wk of age, in the contact-exposed brown or white layers at the termination of the study at 53 wks of age, or in the contact-exposed SPF chickens at the end of the study at 12 wk of age. Exogenous avian leukosis-related viruses may still be detected in egg-type chickens, emphasizing the importance of thorough screening before incorporation of experimental genetic material into commercial genetic lines of egg-type chickens.

Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2010, and they were genotyped by comparison with 19 reference strains in the present study. The results showed that LX4-type isolates are still the predominant IBVs circulating in chicken flocks in China, and these isolates could be grouped further into two clusters. Viruses in each cluster had favored amino acid residues at different positions in the S1 subunit of the spike protein. In addition, a recombination event was observed to have occurred between LX4- and tl/CH/LDT3/03I-type strains, which contributed to the emergence of a new strain. The most important finding of the study is the isolation and identification of Taiwan II–type (TW II–type) strains of IBV in mainland China in recent years. The genome of TW II–type IBV strains isolated in mainland China has experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. Pathogenicity testing and sequence analysis of the 3′ terminal untranslated region revealed that TW II–type IBV strains isolated in mainland China have a close relationship with the embryo-passaged, attenuated TW2296/95.

A comparison on the prevalence of Salmonella infection in layer hens from commercial layer farms with high and low rodent densities was investigated. Out of 280 laying hens sampled from three commercial layer farms with high rodent densities, Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) was isolated from 20 (7.14%) hens and Salmonella enterica subsp. enterica serovar Infantis (Salmonella Infantis) from three (1.07%) hens. In contrast, layer hens sampled from four commercial layer farms with low rodent densities were negative for any salmonellae. Significant differences (P < 0.05) in the isolation rates of Salmonella from various organs of infected layer hens were also noted. For Salmonella Enteritidis, liver (55.0%) and the oviduct (55.0%) had the highest isolation rates while all Salmonella Infantis isolates were from the oviduct. Pulsed field gel electrophoresis (PFGE) analysis of BlnI-digested chromosomal DNA of Salmonella Enteritidis isolated from layer hens and rodents showed similar patterns. PFGE analysis of Salmonella Infantis isolated from layer hens, rodents, eggs, and the environment yielded identical patterns. In this study, the significantly higher prevalence rate (P < 0.05) of Salmonella Enteritidis and Salmonella Infantis in layer hens from high rodent density farms could be attributed to the high rodent population density. The persistent Salmonella Enteritidis and Salmonella Infantis infection inside layer houses may have been amplified by the increasing numbers in the rodent population over the years, which increased the opportunity for environment-rodent-chicken interaction and the transmission of salmonellae to chickens. Monitoring of salmonellae from rodents inside poultry premises is recommended to be an effective additional tool in the assessment of the Salmonella status of layer flocks.

Peripheral nerve sheath tumors (PNSTs) are rare in chickens and their etiology remains to be elucidated. In this study, a naturally occurring PNST in a Japanese native fowl (Gallus gallus domesticus) was pathologically examined and the strain of avian leukosis virus (ALV) isolated from the neoplasm was characterized by molecular biological analysis. The fowl presented with a firm subcutaneous mass in the neck. The mass, connected to the adjacent spinal cord (C9–14), was microscopically composed of highly cellular tissue of spindle cells arranged in interlacing bundles, streams, and palisading patterns with Verocay bodies and less cellular tissue with abundant collagen. Immunohistochemically, neoplastic cells were divided into two types: perineurial cells positive for vimentin, glucose transporter 1 (GLUT1), and claudin1; and Schwann cells positive for vimentin, occasionally positive for S-100 α/β but negative for GLUT1. Based on these findings, a diagnosis of neurofibrosarcoma was made. The complete nucleotide sequence of an ALV strain, CTS_5371, isolated from the neoplasm was determined and phylogenetic analysis indicated that the strain was a novel recombinant virus from avian leukosis/sarcoma viruses previously reported. Additionally, experimental infection revealed that CTS_5371 induced the proliferation of Schwann cells and perineurial cells. These results suggest that this ALV strain has the ability to induce PNSTs in chickens.

