Sigma C protein-coding sequences have been used to phylogenetically classify avian reovirus (ARV) strains. However, the relationship between serotype and phylogenetic cluster classification of the five prototype serotype strains of ARV in Japan has not been established. Thus, we used sigma C protein-coding sequences to characterize avian reoviruses (ARVs) isolated from chickens with tendonitis in Japan together with the five prototype serotype strains of ARV in Japan. Phylogenetic analysis of ARVs based on the sigma C protein-coding sequences revealed that the five prototype serotype strains of ARV were each classified into different, independent clusters. Two field isolates (JP/Tottori/2016 and JP/Nagasaki/2017) that were isolated from chickens with arthritis/tenosynovitis were classified into different clusters. JP/Tottori/2016 was classified into cluster VI with the CS-108 strain, and JP/Nagasaki/2017 was classified into cluster I with strain TS-142. Serologically, JP/Tottori/2016 was well-neutralized by antisera against the CS-108 strain, whereas JP/Nagasaki/2017 cross-reacted with antisera against both the CS-108 and TS-142 strains. Embryo lethality test revealed that the two field isolates induced 80% and 67% embryo mortality, respectively, whereas the five prototype strains induced 0%–33% embryo mortality. Our findings will contribute to understanding the characteristics of ARV strains in Japan.

Avibacterium paragallinarum has been subtyped into three serogroups (A, B, and C) and nine serovars (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4) according to the Page and Kume schemes. Both schemes use the hemagglutination inhibition test for serotyping. However, the relationship between the hemagglutinin gene (HMTp210) sequences and serotypes of A. paragallinarum is still unclear. This problem is partly due to the lack of information on the complete HMTp210 sequence from the formal reference strain of Page serogroup B (strain 0222 or Spross). In this study, we determined the complete HMTp210 sequence of strain Spross. The sequence of Spross and those of other HMTp210 sequences retrieved from GenBank were used to conduct phylogenetic analyses to investigate the relationship between the serotypes and HMTp210 sequences of A. paragallinarum. Four phylogenetic clusters, designated clusters A-1, A-2, B, and C, were identified. Clustering based on complete HMTp210 sequences correlates with serotyping based on hemagglutination inhibition tests. Serovar A-2 was found to contain a chimeric HMTp210 gene that might have resulted from recombination between serovar A-1 and serovar C-1. In addition, phylogenetic analysis based on partial sequences (approximately nucleotides 1–1200) of HMTp210 was sufficient to discriminate between serogroups A, B, and C. These findings could be valuable for developing a molecular method for serotyping of A. paragallinarum.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes immunosuppression, T cell lymphomas, and neuropathic disease in infected chickens. To protect chickens from MDV infection, an avirulent live vaccine of turkey herpesvirus (HVT) has been successfully used in chickens worldwide. Many vaccine manufacturers have used chicken embryo fibroblast (CEF) cells to produce the HVT vaccine. Generally, it has been suggested that HVT is a highly cell-associated herpesvirus that spread via cell-to-cell contact, but it is unclear how HVT is transmitted from infected cells to uninfected target cells. Here, we show via immunofluorescence analysis that nanotubes containing the actin cytoskeleton and HVT antigens from infected CEF cells were observed to contact neighboring cells. When the infected cells were treated with inhibitors for actin polymerization or depolymerization, the formation and extension of the nanotubes from infected cells were greatly inhibited and the intercellular contact was abolished, leading to a drastic reduction in plaque formation and viral titers of the cell-associated virus. Our data indicate that cell-to-cell contacts via nanotubes composed of actin filaments are essential for efficient viral spreading and replication. This finding might contribute to the further improvement of efficient HVT vaccine production.

