致病性马兽疫链球菌的分离鉴定、生物学特性及其M-like基因的序列分析

为鉴定锦州某猪场引起关节炎病疫情的病原,本研究将患猪关节液在血平板培养基上培养进行细菌分离;对分离细菌进行形态学观察、生化反应、致病性试验及药物敏感性试验;并对其M-like基因进行克隆和测序分析.结果在显微镜下观察到周围具有透明的β溶血环的革兰氏阳性中长链球菌,经生化反应及M-like基因鉴定为马兽疫链球菌(命名为10JZ12);分离株10JZ12对小鼠的致死剂量为1.75× 108 cfu;并对苯唑西林、青霉素G、红霉素、克拉霉素、克林霉素、米诺环素、环丙沙星、左氟沙星、多粘菌素B、呋喃妥因敏感,对氯霉素、四环素、诺氟沙星、复方新诺明不敏感.     

Identification and molecular characterization of Streptococcus equi subsp. zooepidemicus isolated from camels (Camelus dromedarius) and camel milk in Kenya and Somalia

Seventeen Streptococcus equi subsp. zooepidemicus strains isolated from camels and camel milk in Kenya and Somalia were identified by their cultural characteristics, by biochemical and serological reactions with the help of commercial identification systems and by molecular studies using a multiplex PCR. The isolates were further characterized by a PCR-mediated detection of size polymorphisms in the 16S-23S rDNA intergenic spacer region and the virulence gene szp and by amplification of the virulence gene cne. These molecular analysis are potentially useful in identifying and characterizing S. equi subsp. zooepidemicus strains of this origin and could possibly be valuable in epidemiological investigations.     

Phenotypic and genotypic properties of Streptococcus equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002

The present study was designed to identify and compare 32 beta-hemolytic streptococci isolated from 28 different harbor seals of the German North Sea during the phocine distemper outbreak in 2002. The bacteria were identified as Streptococcus equi subsp. zooepidemicus based on cultural, biochemical, serological and molecular studies. Epidemiological investigations by PCR restriction fragment length polymorphism analysis of the 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 32 strains appeared to be identical. These results indicate that a single bacterial clone seemed to be distributed among the harbor seal population of the German North Sea during this outbreak.     

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) of various origins of the North Sea during 1988-2005

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., L?mmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.     

Studies on the parasites of zebras. IV. Cylicodontophorus reineckei n.sp. (Nematoda: Strongylidae) from the Burchell's zebra, Equus burchelli antiquorum H. Smith, 1841 and the mountain zebra, Equus zebra hartmannae Matschie, 1898

A new species of nematode, Cylicodontophorus reineckei, was collected from Burchell's zebra, Equus burchelli antiquorum H. Smith, 1841, in both the Etosha National and Kruger National Parks and from mountain zebra, Equus zebra hartmannae Matschie, 1898, in the Namib Naukluft Park in South West Africa/Namibia. These nematodes have an external leaf-crown with longer elements than those of the internal leaf-crown and a well-developed dorsal gutter. The very well-developed oesophageal funnel is both wider and deeper than the buccal capsule.     

Characterization of enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC) and necrotoxigenic E. coli (NTEC) isolated from diarrhoeic Mediterranean water buffalo calves (Bubalus bubalis)

Two hundred and twenty Escherichia coli isolates from 314 Mediterranean water buffalo calves less than 4 weeks old affected by severe diarrhoea with a lethal outcome were characterized for the presence of the virulence factors LT, ST, Stx1, Stx2, haemolysins, intimin, CNF1, CNF2, CDT-I, CDT-II, CDT-III, CDT-IV, and F17-related fimbriae (F17a, F17b, F17c, F17d). The prevalence of ETEC, STEC and NTEC were 1.8%, 6.8% and 20.9%, respectively. The ETEC isolates were all LT-positive and ST-negative. The STEC isolates were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%). The NTEC isolates were all CNF and Hly-positive, with CNF2 (83%) more frequent than CNF1 (22%). Susceptibility assays to 11 antimicrobials displayed high rates of resistance (>30%) to antimicrobials tested. These data show that the most prevalent strains in diarrhoeic water buffalo calves were NTEC, mostly CNF2 and HlyA-positive, with strong associations CNF2/CDT-III and CNF2/F17c.     

猪轮状病毒中国分离株JL94 VP7基因的克隆与序列分析

用MA104细胞培养增殖了轮状病毒地方分离株JL94,利用一对根据Genebank中已发表的轮状病毒VP7基因cDNA序列而设计并合成的引物，通过反转录-聚合酶链反应(RT—PCR)从JL94毒株扩增出VP7基因全长cDNA。将其插人pMD18-T载体中，构建了重组质粒pMD18-T—JL94／VP7，并对其进行了Hind Ⅲ，HindⅡ和BamH I的单、双酶切初步鉴定及其核苷酸序列测定，证明克隆的pMD18-T—JL94／VP7为轮状病毒的VP7基因。通过核苷酸序列比较，JL94／VP7与A群猪轮状病毒C134／VP7、OSU／VP7、ICB2185／VP7、YM／VP7核苷酸序列同源性分别为87％、99％、74％和83％。     

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.     

Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries

Streptococcus suis strains (n=411), isolated from diseased pigs in seven European countries were serotyped using specific antisera against serotype 1 to 28, and were phenotyped on the basis of their muramidase-released-protein (MRP) and extracellular-factor protein (EF) production. Overall, S. suis serotype 2 appeared to be most prevalent (32%), followed by serotype 9 (20%) and serotype 1 (12%). Serotype 2 was most frequently isolated in France, Italy and Spain, whereas serotype 9 was most frequently isolated in Belgium, The Netherlands and Germany. In the United Kingdom serotypes 1 and 14 were most frequently isolated. High percentages of S. suis serotype 1, 2, 1/2 and 14 strains, isolated from tissues associated with S. suis infections such as brain, serosa, joint, heart and organs expressed the EF-protein, indicating that in these serotypes expression of EF is likely to be associated with virulence. In contrast, strains belonging to serotype 7 and 9, isolated from tissues associated with S. suis infections did not produce EF. These results strongly suggest that in the serotypes 7 and 9 EF expression is not related to virulence. More than 80% of the S. suis serotype 9 strains produced an MRP* protein, a high molecular variant of the 136kDa MRP. Expression of MRP* in serotype 9 strains is possibly associated with virulence.     

半番鸭源禽1型副粘病毒FM01株的分离鉴定与F蛋白基因分析

从表现类似新城疫症状的病死半番鸭中分离到1株病毒FM01株，经血凝及血凝抑制试验证实为禽1型副粘病毒、以SPF鸡胚测定其平均致死时间为113，4h，对1d雏鸡脑内接种致病指数为0．23，表明为温和型毒株。利用RT-PCR技术一次性扩增其F蛋白全基因，克隆到pMD 18-T质粒载体，测序后获得F基因全序列，并推导出其相应的氨基酸序列。FM01株的F蛋白基因完整的编码区全长1662bp，编码553个氨基酸，其裂解位点的氨基酸序列为112G-R-Q-G-R-L117，具有温和型毒株特有的氨基酸序列结构．与常见新城疫毒株的核苷酸同源性在88．4％～99.6％之间，氨基酸同源性在89．2％～99.1％之间。     

Production of muraminidase-released protein (MRP), extracellular factor (EF) and suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France

A total of 323 isolates of Streptococcus suis recovered from diseased or healthy pigs in France were serotyped. The presence of virulence-related proteins, Muraminidase-Released Protein (MRP), Extracellular Factor (EF) and Suilysin was also studied in 122 isolates of capsular types 2, 1/2, 9, 7 and 3 to evaluate their implication in virulence of S. suis. Capsular types 2, 1/2, 9, 7 and 3 were the most frequently detected (93%), with 69% for the capsular type 2 alone. Capsular types 2, 1/2, 9, 7, 3, 1, 4, 8, 18, 10 and 12 were isolated from diseased pigs, whereas types 2, 7, 9, 1/2, and 3 originated from the nasal cavities or tonsils of healthy animals. Most of the S. suis type 2 isolates recovered from diseased pigs carried MRP+ EF- Suilysin- (46%) or MRP+ EF+ Suilysin+ (28%) phenotypes. The MRP+ EF- Suilysin- phenotype was also detected in 67% of S. suis type 2 strains isolated from healthy pigs. The production of the virulence-related proteins was less frequently found in S. suis types 1/2, 9, 7 and 3 recovered either from diseased or healthy pigs. In this study, all the capsular type 1/2 strains were MRP+ EF- Suilysin- and all the S. suis type 7 harboured an MRP- EF- Suilysin- phenotype. The MRP- EF- Suilysin- phenotype was found in S. suis types 2, 3, 7 and 9 isolated from septicaemia, meningitis, pneumonia, and pleurisy. These results suggest that the presence of these proteins should not be used as a single condition for classifying the virulence of a field isolate in France.     

Comparative sequence analysis of major envelope protein gene (B2L) of Indian orf viruses isolated from sheep and goats

Orf virus (ORFV), the type species of Parapoxvirus, is responsible for contagious ecthyma in sheep and goats. In the present report, sequence analysis of major envelope gene (B2L) of four Indian orf virus isolates originating two each from sheep and goats was carried out. These recent isolates belonged to different outbreaks that occurred in Kumaon hills and adjoining plains during 2004-2005. Preliminary screening of the scab samples was carried out by diagnostic PCR. Full-length B2L gene encoding for immunogenic major envelope protein from all the four ORFV isolates was amplified by PCR and the amplicons (1206 bp) were cloned and sequenced. Comparative sequence analysis revealed an open reading frame of 1137 nucleotides (nt) encoding a polypeptide of 378 amino acids (aa). Indian isolates were highly related amongst themselves with sequence identity of over 97% at the nt and aa level. Further, they showed 97-98% sequence identity with sequences of other ORFV isolates from around the world; while 94-95 and 82.7-83.8% sequence identity was observed, respectively, with pseudocowpox and bovine papular stomatitis viruses--the other members of the genus. Phylogenetic analysis also showed that these Parapoxviruses from sheep and goats are closely related to other orf viruses reported worldwide.     

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province,Iran

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon’s and crow’s samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.