We evaluated the pathogenicity of three live Mycoplasma gallisepticum (MG) vaccine candidates by infection via aerosol of 3-wk-old chickens with log phase broth cultures (trial 1). Two of the candidates (K3020 and K4649A) colonized only 10% and 20% of the chickens, respectively, unlike K2101 (K-strain), which was reisolated from all of the vaccinated chickens tested. K-strain inoculation did not result in significant air sac or tracheal lesions in chickens at 10 and 39 days postinfection (P ≤ 0.05). The efficacy of K-strain as a live vaccine was evaluated in trial 2, by challenge of vaccinated chickens with virulent R-strain via aerosol at 6 wk postvaccination. K-strain vaccination resulted in significant protection from air sac and tracheal lesions (P ≤ 0.05). The K-strain was further investigated to evaluate transmissibility (trial 3), colonization and persistence of infection following aerosol administration (trial 4), genetic and phenotypic stability following back passage through chickens (trial 5), and vertical transmission (trial 6). The K-strain had a low rate of horizontal transmission; it remained primarily in the respiratory system of inoculated birds and persisted in the upper respiratory tract for the duration of the trial 4 (5 mo). There was no increase in virulence of K-strain when it was back passaged five times through chickens, and no vertical transmission of K-strain was detected. K-strain showed great potential as a safe and effective live MG vaccine.

The quality of day-old chick placement and management upon arrival have a major impact on first-week mortality (FWM) and subsequent welfare in layers. The present study investigated FWM and causes of FWM in 50 flocks of layers. Post mortem results from 983 chickens showed that 50% died from infections, whereas noninfectious causes, in particular dehydration and nephropathy with visceral gout, made up the remaining causes of mortality. Escherichia coli and Enterococcus faecalis were identified as the most significant bacterial pathogens associated with FWM. Statistical analysis demonstrated a significant correlation between FWM and total mortality during rearing, and a model predicting total mortality in the rearing period based on FWM was established. A statistically significant correlation between FWM and uniformity of the flock was not demonstrated at 1–2 wk of age or at approximately 15 wk of age. Genetic characterization of E. coli and E. faecalis provided evidence for a polyclonal nature of these infections in affected flocks, indicating different sources of infection. Results obtained underline the importance of minimizing FWM to a level less than 1%.

Cryptococcus neoformans (C. neoformans) is a frequent cause of invasive fungal disease in immunocompromised human hosts. Ninety-eight samples of pigeon droppings were collected from the pigeon shelters in Seoul, and cultured on birdseed agar (BSA) and Sabouraud dextrose agar (SDA). One hundred yeast-like colonies were selected and identified via phenotype characteristics, such as colony morphology and biochemical characteristics. This was then followed with genotyping via sequencing of the internal transcribed spacer (ITS) region. The colonies were classified into four kinds of colony color types: brown type (BrT), beige type (BeT), pink type (PT), and white type (WT). Numbers of isolated BrT, BeT, PT, and WT colonies were 22 (22%), 30 (30%), 19 (19%), and 39 (39%), respectively. All BrT colonies were identified as C. neoformans. BeT were identified as 19 isolates of Cryptococcus laurentii, 10 isolates of Malassezia furfur, and 1 isolate of Cryptococcus uniguttulatus. PT was divided into two colony color types: light-PT (l-PT) and deep-PT (d-PT). Eighteen of l-PT and one of d-PT were identified as Rhodotorula glutinis and Rhodotorula mucilaginosa, respectively. WT were identified as 34 isolates of Cryptococcus guilliermondii, 3 isolates of Cryptococcus zeylanoides, 1 isolate of Cryptococcus sake, and 1 isolate of Stephanoascus ciferrii. Most strains were classified identically with the use of either phenotype or genotyping techniques, but C. uniguttulatus and C. sake classified by phenotyping were Pseudozyma aphidis and Cryptococcus famata by genotyping. This rapid screening technique of pathogenic yeast-like fungi by only colony characteristics is also expected to be very useful for primary yeast screening. Additionally, we investigated the seasonal variations of C. neoformans and other yeast-like fungi from 379 pigeon-dropping samples that were collected from February 2011 to March 2011. We isolated 685 yeast-like fungi from the samples. Almost all C. neoformans and yeast-like fungi were isolated in the fall (298 strains, 43.5%) and spring (244 strains, 35.6%). A few yeast-like fungi were isolated in winter (98 strains, 14.3%) and summer (45 strains, 6%). These results would be used as an important indicator related to epidemiology and prevention of pathogenic yeast-like fungi infections transmitted through pigeon droppings.