The present case is an unusual report of cutaneous fowlpox with an atypical appearance and incidence in broilers. Gross skin lesions were noticed in 41-day-old commercial broilers during the veterinary inspection at a processing plant in the north of Iran. The skin lesions were only observed on feathered skin areas of the broilers and remained unnoticed until slaughter. Round, nodular or coalescent, elongated, reddish-brown proliferative lesions were mainly located on the back, thighs, and proximal areas of the neck of broilers. Nonfeathered skin, including the wattle, comb, eyelids, and legs, were not affected. This condition incurred high losses due to a 5.3% condemnation and trimming of carcasses. Cutaneous lesions were sampled for histopathology and molecular virology for further investigations. Histopathology revealed multifocal necrotic dermatitis with epidermal eosinophilic cytoplasmic inclusion bodies in the skin lesions. Molecular investigations confirmed the presence of fowlpox virus (FWPV) in the proliferative lesions, with further investigations identifying two FWPV genome populations, one carrying a portion of the reticuloendotheliosis virus (REV) and the other a nearly complete REV provirus. Furthermore, the 4b core protein gene-based molecular analysis clustered the field virus into clade A of the genus Avipoxvirus.

Sigma C protein-coding sequences have been used to phylogenetically classify avian reovirus (ARV) strains. However, the relationship between serotype and phylogenetic cluster classification of the five prototype serotype strains of ARV in Japan has not been established. Thus, we used sigma C protein-coding sequences to characterize avian reoviruses (ARVs) isolated from chickens with tendonitis in Japan together with the five prototype serotype strains of ARV in Japan. Phylogenetic analysis of ARVs based on the sigma C protein-coding sequences revealed that the five prototype serotype strains of ARV were each classified into different, independent clusters. Two field isolates (JP/Tottori/2016 and JP/Nagasaki/2017) that were isolated from chickens with arthritis/tenosynovitis were classified into different clusters. JP/Tottori/2016 was classified into cluster VI with the CS-108 strain, and JP/Nagasaki/2017 was classified into cluster I with strain TS-142. Serologically, JP/Tottori/2016 was well-neutralized by antisera against the CS-108 strain, whereas JP/Nagasaki/2017 cross-reacted with antisera against both the CS-108 and TS-142 strains. Embryo lethality test revealed that the two field isolates induced 80% and 67% embryo mortality, respectively, whereas the five prototype strains induced 0%–33% embryo mortality. Our findings will contribute to understanding the characteristics of ARV strains in Japan.

Avian influenza vaccines are commonly used in the poultry industry. The objective of this study was to compare, under experimental conditions, the protective efficacy of four imported commercial inactivated H9N2 vaccines (A, B, C, and D) in broiler chickens. A total of 150 one-day-old chicks were divided into six groups: four experimental groups, each containing 30 chicks, received one of the vaccines (A, B, C, or D) delivered in a 0.3-ml dose subcutaneously at 1 day of age, whereas the control, Group T, was not vaccinated but challenged and Group E was kept unvaccinated and unchallenged. At 21 days postvaccination, Groups A, B, C, D, and T were challenged with 10⁷ embryo infective dose 50% of A/Chicken/Morocco/01/2016 (H9N2). All chicks were observed daily for clinical signs during the 12 days postchallenge (dpc). At 5 and 12 dpc, chicks were euthanatized for necropsy examination. Blood samples were collected weekly for serologic analysis and oropharyngeal swabs were collected for virus detection by real-time RT-PCR. Respiratory signs started at 48 hr pc and maximum severity was observed on 9 dpc. Chiefly, the birds vaccinated with vaccine B showed significantly more respiratory signs than did their counterparts. Serologic analysis revealed that the sera of Groups A and D birds showed a decrease in antibody (Ab) levels up to day 26; then a slight increase of Ab level was observed until day 31, while Group B and C birds showed a stabilization of the titers from day 21 until the end of the experiment. The viral shedding rate was significantly lower in Groups C and A (40%–50% of the birds shed virus for <7 days) compared with other challenged groups (60%–75% of the birds shed virus for ≥9 days). This experiment illustrated that vaccination applied on the first day in the hatchery with the four vaccines tested did not provide an acceptable protection against H9N2 in comparison with the controls that did not receive any vaccine. However, at first glance, we might favor vaccines A and C for their ability to reduce and shorten viral shedding as compared with vaccines B and D.

