Conventional and molecular epidemiologic studies have confirmed the ability of infectious bronchitis virus (IBV) to rapidly evolve and successfully circumvent extensive vaccination programs implemented since the early 1950s. IBV evolution has often been explained as variation in gene frequencies as if evolution were driven by genetic drift alone. However, the mechanisms regulating the evolution of IBV include both the generation of genetic diversity and the selection process. IBV's generation of genetic diversity has been extensively investigated and ultimately involves mutations and recombination events occurring during viral replication. The relevance of the selection process has been further understood more recently by identifying genetic and phenotypic differences between IBV populations prior to, and during, replication in the natural host. Accumulating evidence suggests that multiple environmental forces within the host, including immune responses (or lack thereof) and affinity for cell receptors, as well as physical and biochemical conditions, are responsible for the selection process. Some scientists have used or adopted the related quasispecies frame to explain IBV evolution. The quasispecies frame, while providing a distinct explanation of the dynamics of populations in which mutation is a frequent event, exhibits relevant limitations which are discussed herein. Instead, it seems that IBV populations evolving by the generation of genetic variability ^{and selection on replicons follow} the evolutionary mechanisms originally proposed by Darwin. Understanding the mechanisms underlying the evolution of IBV is of basic relevance and, without doubt, essential to appropriately control and prevent the disease.

Wild waterfowl undertake a variety of long-distance flights during their migration. These flights provide birds with the opportunities to both acquire and disseminate avian influenza viruses (AIVs). The Asian portion of Russia is crossed by four major migration routes and represents the major breeding area for many wild bird species in the Palearctic. The Asian territory of Russia plays an important role in distribution, persistence, and evolution of AIVs due to the ecologic relationships of bird populations from Russia and different Asian, European, African, and North American countries. Our study highlights the results of surveillance conducted in 2008 for AIVs in wild birds in the Asian portion of Russia. During this study, our team collected and tested 5678 samples from wild birds. Among them, 41 samples tested positive for AIV with an isolation rate of 0.72%. The highest AIV prevalence, 1.49%, was found in Anseriformes. In Ardeidae and Laridae, the AIV prevalence was 1.23% and 0.64%, respectively. Rallidae showed the lowest AIV prevalence of 0.61%. Phylogenetic analysis of H3 and H4 subtypes represented close relationships of AIVs isolated from the Asian portion of Russia to the AI strains from Asia, Africa, and Europe. These findings were confirmed by the wild bird migration routes that affect bird populations from Eurasian, African, Australian, and North American continents.

Newcastle disease (ND) is a major threat to the international poultry industry, causing bird mortality, reduction in growth and egg production, and trade restrictions. The primary strategy available to the poultry industry to control virulent Newcastle disease virus (NDV), the causative agent of ND, is vaccination. LaSota and other commonly used live-virus NDV vaccine strains were developed in the 1950s and 1960s and show a great degree of genetic divergence from currently circulating NDV strains. In order to characterize protective immunity induced by LaSota against a heterologous NDV strain, we vaccinated groups of specific-pathogen-free (SPF) chickens with LaSota (virus titers ranging from 10² to 10⁸ egg infective dose 50 [EID₅₀] in 10-fold increments) and challenged the birds 14 days later with ZJ1 strain, an NDV strain that was isolated in the year 2000 from geese in China. We monitored multiple parameters of immunity, including serum antibody titers, antigen-specific lymphocyte proliferation, and splenic cytokine expression and determined that SPF birds vaccinated with an adequate titer of LaSota strain live vaccine are fully protected from morbidity and mortality due to challenge with ZJ1 strain NDV, and we concluded that in the absence of interfering maternal antibody, protection due to vaccination increases with vaccine titer until a threshold titer is reached, beyond which, little or no further benefit can be elucidated.