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n  =  48) and 2-wk-old (n  =  56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

A study was performed in 2007 to isolate and characterize infectious bursal disease viruses (IBDVs) in commercial broilers grown in the Delmarva (DMV) Peninsula region of the United States. Bursae of Fabricius were collected weekly from 1 to 4 wk of age from broilers on 10 farms with a history of poor performance. Microscopic pathology was used to determine the infectious bursal disease (IBD) status of the broilers. Bursae from 1- and 2-wk-old broilers did not show IBD microscopic lesions. Moreover, broilers on 1 of the 10 farms were IBD lesion free at 3 and 4 wk of age. However, 3 of 9 and 9 of 9 farms yielded broilers with IBD-affected bursae from 3- and 4-wk-old commercial broilers, respectively. Ten IBDV isolates were recovered from 3 of 3 lesion-positive bursal pools at 3 wk of age and 7 of 9 lesion-positive bursal pools at 4 wk of age. Analysis of the viral protein (VP) 2 genes identified all isolates as serotype 1 Delaware (Del) variant viruses. Five field isolates, each representing different molecular clades of the Delaware variant viruses, were selected for further study. Experimental infection of specific-pathogen-free white leghorn chickens with isolates DMV/4813/07, DMV/4947/07, DMV/4955/07, DMV/5038/07, and DMV/5041/07 produced gross and microscopic pathology of the bursa consistent with Delaware variant infection. Monoclonal antibody testing showed DMV/4813/07, DMV/4947/07, DMV/4955/07, and DMV/5041/07 to be similar to previous recognized variant viruses. However, DMV/5038/07 was found to be unreactive with the monoclonal antibodies that typically recognize reference strains STC, Del E, GLS, RS593, and AL2. In a challenge of immunity study, 10-day-old progeny from breeders immunized with a commercially available inactivated IBDV vaccine containing the Del E and classic strains were protected to a lesser degree against isolate DMV/5038/07 compared to Del E challenge based on microscopic lesion scores (P < 0.01) of the bursa. This result suggests the virus is antigenically different from the Del E strain contained in the vaccine. Collectively, the monoclonal antibody and progeny challenge of immunity findings suggest DMV/5038/07 is antigenically different from the Del E strain contained in the vaccine.

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase–PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 × 10⁸ to 1 × 10⁹ copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 × 10^2.5 to 1 × 10^3.2 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

This study investigated the ability of two novel adjuvant formulations, QCDC (Quil A/cholesterol/DDA/Carbopol) and QCDCR (QCDC/Bay R1005), in combination with a recombinant profilin vaccine, to modulate host protective immunity and to alter gene expression during experimental avian coccidiosis. Vaccination with profilin plus QCDCR significantly reduced the severity of intestinal lesions and increased mitogen-induced lymphocyte proliferation in infected chickens compared with immunization with profilin alone or profilin plus QCDC. Immunization with profilin plus QCDC or profilin plus QCDCR increased body weight gain but had no effect on fecal oocyst shedding of chickens infected with Eimeria acervulina compared with birds vaccinated with profilin alone. The results of global gene expression analysis revealed that, compared with PBS controls, (a) chickens vaccinated with profilin alone had 71 up-regulated and 56 down-regulated mRNA transcripts, (b) chickens immunized with profilin plus QCDC had 198 up-regulated and 247 down-regulated mRNAs, and (c) birds immunized with profilin plus QCDCR had 210 up-regulated and 267 down-regulated mRNAs. Compared with birds vaccinated with profilin alone, (a) chickens given profilin plus QCDC had 60 up-regulated and 104 down-regulated transcripts and (b) chickens immunized with profilin plus QCDCR had 103 up-regulated and 130 down-regulated mRNAs. Finally, chickens vaccinated with profilin plus QCDCR had 193 up-regulated and 204 down-regulated transcripts compared with birds given profilin plus QCDC. Biological function and network analysis revealed that the majority of altered transcripts were encoded by immune-related genes.

We studied the pathologic features of neurons that contain intracytoplasmic acidophilic droplets (IADs) in chicken spinal cords. The IADs were lustrous spheroid bodies scattered in the cytoplasm of neurons, variable in size, and protein-rich bodies stained eosinophilic with hematoxylin-eosin, acidophilic with Azan, blue indigo with phosphotungstic acid hematoxylin, and yellow-green with Elastica van Gieson stain histopathologically. Ultrastructurally, almost all IADs were observed as homogeneous highly electron-dense spheroid bodies enclosed by double-limited membranes. Small IADs were observed in mitochondria. Anatomically, IAD-CNs were observed only in the ventral horn of the spinal cord between the fourth sacral and third lumbal vertebrae, and they were particularly frequent in the third sacral vertebrae. Their appearance and accumulative amount were likely to increase with age, while the clinical and pathologic significances of IAD-CNs remain unclear.