Extensive usage and the limited number of approved poultry anticoccidial drugs has led to some degree of resistance being developed to all drugs. Three independent battery trials were conducted to evaluate the anticoccidial drug sensitivity (AST) of species/strains of Eimeria within three commercial coccidia vaccines. The vaccines used for these studies were Immucox 3^® (broiler vaccine) (IM3), Immucox 5^® (broiler breeder vaccine) (IM5), and Immucox T^® (turkey vaccine) (IMT). Each trial used a randomized block design with six replications. Each cage contained eight male Cobb 500 broilers (IM3 and IM5) or eight male Hy-Line turkeys (IMT). At 12 days of age (D0), birds were group weighed, allocated to treatment cages, and issued weighed treatment feeds. Birds and remaining feed were weighed on D8 (20 days of age) (IM3 and IM5) or D9 (21 days of age (IMT)). Anticoccidial drugs were blended at commercial use levels. The treatments were as follows. IM3: 1) Nonmedicated nonchallenged (NMNC); 2) Nonmedicated challenged (NMC); challenged and medicated with 3) robenidine 33 parts per million (ppm); 4) decoquinate 30 ppm; 5) clopidal 125 ppm; 6) zoalene 125 ppm; 7) diclazuril 1 ppm; 8) amprolium 125 ppm; 9) salinomycin 66 ppm; 10) narasin 39.6 ppm + nicarbazin 39.6 ppm; and 11) narasin 74.25 ppm. IM5 used the same IM3 treatments 1–11 plus 12) nicarbazin. IMT: 1) NMNC; 2) NMC; 3) lasalocid 79.2 ppm; 4) monensin 66 ppm; 5) amprolium 125 ppm; 6) zoalene 125 ppm; and 7) nicarbazin 125 ppm. The challenge for each vaccine was 100 times the commercial recommended dose given orally on D2 (14 days of age). For trials IM3 and IM5, on D8 (6 days postchallenge) all chickens were coccidia lesion scored. IMT oocysts per gram (D7–9) and dropping scores (D8) were determined instead of lesion scores. For IM3 and IM5 vaccines, the results showed that all strains were sensitive to all anticoccidial drugs. For turkey IMT vaccine, the results showed that all strains were highly sensitive to all anticoccidial drugs. This information can serve as a foundation for the use of these coccidia vaccines in the restoration of coccidia sensitivity in the field.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein–coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Infectious bronchitis virus (IBV) is a gammacoronavirus that primarily induces an upper respiratory disease in chickens, also affecting the urogenital tract and occasionally leading to a condition called false layer syndrome (FLS), where sexually mature hens ovulate normally but are unable to lay eggs. Here, we describe an outbreak of FLS in Arizona from which an IBV variant that is nearly 90% homologous to DMV/1639 using the Spike subunit 1 gene, named AZ/FLS/17, was isolated and used in challenge experiments. Three-day-old specific-pathogen-free chicks were challenged with AZ/FLS/17 or M41 in high and low doses, and the disease outcomes were compared. Overall, no differences in microscopic lesions or viral loads in the reproductive tract were detected between AZ/FLS/17- and M41-infected birds. To minimize the losses linked to FLS in the problematic flocks, an updated live-attenuated IBV vaccine protocol including the use of the Ma5 strain at the hatchery was implemented, resulting in a drastic reduction of false layers in the subsequent flocks. To monitor the circulation of wild-type and vaccine strains in this population, a molecular surveillance study was performed. Samples were collected at 1, 7, 14, and 21 days of age, and from laying hens at 30 and 36 wk. In older birds, the IBV strains detected were more diverse than at 1 and 7 days of age. Nevertheless, live vaccine combinations are still widely used to decrease the losses caused by FLS in commercial egg laying flocks worldwide.

Avian erythroblastosis (AE; erythroid leukosis) was detected in a 78-day-old Japanese native chicken. At necropsy, the liver was enlarged and diffusely dark red in color. Moderate splenomegaly was observed. Histologically, round to polygonal tumor cells were observed only in the blood vessels of the liver and other organs. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) sections to characterize tumor cells. Tumor cells, as well as normal erythrocytes as positive controls, were consistently positive for IHC by using the commercially available anti-human hemoglobin antibody. Exogenous avian leukosis virus (ALV) subgroup B and endogenous ALV-E genes were detected by PCR, although ultrastructural observation revealed no viral particles associated with tumor cells. Results suggest that the commercial anti-human hemoglobin antibody used in the study cross-react to chicken erythrocytes on FFPE sections by IHC and can also be used for definitive diagnosis of the spontaneous case of AE in chickens.

