牛瑟氏泰勒虫吉林株18S rRNA基因的扩增及系统发育研究

本研究根据GenBank上发表的牛瑟氏泰勒虫18S rRNA基因的核苷酸序列设计并合成1对特异性引物,对寄生于牛体内的瑟氏泰勒虫基因组DNA进行扩增,得到1 356 bp的18S rRNA基因片段,测序后blast分析表明该虫种属牛瑟氏泰勒虫。将该基因片段序列与GenBank中8种已知泰勒虫的相应序列进行比较分析,建立系统发育树。结果表明,牛瑟氏泰勒虫吉林分离株与水牛泰勒虫亲缘关系最近,与小泰勒虫亲缘关系较远。这一结果说明宿主因素对泰勒虫的基因型影响较大。     

Babesia bigemina infection in yak (Poephagus grunniens L.): Molecular detection and characterization

Yaks contribute significantly in the Himalayan high land economy. Specific information on prevalence of babesiosis in yaks is lacking. A fast and reliable PCR assay targeting Babesia bigemina small subunit ribosomal RNA sequence (SS rRNA) was laboratory standardized for molecular detection of B. bigemina in yaks. Restriction digestion of the PCR amplified 675 bp target sequence with Vsp I confirmed the prevalent species of Babesia as B. bigemina. Nucleotide sequencing and phylogenetic analysis of PCR amplified 675 bp SS rRNA sequence revealed a close genetic relationship with other bovine isolates of B. bigemina. A PCR based survey involving 94 blood samples of yak from the National Research Centre on Yak, Dirang, Arunachal Pradesh detected infection in 5.32% of yak blood samples, which was significantly higher in comparison to microscope based detection of infection in 2.13% blood smears. This is the first report on sensitive PCR based detection of B. bigemina infection in yaks and PCR-RFLP and nucleotide sequence analysis based molecular characterization of the B. bigemina isolated from yaks.     

四川省汶川县两种牛梨形虫的分离鉴定

自四川省汶川县黄牛体表采集长角血蜱的饥饿或半饱血成虫,带回实验室感染除脾牛.血液涂片检查发现,感染后第10 d,病牛血液中出现一种大型巴贝虫,感染后第24 d,出现一种小杆形的泰勒虫.形态学观察和18 S rRNA基因测序和进化关系分析证明,它们分别为卵形巴贝虫与瑟氏泰勒虫.     

Clinical and Laboratory Findings in Equine Piroplasmosis

The objective of this study was to evaluate equine piroplasmosis (EP) as a cause of morbidity in horses in Sardinia (Italy), describe the clinical signs and altered hematologic and biochemical parameters, and illustrate response to different treatments. Among 44 horses suspected of tick-borne disease, 38 were polymerase chain reaction (PCR) positive for Theileria equi (n = 27) or Babesia caballi (n = 6), whereas five were positive for both protozoans. Typical clinical features of piroplasmosis were seen in some of the horses, whereas others had nonspecific mild symptoms. Hematologic findings revealed involvement of the three blood cell lineages (anemia, leukopenia or leukocytosis, thrombocytopenia), and biochemical variations were related to increased bilirubin, alteration of serum phosphorus, and hypoalbuminemia. We suggest that the two protozoans are the most important causative agents of equine tick-borne disease in this geographic area, and we observe that different clinical features are associated with the disease; in addition to the typical aspects of piroplasmosis, characterized by fever, pale mucous membranes, and icterus, we can signal other nonspecific mild signs such as weight loss, weight loss associated with an insignificant leukopenia, or weight loss associated with depression, anorexia, and mild hyperbilirubin. The study is intended as a practical contribution for veterinary practitioners because it describes different clinical presentations and laboratory findings of EP, suggests diagnostic and therapeutic approaches to the disease, and shows diffusion of the disease in a Mediterranean region.     

