A filarial parasite potentially associated with the health burden on domestic chickens in Japan.

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed, paraffin-embedded tissues of 53 avian species using commercial antibodies.

Providing a method to detect avian lymphocytes by immunohistochemistry (IHC) would be helpful for analyzing immune function and diagnosing diseases in birds. In this study, we comprehensively examined the immunohistochemical identification of avian T and B lymphocytes in formalin-fixed, paraffin-embedded tissues from 53 avian species across 15 orders, using eight commercially available lymphocyte markers. T lymphocytes from all 53 avian species tested were specifically detected by IHC using the anti-CD3 antibody (clone F7.2.38). The appropriate antibody for detecting avian B lymphocytes in IHC varied depending on the avian species. B lymphocytes were specifically labeled by IHC in 46 of 53 avian species (86.8%) using any of seven B cell markers. The anti-PAX5 antibody (clone SP34) immunohistochemically detected B lymphocytes from the majority of avian species (41 out of 53 species), excluding those in the orders Falconiformes (falcons) and Passeriformes (oscines). The anti-BAFF-R antibody (clone 2C4) proved suitable for detecting B lymphocytes in the orders Galliformes (landfowls) and Anseriformes (waterfowls) in IHC. Caution is advised when using the anti-BLA36 (clone A27-42) and two anti-CD20 (clone L26 and product No. PA5-16701) antibodies, which are commonly used as B cell markers in mammals, for detecting avian B lymphocytes. These antibodies reacted with cells located in both T and B cell areas in certain avian species. The anti-Bu-1a/b (clone AV20) and anti-CD79a (clone HM57) antibodies were found not to bind to B lymphocytes in various avian species in IHC.

Susceptibility and Pathogenesis of Eurasian Tree Sparrows Experimentally Inoculated with Velogenic Newcastle Disease Virus.

Wild-caught Eurasian tree sparrows (Passer montanus) were experimentally inoculated with genotype VII velogenic Newcastle disease virus (NDV) APMV1/chicken/Japan/Fukuoka-1/2004 to investigate the susceptibility and pathogenesis of infected sparrows. Intranasal inoculation of two groups with high or low doses of the virus resulted in the mortality of some birds in both groups on days 7-15 postinoculation. Neurologic signs, ruffled feathers, labored breathing, emaciation, diarrhea, depression, and ataxia were observed in a few birds that eventually succumbed to death. The inoculation of the higher viral load resulted in higher mortality and hemagglutination inhibition antibody detection rates. Tree sparrows that survived the 18-day observation period after inoculation exhibited no apparent clinical signs. Histologic lesions in dead birds were observed in the nasal mucosa, orbital ganglion, and central nervous system, accompanied by NDV antigens detected by immunohistochemistry. Viral inclusion bodies were rarely observed in the cytoplasm of neurons. NDV was isolated from the oral swab and brain of dead birds but not from other organs, including the lung, heart, muscle, colon, and liver. In another experimental group, tree sparrows were intranasally inoculated with the virus and then examined 1-3 days later to examine the early pathogenesis of the disease. Inoculated birds exhibited inflammation of the nasal mucosa with viral antigens, and virus was isolated from some oral swab samples on days 2 and 3 postinoculation. The results of the present study suggest that tree sparrows are susceptible to velogenic NDV, and the infection could be fatal, although some birds can exhibit asymptomatic or mild infection. The unique pathogenesis regarding the neurologic signs and viral neurotropism of velogenic NDV was characteristic in infected tree sparrows.

