Partial characterization of an adenovirus-like virus isolated from broiler chickens with transmissible viral proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.     

Retrospective and prospective studies of transmissible viral proventriculitis in broiler chickens in Brazil

We investigated the occurrence and pathologic findings of transmissible viral proventriculitis (TVP) associated with the chicken proventricular necrosis virus (CPNV) in commercial broiler chickens in southeastern Brazil. Seventy-three broilers, 25–36 d old, with a history of reduced growth, were referred to our veterinary pathology services from 2013 to 2017. Broilers were clinically examined, weighed, and euthanized for postmortem examination. Broilers of different ages with proventricular histologic lesions were positive for CPNV by RT-PCR; however, the intensity of histologic lesions was higher among 33-d-old animals, and viral RNA detection was more frequent among those that were 28 d old. In the proventriculi of 35 of 73 (48%) broilers, lesions were characterized by glandular epithelial necrosis, lymphoplasmacytic and histiocytic infiltrates, and metaplasia of glandular epithelium to ductal epithelium. In 24 of 73 (36%) broilers with histologic TVP-compatible lesions, CPNV was detected by RT-PCR for the viral protein 1 (VP1) gene. Broilers with histologic lesions were lighter than expected compared to the Cobb 500 standard weight. TVP has not been reported previously in broiler chickens in Brazil, to our knowledge.     

Pathogenicity of serotype 1 fowl adenovirus in commercial broiler chickens.

The pathogenicity of a serotype 1 fowl adenovirus (FAV-99ZH), isolated from broiler chickens exhibiting gizzard erosion, was investigated in commercial broiler chickens. Five-, 3-, and 1-wk-old commercial broiler chickens were inoculated with FAV-99ZH by both oral and ocular routes. In the 5-wk-old chickens (trial 1), none of which had the maternal antibody to FAV-99ZH, severe gizzard erosions were observed on days 5, 7, and 10 postinoculation (PI). Among the 3-wk-old chickens (trial 2), which were separated into a control group and three treatment groups according to their maternal antibody titer levels, some chickens showed clinical signs such as depression and anorexia. Compared with the control group, all the treatment groups showed decreased weight gain. One treatment group, moreover, showed significantly decreased (P < 0.05) weight gain on day 10 PI. Severe gizzard lesions, such as erosion or ulcers, were observed from day 4 PI in all treatment groups regardless of their maternal antibody levels. The 1-wk-old chickens (trial 3) were separated into a control group and two treatment groups according to their titer levels of the inoculated virus. In spite of high maternal antibody levels, severe gizzard lesions were observed in both treatment groups, which also showed decreased weight gain. One treatment group, inoculated with the higher dose, showed significantly decreased (P < 0.05) weight gain on days 10 and 14 PI compared with the control group. Fowl adenovirus was recovered mainly from gizzard and rectal (including feces) samples from inoculated chickens but was not recovered from liver samples in any of the trials or in any of the control chickens. Although the reproduced disease was similar to that described in a previous report of experimental infection of specific-pathogen-free (SPF) white leghorn chickens with fowl adenovirus, the pathogenicity of FAV-99ZH in commercial broiler chickens was more severe than that in the SPF white leghorn chickens. The results of the present study indicate that FAV-99ZH isolated from gizzard erosion had pathogenicity and produced severe lesions in the gizzards of broiler chickens and that FAV-99ZH could infect and produce illness in broiler chickens with maternal antibodies against this virus.     

Isolation of avian reovirus as a possible etiologic agent of osteoporosis ("brittle bone disease"; "femoral head necrosis") in broiler chickens

Avian reovirus was isolated from intestines of 3-to-7-day-old broiler chickens with enteritis from broiler houses where osteoporosis was a problem. The virus was purified in a cesium chloride gradient (buoyant density 1.37 gm/ml) and identified as a reovirus by electron microscopy. Specific-pathogen-free (SPF) chickens and commercial broiler chickens with anti-reovirus maternal antibodies inoculated at 1 day of age with the reovirus isolate developed lesions of femoral head fractures and/or osteoporosis; reovirus could be reisolated from the bone marrow and intestinal tracts of experimentally infected SPF birds. The reovirus isolate, although isolated from intestines, induced development fo tenosynovitis lesions in SPF and commercial broiler chickens.     

Experimental infection of specific-pathogen-free chickens with serotype-1 fowl adenovirus isolated from a broiler chicken with gizzard erosions

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.     

Detection of serum antibodies in Eimeria tenella-infected chickens by enzyme linked immunosorbent assay (ELISA) with merozoite and oocyst antigens.