Fertilized eggs were obtained from four pairs of sun conures (Aratinga solstitialis) infected with avian bornavirus (ABV) genotype 2, as determined by the sequence of the P24 gene. ABV RNA could be detected in early embryos of all four pairs. ABV RNA also was detected in brain, liver, and eyes of late-stage embryos of one of the pairs (Pair 4) and in blood of a 2-wk-old hatchling of this pair, demonstrating that vertical transmission can occur. ABV RNA could be detected in the liver but not in the brain or eyes of the late-stage embryos of another pair (Pair 3). Although it could be detected in the undeveloped eggs of the female parent and 8-day-old embryos, bornaviral RNA could not be found in the brain and liver of the late-stage embryos or in feathers and blood of young (5–9-wk-old) hatchlings of a third pair (Pair 2). At 11 wk, ABV RNA could be detected again in feathers and blood of these hatchlings and in the brain of one of the hatchlings of Pair 2 that suddenly died. ABV RNA could however be detected in throat swabs of the 5- and 9-wk-old hatchlings and their parents (Pair 2). Although the continued presence of ABV RNA in feathers and blood below the detection level of the reverse transcription-PCR used cannot be excluded, this result also may be attributable to feeding by the infected parents. Analysis by enzyme-linked immunosorbent assay showed that egg yolks and serum of late-stage embryos contain variable amounts of non-neutralizing anti–ABV-P40, -P10, -P24, and -P16 antibodies, the ratio of which reflected the antibody ratio in the serum of the female parent. Antibodies against the viral glycoprotein, which are considered neutralizing in mammals, and against ABV RNA polymerase were not detected. Whereas 5-wk-old hatchlings of the pair (Pair 2) that produced ABV RNA-free late-stage embryos were free of anti-ABV antibodies, such antibodies could be detected again in the serum of these hatchlings at 9 wk of age, before the age that bornaviral RNA could again be detected in feathers and blood. At 16 wk, these antibodies became abundant. The finding that late-stage embryos, presumably free of ABV RNA, can be obtained from eggs from infected parents suggests that hand- or foster-raising of such birds may be a method to obtain birnavirus-free offspring from some infected birds.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA clade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-α, IFN-γ, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Influenza virus infections can cause respiratory and systemic disease of variable severity and also result in economic losses for the turkey industry. Several subtypes of influenza can infect turkeys, causing diverse clinical signs. Influenza subtypes of swine origin have been diagnosed in turkey premises; however, it is not known how common these infections are nor the likely routes of transmission. We conducted a cross-sectional study to estimate the prevalence of influenza viruses and examine factors associated with infection on Minnesota turkey premises. Results from influenza diagnostic tests and turkey and pig premise location data were obtained from the Minnesota Poultry Testing Laboratory and the Minnesota Board of Animal Health, respectively, from January 2007 to September 2008. Diagnostic data from 356 premises were obtained, of which 17 premises tested positive for antibodies to influenza A virus by agar gel immunodiffusion assay and were confirmed as either H1N1 or H3N2 influenza viruses by hemagglutination and neuraminidase inhibition assays. Influenza infection status was associated with proximity to pig premises and flock size. The latter had a sparing effect on influenza status. This study suggests that H1N1 and H3N2 influenza virus infections of turkey premises in Minnesota are an uncommon event. The route of influenza virus transmission could not be determined; however, the findings suggest that airborne transmission should be considered in future studies.

CVI988 (Rispens) is currently the most effective vaccine used to protect against Marek's disease, a lymphoproliferative disease of chickens. A MEQ-deleted Marek's disease virus strain has shown promise as a vaccine candidate; however, unpublished results from vaccine safety trials suggest that this candidate vaccine induces unwanted lymphoid atrophy. The current study evaluated lymphoid atrophy at multiple time points between 2- and 8-wk postinoculation and attempted to correlate results with virus replication in the thymus. Results confirm reports that MEQ-deleted virus strains are able to cause thymus and bursa atrophy, which is most severe at 2-wk postinoculation. The MEQ-deleted virus strains induced lower body weights and relative thymus and bursa weights compared to uninoculated and Rispens-vaccinated chickens at multiple time points between 2- and 8-wk postinoculation. Both MEQ-deleted virus strains produced high levels of in vivo virus replication in the thymus at rates significantly greater than in Rispens-vaccinated chickens and were comparable to levels of RM1 virus, a MDV previously shown to induce severe thymus and bursa atrophy. Virus replication was highly correlated with relative thymus weights at each time point. Understanding this delicate balance between inducing maximum disease protection and preventing immunodepressive effects is critical for the development of future Marek's disease vaccines.

