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.     

Comparative evaluation of cell culture-adapted and chicken embryo-adapted fowl pox vaccine strains

Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age. The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine. The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age. However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC. Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively. The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.     

鸡源血清4型禽腺病毒的分离鉴定

山东、河北等华北地区12个肉鸡养殖场出现了以心包积液、出血性肝炎为特征的疾病,从采集的病料中进行病毒分离鉴定。本研究对分离的毒株进行PCR扩增、血凝试验、ELD_(50)测定、Hexon基因序列测定与分析以及人工感染试验进行组织病理学观察、抗体水平检测等一系列试验。结果显示,分离的毒株不能凝集鸡和鸭的红细胞,但能够凝集大鼠的红细胞,ELD_(50)为10-3.17/0.2mL。组织学变化显示肝细胞纤维化和脂肪变性,肾小管上皮细胞肿胀、变性,心肌纤维水肿、颗粒变性。对临床采集的血清检测结果显示发病鸡群抗体水平为阳性,发病鸡群中的健康鸡阳性率为40%(40/100),表明鸡群中存在隐性感染。将分离株的Hexon基因组与已发表的血清4型禽腺病毒基因进行同源性比较,发现该分离株与印度株在同一个分支上,同源性在99.6%以上,而与韩国、欧洲、美洲株同源性较远。用分离的毒株感染15日龄的雏鸡能引起心包积液、肝脏出血、肾脏肿大等病变。上述结果表明该分离株为血清4型禽腺病毒。     

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 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.     

Pathogenicity studies with a strain of fowl adenovirus serotype 8 (VRI-33) in chickens

Experiments were undertaken to study the pathogenesis of VRI-33, a strain of fowl adenovirus serotype 8 isolated from the liver of a broiler chicken with inclusion body hepatitis. A 30% death rate resulted from oral infection of one-day-old specific pathogen free chickens with 10⁶ plaque forming units of VRI-33. Chickens 10, 14, 21 and 28 days of age did not die following infection via natural routes but there were some motalities following infection via parenteral routes. Immunodepression by neonatal cyclophosphamide treatment, followed by infection with VRI-33 via non-parenteral routes, caused varying degrees of hepatitis with basophilic intranuclear inclusion bodies in hepatocytes. The mortality rate of cyclophosphamide-treated, VRI-33 infected chickens, was not significantly altered by post-infection temperature stress. Infection with infectious bursal disease virus, followed by infection with VRI-33 via natural routes at 14 days of age, was not associated with mortalities.     

Inclusion body hepatitis caused by fowl adenovirus in broiler chickens in Japan, 2009-2010

From January 2009 to June 2010, many broiler chicks suddenly died without clinical signs. The mortality rates were from 1.2% to 17.0% in affected flocks. Inclusion body hepatitis (IBH) was detected in 13 prefectures (northern, eastern, western, and southern areas) in Japan. The livers were enlarged and pale. The bursa of Fabricius and thymus had not atrophied. Multifocal necroses of hepatocytes with basophilic intranuclear inclusions were seen in the liver. Eosinophilic intranuclear inclusion bodies in hepatocytes were rare. Focal necrosis of acinar cells with basophilic intranuclear inclusions was found in the pancreas. Basophilic intranuclear inclusion bodies were detected in intact surface epithelial cells of gizzard and epithelial cells of the small intestine. The intranuclear inclusions of liver, pancreas, gizzard, and small intestine were stained positively for immunohistochemistry of fowl adenovirus (FAV) antigen. Ultrastructurally, basophilic intranuclear inclusions consisted of viral particles approximately 70 nm in diameter and arranged in a crystalline array. FAV was isolated from the liver of chickens affected with IBH. The serotype of most isolates was 2. This study suggests that IBH produced by FAV is epidemic in broiler chicks in Japan and that the present cases occurred as the primary disease without the association of infectious bursal disease virus or chicken anemia virus.     

Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis from broiler chickens in Egypt

Tropical Animal Health and Production - A case-control study was performed to assess prescence of inclusion body hepatitis (IBH) caused by fowl adenoviruses (FAdVs) at Kafr EL-Shiekh Governorate,...     