Hepatitis hydropericardium syndrome (HHS) is an acute infectious disease caused by fowl adenovirus serotype-4 (FAdV-4), which mainly affects broilers aged 4–5 wk. During the winter of January 2021, a 32-day-old broiler flock (Cobb-500) suffered from unusually high mortality (15%) in the Alexandria Governorate, Egypt. The chickens showed depression, ruffled feathers, and greenish diarrhea besides the typical pathologic features of suspected HHS involving flabby hearts, accumulation of a straw-colored fluid in the pericardial sacs, and pale, enlarged hemorrhagic and friable livers with necrotic foci. The kidneys exhibited edema with uric acid depositions. Histopathologic examination of bird livers naturally infected with HHS showed multifocal areas of necrosis, vascular changes, and basophilic intranuclear inclusion bodies (INIB) in the hepatocytes. Molecular identification of the causative agent was accomplished by PCR and sequence analysis of the hyper-variable regions of loop 1 of the hexon gene of fowl aviadenovirus. A pathogenic strain of the novel genotype-4 (FAdV-4) was demonstrated, closely similar to the Israeli strain IS/1905/2019, with an identity of 98%. This is the first report to identify FADV-4 in Egypt, prompting further studies to elucidate its epidemiologic role in all poultry sectors and associated economic losses to provide insights to its control and prevention.

The present study was designed to evaluate the utility of environmental samples for convenient but accurate detection of avian influenza virus (AIV) in commercial poultry houses. First, environmental samples from AIV-negative commercial layer facilities were spiked with an H5N2 low pathogenic AIV and were evaluated for their effect on the detection of viral RNA immediately or after incubation at –20 C, 4 C, 22 C, or 37 C for 24, 48, or 72 hr. Second, Swiffer pads, drag swabs, and boot cover swabs were evaluated for their efficiency in collecting feces and water spiked with the H5N2 LPAIV under a condition simulated for a poultry facility floor. Third, environmental samples collected from commercial layer facilities that experienced an H5N2 highly pathogenic AIV outbreak in 2014–15 were evaluated for the effect of sampling locations on AIV detection. The half-life of AIV was comparable across all environmental samples but decreased with increasing temperatures. Additionally, sampling devices did not differ significantly in their ability to collect AIV-spiked environmental samples from a concrete floor for viral RNA detection. Some locations within a poultry house, such as cages, egg belts, house floor, manure belts, and manure pits, were better choices for sampling than other locations (feed trough, ventilation fan, and water trays) to detect AIV RNA after cleaning and disinfection. Samples representing cages, floor, and manure belts yielded significantly more PCR positives than the other environmental samples. In conclusion, environmental samples can be routinely collected from a poultry barn as noninvasive samples for monitoring AIV.

Colibacillosis, an infectious disease of chickens, is caused by avian pathogenic Escherichia coli (APEC); however, in addition to APEC, other pathogens are also frequently isolated from chickens affected with colibacillosis. Therefore, experimental infections in chickens are necessary to evaluate the pathogenicity of APEC isolates. Recent studies have shown that embryo lethality assays can be used as an alternative method to evaluate the pathogenicity of E. coli. In this study, to determine the important virulence genes associated with the pathogenicity of E. coli, 67 strains of E. coli that possessed different combinations of eight representative virulence genes (cva/cvi, vat, tsh, iucD, papC, irp2, iss, and astA) were isolated from broilers with colibacillosis in Japan, and the chicken embryo lethal assay was conducted. The genes vat, papC, and irp2 showed strong correlation with the level of virulence in E. coli. Our study provides useful information about the important virulence-associated genes in relation to the pathogenicity of E. coli in Japanese chickens.