Historically, avian influenza viruses have been isolated from cloacal swab specimens, but recent data suggest that the highly pathogenic avian influenza (HPAI) H5N1 virus can be better detected from respiratory tract specimens. To better understand how swab sample type affects the detection ability of low pathogenic avian influenza (LPAI) viruses we collected and tested four swab types: oropharyngeal swabs (OS), cloacal swabs (CS), the two swab types combined in the laboratory (LCS), and the two swab types combined in the field (FCS). A total of 1968 wild waterfowl were sampled by each of these four methods and tested for avian influenza virus using matrix gene reverse-transcription (RT)-PCR. The highest detection rate occurred with the FCS (4.3%) followed by the CS (4.0%). Although this difference did not achieve traditional statistical significance, Bayesian analysis indicated that FCS was superior to CS with an 82% probability. The detection rates for both the LCS (2.4%) and the OS (0.4%) were significantly different from the FCS. In addition, every swab type that was matrix RT-PCR positive was also tested for recovery of viable influenza virus. This protocol reduced the detection rate, but the ordering of swab types remained the same: 1.73% FCS, 1.42% CS, 0.81% LCS, and 0% OS. Our data suggest that the FCS performed at least as well as any other swab type for detecting LPAI viruses in the wild ducks tested. When considering recent studies showing that HPAI H5N1 can be better detected in the respiratory tract, the FCS is the most appropriate sample to collect for HPAI H5N1 surveillance while not compromising LPAI studies.

Fumonisins (FBs) are mycotoxins that are found worldwide in maize and maize products. Their main toxic effects have been well characterized in poultry, but differences between species have been demonstrated. Ducks appeared very sensitive to toxicity, whereas turkeys are more resistant. At the same time, alterations of sphingolipid metabolism, with an increase of the concentration of the free sphinganine (Sa) in serum and liver, have been demonstrated in the two species, but the link between the toxicity of FBs and Sa accumulation remains difficult to interpret. The aim of the present work was to compare the effects of FBs (10 mg FB1 FB2/kg body weight) on sphingolipid metabolism in ducks and turkeys. Growth, feed consumption, and serum biochemistry were also investigated to evaluate toxicity. The main results showed that FBs increased Sa concentrations in liver and serum in ducks and turkeys, but these accumulations were not directly correlated with toxicity. Sa accumulation was higher in the livers of turkeys than in ducks, whereas Sa levels were higher in the sera of ducks than in turkeys. Hepatic toxicity was more pronounced in ducks than in turkeys and accompanied a decrease of body weight and an increase of serum biochemistry in ducks but not in turkeys. So, although FBs increase Sa concentration in the livers of both species, this effect is not directly proportional to toxicity. The mechanisms of FB toxicity and/or the mechanisms of protection of ducks and turkeys to the Sa accumulation within the liver remain to be established.

Newcastle disease virus (NDV), a member of the genus Avulavirus of the family Paramyxoviridae, is the causative agent of Newcastle disease (ND), a highly contagious disease that affects many species of birds and which frequently causes significant economic losses to the poultry industry worldwide. Virulent NDV (vNDV) is exotic in poultry in the United States; however, the virus has been frequently associated with outbreaks of ND in cormorants, which poses a significant threat to poultry species. Here, we present the characterization of 13 NDV isolates obtained from outbreaks of ND affecting cormorants and gulls in the states of Minnesota, Massachusetts, Maine, New Hampshire, and Maryland in 2010. All 2010 isolates are closely related to the viruses that caused the ND outbreaks in Minnesota in 2008, following the new evolutionary trend observed in cormorant NDV isolates since 2005. Similar to the results obtained with the 2008 isolates, the standard United States Department of Agriculture F-gene real-time reverse-transcription PCR (RRT-PCR) assay failed to detect the 2010 cormorant viruses, whereas all viruses were detected by a cormorant-specific F-gene RRT-PCR assay. Notably, NDV-positive gulls were captured on the eastern shore of Maryland, which represents a significant geographic expansion of the virus since its emergence in North America. This is the first report of vNDV originating from cormorants isolated from wild birds in Maryland and, notably, the first time that genotype V vNDV has been isolated from multiple wild bird species in the United States. These findings highlight the need for constant epidemiologic surveillance for NDV in wild bird populations and for consistent biosecurity measures to prevent the introduction of the agent into domestic poultry flocks.