Infectious bronchitis coronavirus (IBV) shows extensive genotypic and phenotypic variability. The evolutionary process involves generation of genetic diversity by mutations and recombination followed by replication of those phenotypes favored by selection. In the current study, we examined changes occurring in a wild Arkansas (Ark) challenge strain in chickens that were vaccinated either ocularly with commercially available attenuated ArkDPI-derived vaccines or in ovo with a replication–defective recombinant adenovirus expressing a codon-optimized IBV Ark S1 gene (AdArkIBV.S1_ck). Commercial IBV Ark vaccines A, B, and C provided slightly differing levels of protection against homologous challenge. Most importantly for the current study, chickens vaccinated with the different vaccines displayed significant differences in specific B-lymphocyte responses in the Harderian gland (i.e., the challenge virus encountered differing immune selective pressure during invasion among host groups). Based on S1 sequences, five predominant populations were found in different individual vaccinated/challenged chickens. Chickens with the strongest immune response (vaccine A) were able to successfully impede replication of the challenge virus in most chickens, and only the population predominant in the challenge strain was detected in a few IBV-positive birds. In contrast, in chickens showing less than optimal specific immune responses (vaccines B and C) IBV was detected in most chickens, and populations different from the predominant one in the challenge strain were selected and became predominant. These results provide scientific evidence for the assumption that poor vaccination contributes to the emergence of new IBV strains via mutation and/or selection. In ovo vaccination with a low dose of AdArkIBV.S1_ck resulted in a mild increase of systemic antibody and reduced viral shedding but no protection against IBV signs and lesions. Under these conditions we detected only virus populations identical to the challenge virus. Possible explanations are discussed. From a broad perspective, these results indicate that selection is an important force driving IBV evolution.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [κ;] value of 0.821) using sera from commercial birds and a moderate level of agreement (κ; value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction with the gG-ve ILTV vaccine in a ‘differentiating infected from vaccinated animals’ approach to the control of ILT.

In total, 40 commercial layer farms and 32 replacement pullet farms with a combined population of 7.5 million adult layers and 6.6 million replacement pullets from six prefectures in eastern Japan were investigated for Salmonella Senftenberg contamination. We randomly collected 17,956 environmental samples, 5816 feed samples, and 218,470 egg samples from commercial layer farms; and 427 feed samples and 2896 environmental samples from replacement pullet farms. We monitored all samples for Salmonella. Samples were primarily enriched in Hajna tetrathinoate broth for 24 hr at 37 C followed by incubation in desoxycholate hydrogen sulfide lactose agar for 18 hr at 37 C. Salmonella colonies were confirmed and identified by biochemical tests and serotyped using Salmonella O and H antigens. We recorded 171 environmental samples (0.95%) and 10 feed samples (0.17%) that were positive for Salmonella spp. in which 36 environmental samples (0.20%) and six feed samples (0.10%) were identified as Salmonella Senftenberg. All Salmonella Senftenberg strains were isolated from nine replacement pullet farms. No Salmonella Senftenberg strains were isolated from adult layer farms and from eggs. Pulse field gel electrophoresis of BlnI-digested chromosomal DNA of 19 Salmonella Senftenberg isolates from feeds and environmental samples yielded a single identical DNA pattern. Traceback information showed that all positive feed samples were from a single feed source. Timeline studies showed that Salmonella Senftenberg contamination occurred first mostly in the feeds and then spread to the environment and other farms. This study demonstrated that the prevalence of Salmonella Senftenberg contamination in commercial layer facilities in eastern Japan is very low. Moreover, feed contamination played a major role in the epizootiology and spread of this pathogen in commercial poultry flocks. Given the resilient and persistent nature of this particular Salmonella serotype, routine monitoring and strict quality control measures at the feed level are recommended to prevent the colonization of poultry facilities with Salmonella Senftenberg that may lead to future outbreaks.

