Chicken dendritic cells (DCs) have been demonstrated to be susceptible to infectious bursal disease virus (IBDV), a causative agent of acute and immunosuppressed disease in young chicks known as infectious bursal disease. Further functional characterization of IBDV-infected DCs of chickens is required to provide a better understanding on the influence of the virus on chicken bone marrow–derived dendritic cells (BM-DCs) following very virulent (vv) IBDV infection. Membrane proteins of BM-DCs were extracted and the proteins were further denatured and reduced before performing labeling with isobaric tags for relative and absolute quantitation. The differential expression protein profiles were identified and quantified using liquid chromatography coupled with tandem mass spectrometry, and later validated using flow cytometry and real-time reverse transcriptase PCR. The analysis has identified 134 differentially regulated proteins from a total of 283 proteins (cutoff values of ≤0.67, ≥1.5, and ProtScore >1.3 at 95% confidence interval), which produced high-yield membrane fractions. The entry of vvIBDV into the plasma membrane of BM-DCs was observed at 3 hr postinfection by the disruption of several important protein molecule functions, namely apoptosis, RNA/DNA/protein synthesis, and transport and cellular organization, without the activation of proteins associated with signaling. At the later stage of infection, vvIBDV induced expression of several proteins, namely CD200 receptor 1-A, integrin alpha-5, HSP-90, cathepsin, lysosomal-associated membrane protein, and Ras-related proteins, which play crucial roles in signaling, apoptosis, stress response, and antigen processing as well as in secretion of danger-associated proteins. These findings collectively indicated that the chicken DCs are expressing various receptors regarded as potential targets for pathogen interaction during viral infection. Therefore, fundamental study of the interaction of DCs and IBDV will provide valuable information in understanding the role of professional antigen-presenting cells in chickens and their molecular interactions during IBDV infection and vaccination.

Vaccine regimes and maternal antibody protection are important in ensuring the health of poultry and critically important in decreasing contamination of poultry products with foodborne pathogens that threaten human health. Here, we assessed the role of passive immunity on the resistance of progeny to early colonization when challenged with Salmonella Heidelberg. Two broiler breeder hen flocks which had received a Salmonella vaccine regime consisting of two live attenuated and two killed vaccines during rearing were selected for study. ELISA titers were used to assess antibody levels in the parent flocks, with one low- and one high- titer flock selected for study. Progeny chicks (1 day of age) were taken from each flock and challenged with Salmonella Heidelberg at low (10³) or high doses (10⁵) at 3 days of age. At 14 days postinoculation, all birds were euthanatized and their liver, spleen, and ceca collected for culture. ELISA analysis found Flock A (30-wk flock) demonstrated higher Salmonella antibody titers in the parents as well as yolk titers in the progeny, resulting in greater early protection from colonization by Salmonella Heidelberg while Salmonella colonization rates were higher in the progeny of the older parent Flock (B) that demonstrated lower antibody titers in the parents and the yolks of the progeny. These results suggest that as the breeder hens' protective antibodies wane with age; the maternal antibody protection in their progeny also becomes less effective in preventing cecal colonization by Salmonella early in life, which has the potential for affecting the health of the bird and contamination of meat products destined for the consumer.

Avian bornaviruses (ABVs) are the causative agents of proventricular dilatation disease (PDD), a fatal neurologic disease considered to be a major threat to psittacine bird populations. We performed a reverse transcription PCR survey to detect the presence of canary avian bornavirus (CnBV) in birds of order Passeriformes related to different clinical manifestations, such as sudden death, neurologic signs, apathy, anorexia, excessive beak growth, and PDD. A total of 227 samples from captive and wild canaries were included, of which 80 samples were captive birds, comprising saffron finches (n = 71) and common canary (n = 9), and 147 samples were wild birds distributed among a variety of several species. Two samples from captive birds (2/80) were positive for ABV, and in wild birds, only one sample was positive for ABV. The positive samples were subjected to DNA sequencing, and only the CnBV-1 serotype was found, which was the first time it was detected outside of Germany (Austria/Hungary), where it was first detected in 2009. Phylogenetic analysis confirmed that avian bornavirus serotype CnBV-1 is present in order Passeriformes in Brazil.

The HB strain of duck Tembusu virus (DTMUV) propagated in the brains of newborn mice was used to prepare antigens for use in the hemagglutination inhibition (HI) test. Results showed that such prepared antigens are highly specific to the serum samples derived from DTMUV-infected animals. No spurious hemagglutination reactions against serum samples specific to avian influenza virus H5, H7, H9 subtypes, Newcastle disease virus, egg drop syndrome virus, duck plague virus, and duck hepatitis A virus were observed. The HI test can detect specific antibodies in the serum samples as early as day 4 after experimental infection of ducks with DTMUV. When compared to a virus neutralization test, the sensitivity is 100%. Overall, the HI test developed is highly specific to DTMUV and can be used in clinical diagnosis of diseases and in vaccine studies to monitor the kinetics of antibody response.