Despite the vast Egyptian poultry production, scanty information is available concerning the infection of haemprotozoan parasites as pathogens in commercial broilers. In the present study, we provided the first detection of leucocytozoonosis in five broiler chicken flocks in El-Beheira Egyptian governorate. Despite the low mortality rates in the affected flocks (0.3%–1% as a 5-day mortality), severe postmortem (hemorrhagic spots and scars) and histopathologic lesions appeared in different organs including skeletal muscles, liver, kidney, pancreas, abdominal cavity, and bursa of Fabricius. Evaluation of blood smears revealed gametocytes in erythrocytes and leukocytes. Conventional reverse transcriptase-PCR and partial sequence analysis of mitochondrial cytochrome oxidase b gene detected Leucocytozoon caulleryi. GenBank accession numbers of the five Egyptian L. caulleryi isolates were obtained. The five L. caulleryi were 99.9% identical to each other and 99.14% similar to the L. caulleryi mitochondrial DNA gene of Asian strains from India, Japan, Malaysia, South Korea, Taiwan, and Thailand.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) are double-stranded RNA (dsRNA)-recognizing receptors that mediate innate immune responses to virus infection. However, the roles played by these receptors in the pathogenesis of avian viruses are poorly understood. In this study, we generated TLR3 and MDA5 single knockout (SKO) and TLR3-MDA5 double knockout (DKO) quail fibroblast cells and examined dsRNA receptor-mediated innate immune responses in vitro. The knockout cells were then stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or influenza A virus. Endosomal stimulation of TLR3 by adding poly(I:C) in cell culture media or cytoplasmic stimulation of MDA5 by transfecting poly(I:C) resulted in significant increases of TLR3, MDA5, interferon (IFN) β, and interleukin 8 gene expression levels in wild type (WT) cells. Endosomal poly(I:C) treatment induced a higher level expression of most of the genes tested in MDA5 SKO cells compared with WT cells, but not in TLR3 SKO and DKO cells. Cytoplasmic transfection of poly(I:C) led to significant upregulation of all four genes in WT, TLR3 SKO, and MDA5 SKO cells at 8 hr posttransfection and negligible gene expression changes in TLR3-MDA5 DKO cells. Upon infection with a strain of influenza virus with compromised IFN antagonistic capability, WT cells produced the highest amount of biologically active type I IFN followed by TLR3 SKO and MDA5 SKO cells. DKO cells did not produce detectable amounts of type I IFN. However, the IFN did not induce an antiviral state fast enough to block virus replication, even in WT cells under the experimental conditions employed. In summary, our data demonstrate that TLR3 and MDA5 are the key functional dsRNA receptors in quail and imply their coordinated roles in the induction of innate immune responses upon virus infection.

Adenoviral pancreatitis has been amply described for decades in guinea fowl. Although its pathologic picture has been characterized fairly well, its etiology still remains only partially clarified. Based on several outbreaks diagnosed on commercial guinea flocks raised in France since 2017, we performed direct whole-genome sequencing from pancreatic lesional tissue by using the Oxford Nanopore Technologies (ONT) sequencing method. We generated 4781 viral reads and assembled a whole genome of 43,509 bp, clustering within fowl adenovirus type 1 (FAdV-1). A phylogenetic analysis based on a partial sequence of the hexon and short fiber genes on viruses collected in France showed 98.7% and 99.8% nucleotide identity, respectively. Altogether, these results confirm that an FAdV-1 closely related to chicken and other avian strains is the agent of pancreatitis in guinea fowl. This study illustrates the potential of ONT sequencing method to achieve rapid whole-genome sequencing directly from pathologic material.

The control of poultry diseases has relied heavily on the use of many live and inactivated vaccines. However, over the last 30 yr, recombinant DNA technology has been used to generate many novel poultry vaccines. Fowlpox virus and turkey herpesvirus are the two main vectors currently used to construct recombinant vaccines for poultry. With the use of these two vectors, more than 15 recombinant viral vector vaccines against Newcastle disease, infectious laryngotracheitis, infectious bursal disease, avian influenza, and Mycoplasma gallisepticum have been developed and are commercially available. This review focuses on current knowledge about the safety and efficacy of recombinant viral vectored vaccines and the mechanisms by which they facilitate the control of multiple diseases. Additionally, the development of new recombinant vaccines with novel vectors will be briefly discussed.

