光敏生物素标记EDSV—DNA探针检测鸡减蛋综合症（EDS）的研究

应用光敏生物素标记ＥＤＳＶ－ＤＮＡ与ｐＵＣ１９重组质粒ＤＮＡ做探针，检测人工发病减蛋综合症蛋鸡的粪便，蛋，输卵管样品，以新城疫病毒（ＮＤＶ），传染性病毒（ＩＢＤＶ），传染性支气管炎病毒（ＩＢＶ），包涵体肝炎病毒（ＩＢＨＶ）作对照。实验结果显示：探针能特异地检出ＥＤＳ病鸡粪便，输卵管，蛋清样品中的病毒，与ＮＤＶ，ＩＢＤＶ，ＩＢＶ及ＩＢＨＶ均呈阴性反应。探针灵敏度达１０ｐｇＥＤＳＶ－ＤＮＡ。     

鸡白色念珠菌、传染性支气管炎病毒、新城疫病毒三重PCR检测方法的建立与应用

根椐Gen Bank中已发表的白色念珠菌(CA)、传染性支气管炎病毒(IBV)、新城疫病毒(NDV)基因序列设计并合成三种病原的特异性检测引物,通过三重PCR反应条件的优化,建立了检测三种病原的PCR检测方法。该方法对同一样品中的CA、IBV和NDV核酸模板可同时扩增出231 bp、417 bp和747 bp的特异性片段,检测光滑念珠菌(CG)、克柔念珠菌(CK)、热带念珠菌(CT)、H9亚型禽流感病毒(AIV H9)、鸡传染性法氏囊病毒(IBDV)、鸡传染性喉气管炎病毒(ILTV)、减蛋综合症病毒(EDSV)等7种禽病病原均为阴性,该方法对CA DNA、IBV c DNA、NDV c DNA的检测最低限度分别为25.0,33.5,36.5 pg,临床样品检测结果表明三重PCR检测方法可以用于这三种病原单独和混合感染的检测和鉴别诊断。说明所建立的三重PCR检测方法具有很好的特异性、敏感性、重复性和临床适用性,为三种病原单独或混合感染的检测和鉴别诊断提供了一种操作简单、检测快速的分子生物学诊断方法。     

胶体金快速诊断方法对鸡减蛋综合征病毒的检测

选择15nm的胶体金颗粒作为标记物，制备了鸡减蛋综合征病毒（EDSV）胶体金试纸条。该试纸条能检出浓度约为1．35μg／mL的纯化EDSV，用该试纸条检测135份临床样本，并与HA方法相比较，结果显示，二者的阳性符合率为89．8％。特异性试验结果显示，与鸡新城疫病毒（NDV）、鸡传染性腔上囊病病毒（IBDV）、鸡传染性支气管炎病毒（IBV）无交叉反应。表明，该试纸条用于检测EDSV具有快速、操作简便、特异性强等优点，可用于大批量抗原样本的检测。     

禽传染性支气管炎病毒一步法RT-PCR检测方法的建立及应用

为建立一种快速检测禽传染性支气管炎病毒(Infectiousbronchitisvirus,IBV)病原的方法,根据GenBank上的禽传染性支气管炎病毒基因组序列,设计1对引物,建立了检测IBV的一步法RT-PCR方法,该方法对20份疑似传染性支气管炎(Infectiousbronchitis,IB)病料、传染性法氏囊病毒(Infectiousbursaldiseasevirus,IBDV)、新城疫病毒(Newcastaldiseasevirus,NDV)进行检测,结果仅IBV为阳性,IBDV、NDV均为阴性。该一步法RT-PCR方法具有良好的特异性、灵敏性和重复性,最低可检测出约4pg的IBVRNA,将为IBV的病原检测及分子流行病学调查等提供早期快速的诊断方法。     

检测鸡新城疫病毒和鸡传染性支气管炎病毒双重RT-PCR的建立与初步应用

为了快速检测临床样品中新城疫病毒(NDV)和鸡传染性支气管炎病毒(IBV)的混合感染,根据NDV的F基因和IBV的N基因保守序列分别设计了2对特异性引物,优化双重RT-PCR反应条件,建立了可同时检测NDV和IBV的快速双重反转录PCR(RT-PCR)诊断方法。敏感性试验表明,此方法对NDV和IBV的RNA最小检出量分别为0.014 3和0.013 6ng;NDV的扩增片段大小为459bp,IBV的扩增片段为282bp。对其他病毒如IBDV、AIV和EDSV的检测结果均为阴性,表明此方法具有较好的特异性。对58份临床疑似样品进行检测,NDV和IBV的阳性率分别为44.82%和31.03%,NDV和IBV混合感染率为20.68%,表明本研究建立的NDV与IBV双重RT-PCR检测方法,可用于临床发病病料中NDV和IBV混合感染的快速鉴别诊断。     