Susceptibilidad y patogenia en los gorriones molineros inoculados experimentalmente con un virus velogénico de la enfermedad de Newcastle. Gorriones molineros (Passer montanus) capturados de la naturaleza se inocularon experimentalmente con un virus velogénico de la enfermedad de Newcastle (NDV) del genotipo VII APMV1/chicken/Japan/Fukuoka-1/2004 para investigar la susceptibilidad y la patogenia de los gorriones infectados. La inoculación intranasal de dos grupos con dosis altas o bajas del virus resultó en la mortalidad de algunas aves en ambos grupos en los días siete a 15 posteriores a la inoculación. Se observaron signos neurológicos, plumas erizadas, dificultad para respirar, emaciación, diarrea, depresión y ataxia en algunas aves que finalmente sucumbieron a la muerte. La inoculación de la carga viral más alta resultó en tasas más altas de detección de anticuerpos inhibidores de hemaglutinación y mortalidad. Los gorriones molineros que sobrevivieron al período de observación de 18 días después de la inoculación no mostraron signos clínicos aparentes. En las aves muerta se observaron lesiones histológicas en la mucosa nasal, ganglio orbitario y sistema nervioso central, acompañadas de antígenos virales detectados por inmunohistoquímica. Rara vez se observaron cuerpos de inclusión virales en el citoplasma de las neuronas. El virus de Newcastle se aisló del hisopo orales y del cerebro de aves muertas, pero no de otros órganos, incluidos los pulmones, el corazón, los músculos, el colon y el hígado. En otro grupo experimental, el virus se inoculó por vía intranasal a los gorriones molineros y luego se examinaron al día uno y tres para examinar la patogenia temprana de la enfermedad. Las aves inoculadas exhibieron inflamación de la mucosa nasal con antígenos virales y se aisló el virus de algunas muestras de hisopos orales en los días dos y tres posteriores a la inoculación. Los resultados del presente estudio sugieren que los gorriones molineros son susceptibles al virus de Newcastle velogénico y que la infección podría ser mortal, aunque algunas aves pueden presentar una infección asintomática o leve. La patogénesis única con respecto a los signos neurológicos y el neurotropismo viral del virus de Newcastle velogénico fue característica en los gorriones molineros infectados.

Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Detection of Gyrovirus galga 1 in Cryopreserved Organs from Two Commercial Broiler Flocks in Japan.

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

Development of monoclonal antibodies specific to Marek disease virus-EcoRI-Q (Meq) for the immunohistochemical diagnosis of Marek disease using formalin-fixed, paraffin-embedded samples.

Marek disease (MD) is a viral disease characterized by the development of lymphoma in poultry. Although morphologic confirmation of lymphoma is used to diagnose MD, immunohistochemical detection of MD virus-EcoRI-Q (Meq), which is a viral protein that is expressed exclusively in MD tumor cells, would further improve the accuracy of diagnosis. We developed monoclonal antibodies (mAbs) that specifically detect Meq by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) sections. We evaluated the sensitivity and specificity of 14 mAbs that we produced, using FFPE samples of MDCC-MSB1 cells, MD tumor tissues, and tissues of uninfected chickens. Four different antigen retrieval conditions were investigated. Thirteen mAbs reacted with Meq in FFPE sections, but immunohistochemical reactivity and specificity varied depending on the mAb and antigen retrieval condition; heat-induced antigen retrieval (HIAR) was more effective at detecting Meq than the other tested conditions. HIAR pH 9 tended to increase immunoreactivity and decrease specificity. Of the 5 mAbs that immunoreacted strongly with Meq without nonspecific reactions under the optimal antigen retrieval conditions, 3 mAbs (1C1-121, 3A3-112, 5F7-82) did not produce background staining of tumor or non-tumor tissues; 2 mAbs (2C5-11, 4A5-54) produced background staining. The mAb 6B5-128 reacted moderately with Meq without nonspecific reactions and background staining. The remaining mAbs showed weak immunoreactivity or problematic nonspecific reactions. Our results suggest that some of our developed mAbs can be used in IHC to detect Meq in FFPE sections with high specificity, and that the use of IHC may greatly improve the diagnosis of MD.

Genetic characterization of chicken anemia viruses newly isolated from diseased chicks in Japan in 2020.

In this study, a total of nine chicken samples obtained from two broiler flocks in Oita and Tottori prefectures in 2020 were examined for Chicken anemia virus (CAV) infection. The samples were collected from clinically suspected flocks and diseased chickens. The CAV genome was detected in all nine samples tested by real-time PCR. Phylogenetic analyses and sequence comparisons of the full-length VP1 gene sequences indicated that all the Japanese CAV strains obtained in this study formed a similar cluster of genotype III and shared high nucleotide (99.62-100%) identity. The current Japanese CAV strains were closely related to Chinese CAV strains but not related to vaccine strains. One positive selection site of VP1 was detected among the Japanese CAV strains.

Spontaneous Avian Erythroblastosis in a Chicken Confirmed by Immunohistochemical Detection of Hemoglobin in Tumor Cells.

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.