Cross-protection and immune responses elicited by infectious bronchitis virus (IBV) vaccination on Day 1 of age or at later time points were examined. Specific-pathogen-free chickens were vaccinated with a Massachusetts-type vaccine and heterologous challenge was performed with an Arkansas (Ark) -type virulent strain. In Trial 1, chickens vaccinated on Day 1 or Day 10 of age were challenged 21 days after vaccination. Analysis of tracheal histopathology and viral load demonstrated less cross protection when vaccination was performed on Day 1 of age. In Trial 2, chickens were vaccinated on Day 1 or Day 14 of age. A somewhat stronger systemic antibody response to IBV was detected in chickens vaccinated at 14 days of age. In addition, avidity of antibodies to Ark-type S1 protein elicited by vaccination at 14 days of age was greater. Differences in immune-cell populations in the Harderian gland (HG) observed at the time of sampling (35 days following vaccination) between chickens vaccinated at 1 day or 14 days of age indicated greater, rather than reduced, immune activity in the chickens vaccinated at 1 day of age. These differences are, perhaps, a result of the higher levels of persisting vaccine virus observed in the younger chickens. Both nonvaccinated/challenged groups showed significantly higher (P < 0.05) proportions of B cells and CD8⁺ T cells 7 days after challenge than age-matched vaccinated/challenged groups or age-matched nonvaccinated/nonchallenged control groups. These results indicate infiltration and/or expansion of B cells and CD8⁺ cells in HGs following challenge of nonvaccinated chickens. A fortuitous finding was that the more immature immune system of chickens vaccinated at 1 day of age was less effective at clearing vaccine virus after vaccination. Collectively, the current results indicate that IBV vaccination at 1 day of age can decrease the potential for heterologous cross protection compared with vaccination at least 10 days after hatch. A lower level of antibody affinity maturation likely contributes to decreased cross protection.

The major histocompatibility complex (MHC) B locus of chickens has been associated with resistance to different viral diseases. We previously provided evidence that chicken lines expressing MHC haplotypes B2 and B19 exhibit different resistance to a challenge with infectious bronchitis virus (IBV) Massachusetts 41 (M41). In the current study, we attempted to determine if those differences were true for genetically diverse IB viruses, i.e., IBV M41 and Arkansas-Delmarva poultry industry (ArkDPI). Clinical, pathologic, molecular, and immunologic outcomes were compared. Our results showed subtle clinical and pathologic differences between the two MHC chicken lines tested. Clinical differences were observed in respiratory signs at 2 days postinfection (dpi) in M41-infected birds. Pathologic differences were detected in viral load at 2 dpi in M41-infected birds and in tracheal epithelial thickness at 6 dpi in ArkDPI-infected birds. Substantial differences were observed in antibody responses at 14 dpi. The transcriptome analysis showed that B19 chickens highly expressed genes related to inflammatory and innate immune responses. This increased immune gene expression detected in B19 birds at 6 dpi did not lead to enhanced antibody production at 14 dpi. On the other hand, B2-haplotype chickens highly expressed genes related to cell responses, suggesting that B2 is able to diligently control the infection. Although not identical, genes triggered by M41 and ArkDPI are part of communal pathways and suggest similar immune and cell responses to both IBV genotypes. This work provides modest evidence for differential resistance to IBV by chickens displaying different MHC haplotypes as well as insights into the expression of a variety of genes after IBV replication in the host.

Enterococcus hirae is a zoonotic Enterococcus species that causes opportunistic infections in both humans and animals and can be transmitted by contact with animals or through contaminated food. The aim of this study was to investigate the importance of E. hirae in broilers with endocarditis, as well as the antimicrobial resistance patterns and genetic relatedness of the isolates. A total of 477 three- to five-week-old broilers were studied during five fattening periods on a farm with mortality due to endocarditis. Endocarditis was observed in 27 chickens (5.66%), and samples were taken for pathological, microbiological, and molecular studies. Lesions were mainly found in the right atrioventricular valve and corresponded with a fibrinous endocarditis. Enterococcus hirae was identified in all cases. Pulsed-field gel electrophoresis results showed clonality among some isolates, with one pulsotype harboring 11 isolates that were found throughout the study. Most of the isolates showed multi-drug-resistant phenotypes. These results confirm that E. hirae is a significant cause of endocarditis in broilers, and suggest that broilers may be important carriers of antimicrobial-resistant E. hirae that might enter into the food chain.

Infectious laryngotracheitis (ILT) is an upper respiratory disease of chickens, pheasants, and peafowl caused by the alphaherpesvirus Gallid alpha herpesvirus 1 (GaHV-1), commonly known as infectious laryngotracheitis virus. ILT is an acute respiratory disease characterized by clinical signs of conjunctivitis, nasal discharge, dyspnea, and lethargy. In severe forms of the disease, hemorrhagic tracheitis together with gasping, coughing, and expectoration of bloody mucus are common. The morbidity and mortality rates of the disease vary depending on the virulence of the strain circulating, the level of virus circulating in the field, and the presence of other respiratory infections. Since the identification of the disease in the 1920s, ILT continues to affect the poultry industry negatively across the globe. The disease is primarily controlled by a combination of biosecurity and vaccination. The first commercial vaccines, introduced in the late 1950s and early 1960s, were the chicken embryo origin live attenuated vaccines. The tissue culture origin vaccine was introduced in late 1970s. Recombinant viral vector ILT vaccines were first introduced in the United States in the 2000s, and now they are being used worldwide, alone or in combination with live attenuated vaccines. This review article provides a synopsis of what we have learned about vaccines and vaccination strategies used around the world and addresses knowledge gaps about the virus and host interactions that remain unknown.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek's disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%–76% vs. 95%–100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.

