Molecular study and genotyping of Cryptosporidium baileyi and Cryptosporidium parvum from free-range and commercial broiler chickens in Guilan province,Iran

Cryptosporidiosis acutely impacts the digestive and/or respiratory tract of the birds in many species of various orders. More importantly, it is also well known as a significant zoonotic disease, which can lead to diarrhea in humans and livestock. Regarding increasing demand for free-range products and increasing the number of free-range poultry farms, the present paper evaluated histopathological and molecular detection of Cryptosporidium baileyi and Cryptosporidium parvum in free-range and commercial broiler chickens in the north part of Iran. For this purpose, 100 fecal and tissue samples of the chickens in Guilan province were collected. After microscopic examination using Ziehl-Neelsen staining, molecular analyses of the fecal samples were processed by Nested-PCR targeting the 18S rRNA gene followed by sequencing of the amplicons and phylogenetic analyses. Eventually, the tissue samples were studied for histological lesions. Findings demonstrated the presence of Cryptosporidium baileyi and Cryptosporidium parvum in 6 % and 2 % of fecal samples, respectively. This is the first identification of C.parvum in avian hosts in Iran, and for the first time, C.baileyi and C.parvum are shown in native free-range chickens in Iran. All of the PCR positive birds with clinical symptoms showed gross lesions of respiratory infections. There was no significant difference between infection rate in free-range and commercial broiler chickens; however, the infection rate was significantly higher in chickens <25 days old. To conclude, we present here a notable Cryptosporidium infection rate in the free-range chicks in Iran, which notify the role of this host as a reservoir and should be more noted due to the economic and zoonotic importance.     

Suspected Reticuloendotheliosis in Turkeys with Cutaneous Lesions Reminiscent of Fowl Pox

Twelve turkeys from a flock of 2,500 had cutaneous lesions somewhat resembling fowl pox. Lymphoid-like tumors were evident in livers and spleens. Closer examination of cutaneous lesions, and histopathological examinations indicated skin lesions were also infiltrative and not unlike reticuloendotheliosis. Electron micrographs revealed the presence of C-type virus particles.     

奶牛伪牛痘病的研究1.奶牛伪牛痘病的诊断

在北京市某奶牛场，对１９８６年以来疑似存在的伪牛痘病毒感染情况进行了调查。在临床调查的基础上，应用光镜和电镜技术对患病乳头的丘疹进行了病理组织学和病原学检查。丘疹的病理组织学变化符合痘病毒感染的特征。试验证明，该牛场系为伪牛痘病毒感染     

Investigation of Marek’s disease virus from chickens in central Ethiopia

Marek’s disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek’s disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366–76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

     

小尾寒羊绵羊痘病例的诊断

旨在对甘肃某羊场小尾寒羊疑似绵羊痘进行确诊,并深入探讨其病理变化特点。对临诊疑似患绵羊痘的小尾寒羊进行了羊痘间接ELISA抗体检测和PCR检测确诊,并对6只病羊进行病理剖检、病理组织学和超微结构研究。结果如下：羊痘间接ELISA抗体检测显示6例病羊样品均为阳性;同时,对2例病羊样品进行PCR检测,均扩增出绵羊痘病毒（SPV）585 bp的目的基因片段,表明本次小尾寒羊所患疫病为绵羊痘。病理剖检显示,病羊皮肤无毛或少毛处充血,局部有红斑和坏死,尾部常有感染化脓形成溃疡;消化道黏膜,尤其舌和瘤胃黏膜有大小不等痘疹与坏死病灶,局部甚至形成糜烂或溃疡。肺膈叶以及肝、脾等常有痘疹和坏死病灶。病理组织学观察显示,肺痘疹病灶主要呈现渗出-增生结节和坏死-增生结节变化,病灶内血管、细支气管等周围常有大量绵羊痘细胞,胞质内有嗜酸性包涵体;病变消化道黏膜局部,上皮细胞程度不等增生、水泡变性,固有层及黏膜下层充血、水肿,有大量绵羊痘细胞,其细胞质内包涵体明显。肝、脾、肾也有程度不等坏死-增生结节变化。肺超微结构观察显示,肺泡上皮细胞肿大,细胞核浓缩、染色质聚集,线粒体程度不等肿胀、嵴断裂、空泡化,可见细胞质内包涵体。研究证明,除局部皮肤病变外,消化道黏膜和肺等实质器官的痘疹病变是患绵羊痘小尾寒羊具代表性的病理变化,尤其痘疹病灶中大量绵羊痘细胞的出现具有证病性意义。     