Soluble antigens prepared from sporulated oocytes and second generation merozoites of E. tenella were used for enzyme linked immunosorbent assay (ELISA) to investigate antibody in sera of two breeds of chickens, i.e. commercial broilers and SPF single comb white leghorn layers, which were experimentally infected with E. tenella. In broilers inoculated with oocysts at 15 days of age, ELISA values increased rapidly after day 19 post inoculation (PI) and reached the maximum lebel on days 29 and 32 PI against both merozoite and oocyst antigen. The values against merozoite antigen were significantly higher than those against oocyst antigen. In SPF layers infected at 15 days of age, the values increased gradually after 7 days PI. There were no significant differences between values against two antigens. Generally, the values in broilers tended to be higher than those in SPF layers, especially against merozoite antigen. In broilers inoculated with oocysts at 1 and 15 days of age, ELISA values increased rapidly and reached the maximum level on days 11 and 20 post second inoculation (PSI) against merozoite and oocyst antigens respectively and then the values against merozoite antigen decreased. The values against merozoite antigen were markedly higher than those against oocyst antigen. In SPF layers inoculated twice, the values reached the highest on day 11 PSI as in the case of broiler; however, after that day, the values against both antigens decreased. The sera reacted similarly against both antigens. The values against merozoite antigen were significantly higher in broilers than in SPF layers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenoviral gizzard erosion in commercial broiler chickens

Pathologic and immunohistochemical changes caused by group I of the fowl adenovirus (FAV) serotype-1 99ZH strain, isolated from broiler chickens exhibiting gizzard erosion, were investigated in commercial broiler chickens. One hundred twenty-two chickens were inoculated with the strain by both oral and ocular routes at 1, 3, or 5 weeks of age and euthanatized for necropsy within 4-18 days of inoculation. Focal gizzard erosions were observed in the inoculated chickens of each age group. A histologically degenerative koilin layer, necrotic mucosa, intranuclear inclusion bodies in the glandular epithelial cells, inflammatory cell infiltrations in the lamina propria, submucosa, and a muscle layer were seen in the gizzards. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies. These findings were recognized regardless of their maternal antibody levels for FAV serotype-1. Gizzard lesions appeared later in the lower-dose-inoculated chickens than in the higher-dose-inoculated chickens. Numerous CD3-positive cells and IgY-positive plasma cells were seen in the gizzard lesions. In 5-week-old chickens the heterophil infiltrations in the lesions were milder than in younger chickens. Intranuclear inclusion bodies also were observed in the epithelial cells of the ileum or cecal tonsils of some chickens. Thus, this study shows that FAV-99ZH causes adenoviral gizzard erosion in broiler chickens without hepatic or pancreatic lesions and that cell infiltration is more severe than in dietary gizzard erosions.     

Enteropathogenicity of Dutch and German avian reoviruses in SPF white leghorn chickens and broilers

The enteropathogenicity of avian reoviruses (ARVs), isolated from chickens affected with malabsorption syndrome (MAS) from The Netherlands and Germany was studied. In the first trial seven different ARVs isolated from MAS cases were inoculated in 1-day-old specific pathogenic free (SPF) white leghorns. The pathogenicity was compared with 2 ARVs isolated from cases of tenosynovitis, namely reference strain S1133 and a Dutch strain. Although a difference in the severity of the clinical disease was observed, all reoviruses could induce vacuolar degeneration and sloughing of the epithelium of the small intestine at day 2 post inoculation (PI) till day 7 PI. Two Dutch and one German ARV derived from MAS causing the most severe intestinal lesions at day 2 PI, were further studied in the second trial using SPF broilers. These reoviruses did not cause weight gain depression in the broilers although lesions in the small intestine were present from day 1 up to day 4 PI and were more severe than in the white leghorn chickens. In one of the inoculated groups apical denuded villi were already present at day 1 PI. At day 7 PI the small intestine of the infected broilers appeared to be normal. Reovirus antigen was detected in the cytoplasm of the enterocytes at the tip and middle section of the affected villi both in layers and in broilers. To study the role of intestinal CD4+ and CD8+ T-cells and macrophages/monocytes in the pathogenesis of ARV, the numbers of these cells of the jejunal villi of one infected and the control broiler groups were compared. CD4+ T-cells were detected in low numbers and only in the infected broiler group at day 14 PI. The numbers of CD8+ T-cells and macrophages/monocytes were significantly higher in the infected broiler group than in the control broiler group at day 7 and 14 PI and at day 7 PI respectively. Our study indicates that the reovirus alone cannot induce intestinal lesions as found in MAS chickens. Moreover, CD8+ T-cells may play a major role in the pathogenesis and or reovirus clearance in the small intestine.     