鸡J亚群禽白血病病毒与七种常见病毒混合感染的调查

应用PCR方法对2009年6月到2009年9月来自不同地区、不同品种、不同日龄的45个疑似感染J亚群禽白血病病毒(ALV-J)的病鸡病料进行检测,同时对ALV-J与新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、传染性法氏囊病病毒(IBDV)、H9N2亚型禽流感病毒(H9N2 AIV)、包涵体肝炎病毒(IB-HV)、鸡传染性贫血病毒(CIAV)和呼肠病毒(REOV)等病毒的混合感染情况进行了调查。结果表明,采用ALV-J的特异性引物H5/H7,在45份送检病料中,有3份检出ALV-J,阳性率为6.67%。绝大多数病例检测出内源性禽白血病病毒,部分病例检测有其他7种病毒中的一种或几种。混合感染病例中以IBHV和IBV混合感染的比率较高,其中ALV-J与H9N2亚型AIV混合感染多见。     

同胚接种IBDV和NDV制造二联弱毒苗

用鸡传染性腔上囊病病毒（IBDV）和新城疫病毒（NDV）接种同一鸡胚收取种毒，试制IBDV、NDV二联弱毒冻干疫苗，安全性和效力检验均达到这2种单苗《规程》规定的标准，证明2种病毒在鸡体人产生互不干扰抗体。     

传染性法氏囊病病料中MDV、CAV、REV的共感染检测

从江苏、山东、浙江、河南、上海、广西、云南等地收集根据临床表现及病理变化诊断为传染性法氏囊病（IBD）的病鸡法氏囊样品66份，用相应的核酸探针及斑点杂交法分别检测样品中鸡传染性贫血病病毒（CAV）、马立克氏病病毒（MDV）、网状内皮细胞增生病病毒（REV）的感染；用抗IBD血清做琼扩检测IBDV。结果显示，部分地区病料中这些病毒有不同程度的二重感染、三重感染甚至四重感染。提示在研究特定毒株IBDV的毒力时，应考虑多重感染对致病性的影响。     

鸡传染性支气管炎病毒793/B RT-PCR检测方法的建立及应用

根据GenBank已发表的鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）793／BS1基因序列，设计1对引物，扩增891bp特异性核酸片段，建立了检测IBV793／B的RT—PCR方法。特异性试验结果表明，IBV793／B能扩增出891bp的核酸片段，而IBVM41H120H52毒株以及新城疫病毒（NDV）、禽流感病毒（AIV）、传染性法氏囊病病毒（IBDV）均无特异性条带出现。敏感性试验结果表明，该方法的最低检出量为10pg的模板。上述结果表明，本试验所建立的RT—PCR方法敏感性高、特异性强。利用建立的RT—PCR方法对从山东省分离的8株疑似鸡IBV793／B进行检测，结果7株为阳性。该方法的建立为IBV793／B的诊断及流行病学调查提供了可靠的方法。     

鸡传染性支气管炎病毒对新城疫病毒的干扰作用观察

为观察鸡传染性支气管炎病毒（IBV）HN99株对新城疫病毒（NDV）增殖的干扰作用,该试验采用不同浓度的IBV标准株M41和地方株HN99与鸡新城疫病毒（NDV）分别按不同接种顺序同胚增殖,利用病毒血凝试验（HA）测定NDV的效价,观察IBV对NDV的干扰作用,从而为检测IBV地方株HN99提供方法,也为同胚增殖两种病毒提供一系列的数据参考。试验结果表明,鸡传染性支气管炎病毒地方株HN99对NDV的干扰作用与其浓度和接种顺序有关。     

The relationship between the numbers of Salmonella Enteritidis, Salmonella Heidelberg, or Salmonella Hadar colonizing reproductive tissues of experimentally infected laying hens and deposition inside eggs