广西玉林市规模化禽场禽流感病毒检测

为了解广西玉林市2020年规模禽场禽流感病毒感染状况,采用荧光RT-PCR方法,对广西玉林市7个县(市、区)42个规模化禽场采集的1260份禽喉/泄殖腔棉拭子样品进行了通用型禽流感病毒核酸检测(荧光PCR),并对检测为阳性的样本进行H5、H7亚型(双重荧光PCR)和H9亚型(荧光PCR)分型鉴定。结果显示:在42个规模化禽场中,未检出H5和H7亚型高致病性禽流感病毒阳性样品;在2个鸡场中检出18份H9亚型低致病性禽流感病毒阳性样品,在2个鸡场和4个鸭场中检出115份其他亚型低致病性禽流感病毒阳性样品。结果表明:在高致病性禽流感(H5+H7)三价灭活疫苗强制免疫政策下,广西玉林市规模化禽场的高致病性禽流感病毒感染风险较小,但仍须加强禽流感的免疫、监测,做好综合防控,以降低禽流感病毒由低致病性重组变异为高致病性的风险。本检测为指导广西玉林市禽流感防控提供了依据。     

Health survey of backyard poultry and other avian species located within one mile of commercial California meat-turkey flocks.

A survey was conducted to characterize domestic and exotic bird populations, estimate seroprevalence to selected disease agents, and describe health management practices on 62 premises containing "backyard" flocks located within one mile of 22 commercial California meat-turkey flocks participating in National Animal Health Monitoring System (NAHMS). Chickens were present on 56 backyard premises and turkeys on seven. Antibodies were identified against Mycoplasma gallisepticum, M. synoviae, M. meleagridis, Salmonella pullorum, Newcastle disease virus, avian encephalomyelitis virus, Bordetella avium, hemorrhagic enteritis virus, infectious bronchitis virus, and infectious bursal disease virus in 367 blood samples from 32 backyard premises. Twenty-two owners of backyard premises said they restricted visitor contact with their birds, and two required visitors to wear rubber boots and use boot disinfectant. Owners of seven premises used biologics and/or pharmaceutics for disease prevention. One family member worked on a commercial turkey ranch, but no other contact between owners, relatives, or employees and commercial poultry was reported.     

Full sequence analysis of hemagglutinin and neuraminidase genes and proteins of highly pathogenic avian influenza H5N1 virus detected in Iran, 2015

Over the last two decades, the highly pathogenic avian influenza H5N1 virus has gained a lot of attention due to its zoonotic and mutative nature. Iran is among the countries significantly affected by the virus as it hosts migratory birds during seasonal migration. In this study, the molecular characterizations of hemagglutinin (HA) and neuraminidase (NA) genes and proteins of H5N1 strain A/chicken/Iran/8/2015 detected in backyard poultry, Mazandaran province, were investigated. Phylogenetic analysis classified this virus as a member of subclade 2.3.2.1c, with the cleavage site motif of “PQRERRRK-R/GLF”. HA carried a few mutations altering affinity to mammalian cells; however, the virus was categorized as avian. NA protein had the 20-amino acid deletion at aa position 49–69 similar to those isolated since 2000. Mutations of H253Y and H274Y contributing to antiviral resistance were present in NA. From this analysis, it can be concluded that the wild migratory birds flying from Western Asia to Eastern Africa are probably the main carriers of seasonal H5N1 in the country.

     

Clinical and Histopathological Survey of Lesions Similar to Pox Skin Lesions in Three Flocks of a Large Commercial Layer Farm

In this study, we report an outbreak of fowl pox disease in a large commercial layer farm in Iran. Three flocks of this farm were affected. Flock A (23 wk of age), flock B (44 wk of age), and flock C (71 wk of age) showed cutaneous lesions. Beak necrosis was seen at the posterior part of the upper or lower beak. Also, some vesicles were seen in combs, wattles, and eyelids. Beak and comb lesions were scraped and stained with Wright's staining for histopathological examination. Samples showed hyperplasia of epidermis but no Bollinger bodies were seen. Histopathologically, the epidermis had marked hyperplasia (acanthosis) caused by the swelling and increased number of cells in the stratum spinosum. Clinical observations, as well as histopathological findings, led to a diagnosis of fowl pox disease.     

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.     

