Development and Preliminary Application of a Semi-nested PCR Assay for Detection of Chicken Parvovirus

In order to set up and optimize a semi-nested PCR for rapid detection of chicken parvovirus (ChPV), three specific primers were designed according to conserved sequences of NS 1 gene of ChPV. The specificity and sensitivity of ChPV semi-nested PCR were tested, and the assay was applied to detect 48 clinical samples. The specificity and sensitivity tests showed that this semi-nested PCR was only sensitive to ChPV for amplifying specific band of 186 bp and it could detect 5.62 fg/μL of ChPV DNA, without any sensitivity to other viruses, such as Newcastle disease virus, H9 subtype avian influenza virus, Marek's disease virus, infectious laryngotracheitis virus and infectious bronchitis virus. 48 chicken samples were detected and the positive rate was 16.67% (8/48). The results of our study demonstrated that the optimized semi-nested PCR could be a method that was suitable for clinical detection of ChPV.     

鸡细小病毒半巢式PCR检测方法的建立及初步应用

为建立一种快速、特异、灵敏的检测鸡细小病毒（chicken parvovirus,ChPV）的方法,根据ChPV的保守基因NS1设计了3条特异性引物,建立并优化了能快速检测ChPV的半巢式PCR方法,对其进行特异性和敏感性试验,并用所建立的方法对48份临床样品进行了检测。特异性和敏感性试验结果显示,建立的半巢式PCR只对ChPV敏感,扩增产物为186 bp的特异性条带;其最低能检测到5.62 fg/μL的ChPV DNA;而对鸡新城疫病毒、H9亚型禽流感病毒、马立克氏病病毒、鸡传染性喉气管炎病毒、鸡传染性支气管炎病毒不敏感。临床检测结果显示,同时对48份临床样品进行检测,检出率为16.67%（8/48）,提示广西区内鸡群存在ChPV感染。本研究建立的ChPV半巢式PCR方法适用于ChPV的临床检测。     

Infections Investigation of ALV and REV in Guangxi Local Poultry Breeds and Sequence Analysis of gp85 Gene of ALV Isolates

In order to investigate the infection status of avian leukosis virus (ALV) and avian reticuloendotheliosis virus (REV) in the major local breeds of Qinzhou,Guangxi,totally 953 samples of egg white,cloaca swab and serum of Ma duck,Shitou goose,Tiejiao-Ma chicken,turkey and pigeon were collected from the representing flocks and detected by the commercial ELISA kits.ALV was isolated for the ALV p27 positive samples by culturing on DF-1 cells,and gp85 gene was sequenced.The results showed that the detections of ALV were negative in the samples except those of Tiejiao-Ma chicken,while REV antibody was found positive in Ma duck,Tiejiao-Ma chicken and turkey.The nucleotide sequences of gp85 gene of two isolates shared 94.5% identity with each other,and shared 86.9% to 94.9% with reference strains.The amino acid sequences of gp85 gene of two isolates shared 91.5% identity with each other,and shared 84.0% to 91.6% with reference strains.There were many variable sites in the hyper variable region hr1 and hr2,and the vr2 and vr3 variable regions were relatively conservative.Phylogenetic tree analysis showed that the two isolates shared the highest homology with SCAU11-XG strain.     

广西地方禽类品种ALV和REV感染情况的调查及ALV分离株gp85基因序列分析

为了解广西钦州地区主要地方品种商品禽类中禽白血病病毒(ALV)、禽网状内皮组织增殖症病毒(REV)的感染情况,本试验随机采集了广西钦州地区代表饲养场的麻鸭、狮头鹅、铁脚麻鸡、火鸡、鸽子共5个品种的蛋清、肛拭子及血清样品共953份,使用ELISA商品试剂盒进行检测;然后对部分ALV p27阳性的个体采集其血清样品接种DF-1细胞进行病毒分离并测定其gp85基因的序列。结果发现,除铁脚麻鸡外其他4个非鸡禽类品种的ALV检测均为阴性;除鸽子和狮头鹅外,麻鸭、铁脚麻鸡、火鸡均检测到REV抗体阳性;获得的两株铁脚麻鸡ALV分离株gp85基因之间核苷酸同源性为94.5%,与参考株之间核苷酸同源性为86.9%~94.9%;两株分离株gp85基因之间氨基酸同源性为91.5%,与参考株之间氨基酸同源性为84.0%~91.6%。高变区hr1和hr2区存在较多可变位点,可变区vr2和vr3相对较保守。系统进化树分析结果表明这两个毒株与参考株SCAU11-XG的亲缘关系最近。     

Serologic evidence in commercial chicken and turkey flocks of infection with reticuloendotheliosis virus