We analyzed factors involved in the introduction of infectious laryngotracheitis (ILT) virus (ILTV) onto broiler farms during a localized outbreak in an immunologically naïve broiler population. The outbreak occurred in the state of Mississippi, United States in 2002–2003. From the responses to a retrospective survey questionnaire administered via personal interviews, 181 farm-level risk factors were defined and analyzed for their association with ILTV introduction using logistic regression. There were 27 case farms (93% of all the infected broiler farms) and two sets of controls: farms matched to the cases by location and those randomly selected among the broiler farms in Mississippi. We found that farm suppliers such as gas company representatives, who are likely to visit other farms, and farm-workers who visit other chicken farms, are likely vehicles of ILTV introduction onto broiler farms. These risks can be greatly reduced by following biosecurity procedures, in particular if farm workers bathe and change footwear prior to entering broiler houses on their own farm. Footbaths for farm visitors can provide a false sense of security during an ILT outbreak when, indeed, other practices such as plastic boots or changing boots are more effective in preventing ILTV transmission. Sharing of equipment used for removal of caked broiler litter between subsequent flocks may also serve as an important vehicle of ILTV transmission. During the 2002–2003 outbreak, shared litter removal equipment was associated with ILTV transmission despite a requirement being put in place for litter decontamination. We also found that tunnel-ventilated broiler houses with inlets toward a neighboring poultry farm are more likely to get infected with ILTV. In addition to this analysis, the data collected provide a good overview of the actual practices and deficiencies of biosecurity undertaken on broiler farms in this part of the United States.

Molecular epidemiologic analyses of the 42 clinical isolates of Pasteurella multocida from various avian hosts (geese, ducks, turkeys, and laying hens) in Poland from 2001 to 2011, including a single reference strain, were performed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, single primer PCR, and repetitive extragenic palindromic (REP)–PCR. Forty-two isolates were identified as P. multocida (serotype A). The majority of P. multocida strains were obtained from waterfowl clustered within one genotype, and they were not consistent with the genotypes obtained from the turkey strains. Pasteurella multocida showed genetic homogeneity between the host species, especially when isolated on the same farm. Some of the clones also were characteristic to the particular farm. The strains obtained in different regions represent distinct molecular patterns. The present findings demonstrate that some clones of P. multocida are restricted in geographical and host distribution. In addition, this study suggests that ERIC-PCR, single primer PCR, and REP-PCR are suitable techniques for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Whole-genome sequencing analysis showed that A. paragallinarum strain H18 contains an RTX toxin–like operon with strong similarity to the RTX operons of other members of the Pasteurellaceae. Four genes, designated avxIC, avxIA, avxIB, and avxID, were found in this operon. The avxIA gene encodes the structural RTX toxin–like protein, which has a predicted molecular mass of about 250 kDa. The AvxIA protein contains a peptidase S8 domain and a proprotein convertase P-domain, neither of which has been found in other RTX toxins. Recombinant AvxIA proteins expressed in Escherichia coli showed neither hemolytic nor cytotoxic activity. Polymerase chain reaction and sequencing analysis revealed that the avxIA gene was present in all strains and field isolates of A. paragallinarum examined in this study. Sera collected from chickens exposed to A. paragallinarum exhibited strong reactivity to the AvxIA protein, which suggests that AvxIA is immunogenic. This is the first report of the identification of an RTX toxin–like operon from A. paragallinarum. The gene products of this operon may be related to disease pathogenesis and potentially represent a useful vaccine target of A. paragallinarum.