Characterization of fowl adenovirus serotype-4 associated with hydropericardium syndrome in chicken

The polypeptides of three fowl adenovirus-4 (FAV-4) field isolates of hydropericardium syndrome from various geographical areas of the country and the standard FAV-1 (CELO virus) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by protein immunoblotting with polyclonal antibodies to FAV-4 and FAV-1. Protein profile analysis of FAV-4 isolates revealed similarity of all the eight polypeptides with molecular weight ranging from 20 to 107 kDa but differed from CELO, particularly in their 24.2 kDa protein. Subsequent immunoblotting showed relatedness of at least five protein fractions of FAV-4 to CELO virus.     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

禽腺病毒Ⅰ群C种4型病毒的荧光PCR诊断方法的建立

心包积水-肝炎综合征(HHS),是由腺病毒科禽腺病毒属Ⅰ群C种4血清型病毒引起的一种鸡新型传染病,对养鸡业危害较大。当前该病的诊断主要依靠普通PCR,敏感性和高通量受到限制。本研究建立了一种Taqman荧光PCR方法,该方法特异性强,敏感性高较普通PCR提高1 000倍,经临床样品验证,本方法适用于临床发病病例的诊断和临床正常鸡只的流行病学调查,并适用于高通量检测。为该病的防控提供了一种有效的手段。     

禽腺病毒血清4型RPA-LFD快速检测方法的建立

为了建立一种快速、灵敏、简便的禽腺病毒血清4型(FAdV-4)现场检测方法,本试验将重组酶聚合酶扩增(RPA)与胶体金侧向流试纸条(LFD)技术联合,设计针对FAdV-4hexon基因的特异性RPA引物,上、下游引物的5′端分别进行6-羧基荧光素(FAM)和生物素(Biotin)标记。通过对引物浓度、RPA反应时间和温度等条件的优化,建立了一种可用于FAdV-4现场检测的RPA-LFD方法。该检测方法在25～45℃恒温反应15 min即可实现对FAdV-4目的基因片段的有效扩增,RPA扩增产物用胶体金侧向流试纸条进行检测,检测结果肉眼可见。该方法的最低检测限为100拷贝,敏感性是常规PCR的100倍,且与其他常见禽病病原核酸检测无交叉反应。结果显示,本试验建立的RPA-LFD可用于FAdV-4的临床快速检测,为FAdV-4感染的流行病学检测提供了一种有效方法。     

Molecular characterization of Malaysian fowl adenovirus (FAdV) serotype 8b species E and pathogenicity of the virus in specific-pathogen-free chicken

BackgroundInclusion body hepatitis (IBH) is an economically important viral disease primarily affecting broiler and breeder chickens. All 12 serotypes of fowl adenovirus (FAdV) can cause IBH.ObjectivesTo characterize FAdV isolates based on phylogenetic analysis, and to study the pathogenicity of FAdV-8b in specific-pathogen-free (SPF) chickens following virus inoculation via oral and intramuscular (IM) routes.MethodsSuspected organ samples were subjected to virus isolation and polymerase chain reaction (PCR) for FAdV detection. Hexon gene sequencing and phylogenetic analysis were performed on FAdV-positive samples for serotype identification. One FAdV-8b isolate, UPM/FAdV/420/2017, was selected for fiber gene characterization and pathogenicity study and was inoculated in SPF chickens via oral and IM routes.ResultsThe hexon gene phylogenetic analysis revealed that all isolates belonged to FAdV-8b. The fiber gene-based phylogenetic analysis of isolate UPM/FAdV/420/2017 supported the grouping of that isolate into FAdV species E. Pathogenicity study revealed that, chickens infected with UPM/FAdV/420/2017 via the IM route had higher clinical score values, higher percent mortality, higher degree of the liver lesions, higher antibody response (p < 0.05), and higher virus shedding amounts (p < 0.05) than those infected via the oral route. The highest virus copy numbers were detected in liver and gizzard.ConclusionsFAdV-8b is the dominant FAdV serotype in Malaysia, and pathogenicity study of the FAdV-8b isolate UPM/FAdV/420/2017 indicated its ability to induce IBH in young SPF chickens when infected via oral or IM routes.     

Isolation of chicken anaemia virus from broiler chickens.

Chicken anaemia virus (CAV) infection was demonstrated, by both serology and virus isolation, in 1- to 6-week-old broiler chickens originated from various parent flocks in Hungary. Total losses in the broiler flocks were estimated at 7 to 8% and about 25% of the chickens failed to reach target body mass by the 7th week of life. The clinical signs, postmortem lesions and histopathological changes of the affected chickens were similar to those of naturally occurring CAV-induced infectious anaemia of young chickens. In MDCC-MSB1 cell cultures, a chloroform-resistant virus smaller than 50 nm in diameter, resistant to heating at 70 degrees C for 30 min, and antigenically very closely related to the Cux-1 strain of CAV was isolated from the liver of naturally diseased broilers. This virus isolate was designated the Bia strain of CAV. Inoculation of susceptible 1-day-old SPF chicks with a CAV-positive liver extract from naturally diseased broilers caused pathological changes characteristic of CAV infection, namely impaired growth, severe anaemia with atrophy of the bone marrow, marked atrophy of the lymphoid organs and petechial haemorrhages throughout the body. A quite similar pathological syndrome was also induced by inoculation of 1-day-old SPF chicks with the MDCC-MSB1 cell-culture-propagated new Bia strain of CAV. The CAV was successfully reisolated from the livers of experimentally inoculated birds, and antibodies to the reference Cux-1 strain of CAV were also demonstrated by the indirect immunofluorescence test in sera of naturally diseased and experimentally inoculated chickens. No antibodies were found against infectious bursal disease virus, reticuloendotheliosis virus, Marek's disease herpesvirus as well as avian adenoviruses and reoviruses. The reported disease of young broiler chickens was associated with natural infection of a new isolate of CAV. On the basis of its physicochemical, antigenic and pathogenic characteristics, this virus is similar to other strains of CAV isolated from chickens in other countries.     