Maedi in slaughtered sheep: A pathology and polymerase chain reaction study in southwestern Iran

Maedi-visna (MV) is an important slow viral disease of sheep leading to a progressive lymphoproliferative disease. It affects multiple organs primarily the lungs, where it causes interstitial pneumonia (maedi). In this study, the lungs of 1,000 sheep carcasses were grossly inspected and those suspected to have maedi were studied at histopathological and molecular levels. A polymerase chain reaction (PCR) technique that amplified a 291-base pair DNA in the long terminal repeat (LTR) sequence of MV provirus was conducted on all the 50 suspected lungs together with 10 normal appearing lungs as controls. Amplicons of the expected size were detected in 11 (n = 11/50) suspected sheep, and one of the 10 control sheep. Histopathologic study of the pulmonary lesions of all 11 (n = 11/11) positive sheep showed MV lesions, including hyperplasia of the perivascular and peribronchiolar lymphoid cells, interstitial lymphoplasmacytic infiltration and smooth muscle hyperplasia and the histopathologic findings were correlated with PCR results. In contrast, the tissue sections of control animals were almost normal at histopathological level; however, PCR technique demonstrated that one of them was affected by maedi. This study showed that the LTR-PCR had high specificity and sensitivity in diagnosis of this viral infection. This study is the first to evaluate the prevalence of MV virus infection in sheep in Iran.     

Rapid detection of group I avian adenoviruses by a loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was optimized for the rapid detection of Group I avian adenoviruses. A set of six primers was designed from the DNA sequences of hexon genes from Group I avian adenovirus. The assay was performed in a water bath for 60 min at 63 C, and the amplification result was visualized by adding a fluorescence dye reagent or by inspecting the white sediment. The results showed that the LAMP assay could detect all 12 serotypes of Group I avian adenovirus and nine Guangxi Group I avian adenovirus isolates. This avian adenovirus Group I-specific LAMP assay could detect 238 copies of avian adenovirus. No cross-reactions were detected using the LAMP assay with avian adenoviruses type II and III or with other avian viruses. The ability of LAMP to detect Group I avian adenovirus isolates was further evaluated with 184 cloacal swab samples from poultry. In total, 72 out of 184 cloacal swab samples from poultry were identified as positive by LAMP, whereas 45 out of 184 were identified as positive by conventional PCR test. The Group I avian adenovirus specific LAMP results were further confirmed by real-time PCR. This specific LAMP method holds promise as a rapid and specific diagnostic assay for detection of samples from birds suspected of adenovirus infection.     

Expornitic of avian pox in a zoo.

During a 6-week period at the San Diego Zoo, avian pox occurred in 9 pheasants representing 5 species. Lesions were limited to facial skin and consisted of epithelial cell hyperplasia, secondary inflammatory changes, and intracytoplasmic eosinophilic inclusion bodies which, by electron microscopy, were shown to contain pox virus. The disease was self-limiting in 7 pheasants, but 2 pheasants died. Free-ranging Indian red junglefowl were implicated as the source of the infection.     

Cowpox in cats

Orthodox virus infection of domestic cats in the United Kingdom was first reported in 1978. Outbreaks of a disease considered to be due to cowpox (orthopox) infection had been reported in a number of species of zoo animals in Europe between 1971 and 1977 and a serious outbreak occurred among cheetahs in a United Kingdom zoo. In this species the clinical signs were ülcerating and crusted skin lesions, in some cases complicated by respiratory signs and death. An increasing number of cases of pox infection of domestic cats has been reported in Great Britain. Affected animals have shown ulcerating and crusted skin lesions particularly of the head and limbs; systemic illness was uncommon. Immunologically compromised cats, ie, FeLV positive or those receiving corticosteroid of megestrol acetate therapy, were found to be more likely to show extensive or generalised pox lesions. The source of infection is thought to be small wild mammals, ie, voles, acting as a reservoir of virus, which are preyed upon by domestic cats. Feline orthopox virus infection is a zoonosis. Transmission of the disease from affected cats to incontact owners has been reported from Great Britain and the continent of Europe. The differential diagnosis of the condition in cats includes those skin disorders commonly treated with steroidal anti-inflammatory preparations. Definite diagnosis is important as these drugs are contraindicated in viral infections. Confirmation of diagnosis may be by virus isolation and serology. Histological examination of skin biopsy specimens is also helpful. Whether or not the causal agent of this disease should be referred to as cowpox virus or feline orthopox virus continues to be a matter of debate.     