Frequency of Rhodococcus equi in Feces of Mares in Central Kentucky

Rhodococcus equi (R equi) pneumonia is an important cause of disease and death in foals. Feces from mares can contain R equi, including virulent R equi, and thus may act as a source of the bacteria at horse breeding farms. A previous report documented that every mare at a farm in central Kentucky shed virulent R equi in at least one of four fecal samples collected serially during the periparturient period. The objective of this study was to assess the extent to which this high prevalence of fecal shedding could be replicated at other horse breeding farms. The frequency of detection of R equi and virulent R equi in fecal samples was studied among 131 mares from 24 farms in central Kentucky. The proportions of fecal samples from mares containing R equi and virulent R equi were 95% and 76%, respectively. These findings indicate that R equi and virulent R equi may be isolated with high frequency from feces of mares at breeding farms in central Kentucky, and that mares are a source of virulent R equi for the environment of their foals.     

云南鸭疫里默氏杆菌外膜蛋白A 及16S rRNA序列分析

为了探讨鸭疫里默氏杆菌(Riemerella anatipestifer, RA)云南流行株的外膜蛋白A (OmpA)的基因序列差异及其与16S rRNA序列的相关性,PCR扩增18株云南流行株鸭疫里默氏杆菌OmpA基因及16S rRNA核苷酸序列,分别构建其系统进化树,分析其系统进化关系。结果表明,18株鸭疫里默氏杆菌OmpA基因分为2个群,其同源性分别为86%~99.2%和92.6%~100%。18株鸭疫里默氏杆菌16S rRNA基因同属1个群,同源性高达96.1%~100%。 RA-1、RA-2、RA-11和RA-39 4株分离株的OmpA基因位于进化树的同一个亚群,其16S rRNA基因也位于进化树的同一亚群,两者呈现出明显的相关关系,其他14株分离株的OmpA基因系统进化树与16S rRNA基因系统进化树无明显的相关关系。     

多浆旱生植物霸王18SrRNA基因的克隆及序列分析

为探讨多浆旱生植物霸王(Zygophyllum xanthoxylum)的生物进化历程及与其他植物的亲缘关系,本研究以霸王叶基因组DNA为模板,使用通用引物扩增其18S rRNA 基因片段,并克隆到pGEM T载体,阳性克隆经鉴定后进行测序。核苷酸序列分析结果表明,该片段长1 808 bp,所得序列与GenBank中注册的18S rRNA基因序列的同源性均在96%以上。可见,高等植物18S rRNA 的基因非常保守。同源性分析与Blast比较结果表明,霸王与小盘木(Galearia filiformis)、驱虫苋(Cnidoscolus aconitifolius)及橡胶树(Hevea brasiliensis)同源性最高。系统进化树分析表明,霸王与三七(Panax notoginseng)的亲缘关系最近。     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Phylogenetic analysis of the genus Anaplasma in Southwestern China based on 16S rRNA sequence

To identify the species within the genus Anaplasma circulating among ruminants in the Southwest of China, we performed the phylogenetic analysis of the 16S rRNA gene of two Anaplasma isolates from cattle and seven from goats. The two sequences obtained from cattle strains belonged to the A. marginale cluster, whereas the other seven sequences from caprine strains formed two Anaplasma spp. clusters, which diverged earlier than the clusters of A. marginale, A. centrale and A. ovis. These results indicate that there are at least two Anaplasma species circulating among ruminants in Southwestern China.     