Salmonella living in biofilms are more resistant to chemical and physical stresses. However, information regarding the regulation of genes involved in biofilm formation for Salmonella enterica serovar Pullorum remains limited. In this study, eight mutants with knockout of genes ompR, rpoS, rfaG, rfbH, rhlE, metE, spiA, or steB from the Salmonella enterica serovar Pullorum strain S6702 were constructed. Phenotypic analysis revealed that all mutants were similar to the wild-type strain in growth rate. Only the ompR mutant showed a complete loss of production of curli and biofilm formation. The other mutants showed a modified production of curli and cellulose with less effect related to biofilm formation. The results of animal experiments indicated that the deletion of genes ompR, spiA, rfaG, or metE in wild-type strains contributed to attenuation of virulence in 1-day-old chickens. This study may bring new insights into novel vaccines or therapeutic interventions against Salmonella enterica serovar Pullorum infections.

During the outbreak of highly pathogenic avian influenza (HPAI) H5N1 in Sweden in 2006, disease and mortality were observed in a number of wild bird species. Encephalitis was one of the most consistent and severe findings in birds submitted for postmortem examination. However, the distribution and severity of the inflammation varied among individuals. This study characterized the encephalitis and the phenotype of the cellular infiltrate in brains of 40 birds of various species naturally infected with HPAI H5N1. Brain sections stained with hematoxylin and eosin and immunostained for influenza A viral antigen were evaluated in parallel to brain sections immunostained with antibodies against T lymphocytes (CD3 ), B lymphocytes (CD79a ), macrophages (Lectin RCA-1 ), and astrocytes expressing glial fibrillary acidic protein. The virus showed marked neurotropism, and the neuropathology included multifocal to diffuse areas of gliosis and inflammation in the gray matter, neuronal degeneration, neuronophagia, vacuolation of the neuropil, focal necrosis, perivascular cuffing, and meningitis. Broad ranges in severity, neuroanatomical distribution, and type of cellular infiltrate were observed among the different bird species. Since neurotropism is a key feature of HPAI H5N1 infection in birds and other species and because the clinical presentation can vary, the characterization of the inflammation in the brain is important in understanding the pathogenesis of the disease and also has important diagnostic implications for sample selection.

To study the course of natural avian bornavirus (ABV) infection, 63 psittacines of three bird collections where ABV had been demonstrated were investigated over a period of 1 yr. The psittacines were clinically observed and swabs of crop and cloaca as well as serum samples were collected three separate times at intervals of 2–6 mo. According to the results of detection of ABV RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and of anti-ABV antibodies by indirect immunofluorescence assay (IIFA), 43 of the birds were found to be infected with ABV. Based on variations in virus shedding and antibody production in combination with the occurrence of proventricular dilatation disease (PDD) –related clinical signs, pathological findings, and lethal outcome, four different groups of infected psittacines and a fifth group of noninfected psittacines were identified. Group 1 comprised six birds with various courses of ABV infection and forms of clinical PDD. Groups 2–4 included all birds with subclinical ABV infections: Group 2 contained 13 birds that were consistently (subgroup A, 6 birds) or inconsistently (subgroup B, 7 birds) ABV positive by PCR and serology; group 3 was composed of 13 psittacines exhibiting only anti-ABV antibodies; and 8 birds that had positive ABV RNA detection in crop and cloaca, but did not develop anti-ABV specific antibodies, were classified in group 4. Twenty-three out of the 63 psittacines remained free of detectable ABV RNA or anti-ABV antibodies over the whole observation period (group 5). Based on the results, it seems that birds with high ABV RNA load in crop and cloaca combined with high anti-ABV antibodies have a high risk of the development of PDD, indicating that the humoral antibodies do not protect against the disease. The meaning of the detection of ABV RNA and antibodies at a low and inconsistent level for the single bird as well as for the epidemiology of the ABV infection remained unclear in this field study and needs to be further investigated.