A serologic survey has documented probable infection with reticuloendotheliosis (RE) virus in 21.0% of 101 layer flocks, 23.5% of 85 broiler and broiler-breeder flocks, 2.3% of 43 backyard chicken flocks, and 4.8% of 125 turkey production and breeder flocks. However, no infection was detected in 72 grandparent lines of chicken breeding stocks representing meat-type and layer strains. The existence of natural infection was further supported by isolation of RE virus from one experimental chicken flock and two commercial turkey flocks. This study supports earlier but subsequently discounted data by Aulisio and Shelokov that exposure to RE virus occurs commonly among commercial chickens in the United States, as has also been reported in other countries.     

鸡细小病毒与禽呼肠病毒二重PCR检测方法的建立

建立可同时检测鸡细小病毒(Chicken parvovirus,ChPV)与禽呼肠病毒(Avian reovirus,ARV)的二重PCR方法,为防控ChPV与ARV提供技术支撑。根据鸡细小病毒NS1基因和禽呼肠病毒σC基因的保守序列,设计合成两对引物用于检测ChPV和ARV,通过优化二重PCR的反应体系,特异性、敏感性试验评价建立的ChPV与ARV二重PCR。优化后的二重PCR反应体系为:2×PCR Mix 12.5μL,其中ChPV与ARV的上、下游引物各1.0μL,混合模板2.0μL,ddH2O补足25μL;最佳的反应程序为:95℃5min;95℃1min,56.1℃1min,72℃1min,35个循环;最后72℃延伸10min。结果显示,建立的二重PCR能够同时扩增出204bp ChPV和405bp ARV片段;该方法对ChPV与ARV的检测敏感性分别达到58fg和53fg,但对鸡新城疫病毒、H9亚型禽流感病毒、马立克病病毒、鸡传染性喉气管炎病毒、鸡传染性支气管炎病毒等病原体均无特异性扩增,对ChPV与ARV混合感染的临床阳性病料的检测结果与各病毒单项PCR检测结果符合率为94%以上。建立的二重PCR可用于ChPV与ARV感染的快速鉴别诊断。     

Hemorrhagic enteritis of turkeys in California: serologic study of hemorrhagic enteritis virus antibody with an enzyme-linked immunosorbent assay

The incidence of hemorrhagic enteritis (HE) infection in California turkeys was studied by testing 2220 turkey blood samples from 173 flocks for HE virus (HEV) antibody by the enzyme-linked immunosorbent assay (ELISA). Maternal antibody was detected at 1 day of age in all flocks tested, and it vanished after 3 weeks. Acquired HEV antibody appeared at 8 to 10 weeks, and 100% of the meat and breeder turkey flocks were positive after 11 weeks of age. HEV infection occurred earlier in the meat flocks than in the breeder flocks, and it also occurred earlier during summer than during the fall and winter months.     

Drug use and antimicrobial resistance among Escherichia coli and Enterococcus spp. isolates from chicken and turkey flocks slaughtered in Quebec,Canada

An observational study was conducted of chicken and turkey flocks slaughtered at federal processing plants in the province of Quebec, Canada. The objectives were to estimate prevalence of drug use at hatchery and on farm and to identify antimicrobial resistance (AMR) in cecal Escherichia coli and Enterococcus spp. isolates and factors associated with AMR. Eighty-two chicken flocks and 59 turkey flocks were sampled. At the hatchery, the most used antimicrobial was ceftiofur in chickens (76% of flocks) and spectinomycin in turkeys (42% of flocks). Virginiamycin was the antimicrobial most frequently added to the feed in both chicken and turkey flocks. At least 1 E. coli isolate resistant to third-generation cephalosporins was present in all chicken flocks and in a third of turkey flocks. Resistance to tetracycline, streptomycin, and sulfisoxazole was detected in > 90% of flocks for E. coli isolates. Antimicrobial resistance (AMR) was observed to bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, and tetracycline in both chicken and turkey flocks for Enterococcus spp. isolates. No resistance to vancomycin was observed. The use of ceftiofur at hatchery was significantly associated with the proportion of ceftiofur-resistant E. coli isolates in chicken flocks. In turkey flocks, ceftiofur resistance was more frequent when turkeys were placed on litter previously used by chickens. Associations between drug use and resistance were observed with tetracycline (turkey) in E. coli isolates and with bacitracin (chicken and turkey), gentamicin (turkey), and tylosin (chicken) in Enterococcus spp. isolates. Further studies are needed to provide producers and veterinarians with alternative management practices and tools in order to reduce the use of antimicrobial feed additives in poultry.     