This study examined the effects of fumonisin B₁ (FB₁) and moniliformin (M) on the heart of Japanese quail (Coturnix coturnix japonica). Three hundred and ninety day-old Japanese quail were randomly divided into four groups: 1) FB₁ alone (FX), 2) M alone (MX), 3) FB₁ and M (FM), and 4) chick mash alone (CX). We used three pen replicates of 35 quail per pen in groups FX, MX, and FM and three pen replicates of 25 quail per pen in group CX. Gross and microscopic changes in the heart were studied in nine birds (three birds per replicate) from each group at weekly intervals up to 28 days postfeeding (DPF). Ultrastructural changes were studied in the heart of three birds (one bird per replicate) from each group at 21 DPF. Thinning of the heart was the only significant gross lesion in group FX. In contrast, mild-to-severe cardiomegaly was a significant finding in groups MX and FM throughout the study. Microscopically, thinning of cardiomyocytes was evident at 7 DPF in group FX. In addition to the hypertrophy of cardiomyocytes evident as early as 7 DPF, myocardial karyomegaly, nuclear hyperchromasia, and myofibril disarray exhibiting a wavy pattern were more pronounced at 28 DPF in group MX. Similar but more severe lesions were observed in the FM combination group that included myocardial hemorrhages, vacuolar changes, hypertrophy of cardiomyocytes, focal myocarditis, and loss of myofibrils cross-striations. Via transmission electron microscopy, the maximum effect of FB₁ toxicity was observed on mitochondria. In addition to an increase in the number of mitochondria, the mitochondria seemed invariably swollen and pleomorphic, although the outer membrane was intact, and the membrane cristae were usually distinct. Myofibrils seemed thinner, without much disruption in their architecture. Large numbers of vacuolar bodies of irregular size, both in the sarcoplasm and in between the myofibrils, were conspicuous in group FX. In contrast to group FX, the increase in number of mitochondria resulted in widespread separation of muscle fibers in group MX. In addition, the mitochondria were swollen and varied from round to oval to slightly elongated and occasionally forked, and vacuolation was rarely noticed in group MX. In the FM combination group, a significant increase in the number of mitochondria caused muscle fibers to look much thinner and assume a wavy pattern. We conclude that the effect of M on the heart is exaggerated in the presence of FB₁. Although the overall interactive effect of FB₁ and M was less than additive, the interactive effects between the two toxins for cardiac lesions were greater than additive to synergistic up to the second week, raising serious concerns on early age exposure to a combination of these two mycotoxins.

Worldwide, Newcastle disease (ND) remains one of the most economically important diseases of poultry. Current vaccination strategies for commercial poultry include the use of inactivated and live ND vaccines that typically induce protection against virulent field viruses. Here, we tested the efficacy of an antigen–antibody complex (AAC) ND vaccine delivered in ovo. Commercial maternal antibody–positive broiler chickens (Gallus domesticus) were vaccinated in ovo with an AAC vaccine composed of live B1-LaSota Newcastle disease virus (NDV) complexed with NDV-specific antiserum, and then they were challenged at weekly intervals after hatch. Challenge viruses included three exotic ND disease (END) viruses: the neurotropic strain Texas GB NDV-92-01 (TxGB) and two viscerotropic isolates, one isolate from the 2002–2003 outbreak in California (California 2002 isolate S212676 [CA]) and the other isolate from a 1997 END outbreak in South Korea (South Korea 94-147 [SK]). Results demonstrate that maternal antibody was able to provide approximately 50% protection in either vaccinated or control chickens at 7 days of age after TxGB challenge. However, with challenge at ≥14 days, most control birds died, whereas all AAC-vaccinated birds were protected. Challenge with the CA or SK viruses in chickens at 28 days of age resulted in 100% protection of vaccinated birds, whereas all control birds died. In addition, AAC-vaccinated birds displayed decreased incidence of viral shedding in oral and cloacal swabs than control birds. Antibody titers were significantly (P < 0.05) higher in vaccinated chickens, as determined by enzyme-linked immunosorbent assay and hemagglutinin-inhibition tests, than in nonvaccinated controls. Together, these results demonstrate the efficacy of AAC vaccines delivered in ovo to protect commercial poultry.

Infections of poultry due to Streptococcus equi subsp. zooepidemicus have been rare during the past decades and dissimilarities have been reported as to symptoms and lesions; likewise, the source of serious outbreaks has remained speculative. An outbreak affecting 11,000 free-range chickens at the age of 47 wk is reported. The outbreak manifested itself as acute at the onset and was followed by a chronic stage, resulting in some 80% mortality within 21 wk. Small-colony variants (SCVs) of S. equi subsp. zooepidemicus associated with the chronic phase are reported for the first time, and it is discussed whether SCVs might explain the change in lesions observed. Comparison of partial sequences of rpoB, multilocus sequence typing, and pulsed-field gel electrophoresis of isolates from chickens and horses kept at the farm showed the isolates to be identical and horses a likely source of infection. The present findings underline the importance of protecting free-range chickens from contact with other animals and birds known to host pathogens of importance to poultry.