Pathogenicity of serotype 8 fowl adenovirus isolated from gizzard erosions of slaughtered broiler chickens

The pathogenicity of serotype 8 fowl adenovirus (FAV), isolated from gizzard erosions of slaughtered broiler chickens, was investigated. In experiment 1, 29 5-day-old specific-pathogen-free (SPF) chickens were inoculated with the isolates of serotype 8 FAV, M013 (group 1) or G0054 (group 2) strain, via an oral route. There were no clinical signs in any of chickens after inoculation, and mild gizzard erosions were observed macroscopically and microscopically in three inoculated chickens of group 2. FAV was recovered from gizzards and rectums but was not recovered from pancreas and livers from chickens in both inoculated groups. In experiment 2, 27 1-day-old SPF chickens were inoculated with the G0054 strain by intramuscular route. Five, 6, and 3 inoculated chickens died on days 3, 4, and 5 postinoculation (PI), respectively. Four, 3, 1, and 1 inoculated chickens became moribund with severe clinical signs such as ruffled feathers, severe depression and closed eyes from days 3 to 6 PI, respectively. Macroscopically, the common characteristic of the gross lesions of dead chickens and euthanized moribund chickens was discoloration of liver. FAV was recovered from the gizzard, liver, pancreas and rectum. Virus titers in the liver and pancreas were high until day 6 PI. Histologically, necrotizing hepatitis and pancreatitis with intranuclear inclusion bodies were observed in the inoculated chickens. These results indicate that some strains of serotype 8 FAV are able to reproduce not only gizzard erosion by oral inoculation but inclusion body hepatitis (IBH) by intramuscular inoculation.     

Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specific-pathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.     

Experimental gizzard erosions in specific-pathogen-free chicks by serotype 1 group I avian adenoviruses from broilers

The pathogenicity of serotype 1 group I avian adenovirus (GIAAV) strains isolated from gizzard erosions for 1-wk-old specific-pathogen-free (SPF) chickens was investigated. In Experiment 1, SPF chickens inoculated orally with a serotype 1 strain (Tokushima2000/GE) of GIAAV isolated from gizzard erosion in broilers were euthanatized and necropsied from 3 to 12 days after inoculation. Clinically, there were no significant signs in the chickens inoculated with Tokushima strain. The gizzards were found macroscopically to be dilated with liquefied contents. The koilin layer of the gizzard exhibited multifocal white lesions with occasional erosions. Gross lesions of the koilin layer (white foci and erosion) appeared at 3 days after inoculation and persisted until 12 days after inoculation. The gross lesions were most severe at 9 days after inoculation. Histologically, multifocal degeneration, necrosis, and loss of the glandular epithelial cells with intranuclear inclusions, liquefactive change of koilin substance, and erosive lesions of koilin layers and surface epithelium of the gizzards were observed. Macrophages increased in the focal areas of the lamina propriabelow the liquefactive koilin substances. Immunohistochemically, intranudear inclusions were positive against GIAAV antigen. Histologic lesions of gizzards were most severe at 7 and 9 days after inoculation, but the frequency of intranuclear inclusions was greatest at 5 days after inoculation. In Experiment 2, the pathogenicities of four strains of serotype 1 GIAAV for 1-wk-old SPF chickens were compared. Three strains isolated from gizzard erosion had similar pathogenicities but one strain from a normal chicken was not pathogenic for chickens. These results suggest that gizzard erosions by serotype 1 GIAAV infection may occur in broiler chickens in Japan.     

Pathogenicity of an avian influenza virus serotype H9N2 in chickens

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity ofAIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P = 0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2(8.2) and 2(9.3), respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.     

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology

Age-related susceptibility patterns of turkeys, broilers, and specific pathogen-free (SPF) White Leghorn chickens to experimentally induced infection with turkey or chicken rotavirus isolates were compared. The following determinants were evaluated: clinical signs, onset and duration of virus production, viral titers, involvement of intestinal villi in the replication of the virus, and the development of antibodies against the virus. Older turkeys and chickens were more susceptible than were their younger counterparts, turkeys were more susceptible than were broiler and White Leghorn chickens (regardless of age), and broiler chickens were slightly more susceptible than were age-matched White Leghorn chickens. Turkeys developed diarrhea, accompanied by high viral titers within 1 day after inoculation with virus. Viral antigen was found in the epithelial cells of the intestinal villi throughout the intestinal tract and some cells of the cecal tonsils. Antibodies could be detected as early as 4 to 5 days after inoculation. These findings were more pronounced in turkeys inoculated at 112 days of age than in birds inoculated at a younger age. Age-related susceptibility patterns were similar in White Leghorn and broiler chickens. Infection was subclinical in birds less than 56 days old, whereas older birds developed soft feces. Egg production in the White Leghorn chickens decreased after being inoculated with virus at 350 days of age.     

IMC10200株ALV-J实验诱发禽骨髓性白血病的研究

对分离自肉种鸡群亚临床感染的J亚群禽白血病病毒IMC1o200株进行了实验感染诱发禽骨髓性白血病的病理学研究.IMC10200株J亚群禽白血病病毒尿囊腔接种11日龄肉鸡胚和SPF蛋鸡胚,孵出后跟踪观察.9周龄时随机抽检感染鸡,感染肉鸡特异引物PCR检测全部为阳性;组织病理学观察感染鸡无明显病变.至21周龄时感染肉鸡出现第一例典型骨髓性白血病病例;感染SPF蛋鸡未见明显J亚群禽白血病相关病变.     

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.     

Safety and efficacy of an experimental reovirus vaccine for in ovo administration

A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2-5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response. In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In evo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus-antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.