Contamination of eggs by Salmonella Enteritidis has been a prominent cause of human illness for several decades and is the focus of a recently implemented national regulatory plan for egg-producing flocks in the United States. Salmonella Heidelberg has also been identified as an egg-transmitted pathogen. The deposition of Salmonella strains inside eggs is a consequence of reproductive tract colonization in infected laying hens, but prior research has not determined the relationship between the numbers of Salmonella that colonize reproductive organs and the associated frequency of egg contamination. In the present study, groups of laying hens in two trials were experimentally infected with large oral doses of strains of Salmonella Enteritidis (phage type 13a), Salmonella Heidelberg, or Salmonella Hadar. Reproductive tissues of selected hens were cultured to detect and enumerate Salmonella at 5 days postinoculation, and the interior contents of eggs laid between 6 and 25 days postinoculation were tested for contamination. Significantly more internally contaminated eggs were laid by hens infected with Salmonella Enteritidis (3.58%) than with strains of either Salmonella Heidelberg (0.47%) or Salmonella Hadar (0%). However, no significant differences were observed between Salmonella strains in either isolation frequency or the number of colony-forming units (CFU) isolated from ovaries or oviducts. Salmonella isolation frequencies ranged from 20.8% to 41.7% for ovaries and from 8.3% to 33.3% for oviducts. Mean Salmonella colonization levels ranged from 0.10 to 0.51 log CFU/g for ovaries and from 0.25 to 0.46 log CFU/g for oviducts. Although parallel rank-orders were observed for Salmonella enumeration (in both ovaries and oviducts) and egg contamination frequency, a statistically significant relationship could not be established between these two parameters of infection.     

Presence and recovery of Ascaridia dissimilis ova on the external shell surface of turkey eggs.

Ascaridia dissimilis, a roundworm in turkeys, has been noted with increased frequency in commercial turkeys. Because infected turkeys can shed A. dissimilis ova in their feces, the potential exists for the external surface of turkey eggshells to be contaminated with A. dissimilis ova. The objectives of this study were to determine the presence of and recover A. dissimilis ova on the external surface of the turkey egg. In Experiment 1, turkey eggs were collected from naturally infected flocks, and eggs were processed by a sodium hydroxide procedure to recover any A. dissimilis ova on the external egg surface. In Experiment 2, the external surface of the turkey eggs was inoculated with 116 A. dissimilis ova/g feces, and eggshells were sampled every 3 days until 28 days of incubation to assess the recovery of A. dissimilis ova from the eggshell. In Experiment 1, of the 36 eggs examined from a flock naturally infected with A. dissimilis, one egg had an A. dissimilis ovum on its external eggshell surface. Experiment 2 demonstrated that A. dissimilis ova can be recovered from the external egg surface after a 28-day incubation period in the incubator. Ova recovery declined from an average of 62 A. dissimilis ova/turkey egg at day 2 of incubation to an average of 3 A. dissimilis ova/turkey egg at day 28 of incubation.     

Anthelmintic efficacy of milbemycin D against Toxocara cati and Ancylostoma tubaeforme in domestic cats.

Anthelmintic efficacy of milbemycin D was evaluated against Toxocara cati and Ancylostoma tubaeforme in domestic cats. Twelve cats naturally infected with each nematode species were allocated among 2 groups of 6 animals each, and milbemycin D was orally administered to the 2 groups of cats in doses of 0.05 mg/kg and 0.1 mg/kg body weight, respectively. In all the cats infected with T. cati, fecal egg counts decreased followed by their disappearance from the feces and 2-35 worms were excreted into the feces after the medication in both doses of 0.05 mg/kg and 0.1 mg/kg. At postmortem of these medicated groups, no worms were detected from 4 cats of each group, but 1 and 2 immature worms were recovered from the other 2 cats respectively. In the cats infected with A. tubaeforme, fecal egg counts decreased followed by the disappearance from the feces and 2-62 worms were excreted into the feces in all the cats of the 2 groups, no nematodes remaining at postmortem. These results indicate that milbemycin D is fully effective against T. cati and A. tubaeforme in cats in a dose of 0.05-0.1 mg/kg.     

Isolation and characterization of Chlamydophila psittaci isolated from laying hens with cystic oviducts

The objective of this study was to isolate and identify a hypothetical Chlamydiaceae pathogen from laying hens with an oviduct cyst, and to characterize its potential causal relation with decreased egg production. Our clinical survey showed that cystic oviducts were prevalent at rates of 10% and 15.1% in breeder and commercial hen flocks, respectively. Chlamydial antigens were detected in 20 of 50 pharyngeal swabs (40%) and in 17 of 20 oviduct tissues (85%) using enzyme-linked immunosorbent assay (ELISA) antigen detection kits. The isolated pathogen was identified as Chlamydophila psittaci via complement fixation test, PCE-ELISA, and immunofluorescence assay. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were excluded after oviduct tissues were inoculated onto the chorioallantoic membrane of embryonating eggs. The nucleotide sequence of the omp1 gene (accession no. EF202608) from the isolate was similar to that of C. psittaci avian type C (accession no. L25436). Typical cystic oviducts were observed in specific-pathogen-free hens inoculated intraperitoneally with the isolate. The high presence of chlamydial antigen is consistent with the cystic oviducts and poor egg production. We conclude that the isolated C psittaci is most likely associated with cystic oviducts in laying hens.     