Sera samples from commercial broiler chickens and turkeys diagnosed with respiratory and disseminated aspergillosis were tested for the presence of antigen and antibody to Aspergillus. Antigen detection consisted of testing for two cell-wall components, beta-glucan and galactomannan, which have been used extensively in human medicine. There were significantly higher levels of galactomannan in all broiler chicken submissions (100%) and antibody to Aspergillus in 6 out of 9 submissions (66.6%) vs. control birds. Beta-glucan analyses did not show any differences among levels in the broiler chicken groups. There were significantly higher levels of galactomannan antigen in 4 out of 7 submissions (57.1%) of aspergillosis in commercial turkeys, while only 2 out of 7 submissions (28.5%) had higher levels of antibody to Aspergillus vs. the control group. This study shows that diagnosis of respiratory and disseminated aspergillosis may be performed by detection of galactomannan antigenemia and antibodies in broiler chickens and to an extent in turkeys.

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.

Astroviruses (AstVs) are nonenveloped RNA small round viruses (SRVs) with a genome of 6.8–7.9 kb. Known avian AstVs are spread worldwide; they have been associated with poult enteritis and mortality syndrome in the United States and reported in Italy in intensive turkey and guinea fowl flocks. Nevertheless, their real prevalence and their pathogenic role in avian enteritis affecting Italian flocks is far from clear. Negative staining electron microscopy (nsEM) is used for the routine diagnosis of avian enteric SRVs, although it cannot distinguish morphologically similar particles. Enzyme-linked immunosorbent assay (ELISA), reverse-transcription PCR (RT-PCR), and genomic sequencing are now used for this specific purpose. We analyzed 329 samples of chicken, turkey, and guinea fowl intestinal contents from Italian poultry flocks. Most samples were from enteritis outbreaks, but we also included samples from three longitudinal studies (one on 11 broiler flocks and the other two on a guinea fowl flock). We first examined the samples with nsEM. SRVs, including AstVs, are often associated with rotaviruses and were the most commonly detected morphotypes in avian enteric diseases. We then analyzed 124 of the samples with an RT-PCR targeting the open reading frame (ORF)-1b of AstV. This gene codes for an RNA-dependent polymerase. We then sequenced and genetically analyzed the RT-PCR positive samples. Phylogenetic analysis distinguished three defined clusters: the first included guinea fowl AstVs and turkey AstVs-2; the second, chicken AstVs; and the third was formed by avian nephritis viruses (ANVs). No strains clustered with turkey AstVs-1. The results indicate that ORF-1b presents certain genetic variability, even among AstVs from the same species. In longitudinal studies, samples retrieved from the same shed were homogeneous, with some exceptions suggesting possible coexistence of different genetic types in the same unit. The finding of ANV-like viruses in commercial guinea fowls underlines the genetic variability of AstVs and strengthens the hypothesis of a varied intraherd situation.

Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1→3)-β-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell®, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M  =  3098.7 pg/dl, SD  =  5022.6, n  =  22) followed by companion avian species and raptors with confirmed aspergillosis (M  = 1033.8 pg/dl, SD  =  1531.6, n  =  19) and experimentally infected Japanese quail (Coturnix japonica; M  =  1066.5 pg/dl, SD  =  1348.2, n  =  17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-off value of ≥461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 µg of GST-Iss, and 1/12 chickens immunized with 50 µg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA®) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n  =  6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anas platyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 × 10^4.7 median embryo infectious dose (EID₅₀)/ml (range: 1 × 10^4.3 to 1 × 10^5.4 EID₅₀/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Even though Newcastle disease virus (NDV) live vaccine strains can be applied to 1-day-old chickens, they are pathogenic to chicken embryos when given in ovo 3 days before hatch. Based on the reverse genetics system, we modified recombinant NDV (rNDV) established from lentogenic vaccine strain Clone 30 by introducing specific mutations within the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, which have recently been suggested as being responsible for attenuation of selected vaccine variants (Mast et al. Vaccine 24:1756–1765, 2006) resulting in rNDV49. Another recombinant (rNDVGu) was generated to correct sequence differences between rNDV and vaccine strain NDV Clone 30. Recombinant viruses rNDV, rNDV49, and rNDVGu have reduced virulence compared with NDV Clone 30, represented by lower intracerebral pathogenicity indices and elevated mean death time. After in ovo inoculation, hatchability was comparable for all infected groups. However, only one chicken from the NDV Clone 30 group survived a 21-day observation period; whereas, the survival rate of hatched chicks from groups receiving recombinant NDV was between 40% and 80%, with rNDVGu being the most pathogenic virus. Furthermore, recombinant viruses induced protection against challenge infection with virulent NDV 21 days post hatch. Differences in antibody response of recombinant viruses indicate that immunogenicity is correlated to virulence. In summary, our data show that point mutations can reduce virulence of NDV. However, alteration of specific amino acids in F and HN proteins of rNDV did not lead to further attenuation as indicated by their pathogenicity for chicken after in ovo inoculation.