A free-ranging, adult male ruffed grouse (Bonasa umbellus) was harvested by a hunter during November 2019 in Forest County, PA. The bird was submitted for necropsy due to a skin mass on its left leg. Upon necropsy, two proliferative skin masses were grossly visible, one on the left leg and one on the cere. An additional mass was present on the oropharyngeal mucosa covering the hard palate. These masses were diagnosed as avian pox based on histopathologic and cytologic findings, including marked epithelial hypertrophy, hyperplasia, vacuolar degeneration with eosinophilic stippling, and intracytoplasmic inclusion bodies. An avipoxvirus was detected using PCR and was identified as fowlpox virus through sequencing of the 4b core gene segment. The avipoxvirus from this case showed genetic similarity to isolates from Eastern wild turkeys (Meleagris gallopavo silvestris).

In this study we evaluated the effectiveness of adding serotype 793B vaccine to an immunization program in order to control the infectious bronchitis virus (IBV) GI-16 lineage. Therefore, two different experiments were performed. First, a virus cross-neutralization test was carried out, which indicated that neither the Massachusetts (Mass) nor 793B serotypes are antigenically related to the field isolate A13 (GI-16). We also performed a challenge trial to evaluate if the Mass/793B combination is more efficient than Mass/Connecticut (Conn) to protect chickens against the Argentinian variant A13. Thus, 40 chickens were organized in four groups. Chickens in Group A were vaccinated at 1 day of age with Mass serotype and then at 14 days old with Mass plus Conn serotypes. Chickens in Group B received Mass and 793B serotypes at 1 and 14 days old, respectively. Groups C and D remained unvaccinated. At 28 days of age, Groups A, B, and C were challenged with the A13 isolate, while Group D remained as the negative control. The statistical analysis of the ciliostasis evaluation, performed at 7 days postchallenge (dpch), indicated that the difference between Mass/793B and Mass/Conn was not significant (p > 0.05). However, the comparison against the negative control showed that only Group A was significantly different, suggesting a slightly better performance on blocking ciliostasis for the Mass/793B combination. On the other hand, no significant differences were observed in the viral load, quantified by reverse-transcription quantitative real-time PCR (RT-qPCR) in tracheal swabs and kidneys (at 3 and 7 dpch, respectively) between vaccinated groups. Furthermore, some amounts of the viral genome were found in both vaccinated groups that could indicate that neither the Mass/793B nor the Mass/Conn combinations totally inhibited the viral replication. Such viral replication in vaccinated chickens should seriously be taken into consideration because it could promote the selection of new variants in the future.

Avian favus (dermatophytosis) is a superficial mycosis caused by Microsporum gallinae in poultry. This disease is an important problem in poultry husbandry, but the standard antifungal treatment can leave drug residues in farm products. The aim of this study was to compare the efficacy of a clove essential oil ointment (3%, w/w) with commercially available ketoconazole cream (2%, w/w) for the treatment of M. gallinae infection in chickens. An in vitro time-kill assay showed that clove essential oil ointment reduced the number of viable M. gallinae ATCC 90749 by 99.99% within 1 hr. A randomized controlled trial showed that the therapeutic efficacy of clove essential oil ointment (3%, w/w) was noninferior to ketoconazole cream (2%, w/w) in M. gallinae–infected chickens. The percentage of completely recovered (culture-negative) animals in both treatment groups was 90% in day 35 after initial treatment. This study indicates that clove essential oil is suitable for preparation as an alternative topical treatment for avian dermatophytosis.

Coccidiosis is an enteric disease caused by protozoa of the Eimeria genus and is of great economic relevance in industrial aviculture. The objective of this work was to determine the prevalence of Eimeria sp. in broiler poultry houses with positive (System 1) and negative (System 2) pressure ventilation and assess the associated factors. A transversal study was conducted using 8 random broiler chickens from 64 houses (n = 512) and macroscopic and histologic evaluation of the intestines, as well as PCR for Eimeria sp. The prevalence of Eimeria sp. was 90.6% (95% confidence interval [CI]: 97.8–83.5), with 93.8% (95% CI: 100–85.4) in System 1 and 87.5% (95% CI: 99.0–76.0) in System 2. The most prevalent species was Eimeria acervulina, and the most common combination was Eimeria acervulina, Eimeria maxima, and Eimeria tenella. System 2 and the negative Eimeria subgroup showed the best results for feed conversion and daily weight gain. By evaluating litter treatment, we found that quicklime reduced the risk of presence of Eimeria maxima and Eimeria tenella. In conclusion, Eimeria sp. had a high prevalence in both systems, with a predominance of mixed infections. System 2 and negative flocks showed the best zootechnical results.