禽流感病毒与鸡细小病毒二重PCR检测方法的建立

为建立一种能同时鉴别诊断禽流感病毒（avian influenza virus,AIV）和鸡细小病毒（chicken parvovirus,ChPV）的检测方法,本研究根据GenBank中AIV的M基因和ChPV的NS基因保守序列,分别设计并筛选出两对特异性引物,用于AIV和ChPV的检测。通过优化反应条件,建立了AIV和ChPV二重PCR检测方法。试验结果表明,该方法特异好,能同时检测AIV和ChPV,对其他常见的禽病病原体均未反应;该法对AIV和ChPV的检测下限均为100 fg;对159份临床样品检测结果与PCR阳性产物测序结果一致。本研究建立的AIV和ChPV二重PCR检测方法具有特异性好、灵敏度高的特点,对AIV和ChPV的防制具有重要意义。     

Development of a Duplex PCR Assay for Detection of Avian Influenza Virus and Chicken Parvovirus

In order to establish a method to simultaneously detect avian influenza virus (AIV) and chicken parvovirus (ChPV),two pairs of specific primers were designed according to the sequences of AIV M gene and ChPV NS gene in GenBank. The duplex PCR assay was established by optimizing the reaction conditions.The tests showed that this method had high specificity, could simultaneously detect AIV and ChPV and no specific band was amplified for other subtypes avian pathogenic virus. The sensitivity result showed that the lower detection limit of this method was 100 fg. The results of 159 clinical samples were consistent with the sequencing results of PCR positive product. The double PCR methods for detection of AIV and ChPV established in this study had the characteristics of good specificity and high sensitivity, which was of great significance to the prevention control of AIV and ChPV.     

Serological investigations on reticuloendotheliosis in turkey flocks

Four meat turkey and one turkey breeding flocks were surveyed for antibodies against reticuloendotheliosis virus (REV) at different intervals using commercial enzyme-linked immunosorbent assays. In addition, serum samples collected from 18 flocks at different ages were also tested for antibodies against REV. No antibodies were detected in any of the four meat turkey flocks that were surveyed. In the breeder flock, 20%) of tested samples from 1-day-old poults were positive. Between the fourth and 12th weeks all samples that were tested yielded negative results. At 16 weeks of age 15% of samples yielded a positive reaction, but antibodies could not be detected 4 weeks later. Examination of serum samples from 18 different flocks at various ages revealed that antibodies could be detected in five flocks. The percentage of positive sera per flock ranged between 10 and 40%.     

Risk factors for foot-pad dermatitis in chicken and turkey broilers in France

A prospective cohort study was conducted in the Brittany region to identify the risk factors related to foot-pad dermatitis (considered an indicator of animal well-being) in chicken and turkey broilers reared under commercial conditions. Factors related to the shed, equipment, litter management and stocking density were recorded; the dependent variable was the prevalence of lesions observed on the slaughterhouse chain. Lesions were scored from 0 (no lesion) to 3 (severe lesion). Our survey lasted from May 1999 to October 2000. Fifty flocks of chicken broilers (15 farms), 27 flocks of female turkey broilers (21 farms) and 41 flocks of male turkey broilers (27 farms) were surveyed. In chicken broilers, 10% of flocks were of high quality (80% of birds with score 0) and this was related to the use of concrete floors with a thin layer of wood shavings. In turkey broilers, 48% of female and 46% of male flocks were of bad quality (>10% of birds with score 3). A poor fan ventilation system (<150 m³/h/kg) was a significant risk factor. Turkey flocks of high quality were not observed. Stocking density had no influence on the prevalence of foot-pad dermatitis. We concluded that it is possible under high commercial stocking densities to have flocks with a low prevalence of foot-pad dermatitis in chicken broilers, whereas it is not in turkey broilers. Hence in chicken broilers, implementing a monitoring system based on the observation of foot-pad dermatitis prevalence at slaughter appears to be more appropriate than to legislate stocking density. In turkey broilers, it would probably be necessary either to reduce the stocking density drastically or to investigate new systems of floor drainage.     

In vivo events of retroviral long terminal repeat integration into Marek's disease virus in commercial poultry: detection of chimeric molecules as a marker

The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV. A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HS-cPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.     