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

油脂类型和水平对肉鸡组织18S rRNA表达的影响

本试验旨在探究不同类型和水平的油脂对肉鸡肝脏、胸肌中18S rRNA表达的影响。试验选用240只1日龄科宝肉鸡母雏,随机分为8个组(分别在饲粮中添加2.50%、5.00%的亚麻油、玉米油、芝麻油和猪油),每个组6个重复,每个重复5只鸡,于42日龄分别从每个重复随机选取1只屠宰取样。结果表明:1)油脂类型和水平对肉鸡胸肌、肝脏中18S rRNA相对表达量影响极显著(P<0.01),且两者的交互作用对肝脏中18S rRNA相对表达量影响也极显著(P<0.01);2)与2.50%油脂水平相比,除5.00%亚麻油水平对肝脏中18S rRNA相对表达量影响不显著(P>0.05)以外,提高油脂水平(5.00%)可显著或极显著上调肉鸡胸肌、肝脏中18S rRNA相对表达量(P<0.05或P<0.01);3)在肉鸡胸肌、肝脏中,亚麻油组18S rRNA相对表达量不同程度地高于玉米油组、芝麻油组、猪油组(P<0.05或P>0.05)。研究表明,肉鸡肝脏和胸肌18S rRNA表达受饲粮添加油脂类型和水平的影响。     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

Associations between the Exposure to Airborne Virulent Rhodococcus equi and the Incidence of R equi Pneumonia among Individual Foals

Rhodococcus equi is a significant cause of pneumonia, resulting in disease and sometimes death of foals. It is believed that infection occurs by inhalation of dust contaminated with virulent R equi. Although association between the airborne concentration of virulent R equi and the incidence of foal pneumonia at breeding farms has been documented, studies at the level of individual foals have not been reported. Thus, the objective of this study was to determine whether the magnitude of airborne virulent R equi was significantly associated with risk of R equi pneumonia for individual foals. The concentration of virulent R equi was significantly (P < .001) greater in stalls than paddocks among samples collected from 47 foals at a breeding farm in central Kentucky. The presence of airborne virulent R equi in stalls was significantly (P = .045) more likely at 7 days of age for foals subsequently found to be affected by rhodococcal pneumonia. Additionally, airborne concentrations of virulent R equi in stalls were significantly greater at 7 and 14 days of age than at birth. Presence of the mare and foal at the time of sampling was significantly (P < .001) associated with increased airborne concentrations of virulent R equi in stalls. These findings suggest that environments containing airborne virulent R equi during the first week of life may influence the risk of subsequent disease for a foal.     

Investigation of seroprevalence of <Emphasis Type="Italic">Theileria equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> in horses in Nigde province,Turkey

The prevalence of equine piroplasmosis caused by Theileria equi and Babesia caballi in Nigde, in central Anatolia, Turkey has remained unknown. Serum samples were obtained from a total of 125 horses and were tested for antibodies to T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). Twenty-three (18.4%) horses were seropositive for equine piroplasmosis. Anti-T. equi was observed in 16 horses (12.8%) while anti-B. caballi was detected in 12 horses (9.6%). In addition, 5 serum samples were positive for both parasites. The prevalence rates of antibodies to T. equi and B. caballi for female and male horses were statistically indifferent (p = 0.19 and 0.90). The difference between the seropositivity rates to T. equi among age groups was statistically insignificant (p = 0.44) while the difference to B. caballi among age groups is statistically significant (p = 0.01). Seropositivity rates ranged from 2.9% to 25.7% for T. equi and 2.9% to 14.3% for B. caballi from the selected districts in Nigde. A statistically significant difference on seropositivity rates for the study sites was observed for only T.equi (p = 0.03). This study indicates that T. equi is higher than B. caballi in Nigde. This study was supported by the Scientific Research Projects Unit of Nigde University (FEB 2007/08).     