The 2015 highly pathogenic avian influenza (HPAI) H5N2 outbreak affected more than 200 Midwestern U.S. poultry premises. Although each affected poultry operation incurred substantial losses, some operations of the same production type and of similar scale had differences between one another in their ability to recognize evidence of the disease before formal diagnoses and in their ability to make proactive, farm-level disease containment decisions. In this case comparison study, we examine the effect of HPAI infection on two large egg production facilities and the epidemiologic and financial implications resulting from differences in detection and decision-making processes. Each egg laying facility had more than 1 million caged birds distributed among 18 barns on one premises (Farm A) and 17 barns on the other premises (Farm B). We examine how farm workers' awareness of disease signs, as well as how management's immediate or delayed decisions to engage in depopulation procedures, affected flock mortality, levels of environmental contamination, time intervals for re population, and farm profits on each farm. By predictive mathematical modeling, we estimated the time of virus introduction to examine how quickly infection was identified on the farms and then estimated associated contact rates within barns. We found that the farm that implemented depopulation immediately after detection of abnormal mortality (Farm A) was able to begin repopulation of barns 37 days sooner than the farm that began depopulation well after the detection of abnormally elevated mortality (Farm B). From average industry economic data, we determined that the loss associated with delayed detection using lost profit per day in relation to down time was an additional $3.3 million for Farm B when compared with Farm A.

Migratory waterfowl are the primary reservoir of avian influenza viruses (AIV), which can be spread to commercial poultry. Surveillance efforts that track the location and abundance of wild waterfowl and link those data to inform assessments of risk and sampling for AIV currently do not exist. To assist surveillance and minimize poultry exposure to AIV, here we explored the utility of Remotely Sensed Moderate Resolution Imaging Spectroradiometer (MODIS) satellite imagery in combination with land-based climate measurements (e.g., temperature and precipitation) to predict waterfowl location and abundance in near real-time in the California Central Valley (CCV), where both wild waterfowl and domestic poultry are densely located. Specifically, remotely collected MODIS and climate data were integrated into a previously developed boosted regression tree (BRT) model to predict and visualize waterfowl distributions across the CCV. Daily model-based predictions are publicly available during the winter as part of the dynamic California Waterfowl Tracker (CWT) web app hosted on the University of California's Cooperative Extension webpage. In this study, we analyzed 52 days of model predictions and produced daily spatiotemporal maps of waterfowl concentrations near the 605 commercial poultry farms in the CCV during January and February of 2019. Exposure of each poultry farm to waterfowl during each day was classified as high, medium, low, or none, depending on the density of waterfowl within 4 km of a farm. Results indicated that farms were at substantially greater risk of exposure in January, when CCV waterfowl populations peak, than in February. For example, during January, 33% (199/605) of the farms were exposed for ≥1 day to high waterfowl density vs. 19% (115/605) of the farms in February. In addition to demonstrating the overall variability of waterfowl location and density, these data demonstrate how remote sensing can be used to better triage AIV surveillance and biosecurity efforts via the utilization of a functional web app–based tool. The ability to leverage remote sensing is an integral advancement toward improving AIV surveillance in waterfowl in close proximity to commercial poultry. Expansion of these types of remote sensing methods, linked to a user-friendly web tool, could be further developed across the continental United States. The BRT model incorporated into the CWT reflects a first attempt to give an accurate representation of waterfowl distribution and density relative to commercial poultry.