A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either ¹¹²GKQGRL¹¹⁷ (n  =  8) or ¹¹²GRQGRL¹¹⁷ (n  =  2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n  =  8) and II (n  =  2). Genotype I viruses were most closely related to the NDV V4 strain (n  =  7) or the NDV Ulster 2C strain (n  =  1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.

Histomonas meleagridis is the causative agent of blackhead disease or histomonosis in turkeys, and previous research suggests that this parasite survives poorly outside of hosts except within heterakid nematodes. However, we investigated the viability of H. meleagridis in or on several artificially contaminated materials kept at ambient room temperature (22 ± 2 C) to mimic the situation in the field. The protozoan survived for up to 1 hr on wood, rubber, and metal; up to 3 hr on egg-tray cartons, egg shells, and bricks; up to 6 hr on straw, turkey feathers, and feed; and up to 9 hr in nonchlorinated tap water and fecal matter. Therefore, contaminated water, fresh fecal matter, or both could play a role in transmission of the parasite within and among poultry houses rather than other materials tested in this study.

The effect of pooling 11 or 5 oropharyngeal (O/P) swabbings on detecting avian influenza virus (AIV) by real-time reverse transcription (RRT)–PCR was evaluated. The model used for the evaluation was designed to minimize viral load and, thus, assess the effect of the pooling on detection. Two-week-old broiler chickens were inoculated via the intranasal route with the low pathogenicity chicken/Maryland/Minh Ma/04 H7N2 strain or remained uninoculated. On days 2, 3, 4, 5, 7, 9, 11, and 14 postinoculation (PI), O/P swabbings were collected from individual infected birds and pooled with either 10 or 4 O/P swabs from uninfected broilers to produce 10 replicate pools of 11 or 5 swabbings, respectively. AIV was readily detected (80%–100%) by RRT-PCR in the pools of 11 and pools of 5 swabbings from days 2 through 5 PI. Detection in pools of both types decreased to similar levels on day 7 (40% for the pools of 11 and 50% for the pools of 5). AIV was not detected on day 9, 11, and 14 PI in pools of either size. On a given sample day PI, mean cycle threshold (Ct) values were consistently higher (lower genome levels) in the pools of 11 compared to the pools of 5. These differences were statistically significant on days 3 and 5 PI, yet Ct values associated with both types of pools were clearly interpretable as AIV positive.

Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C. perfringens are its four major toxins (α, β, ε, ι;) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C. perfringens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C. perfringens α-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C. perfringens α-toxin and NetB toxin that were increased in birds experimentally co-infected with Eimeria maxima and C. perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

The study provides the results of avian influenza virus surveillance in Central Asia during 2003–2009. We have analyzed 2604 samples from wild birds. These samples were collected in Kazakhstan (279), Mongolia (650), and Russia (1675). Isolated viruses from samples collected in Mongolia (13 isolates) and in Russia (4 isolates) were described. Virological analysis has shown that six isolates belong to the H3N6 subtype and five isolates belong to the H4N6 subtype. Two H1N1 influenza viruses, one H10N7 virus, two H3N8 viruses, and an H13N8 virus that is new for Central Asia have been also isolated. Samples were taken from birds of six orders, including several species preferring water and semiaquatic biotopes, one species preferring dry plain regions, and one more species that can inhabit both dry and water biotopes.

There are only a few reports about the occurrence of coccidia in peafowl and no reports about the occurrence of Eimeria spp. in peafowl kept in Europe. Here, we describe the occurrence of Eimeria pavonina in diseased peafowl from Germany. In January 2011, one young peacock kept in an aviary showed a marked depression. No parasites were detected in samples from the diseased bird, but in samples of birds from the same and other aviaries, coccidian counts were between 400/g and 66,000/g. All peacocks were treated with toltrazuril. After treatment, the clinical condition of the diseased bird improved but, two weeks afterwards, other birds in the aviary were still shedding coccidia in their feces. Based on morphology, the coccidia were identified as E. pavonina. Parts of the 18s rRNA gene and the cytochrome oxidase subunit 1 (cox-1) gene were sequenced. A phylogenetic tree based on the 18s rRNA sequence placed the Eimeria sp. from peafowl closest to Eimeria spp. found in pheasants and partridges as well as to Eimeria meleagrimitis. A phylogenetic tree based on the sequence of cox-1 in contrast suggested a closer relationship to Eimeria necatrix and Eimeria tenella.