The population of ruddy turnstones (Arenaria interpres morinella) that migrates through Delaware Bay has undergone severe declines in recent years, attributable to reduced availability of horseshoe crab (Limulus polyphemus) eggs at this critical spring migration stopover site. Concurrently, this population has experienced annual low pathogenicity avian influenza virus (AIV) epidemics at this same site. Using a prospective cohort study design with birds individually flagged during May–June 2006–2008, we evaluated resighting rates (a proxy for annual survival) between AIV-infected and uninfected birds at 1 yr after capture, testing, and measurement. Overall resighting rate was 46%, which varied by year and increased with relative mass of the bird when captured. Resighting rates were not different between AIV-infected and uninfected birds in any period. In multivariate analyses, infection status was also unrelated to resighting rate after controlling for year, day, state, sex, body size, mass index, or whether the bird was blood-sampled. Thus, apparent annual survival in ruddy turnstones was not reduced by AIV infection at this migratory stopover. However, it is unknown whether intestinal AIV infection might cause subtle reductions in weight gain which could negatively influence reproduction.

Nucleotide and predicted amino acid sequences of the infectious bursal disease virus (IBDV) surface protein VP2 have been used to identify strains of the virus and place them into phylogenetic groups. The amino acids across the hypervariable sequence region of VP2 (hvVP2) vary, but typically variant viruses have amino acids 222T, 249K, 286I, and 318D and classic viruses have 222P, 249Q, 286T, and 318G. A molecular epidemiologic study was conducted from 2001 to 2011 in commercial chickens (Gallus gallus) from Mexico, Colombia, and Venezuela. Although many IBDVs were identified, most had the typical variant or classic amino acid sequences across the hvVP2 region. Four viruses identified in 2004, one in 2006, and 10 in 2011 from Mexico had the amino acids 222T, 249Q, 286T, and 318D. Six samples from Venezuela in 2001, one sample from Colombia in 2001, two samples from Venezuela in 2004, and one sample from Venezuela in 2005 had the amino acids 222P, 249K, 286I, and 318G. These combinations of classic and variant amino acid sequence markers had not been identified previously in any IBDV strains. The VP2 amino acid sequences in the P_BC and P_HI loop structures of the Venezuela and Colombia viruses were similar to most classic viruses, whereas their minor P_DE and P_FG loop sequences were typical of Delaware variant strains. The Mexico viruses had VP2 P_BC loop sequences that were typical of variant IBDV strains, but their minor P_DE and P_FG loop structures contained amino acids that were similar but not identical to classic strains. The P_HI loop sequences of the Mexico viruses had 318D that is typical of a Delaware variant virus, but the other amino acids in this loop structure distinguished them from all other IBDV strains. The data suggest that one or more recombination events may have occurred to create this type of sequence diversity. Because of importation regulations, immunologic studies could not be conducted in the United States to determine the antigenicity of the viruses examined in this study. The amino acid sequence data, however, suggest they would contain antigenic epitopes of both variant and classic IBDVs.

Chickens infected with subgroup J avian leukosis virus (ALV J) early in posthatch life develop viremia followed by a neutralizing antibody (Nab) response that may or may not be able to clear the viremia. Occasionally, chickens that do clear viremia by developing an efficient Nab response revert to viremia, and the factors responsible for this reversion are not clear. In this study, it was hypothesized that stress can cause seroconverted viremia-free chickens to revert to viremia. Adult (52-wk-old) male commercial meat-type chickens that were exposed to ALV J at hatch and had since cleared viremia and remained viremia-free for up to 40 wk, when subjected to chronic stress (for 14 days) induced by porcine adrenocorticotrophin (ACTH), reverted to viremia and cloacal shedding (2/6 [33%]). However, chickens that were contact-exposed to ALV J at 32 wk of age and had seroconverted failed to revert to viremia when subjected to similar chronic stress. Stress did not increase the susceptibility of adult meat-type chickens to ALV J infection by contact exposure. The lack of statistical significance due to the small sample size is a limitation of this study. However, in general, the results suggest that treatment of chickens with ACTH can cause reversion of viremia and cloacal shedding in ALV J–seroconverted adult male chickens that had been exposed to the virus at hatch, but not in chickens that were contact-exposed at 32 wk of age. The results warrant further studies with greater sample size to examine the role of stress in ALV J epidemiology.