Effect of prior serial in vivo passage on the frequency of Salmonella enteritidis contamination in eggs from experimentally infected laying hens

Experimental infection models are valuable tools for understanding and preventing the deposition of Salmonella enteritidis inside eggs. Oral inoculation is believed to closely simulate naturally occurring S. enteritidis infections of chickens, but oral infection studies have often generated relatively low frequencies of egg contamination. The present study assessed whether repeated in vivo passage of an S. enteritidis strain could affect its ability to cause egg contamination in experimentally infected hens. The incidence of egg contamination was determined in groups of hens inoculated orally with either a phage type 13a S. enteritidis strain or derivatives of this parent strain that were obtained by three successive rounds of passage and reisolation from tissues of infected hens. Passaged S. enteritidis isolates recovered from ovaries and oviducts induced a significantly higher incidence of egg contamination (16.97%) than was attributed to the parent strain (8.27%). However, passaged S. enteritidis isolates recovered from livers and spleens were not associated with a significantly increased frequency of deposition in eggs. By either inducing or selecting for the expression of relevant microbial properties, passage of S. enteritidis through reproductive tissues of chickens may be useful for improving the efficiency at which experimental infection models produce egg contamination.     

文昌鸡生态放养过程中驱虫程序的研究

采用虫卵漂浮法、麦克马斯特氏虫卵计数法对文昌鸡生态放养期间感染寄生虫的种类与粪便中虫卵数量进行监测。结果表明,文昌鸡生态放养感染的寄生线虫主要为鸡蛔虫和几种异刺线虫。自然条件下生态放养35 d后,每克粪便中寄生虫虫卵数（eggs per gram, EPG）达到了700,呈显著增长趋势。抽样宰杀30只鸡,检出18只感染鸡蛔虫成虫,感染率为60％,平均感染强度为8条/只。因此,建议整个生态放养期间驱虫3～4次为宜,首次驱虫时间为放养后25～30 d。     

Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus

The ovicidal activity of the essential oil of Ocimum gratissimum Linn. (Labideae) and its main component eugenol was evaluated against Haemonchus contortus, gastrointestinal parasite of small ruminants. The oil and eugenol were diluted in Tween 20 (0.5%) at five different concentrations. In the egg hatch test, H. contortus eggs were obtained from feces of goats experimentally infected. At 0.50% concentration, the essential oil and eugenol showed a maximum eclodibility inhibition. These results suggest a possible utilization of the essential oil of O. gratissimum as an aid to the control of gastrointestinal helmintosis of small ruminants.     

The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.     

Determining Treatment to Control Two Multidrug-Resistant Parasites on a Texas Horse Farm

A study was undertaken at the Texas A&M Horse Center to evaluate and compare the effectiveness of three anthelmintics—ivermectin, fenbendazole, and a combination of ivermectin and pyrantel pamoate—on fecal egg count reductions of cyathostomes and Parascaris equorum in 30 naturally infected foals. The foals were randomized into three treatment groups, with individuals being rerandomized after each 8-week observation period. The treatments of ivermectin and fenbendazole were given at the manufacturer's recommended doses, and the pyrantel treatment was given at two times the manufacturer's recommended dose. Fecal egg counts were performed at the time of treatment and at 2-week intervals after treatment for a total of 8 weeks. Each foal received a total of three treatments during the course of the study. Fecal egg counts were performed by a modified McMaster's test, with a sensitivity of 25 eggs per gram of feces, and by the modified Wisconsin double centrifugal flotation technique, with a sensitivity of 0.2 eggs per gram of feces. Fecal egg reduction percentages were calculated. Analysis of the results showed that ivermectin, either used alone or with pyrantel, was a more effective anthelmintic for cyathostome (small strongyle) control than fenbendazole. Fenbendazole and pyrantel showed a higher initial reduction in Parascaris egg counts when compared with the ivermectin-only-treated group, but this difference lessened over time. The use of the combination treatment showed the best results for controlling both parasites, indicating that a combination of anthelmintics may be necessary to control parasites on some equine farms.     

The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens,followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

Three groups of 100 individually marked salmonella-free chickens were followed for a period of 53 wk. The chickens were infected as day olds by crop instillation of 10(8) colony-forming units: one group with Salmonella enteritidis and a second group with Salmonella typhimurium. A third group was kept uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 8 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yolk samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods in surveillance programs, in particular to detect flocks in early stages of infection before a measurable serologic response has been raised.