Pathogenicity and molecular characterization of a fowl adenovirus 4 isolated from chicken associated with IBH and HPS in China

Background

Since July in 2015, an emerging infectious disease, Fowl adenovirus (FAdV) species C infection with Hepatitis-Hydropericardium syndrome was prevalent in chicken flocks in China. In our study, one FAdV strain was isolated from commercial broiler chickens and was designated as SDSX1.The phylogenetic information, genetic mutations and pathogenicity of SDSX1 were evaluated.

Results

The phylogenetic analysis indicated that SDSX1 is a strain of serotype 4, FAdV-C. The amino acid analysis of fiber-2 showed that there were more than 20 mutations compared with the non-virulent FAdV-C strains. The pathogenic evaluation of SDSX1 showed that the mortality of one-day-old chickens inoculated SDSX1 was 100%. The typical histopathological changes of SDSX1 were characterized by the presence of basophilic intranuclear inclusion bodies in hepatocytes. The virus copies in different tissues varied from10⁷ to 10¹¹ per 100?mg tissue and liver had the highest virus genome copies.

Conclusion

In conclusion, the isolate SDSX1, identified as FAdV-4, could cause one-day-old chicks’ typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were the highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1.

     

血清4型禽腺病毒Fiber蛋白在杆状病毒中的表达及其在抗体检测中的应用

为建立检测血清4型禽腺病毒(FAdV-4)抗体快捷特异方法,本研究首先分别构建含FAdV-4纤突蛋白Fiber-1和Fiber-2的两个重组杆状病毒rBv-Fiber-1和rBv-Fiber-2。在利用IFA以及Western blot鉴定重组杆状病毒分别高效表达Fiber-1和Fiber-2蛋白的基础上,以Ni柱纯化蛋白,并作为特异性识别FAdV-4抗体的包被抗原构建间接ELISA方法。特异性试验表明,间接ELISA仅特异地检出FAdV-4阳性血清,而不与Ⅰ群其他血清型禽腺病毒及其他禽源病毒阳性血清反应。间接ELISA检测灵敏度高于常规IFA方法,批间和批内重复性变异系数均小于10%。临床血清检测结果表明,间接ELISA可有效检出感染FAdV-4或免疫FAdV-4灭活疫苗的鸡群中抗Fiber抗体,且检测结果与BioChek商品化ELISA试剂盒一致。结果表明,本研究利用杆状病毒系统表达的Fiber蛋白及基于表达产物所构建的间接ELISA方法在FAdV-4感染预警和免疫评估有良好的应用前景。     

Identification of two novel fowl adenovirus C-specific B cell epitopes using monoclonal antibodies against the capsid hexon protein

The diseases associated with fowl adenovirus (FAdV) infection, such as inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HPS), and gizzard erosion (GE), were first reported in Pakistan in 1987, and subsequent outbreaks have been reported worldwide, especially in China, where severe outbreaks of HPS with high mortality from 30 to 100% were recently reported and resulted in significant economic losses to the poultry industry. The diagnosis methods of FAdVs were mostly limited to the nucleotide sequence of hexon by PCR and DNA sequencing. The aim of this study was to generate B cell epitope maps of the species- and serotype-specific hexon L1 using monoclonal antibodies (mAbs) and bioinformatics tools for the development of novel diagnostic methods. In this study, the hexon L1 (230 amino acids) was expressed and used to generate 10 mAb-expressing hybridoma cell lines against the relative protein peptide. Subsequently, we defined the linear peptide epitopes recognized by these mAbs using a series of partially overlapping peptides derived from the FAdV-C hexon protein amino acid sequence to map mAbs reactivity. Finally, a common B cell epitope (₃₁PLAPKESMFN₄₀) for all species FAdVs and two FAdV-C-specific epitopes (₇₉KISGVFPNP₈₇ and ₁₈₁DYDDYNIGTT₁₉₀) were identified. These mAbs and their defined epitopes may support the development of the universal or species-specific differential diagnostic methods of FAdVs.

     

Isolation of chicken anemia agent with MDCC-MSB1 cells from chickens in the field

An attempt was made to isolate chicken anemia agent (CAA) from chickens suffering from anemia in the field by using MDCC - MSB1 , which was an established cell line derived from Marek's disease lymphoma. When 99 chickens of 15 flocks were examined, CAA was isolated from 58 chickens of 12 flocks. The rate of CAA isolation with MDCC - MSB1 cells was almost the same as that determined by an in vivo method by chick inoculation. It was shown that CAA was more closely concerned with anemic diseases of chickens in the field than fowl adenoviruses.     

Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: nonexculsive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus.

A number of antigenic hybrids of influenza A viruses were produced possessing either the hemagglutinin or the neuraminidase of fowl plague virus and the corresponding antigen derived from another influenza A virus. Other recombinants were obtained carrying both surface antigens of fowl plague virus but differing from the parent in certain biological properties. None of the recombinants isolated were pathogenic for adult chickens. Most recombinants obtained after crosses between reciprocal recombinants carrying both fowl plague virus surface antigens were also apathogenic for chickens. Using the same parent recombinants for double infection some of the progeny "back-recombinants" were pathogenic, whereas others were not. From these results it is concluded that the surface components do not by themselves determine the pathogenicity of influenza A viruses.     

Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens

Hepatitis E virus (HEV) is an important human pathogen. Due to the lack of a cell culture system and a practical animal model for HEV, little is known about its pathogenesis and replication. The discovery of a strain of HEV in chickens, designated avian HEV, prompted us to evaluate chickens as a model for the study of HEV. Eighty-five 60-week-old specific-pathogen-free chickens were randomly divided into three groups. Group 1 chickens (n=28) were each inoculated with 5 x 10(4.5) 50% chicken infectious doses of avian HEV by the oronasal route, group 2 chickens (n=29) were each inoculated with the same dose by the intravenous (i.v.) route, and group 3 chickens (n=28) were not inoculated and were used as controls. Two chickens from each group were necropsied at 1, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, and 42 days postinoculation (dpi), and the remaining chickens were necropsied at 56 dpi. Serum, fecal, and various tissue samples, including liver and spleen samples, were collected at each necropsy for pathological and virological testing. By 21 dpi, all oronasally and i.v. inoculated chickens had seroconverted. Fecal virus shedding was detected variably from 1 to 20 dpi for the i.v. group and from 10 to 56 dpi for the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples from both i.v. and oronasally inoculated chickens. Gross liver lesions, characterized by subcapsular hemorrhages or enlargement of the right intermediate lobe, were observed in 7 of 28 oronasally and 7 of 29 i.v. inoculated chickens. Microscopic liver lesions were mainly lymphocytic periphlebitis and phlebitis. The lesion scores were higher for oronasal (P=0.0008) and i.v. (P=0.0029) group birds than for control birds. Slight elevations of the plasma liver enzyme lactate dehydrogenase were observed in infected chickens. The results indicated that chickens are a useful model for studying HEV replication and pathogenesis. This is the first report of HEV transmission via its natural route in a homologous animal model.     

Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India

In the present study characterisation has been done for six group I fowl adenoviruses (FAV) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years 1994-98. These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values 0.93-0.96). Under the experimental conditions, we have been able to induce IHP using FAV serotype 4 isolate AD: 411 and were also able detect FAV antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FAV serotype 4 strain replicate in myocardial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FAV serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FAV serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, any one of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Observations on the Taiwanese Strain of Leucocytozoon caulleryi (Haemosporina) in Chickens

ABSTRACT. The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25°C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected witt the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the strain isolated in Japan.     

Observations on the Taiwanese strain of Leucocytozoon caulleryi (Haemosporina) in chickens

The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25 degrees C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected with the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the the strain isolated in Japan.     

减蛋综合征病毒100K蛋白基因的克隆与序列分析

用常规方法提取减蛋综合征病毒（ＥＤＳＶ）中国分离株（ＡＡ２株）病毒ＤＮＡ,分别构建了限制性内切酶ＨｉｎｄⅢ、ＳｐｈⅠ、ＰｓｔⅠ水解片段的全基因文库,并对其中１００Ｋ蛋白基因的序列进行了分析。ＥＤＳＶ１００Ｋ蛋白基因位于减蛋综合征病毒基因组５５．７～６４．８物理图谱单位（ｍ．ｕ）,共２０９１个核苷酸（ｎｔ）,其编码产物由６９６个氨基酸（ａａ）组成,推测其分子量为７７．７ｋＤ。编码蛋白氨基酸同源性分析表明,ＥＤＳＶ１００Ｋ蛋白与人腺病毒（Ａｄ２、Ａｄ５、Ａｄ１２、Ａｄ４１）、Ⅰ群禽腺病毒（ＣＥＬＯ和ＦＡＶ１０）的同源性为３２．３～３４．４％之间,而与羊腺病毒（ＯＡＶ）的同源性达到５６．４％。     

Identification of novel avian influenza virus derived CD8+ T-cell epitopes

Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.     

敲除meq的鸡马立克氏病毒强毒株对超强毒的免疫保护作用

【目的】比较和评价一株敲除了meq基因的马立克氏病毒(MDV)的致病性及其诱发的保护性免疫作用。【方法】将1日龄SPF鸡150只随机分为5组,每组30只,分别饲养于正压过滤空气的SPF动物饲养隔离罩内。1日龄时,第1和第5组鸡以2000PFU/只的剂量腹腔接种GX0101Δmeq,第2组鸡以2000PFU/只的剂量腹腔接种CVI988/Rispens疫苗株,第3和第4组鸡不接种任何病毒作为对照组。免疫接种5d后,第1、2、3组分别以500PFU/只的剂量攻击MDV超强毒株vvrMd5。饲养90d,观察死亡情况,对各组死亡鸡只剖检,并取疑似马立克特有病变脏器做石蜡切片,于攻毒后90d处死全部存活鸡并随机取心脏、肝脏、脾脏做病理切片。【结果】单独接种GX0101Δmeq的第5组没有任何马立克氏病临床症状和特有的组织学病变,接种GX0101Δmeq再感染超强毒株vvrMd5的第1组也没有马立克病特有的组织学病变,但CVI988/Rispens免疫后感染超强毒株vvrMd5的第2组显示马立克病特有病变的病理切片比例为9/42,单独接种超强毒株vvrMd5的第3组死亡率为87%,死亡鸡出现可眼观典型肿瘤率为25%,免疫接种GX0101Δmeq和CVI988/Rispens的第1组和第2组对超强毒株vvrMd5攻击的保护指数分别为100%和89%。【结论】本实验构建的MDVmeq基因缺失株-GX0101Δmeq可在体外稳定复制,不仅对SPF鸡没有致病性和致瘤性,而且能诱导比CVI988/Rispens疫苗株更好的对超强毒MDV的免疫保护效果。     