鸡源鸭疫里默杆菌的分离鉴定及ompA基因遗传进化分析

从病死鸡肝脏中分离到8株革兰阴性多形杆菌,对8株分离株进行形态学和16S rDNA基因鉴定并分析ompA基因及其推导的蛋白序列遗传进化特征。16S rDNA进化分析显示,8株鸡源分离株与22株鸭疫里默杆菌(Riemerella anatipestifer,RA)参考株形成一个大的进化分支,与RA模式菌株ATCC11845和22株RA参考株之间的同源性分别为99.3%~99.8%和98.8%~100.0%。根据形态学和16S rDNA基因鉴定结果,将8株分离株鉴定为RA。ompA基因进化分析显示,8株鸡源RA分离株与3株鸭源RA分离株KML1、KML2、KML3以及1株鹅源RA分离株KS9901-G形成一个大的进化分支,同源性为94.0%~99.6%。与ATCC11845株相比较,8株鸡源RA分离株ompA基因均发生99个核苷酸位点的突变。OmpA蛋白进化分析显示,8株鸡源RA分离株形成一个独立的大的进化分支,同源性为100%。与ATCC11845株相比较,8株鸡源RA分离株OmpA蛋白均发生19个氨基酸位点的突变。8株鸡源RA分离株对青霉素、阿莫西林克拉维酸、头孢噻吩等10种抗生物敏感。本试验在国内分离到鸡源RA,证实鸡RA感染在我国的存在,对鸡RA感染的预防和控制具有重要指导意义。     

Serologic evidence of chicken infectious anemia in commercial chicken flocks in southwest Nigeria

Sera samples from seven poultry farms in southwest Nigeria consisting of 7 broiler, 10 pullet, 1 layer, 1 cockerel, and 1 broiler breeder flocks were tested for the presence of chicken infectious anemia virus (CIAV) antibodies using a commercial enzyme-linked immunosorbent assay kit. Eleven of the 20 flocks (55%) and six out of seven (86%) farms were positive for CIAV antibodies. The seroprevalence largely depended on the age of the flocks. Seroprevalence was higher within the older pullet and layer flocks (83%-100%) than in the younger broiler flocks (0%-83%). In essence, all flocks older than 6 to 8 wk became infected. This is the first report of serologic evidence of CIAV in Subsaharan Africa. Since Southwest Nigeria is the main port of entry of imported chicken and the hub of major poultry breeders, the disease can probably be found throughout the country and beyond. Further studies are necessary to assess economic losses due to CIAV and the cost benefit of countermeasures.     

A single subtype of avian pneumovirus circulates among Minnesota turkey flocks.

The recent emergence of avian pneumovirus (APV) infection among US turkey flocks has resulted in a major economic threat to the turkey industry. In order to elucidate the molecular epidemiology of APV, comparative sequence analysis of the fusion (F) protein gene of APV was performed for 3 cell culture-adapted isolates and 10 APV positive clinical samples recovered from US turkey flocks. Relatively modest levels of nucleotide and amino acid sequence divergence were identified, suggesting the prevalence of a single lineage of APV among US turkey flocks. Additionally, numerous polymorphisms were identified that were only represented in the clinical samples but not in the in vitro propagated isolates of APV. Phylogenetic analyses confirm that the subtype of APV circulating in the upper Midwestern United States is evolutionarily related to, but distinct from, European APV subgroups A and B. Overall, the results of the present investigation suggest that there has been only a single recent introduction of APV into US turkey populations in the upper Midwestern United States.     

Investigation of field outbreaks of turkey haemorrhagic enteritis in Hungary

Epidemiological, pathological, serological and virological investigations are reported on turkey haemorrhagic enteritis virus (THEV) infection in Hungarian turkey flocks. The pathogenesis of infection in experimentally infected turkeys and chickens, as well as the usefulness of polymerase chain reaction (PCR)/sequencing method for epidemiological investigation and for the differentiation of vaccine and field strains of THEV was also studied. Since the first recognition of the disease in Hungary in the late 1970s, until recently the disease has been diagnosed sporadically in its mild form. In the last few years (2000-2005), however, the number of outbreaks and the severity of the disease increased (9-23 affected flocks/year). Most of the outbreaks occurred at the age of 6 to 8 weeks and was complicated with Escherichia coli infection. The antibody levels to THEV in turkey flocks gradually declined till 5-7 weeks of age, and then they increased sharply due to natural infection with THEV. The immune response to vaccination (at 5 weeks of age) showed no significant antibody level increase one week postvaccination, but four weeks later the antibody level reached high values and then remained at this high level. The agar gel immunodiffusion (AGID) test to detect turkey adenovirus A (TAdV-A) antigen and PCR methods for THEV-specific DNA gave similarly positive results if spleens with pathognomonic lesions were tested; however, PCR proved to be more sensitive in cases with less characteristic pathological lesions. Nucleotide sequence alignment of PCR products amplified from Hungarian field strains and the Domermuth vaccine strain and that of the published THEV hexon sequences in GenBank database revealed slight differences between the sequences.