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.     

Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus

The hypothesis that an effective protection of progeny chickens against inclusion body hepatitis/hydropericardium syndrome (IBH/HP) can be achieved by dual vaccination of breeders with fowl adenovirus (FAV) serotype 4 and chicken anemia virus (CAV) was tested. Thus, 17-wk-old brown leghorn pullet groups were vaccinated by different schemes including single FAV (inactivated), single CAV (attenuated), FAV and CAV dually, or were not vaccinated (controls). Subsequent progenies of these breeders were challenged with the virulent strains FAV-341 and CAV-10343 following three strategies: 1) FAV-341 intramuscularly (i.m.) at day 10 of age (only FAV-vaccinated and control progenies); 2) FAV + CAV i.m. simultaneously at day 10 of age (all progenies); 3) CAV i.m. at day 1 and FAV orally at day 10 of age (all progenies). The induction of IBH/HP in these progenies was evaluated throughout a 10-day period. Both breeder groups vaccinated against FAV and those vaccinated against CAV increased virus neutralizing specific antibodies. Challenge strategy 1 showed 26.6% mortality in control progeny chickens and 13.3% in the progeny of FAV-vaccinated breeders. Presence of lesions in the liver of these groups showed no significant differences (P > 0.05), suggesting a discreet protective effect of the vaccine. Challenge strategy 2 showed 29.4% mortality in controls and 94% of chickens showed hepatic inclusion bodies (HIB). Single CAV vaccination of breeders did not demonstrate a beneficial effect, with both mortality and liver lesions resembling the nonvaccinated controls. FAV vaccination of breeders significantly reduced both mortality (7.4%) and liver lesions (26% HIB) (P < 0.05), providing protection against this challenge strategy. Dual vaccination of breeders with FAV and CAV proved to be necessary to achieve maximum protection of the progeny (no mortality and 7% HIB). Challenge strategy 3 produced no mortality but consistent liver damage in controls (96% HIB). In this case, both CAV and FAV + CAV-vaccinated breeders showed best protection results in terms of liver histopathology (8% and 0% HIB, respectively). FAV vaccination alone produced 24% HIB, similar to challenge strategy 2, demonstrating a lower protective effect.     

Isolation of chicken anaemia virus from broiler chickens in New Zealand

Chicken anaemia virus was isolated for the first time in New Zealand from the New Zealand domestic chicken population. The virus was recovered from diseased birds in five separate flocks of broiler chickens aged between 14 and 33 days of age. Six isolates were obtained from bone marrow and lymphoid tissues using the MDCC-MSB1 cell line derived from Marek's disease lymphoma. All isolates were resistant to chloroform and survived exposure to 70 degrees C for 5 minutes. The main clinical features consistently associated with the disease outbreaks were increased mortality, yolk sac infections, sub-cutaneous haemorrhages and atrophy of the thymus. Fungal pneumonia occurred in two flocks, and gangrenous dermatitis as a result of bacterial infection in another flock. Microscopic examination showed atrophy of the thymus, reduced medullary haematopoiesis and inflammation resulting from secondary infections.     

表达FAdV-8b Fiber蛋白重组FAdV-4的构建与鉴定

为了构建能够同时防控鸡肝炎-心包积液综合征(HHS)和包涵体肝炎(IBH)的新型疫苗,本研究利用禽腺病毒4型(FAdV-4)中国流行株CH/HNJZ/2015的反向遗传操作技术,将禽腺病毒8b型(FAdV-8b)中国流行株的全长Fiber基因插入至FAdV-4基因组中1 966 bp自然缺失区,构建感染性克隆并拯救出重组病毒rHNJZ-Fiber/FAdV8b,利用PCR、Western blot和间接免疫荧光试验对重组病毒rHNJZ-Fiber/FAdV8b进行鉴定。结果表明,FAdV-8b的Fiber基因得以准确插入,Fiber蛋白正确并稳定表达,重组病毒rHNJZ-Fiber/FAdV8b具有良好的遗传稳定性,在细胞中具有与亲本毒株相似的高滴度生长特性和复制动态。本研究构建的表达FAdV-8b中国流行株Fiber基因的重组FAdV-4将为同时研发HHS和IBH的有效疫苗提供新的思路。