Multiple gene typing and phylogeny of avipoxvirus associated with cutaneous lesions in a stone curlew

Avipoxvirus (APV) infections have been observed in a wide variety of wild, captive and domestic avian hosts, recently including a range of island endemic and endangered species. However, not enough is known about genome diversity and phylogenetic relationships of APVs, as well as their host-range specificity. A wild stone curlew (Burhinus oedicnemus) was recovered in Sardinia (Italy), showing large wart-like lesions and nodules on both legs and toes, which resulted positive to poxvirus by PCR. Histopathological examination of the lesions showed ballooning degeneration and large intracytoplasmic inclusion bodies consistent with APV infection. A multiple gene sequencing approach was applied to highlight the phylogenetic relationships of this virus with a panel of selected APVs at the clade and subclade levels. This novel isolate was characterized by sequencing partial 4b core protein, P35 (locus fpv140) and DNA polymerase genes and phylogenetic analyses assigned it to clade A, (Fowlpox virus, FWPV), subclade A2. Conservation implications of avian pox presence in Sardinian stone curlews and possibly in other island bird species are discussed.     

山羊痘病毒NM株的分离鉴定

从内蒙古牛场疑似山羊痘病毒感染的病羊中分离到1株病毒,命名为NM株。取病羊的皮肤痘疹和水疱作病料样品,分别接种BHK-21传代细胞和10日龄SPF鸡胚。结果表明,BHK-21细胞出现了明显的、规律的细胞病变,鸡胚接种部位出现了明显的痘斑;NM株细胞毒和胚毒均能够被山羊痘病毒阳性血清所抑制。利用1对山羊痘病毒特异性检测引物对NM株病毒进行PCR扩增,获得445 bp片段,与预期大小一致。将扩增产物提纯后克隆至pMD18-T载体,经酶切鉴定正确后,对阳性克隆株进行序列测定,结果表明,NM株的基因片段与已发表的山羊痘病毒毒株的相应基因片段具有高度同源性,同源性在99.2%~100%之间。     

奶山羊羔羊山羊痘的病理学、血清学及分子生物学诊断

目的 确定内蒙古某规模化奶山羊养殖场部分引进羔羊发病死亡的原因,为该场加强重点疫病免疫防控提供科学依据。方法 对出现典型临床症状的发病羊只进行病理解剖学观察,无菌采集肺脏病变样本,制备病理组织切片;无菌采集发病羊只的血液制备血清,采用高敏荧光技术检测血清中的山羊痘病毒抗体;无菌采集发病羊只的肺组织、鼻拭子、眼拭子,利用PCR技术检测山羊痘病毒P32基因,对测序获得的序列进行BLAST比对并进行同源性分析。结果 剖检可见病羊口腔、鼻腔、喉头、气管黏膜有圆形疹痘;肺部明显水肿,表面存在红色疹痘,切开后呈不透明、灰白色胶冻样;瘤胃、网胃、瓣胃、皱胃黏膜均出现疹痘。病理组织学检查可见肺泡红色深染,间质变宽,腔内存在脱落的上皮细胞,Ⅱ型肺泡上皮细胞增生,并化生为腺泡样状;支气管腔内存在大量的炎性细胞。发病山羊血清经高敏荧光技术检测,判定为山羊痘病毒抗体阳性。采集肺组织（GTPV-YP1）、鼻拭子（GTPV-YP2）、眼拭子（GTPV-YP3）样品各1份,3份样品经P32基因PCR检测均获得983 bp的扩增片段,与预期大小一致;GTPV-YP1、GTPV-YP2样品P32基因序列与山羊痘病毒中国分离株KC951854.1、Oman分离株 MN072621.1 P32基因的同源性为100%,GTPV-YP3与山羊痘病毒中国分离株EF514892.1、HM572329.1、JN596275.1、MG817382.1的同源性为99.0%。结论 经病理解剖学观察、病理组织学检查、血清学检测以及病原PCR鉴定,确定引起该场引进奶山羊羔羊发病的原因为山羊痘病毒感染。     

重庆市禽白血病及禽网状内皮组织增生症的血清流行病学调查

禽白血病和禽网状内皮组织增生症均为禽的肿瘤性免疫抑制疾病,是危害养鸡业的两种非常重要的病毒性传染病。为调查重庆市肉鸡中禽白血病及禽网状内皮组织增生症的流行情况,在北碚区、涪陵区、开县、垫江县、潼南县五个区（县）的8个活禽交易市场采集260份血清样本,采用酶联免疫吸附试验检测了所有血清中禽白血病病毒（ALV）和禽网状内皮组织增生病毒（REV）的抗体。检测结果显示：禽白血病病毒和禽网状内皮组织增生病毒抗体阳性率分别为16.5%（43/260）和5%（13/260）,双抗体阳性率为4.61%（12/260）。与全国其他地区相比,重庆市肉鸡中这两种病原的感染率相对要低,但仍应重视这两种疾病的防控,以控制病原的进一步传播。