Reporte de caso- Eritroblastosis aviar espontánea en un pollo confirmada por detección inmunohistoquímica de hemoglobina en células tumorales. Se detectó eritroblastosis aviar (AE; leucosis eritroide) en un pollo nativo japonés de 78 días de edad. En la necropsia, el hígado estaba agrandado y de color rojo oscuro difuso. Se observó esplenomegalia moderada. Histológicamente, se observaron células tumorales redondas a poligonales solo en los vasos sanguíneos del hígado y otros órganos. Se realizó inmunohistoquímica (IHC) en secciones fijadas con formalina e incluidas en parafina (FFPE) para caracterizar las células tumorales. Las células tumorales, así como eritrocitos normales utilizados como controles positivos, fueron consistentemente positivos para IHC usando el anticuerpo de anti-hemoglobina humana disponible comercialmente. Se detectaron mediante PCR genes de virus exógenos de la leucosis aviar exógena (ALV) del subgrupo B del virus y endógenos ALV-E, aunque la observación ultraestructural no reveló partículas virales asociadas con las células tumorales. Los resultados sugieren que el anticuerpo comercial anti-hemoglobina humana utilizado en el estudio presenta una reacción cruzada con eritrocitos de pollo en secciones fijadas con formalina e incluidas en parafina mediante IHC y también puede utilizarse para el diagnóstico definitivo de caso espontáneos de eritroblastosis aviar en pollos.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed paraffin-embedded chicken tissues using commercial antibodies.

Immunohistochemical method to detect avian lymphocytes is an efficient and reliable tool for accurate diagnosis, and immunological analysis of avian diseases. However, there are scarce studies reporting immunohistochemistry (IHC) using commercially available antibodies in formalin-fixed paraffin-embedded (FFPE) chicken tissues. In the present study, we established an immunohistochemical method to identify chicken T and B lymphocytes in FFPE chicken tissues using commercial antibodies against chicken or human antigens. For this IHC method, the five tested anti-T lymphocyte antibodies reacted with chicken T lymphocytes on the FFPE sections. Further, 10 commercial anti-B lymphocyte antibodies were tested; of these, three successfully detected chicken B lymphocytes for IHC. In particular, anti-human CD3 (clone F7.2.38) antibody was most suitable for the detection of chicken T lymphocytes, whereas anti-chicken B cell activating factor receptor (BAFF-R) antibody (clone 2C4) was most suitable for the detection of chicken B lymphocytes under our IHC staining conditions. These two antibodies reacted with numerous lymphocytes of all representative lymphoid tissues without problematic background staining and nonspecific reactions. Our results indicate that T and B lymphocytes in FFPE chicken tissues can be immunohistochemically detected using commercial antibodies.

Pathological and virological analysis of concurrent disease of chicken anemia virus infection and infectious bronchitis in Japanese native chicks.

A concurrent infection of chicken anemia virus (CAV) and infectious bronchitis virus (IBV) was detected in Japanese native chicks in 2017, in which a high mortality rate (97.7%) was recorded in a small flock of 130 chicks exhibiting poor growth. Histological examination revealed that the affected chicks exhibited two different pathological entities: one was severe hematopoietic and lymphocytic depletion with abnormally large cells containing intranuclear inclusion bodies of CAV, whereas the other was renal tubular necrosis due to IBV infection. Immunohistochemistry detected CAV antigens in the bone marrow, liver, and spleen as well as IBV antigens in the kidneys, trachea, and air sacs. CAV was isolated from the liver sample of the chicks, and the isolated strain was designated as CAV/Japan/HS1/17. A phylogenetic analysis of the CAV VP1 gene revealed that CAV/Japan/HS1/17 is genetically similar to Chinese strains collected from 2014 to 2016. An experimental infection was performed using CAV/Japan/HS1/17 and specific-pathogen-free chicks to determine the pathogenicity of CAV/Japan/HS1/17. The isolate caused 100% anemia and 70% mortality to chicks inoculated at one day old, 80% of chicks inoculated at seven days old also developed anemia, and 10% died from CAV infection. These results suggest that the unusually high mortality in Japanese native chicks can be attributed to dual infection with both CAV and IBV. The results of the experimental infection suggest that CAV/Japan/HS1/17 has a pathogenic potential to specific-pathogen-free chicks and a relatively higher pathogenicity than previous Japanese CAV strains.