滑液囊支原体不同感染途径的致病力比较

【背景】自2010年以来,我国鸡群中滑液囊支原体(Mycoplasma synoviae,MS)的感染率不断增高,目前MS已广泛存在于我国不同的鸡群中,包括蛋鸡、种鸡、白羽肉鸡和地方品种鸡等,其血清阳性率已超过40%,严重危害我国养鸡业,并造成了严重的经济损失。【目的】系统比较MS以不同感染途径对56日龄无特定病原体(Specific Pathogen Free,SPF)鸡的致病力。【方法】采用MS强毒FZ株以点眼、爪垫注射、胸部皮下注射、单次气管注射、连续3次气管注射等不同途径感染56日龄SPF鸡,观察感染后临床症状和解剖病理变化,检测感染后血清中的MS抗体,并且对气管组织进行病原再分离和组织病理学观察。【结果】MS的FZ株以不同途径感染SPF鸡后临床表现和发病率差异较大,爪垫注射和胸部皮下注射可导致100%的鸡发生爪垫肿大或胸部囊肿,而单次或连续3次气管注射可引起33%-50%的鸡发生严重气囊炎,点眼感染途径基本不能引起临床病理变化;爪垫肿大主要为肉芽组织增生和出现大量的黄色干酪样块状物质,胸部囊肿在囊肿部位有大量的血红色样液体和黄色干酪样块状物质;组织病理学结果显示连续3次气管注射方式感染更易对气管造成损伤,表现为气管黏膜固有层/黏膜下层发生轻微至轻度灶性炎细胞浸润,而爪垫肿大和胸部囊肿组织有大量的纤维组织和血管增生,同时伴有大量炎性细胞浸润;点眼和气管注射途径的气管MS病原再分离率可达100%,而胸部皮下注射或脚垫注射也可从气管中分离到MS病原;爪垫注射途径更易引起MS抗体转阳。【结论】系统比较了MS以不同感染途径对56日龄SPF鸡的致病性,并筛选出了相应的评价指征,成功建立了MS人工感染8周龄SPF鸡的发病模型,其中点眼感染途径和气管注射途径以气管病原再分离作为主要指征,辅以气囊炎进行评价,而胸部皮下注射和爪垫注射途径分别以胸部囊肿和爪垫肿胀作为主要病理指征进行评价。     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

3株禽滑液囊支原体分离株致病性的比较和评价

【背景】禽滑液囊支原体是感染鸡的一种重要病原体,给我国的家禽养殖业带来了严重的经济损失。【目的】评价从病鸡中分离到的3株禽滑液囊支原体的致病性,以期丰富不同区域来源的分离株对鸡致病性的认识。【方法】分别用分离株CHN-WF224-2016、CHN-BZJ2-2015、CHN-JNB19-2016感染商品肉鸡,并在攻毒后第10天和21天进行实验室解剖,通过气囊和足垫的临床病变、血清学检测结果及气管粘膜病理形态学改变来比较评价分离株的致病力。【结果】CHN-BZJ2-2015和CHN-WF224-2016分离株能引起明显足垫肿胀和关节滑膜炎,其中CHN-BZJ2-2015分离株致病力最强,感染21 d后的血清学检测和气管粘膜厚度与CHN-JNB19-2016分离株和MS-H疫苗株比较差异显著(P<0.05)。【结论】本研究表明我国目前流行的禽滑液囊支原体菌株其致病力存在明显差异,强调养殖场要积极控制和预防禽滑液囊支原体感染的重要性,同时为今后该疫苗的研发积累了实验数据。     

Rejection of Eimeria by foreign hosts.

Oocysts of Eimeria maxima inoculated into guinea fowl do not develop. Infection occurs when intestinal mucosa or liver tissue from guinea fowl given E. maxima is transferred to susceptible chickens. By transferring material at different times after inoculation, it was shown that most of the stages are lost between 6 and 12 h and few, if any, survive to 48 h. Sporozoites of E. tenella had low infectivity after 48 h contact in vitro with peritoneal macrophages from guinea fowls and turkeys. Sporozoites of E. grenieri from the guinea fowl appeared to be destroyed within macrophages taken from chickens.