Coccidiosis is a major intestinal disease affecting broiler chickens. Tributyrin (TB) is a valid alternative to butyrate acid, which was associated with the improvement of performance and attenuation of intestinal inflammation in animal production. However, there are few reports on TB as a prophylactic treatment against coccidiosis in broilers. The aim of the study was to investigate the effects of TB supplementation on performance and intestinal health of broiler chickens post coccidiosis vaccination with a mixed-species Eimeria. In the first experiment, 612 broiler chicks were randomly assigned to two treatments with six replicates. Treatments included no TB supplementation and coccidiosis vaccination (CV1), or TB supplementation (400 mg/kg) and coccidiosis vaccination (TBCV1). On day 5, all broilers received a single vaccine dose. Performance, intestinal histopathology, clinical severity, and fecal oocyst counts were evaluated from day 1 to day 63. TB supplementation resulted in a nonsignificant effect on body weight gain (BWG), feed intake (FI), and mortality-corrected feed conversion ratio (FCR), except in increased FI on days 22–42 (P < 0.05). The TBCV1 group had increased (P < 0.05) villi heights in the duodenum and increased (P < 0.05) villi widths in the ileum on day 63 of age and reduced oocyst shedding on days 19–26 compared to CV1(P < 0.05). The route of administration in the second experiment was different from the first experiment in which the seeder birds (half of birds from each pen) received a tenfold dose on day 5. TB supplementation in broilers resulted in increased (P < 0.05) BWG and reduced (P < 0.05) FCR on days 22–42, and increased (P < 0.05) villi heights in the duodenum and increased (P < 0.05) villi widths in the ileum on day 63 of age, as well as a lower frequency (P < 0.05) of intestinal hemorrhage on days 13–62 and reduced (P < 0.001) oocyst shedding on day 5 post-Eimeria challenge. In conclusion, the study demonstrated that TB can be considered as a feed additive for protecting broilers from coccidiosis on days 22–42.

To produce more-stable, live attenuated vaccines for infectious laryngotracheitis virus (ILTV), deletion of genes related to virulence has been extensively pursued. Although its function remains unknown, the open reading frame C (ORF C) is among the genes potentially associated with viral virulence that is nonessential for replication in vitro. Earlier results indicated that the ILT virus with deletion of the ORF C gene (BΔORFC) was suitable and safe for eye drop administration but was not sufficiently attenuated for in ovo administration. The objective of this study was to evaluate the safety and protection efficacy of a cell line–adapted, gene-deleted strain (BΔORFC) of ILTV when administered in ovo and/or spray (SP) by itself, or in combination with the recombinant HVT-LT (rHVT-LT) vaccine. Results indicated that vaccination with the BΔORFC strain, either by itself or in combination with an rHVT-LT vaccine, did not affect hatchability, and only marginal signs of respiratory distress were recorded for groups of chickens that received the BΔORFC strain via SP. The replication and seroconversion induced by the BΔORFC strain after in ovo and SP administration was very limited, whereas the replication of the rHVT-LT vaccine was delayed when combined with the BΔORFC strain in ovo. Compared to rHVT-LT or BΔORFC when administered alone, dual vaccination with rHVT-LT + BΔORFC was more effective in mitigating clinical signs of the disease and reducing challenge virus load in the trachea. To our knowledge, this study provides the first proof of concept that ILTV strains can be sufficiently attenuated for early vaccination in ovo or at hatch; also, this study documented the benefits of using a dual (recombinant and live attenuated) hatchery vaccination strategy for ILTV.

The aim of this study was to investigate the sensitivity of three breeds of Japanese native chickens, commercial broilers, and specific-pathogen-free (SPF) white leghorns to three strains of the H5 subtype of highly pathogenic avian influenza viruses (HPAIVs). Chickens were experimentally inoculated with doses of 10², 10⁴, and 10⁶ 50% egg infective dose of A/mandarin duck/Miyazaki/22M-765/2011 (duck-11), A/chicken/Miyazaki/7/2014 (chicken-14), and A/chicken/Kumamoto/1-2C/2016 (chicken-16). The 50% chicken lethal dose of each virus, mean death time, and viral shedding patterns were compared. The Japanese native chickens showed varied susceptibility to the three H5 HPAIV isolates. Although two of the breeds showed some degree of resistance to duck-11 and chicken-14, all three were more sensitive to chicken-16 than commercial broiler chickens. We have shown that Japanese native chickens do not necessarily have resistance to HPAIV and that the pathogenic characteristics of HPAIVs are quite different between native and commercial chickens.