The isolation and molecular characterization of pigeon paramyxovirus type 1 (PPMV-1) from a sick racing pigeon in Uruguay is reported for the first time. Hemagglutination inhibition (HI) tests were performed to detect antibodies against avian paramyxovirus serotype 1 (APMV-1), and a HI titer of 1/32 was obtained. Tracheal and cloacal swabs were processed by real-time reverse transcription–polymerase chain reaction (rRT-PCR) with the use of the National Veterinary Services Laboratory–U.S. Department of Agriculture validated matrix (M) gene assay and were positive for APMV-1. Viral isolation in embryonated chicken eggs confirmed the molecular detection of the isolate. A fragment corresponding to the 3′ region of the fusion (F) protein gene was amplified by RT-PCR, and subsequently sequenced. The deduced amino acid sequence at the F protein cleavage site displayed the motif ¹¹²RRQKR/F¹¹⁷. Phylogenetic analysis of this part of the genome allowed the isolated virus to be grouped in the lineage VIb/4b, which suggests that it shares the same ecologic niche with other PPMV-1 that were found in the region, and it is not imported as other European or North American viruses.

Infectious diseases are common causes of significant morbidity and mortality events of wild aquatic birds (WABs) worldwide. Reports of Australian events are infrequent. A 3-yr passive surveillance program investigating the common causes of morbidity and mortality of WABs was conducted at Billabong Sanctuary near Townsville, North Queensland, from April 2007 to March 2010. Forty-two carcasses were obtained and evaluated by clinico-pathologic, histologic, bacteriologic, and virologic (molecular) examinations. Morbidity and mortality were sporadic and more commonly observed in chicks and juvenile birds in April than other months of the year. Morbid birds were frequently unable to walk. Hemorrhagic lesions and infiltration of lymphocytes in various organs were the most common findings in dead birds. Identified bacterial diseases that could cause bird mortality were colibacillosis, pasteurellosis, and salmonellosis. Salmonella serotypes Virchow and Hvittingfoss were isolated from an Australian white ibis (Threskiornis molucca) chick and two juvenile plumed whistling ducks (Dendrocygna eytoni) in April 2007. These strains have been previously isolated from humans in North Queensland. A multiplex real time reverse transcriptase–PCR (rRT-PCR) detected Newcastle disease viral RNA (class 2 type) in one adult Australian pelican (Pelecanus conspicillatus) and a juvenile plumed whistling duck. No avian influenza viral RNA was detected from any sampled birds by the rRT-PCR for avian influenza. This study identified the public health importance of Salmonella in WABs but did not detect the introduction of the high pathogenicity avian influenza H5N1 virus in the population. A successful network was established between the property owner and the James Cook University research team through which dead birds, with accompanying information, were readily obtained for analysis. There is an opportunity for establishing a long-term passive disease surveillance program for WABs in North Queensland, an important region in Australian biosecurity, thus potentially significantly benefitting public health in the region and the country.

An avian influenza (AI) surveillance was undertaken in Maharashtra state, India during the period 2010–2011. There are no reports of AI surveillance in emus from India. A total of 202 blood samples and 467 tracheal and cloacal swabs were collected from eight emu farms. A hemagglutination inhibition (HI) assay was performed for detection of antibodies against AI H5N1, H7N1, H9N2, and avian paramyxovirus type 1 (APMV-1) viruses. A microneutralization (MN) assay was performed to confirm the presence of neutralizing antibodies against AI H9N2 and to compare with HI assays. A total of 28.2% and 28.7% of samples were positive for antibodies against AI H9N2 by HI and MN assays, respectively, using ≥1∶40 as a cut-off titer; 15.3% samples were positive for APMV-1 by HI assay using a ≥1∶10 cut-off titer. Seropositivity of AI H9N2 was nil in the grower (<1 yr) age group and highest (78%) in the breeder (2–3 yr) age group, whereas seropositivity against APMV-1 was observed in all age groups. Performance of both HI and MN assays was similar, suggesting the utility of using the MN assay along with HI assay for surveillance studies. This is the first report of the seroprevalence of AI H9N2 and APMV-1 in emus in India.