Avian hemosporidian parasites of the genera Haemoproteus, Plasmodium, and Leucocytozoon are transmitted by different dipteran vectors. In the present work, we looked for the presence of these parasites in 47 birds from 12 families, which were sampled in the migratory corridor Paso de Portachuelo, located at the Henri Pittier National Park, Venezuela. The presence of the parasites was evidenced by amplification of a region of 471 bp of their cytochrome b gene. This region of the marker presents enough polymorphism to identify most of the mitochondrial lineages. Therefore, the obtained amplicons were sequenced, not only to identify the genus of the parasites sampled, but also to analyze their genetic diversity in the study area. The overall parasite prevalence was low (11%). We reported, for the first time, Plasmodium in birds of the species Formicarius analis and Chamaeza campanisona (Formicariidae) and Haemoproteus in Geotrygon linearis (Columbidae). A phylogenetic tree was generated using the Haemoproteus, Plasmodium, and Leucocytozoon sequences obtained in this study, together with representative sequences from previous studies. The highest genetic diversities between the two Haemoproteus lineages (11.70%) and among the three Plasmodium lineages (7.86%) found in this study are also similar to those found when lineages reported in the literature were used. These results indicate that in the migratory corridor Paso de Portachuleo, representative parasite lineages are found, making this location an attractive location for future studies.

It has been demonstrated that fhaB2 (filamentous hemagglutinin) is an important virulence factor for Pasteurella multocida in development of fowl cholera disease and that vaccination with recombinant FHAB2 peptides derived from P. multocida, P-1059 (serotype A:3) protects turkeys against P-1059 challenge. Here the hypothesis that vaccination with the same rFHAB2 peptides could cross-protect turkeys against challenge with P. multocida χ73 (serotype A:1) was examined. Three rFHAB2 peptides were purified and pooled, and two doses, consisting of equal amounts of each, were administered subcutaneously to turkeys at 2-wk intervals. Simultaneously, control birds were administered sham inoculations. One week later, vaccinates and controls were challenged intranasally with P-1059 or χ73. The results showed vaccination with rFHAB2 peptides significantly protected turkeys against lethal challenge from both P. multocida serotypes (P < 0.01). The high degree of FHAB2 conservation across serotypes likely allow the observed cross-protection.

This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting “poult enteritis syndrome” were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

Influenza viruses from domestic aquatic birds can be transmitted to chickens, resulting in continued prevalence of the disease. H3 viruses are one of the most frequently identified subtypes in domestic ducks. Results from our previous serologic study suggested that H3 virus infections potentially exist in chickens with a wide geographical distribution in China. To better understand their pathogenic potential, two H3N8 influenza viruses isolated from domestic ducks were selected for experimental infections in chickens. We found that viral shedding lasted for at least 14 days postinfection for both viruses; however, one virus caused mortality in the chickens when coinfected with Escherichia coli. Sequencing of the viral HA gene isolated from the inoculated chickens revealed two amino acid mutations within the gene. These findings demonstrate the pathogenicity of the H3N8 domestic duck influenza viruses to chickens, highlighting the need for routine epidemiologic investigations of H3 subtype influenza viruses in chicken populations.

Surveillance for low pathogenicity avian influenza virus (LPAIV) infections has primarily relied on labor-intensive collection and serological testing of serum, but for many poultry diseases, easier-to-collect yolk samples have replaced serum for surveillance testing. A time-course LPAIV infection study in layers was performed to evaluate the utility of antibody detection in serum vs. egg yolk samples. Layers inoculated with the LPAIV A/Bobwhite Quail/Pennsylvania/20304/98 (H7N2) were tested for antibody levels in the serum and egg yolk by using the agar gel immunodiffusion test (AGID), hemagglutination-inhibition test (HI), and a commercially available enzyme-linked immunosorbent assay (ELISA). Anti-influenza specific antibodies were detected in the serum as early as 7 days postinoculation (DPI), and the majority of the hens remained positive until 42 DPI. Antibodies in the egg yolk were first detected by AGID at 7 DPI, which was also the first day of detection in serum. However, the majority of the eggs were positive by all techniques at 11 DPI and remained positive until 42 DPI, at which time the number of AGID and HI samples declined slightly as compared to ELISA samples. These results suggest that egg yolk can be an alternative to serum for flock serological surveillance against LPAIV infections, and the three methods (AGID, HI, and ELISA) will give similar results for first 42 days after infection, although AGID may give earlier positive response.