The above noted paper is being officially retracted as a publication in the Avian Diseases.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. The disease is caused by Gallid alpha herpesvirus-1 (GaHV-1), commonly known as the infectious laryngotracheitis virus (ILTV). Vaccination remains necessary for the control of the disease. Due to the inherent virulence of live attenuated vaccines, in particular that of the chicken embryo origin (CEO) vaccines, the use of ILT viral vector recombinant vaccines has significantly expanded worldwide as a safer vaccination strategy. However, the protective efficacy of recombinant ILT vaccines can be compromised by the use of fractional doses and improper handling and administration of the vaccine. The objective of this study was twofold: 1) to evaluate the protection efficacy induced by a commercial recombinant HVT-LT (rHVT-LT) vaccine when administered in ovo to broilers at three standardized doses (6000 plaque-forming units [PFU], 3000 PFU, and 1000 PFU), and 2) to assess the potential of rHVT-LT–vaccinated chickens to spread virus to contact chickens after challenge. Independently of the vaccine dose, vaccinated chickens showed reduction in clinical signs, maintained body weight gain after challenge, and lessened the challenge virus replication in the trachea at a rate of 52%–65%. However, in spite of this reduction, transmission of challenge virus from rHVT-LT–vaccinated (6000/Ch, 3000/Ch) to contact-naive chickens was evident. This study is the first to support that rHVT-LT vaccination did not prevent spread of challenge virus to contact birds.

Mycoplasma gallisepticum, the cause of chronic respiratory disease, remains one of the most important pathogens in the poultry industry. Controlling the impact of this disease is done by eradication of positive breeder flocks or by vaccination and medication. Tylosin and tilmicosin are often used in medication programs. However, recent data on the in vivo efficacy of these macrolide antibiotics are scarce. Therefore, two dose titration studies were conducted using a recently isolated M. gallisepticum strain belonging to the wild-type population with regard to its tilmicosin and tylosin minimal inhibitory concentration. In a first trial, broilers were infected with M. gallisepticum and treated with 10 or 20 mg tilmicosin/kg body weight (BW) in the drinking water for five successive days. In a second trial, broilers were infected with M. gallisepticum and treated with 35 or 100 mg tylosin/ kg BW in the drinking water for five successive days. Clinical scoring of respiratory signs, macroscopic scoring of respiratory tract lesions, M. gallisepticum isolation from the respiratory organs, weight gain, and mortality were monitored for efficacy evaluation. All tylosin and tilmicosin treatments significantly reduced the course of clinical respiratory disease, macroscopic lesions in the respiratory organs, and M. gallisepticum numbers in the respiratory tract and obtained higher weight gains compared with the Mycoplasma-infected untreated control group. A treatment of 100 mg tylosin/kg daily for 5 days was not more clinically efficacious than the dosage of 35 mg tylosin/kg daily for 5 days. At final necropsy, in animals treated with 20 mg/kg BW tilmicosin, significantly fewer respiratory tract lesions were present than in the animals treated with 10 mg/kg BW tilmicosin. Therefore, when tilmicosin is used to treat clinical outbreaks of M. gallisepticum in broilers, a dosing scheme of 20 mg tilmicosin/kg BW for five successive days seems to be the most recommended scheme.

Highly pathogenic avian influenza virus (HPAIV) from the goose/Guangdong/1996 clade 2.3.4.4 H5 lineage spread from Asia into North America in 2014, most likely by wild bird migrations. Although several variants of the virus were detected, H5N8 and H5N2 were the most widespread in North American wild birds and domestic poultry. In early 2015, the H5N2 virus spread through commercial poultry in the Midwest, and >50 million chickens and turkeys died or had to be culled. Related H5 HPAIVs are still endemic in much of the Eastern Hemisphere. The wild bird species that were involved with dissemination of the virus in North America are not known. Dabbling ducks, especially mallards (Anas platyrhynchos), typically have the highest detection rates for avian influenza viruses. To better characterize the wild avian species that could spread the virus, American black ducks (Anas rubripes), which are closely related to mallards, were challenged with the North American H5N2 and H5N8 index HPAIV isolates: A/Northern Pintail/WA/40964/2014 H5N2 and A/Gyrfalcon/WA/41088/2014 H5N8. Although the American black ducks could be infected with low doses of both isolates (≤10² 50% egg infective doses), ducks shed the H5N2 longer than the H5N8 (10 vs. 7 days) and the titers of virus shed were higher. Although there were too few ducks available on which to draw definitive conclusions, this suggests that American black ducks could serve as a more efficient reservoir for the H5N2 virus than the H5N8 virus.