Retrospective genetic characterisation of Encephalomyocarditis viruses from African elephant and swine recovers two distinct lineages in South Africa

Encephalomyocarditis virus (EMCV) outbreaks are rare in southern Africa. Only two have been reported to date from South Africa, both coinciding with rodent irruptions. The first outbreak manifested as acute myocarditis in pigs in 1979, whilst the second, occurring from 1993 to 1994, was linked to the deaths of 64 free-ranging adult African elephants (Loxodonta africana). The P1 genome region, inclusive of the flanking leader (L) and 2A genes, of three South African isolates, one from swine and two from elephants, was characterised by PCR amplification and sequencing of up to 11 overlapping fragments. In addition to the resulting 3329 nucleotide dataset, the 3D region that is widely used in molecular epidemiology studies, was characterised, and three datasets (P1, VP1/3 and 3D), complemented with available homologous EMCV data, were compiled for analyses. Phylogenetic inferences revealed the near-identical elephant outbreak strains to be most closely related to a mengovirus from rhesus macaques (Macaca mulatta) in Uganda, differing from the latter by between 11% (3D) and 15% (VP3/1). The South African pig isolate differed by 4% (3D) and 11% (VP3/1) from available European and Asian pig virus sequences. This study confirms the presence of two genetically distinct EMCV lineages recovered from sporadic outbreaks in wild and domestic hosts in southern Africa, and provides valuable baseline data for future outbreak eventualities in the sub-region.     

水貂奇异变形杆菌的分离鉴定及16S rRNA基因序列分析

从辽宁某貂场发病水貂中分离到1株致病菌,命名为PMSD株,通过形态学观察和生化试验等常规鉴定发现符合奇异变形杆菌特性,进一步经VITEK 2Compact 30全自动细菌鉴定及药敏分析系统鉴定该株细菌为奇异变形杆菌。药敏试验结果显示PMSD株对氨基糖苷类药物、喹诺酮类药物等敏感,而对β-内酰胺类药物和磺胺类药物等不敏感。以细菌16SrRNA基因为模板应用通用引物进行PCR扩增,得到PMSD株的16SrRNA基因序列,长约1 504bp,提交到GenBank中,登录号:KM229530。将该序列与GenBank中序列进行BLAST比对,结果发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达99%以上。选取其中前20株作为参考序列,运用生物学软件构建系统发育树并进行同源性比对,结果表明,分离菌PMSD株与20个代表菌株的同源性为98.9%~99.7%,其中与BB2000株、HI4320株、B1株和FCC141株同源性最高,为99.7%。本研究为预防和控制奇异变形杆菌引起的水貂疾病奠定了一定的基础。     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.     

10株火鸡组织滴虫18S rRNA基因的克隆及进化分析

应用聚合酶链反应(PCR)鉴定湖南娄底(简称HMLD1-3)、湘潭(HMXT1-2)、永州(HMYZ1-3)和宁乡(HMNX1-2)共10株火鸡组织滴虫,并用18S rRNA部分序列重构火鸡组织滴虫与其它相近原虫的种群遗传关系.作者通过PCR扩增火鸡组织滴虫虫株的18S rRNA部分序列,将PCR扩增出的片段纯化后克隆...     

Clinicopathological findings, molecular detection and characterization of Babesia gibsoni infection in a sick dog from Italy

A 4-year-old intact female American Pit Bull Terrier from Italy descendant of an American-born bitch was evaluated for anorexia, lethargy, weakness, and intermittent vomiting. On physical examination, the dog was dehydrated, had pale mucous membranes, hunched posture and abdominal pain. A moderate anemia was observed. Splenomegaly and hyperechoic regions suspected as infarcts in the spleen were seen on abdominal ultrasound. Based on the suspicion of splenic torsion, splenectomy was performed. After surgery, the clinical condition deteriorated. A follow-up complete blood count demonstrated severe macrocytic normochromic anemia with evidence of marked regeneration, left shift neutrophilia, monocytosis and marked thrombocytopenia. Blood smear evaluation revealed single to multiple, variable sized (1–3 μm in diameter), and round to oval to band-like piroplasms within many red blood cells consistent with small form Babesia spp. or Theileria spp. A partial segment of the 18S rRNA gene was amplified and the PCR product was analyzed by direct sequencing. The nucleotide sequence was completely identical to that of Babesia gibsoni present in GenBank^®. This is the first molecular detection and characterization of B. gibsoni infection in a sick dog from Italy.