Infectious bursal disease virus (IBDV) is an important immunosuppressive pathogen of chickens worldwide. The introduction and evolution of IBDV in most African countries, especially in Ethiopia, remains unclear. We have investigated IBDV isolates obtained from commercial broilers, indigenous chickens, and pullets. The hypervariable region of the virus protein (VP) 2 and the 5′ two-thirds of VP1 of 11 IBDV isolates were characterized by RT-PCR and further sequencing. All isolates were identified as very virulent (vv) IBDV based on the predicted amino acid (aa) sequences of the VP2 protein. Interestingly, the sequence analysis of the 5′ two-thirds of VP1 indicated that the Ethiopian IBDV strains have aa residues typical for vvIBDV and for attenuated IBDV strains. Among all IBDV strains included in this study for phylogenetic comparison of VP2 nucleotide sequences, Ethiopian strains form a cluster within the vvIBDV lineage. We have also shown that Ethiopian IBDV strains have mutations in the VP1 region. Their roles in IBDV virulence may require further in vivo studies. As depicted in this study, the nucleotide and aa sequence analysis of VP1 in addition to VP2 is necessary to obtain a clear picture of the molecular evolution of IBDV.

In the austral summer of 2011, in the rural area of Villarrica county, southern Chile (39°16′S, 72°19′W), seven black-faced ibis juveniles (approximately 4 mo old) were observed in the field with weakness; they were unable to follow the group and struggling to take flight. Three of these birds were euthanatized, and complete necropsies were performed. Gross examination showed severe infestation with Colpocephalum trispinum and Ardeicola melanopis lice, moderate emaciation, pale musculature, bursal atrophy, and severe hemorrhagic enteritis due to a heavy proventricular and intestinal infection with Porrocaecum heteropterum nematodes. Fungal pneumonia and severe lymphoid depletion on thymus, spleen, and bursa were diagnosed by microscopic examination. Bursal lesions included apoptosis and necrosis of lymphoid cells, and several cystic follicles. The presence of severe lymphoid depletion associated with fungal pneumonia and severe external and internal parasite infections suggest the presence of an immunosuppressive syndrome in these birds that caused the death of several black-faced ibis juveniles in southern Chile during the summer of 2011.

From April 20 to May 10, 2010, multiple species of birds were seen at the Louisiana State University (LSU) Zoological Medicine Department for a disease syndrome characterized by acute lethargy, generalized subcutaneous petechiae, vasculitis, and death caused by a black fly identified as Simulium meridionale. Twenty psittacine birds presented with severe depression and multifocal subcutaneous hemorrhages over the body and especially noted in the featherless areas of the head and neck. Ten out of 20 clinical cases seen survived on a treatment regimen consisting of intravenous fluid therapy, anti-inflammatories, antihistaminic medications, and supportive care. The 10 other birds likely died of cardiopulmonary collapse and anaphylactoid reactions. In all post mortem examinations multifocal to coalescing dermal hemorrhage, eosinophilic dermatitis, severe edema, and vasculitis were observed. The LSU School of Animal Science conducted a brief survey that included 34 of the 64 Louisiana parishes and showed that 17 parishes, at least, were affected by this outbreak. A total of at least 225 poultry bird deaths could be attributed to black fly strikes. Simuliotoxicosis outbreaks have previously been reported in North America, and this report documents the clinical and post mortem findings, treatment response, and extent of such an outbreak in pet and farm birds in Louisiana.

Environmental swabs from pullet houses were tested for the presence of Salmonella Enteritidis (SE) using the RapidChek SE immunoassay test. Of the 1162 samples tested in 2011, 20 samples were positive, but only two samples were confirmed positive for SE by culture. Seventeen positive samples were from pullet houses that had been vaccinated with SE bacterin 2 to 3 days prior to submission to the lab. This study investigated the detection of SE bacterin using the RapidChek SE test system. Swabs were inoculated with different amounts of a commercial SE bacterin. As little as 0.25 ml of the SE bacterin was sufficient to cause positive results with the RapidChek SE test. This finding emphasizes the need to perform bacterial isolation and identification when a positive result occurs using an immunoassay test.