不同动物源脑心肌炎病毒的分离、鉴定和全基因组序列分析

为研究脑心肌炎病毒（encephalomyocarditis virus,EMCV）对不同动物宿主的感染情况。本试验应用改进的＂细胞接种与RT-PCR方法相结合＂技术,成功分离到国内首株地方土猪源EMCV、首株家养野猪源EMCV、1株鼠源EMCV和3株良种猪源EMCV,并进行了全基因组序列测定和分析。结果显示,6株EMCV分离毒的基因组全长从7 724~7 735bp不等,ORF长度均为6 879bp,彼此间核苷酸同源性为99.3%~99.8%,与其他不同动物源EMCV参考毒株的同源性为79.9%~99.9%,与国内猪源、鼠源分离毒的同源性均在99.4%以上;各基因片段中,以VP1和2A变异幅度最大,VP2和3D最为保守;基于EMCV全基因组、ORF和VP1基因序列绘制的系统发育进化树显示,EMCV可分为G1、G2和G3 3个群,猪源EMCV主要分布在G1、G2群,鼠源EMCV在G1、G3群中均有分布;6株EMCV分离毒与其他国内参考毒株同属于G1群,但分布并不完全集中。结果表明,地方土猪和家养野猪可感染EMCV并引起发病,提醒在进行地方品种养殖和野生动物家养时要充分考虑人兽共患疫病传播的生态学;鼠在良种猪、家养野猪和地方土猪EMCV之间的交叉感染、传播与流行中起到重要媒介作用;不同EMCV地方流行株间存在较大的地域差异,其传播具有一定的区域限制性;EMCV在感染不同动物时可能会发生某些突变,以适应新的宿主。     

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.     

Whole genome sequencing of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolated from 4 horses in a veterinary teaching hospital and its ambulatory service

Genomic characterization was conducted on 2 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 2 horses hospitalized during an overlapping period of time and 2 methicillin-sensitive S. aureus (MSSA) strains isolated from 2 distinct horses. Phylogenetic proximity was traced and the genotypic and phenotypic characteristics of the antimicrobial resistance of the strains were compared.Whole genome sequencing of MRSA strains for this report was similar but differed from whole genome sequencing of MSSA strains. The MRSA strains were closely related, belonging to sequence type (ST) 612, spa type t1257, and SCCmec type IVd2B. The MSSA strains were also closely related, belonging to ST1660, spa type t3043, and having no detectable staphylococcal cassette chromosome mec elements. All MSRA and MSSA strains were Panton-Valentine leukocidin negative. There were discrepancies in the genotypic analysis and the antimicrobial susceptibility testing (phenotypic analysis) of MRSA strains for rifampin, trimethoprim-sulfamethoxazole, gentamicin, amikacin, and enrofloxacin.     

Transmission and pathogenicity of encephalomyocarditis virus (EMCV) among rats

Due to the probable role played by rodents as a reservoir for the transmission of the EMC virus to pigs, the experiment reported here was performed in order to assess the transmission rate of EMCV within a rat population. Twenty-five eight-week-old Wistar rats housed in individual plastic cages were experimentally infected either with a Greek myocardial EMCV strain (5 rats with a 0.2 x 10(6) TCID50 dose per rat and 10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally) or a Belgian myocardial EMCV strain (10 rats with a 0.5 x 10(4.5) TCID50 dose per rat, oronasally). Two to five days later, each inoculated rat was moved to a new clean cage and coupled with a contact rat to compare the pathogenicity of the two strains and to estimate the basic reproduction ratio R0, indicating the level of EMCV transmission. During the experiments, faecal virus excretion was measured as well as the serological response against EMCV. After euthanasia, virus isolation was attempted from different rat tissues. Neither strains produced mortality, nor clinical signs and only low titres of neutralising antibodies were found. All contact rats, however, were infected and the virus was isolated from their faeces and from various tissues. Both 10-pair experiments revealed a point estimate for the R0 of infinity (95%-CI for both the Greek and Belgian EMCV strains = 4.48 - infinity), as did the 5-pair experiment with a higher dose of the Greek strain (95%-CI = 1.83 - infinity). Combining the results from the two 10-pair experiments resulted in an estimate for R0 of infinity (95%-CI: 9.87 - infinity). These results indicate that the EMC virus can spread very easily within a rat population by horizontal rat-to-rat transmission (R0 > 1).     

Vaccination of llamas, Llama glama, with an experimental killed encephalomyocarditis virus vaccine

Encephalomyocarditis virus (EMCV) is a pandemic virus that has caused mortality in numerous captive wildlife species worldwide. An experimental killed vaccine was created from two EMCV isolates associated with zoo animal mortality in the southern United States. The vaccine was tested for safety and efficacy in eleven llamas (Llama glama). All animals received an initial vaccination and a second booster vaccination 4 wk later. Serum antibody responses were monitored at initial vaccination and at 4 wks, 8 wk, 6 mo, and 12 mo postvaccination. Eight of the 11 llamas vaccinated experienced at least a 4-fold increase in serum antibody titers to EMCV. Antibody titers of those eight animals remained elevated above prevaccination levels when measured at 12 mo. The experimental killed EMCV vaccine tested may be a useful tool to prevent EMCV infection in llamas when given in 2 doses 4 wk apart, and then revaccinated or with antibody levels monitored annually thereafter.     

Defining issues of space in zoos

The amount of space available to animals in zoos is always limited by a range of factors that has little to do with animal biology. Most zoos exist on confined sites, their absolute boundaries are usually fixed. The starting point in a discussion about an animal's perceived needs is necessarily immediately to be considered in terms of other demands on the space available. Nonetheless, a detailed scientific understanding of the wild biology of a species has come to be regarded as essential to the determination of what constitutes appropriate captive conditions of zoos for that species. An examination of how zoos have confined animals through the last 2 centuries shows a trend away from the closest forms of confinement in cages, toward larger spaces, often with open viewing for visitors. Elephants represent an anomaly. They continue to be maintained in zoos according to traditions involving extraordinarily close human control, as if they were working animals. Looking at traditional captive elephant management practices, however, does not provide any reasonably complete view of elephant needs. Wild biology suggests that zoo elephants should enjoy a naturalistic social life and live more independently than they traditionally have done in zoos. For this, zoo elephants need far more extensive living spaces than those with which they are currently provided.     

猪瘟病毒E2基因部分核苷酸序列的遗传进化关系的研究

本研究利用RT-PCR及测序获得了16株猪瘟病毒E2基因部分核苷酸序列。利用DNAStar软件对其6株已发表的猪瘟病毒毒株序列进行了比较和分析，构建了猪瘟病毒遗传发生树。结果可以看出所绘制的遗传发生树分为2个组群，每个组群分为3个亚组群。13株猪瘟流行毒在2个组群中均有分布，猪瘟石门系强毒和C株属于同一组群、同一亚组群。70～80年代分离的4株猪瘟病毒755和Alfort株（法国）在同一组群，25%和Brescia株（意大利）是同一组群，90年代分离的9株猪瘟病毒33.3%和Alfort株（法国）在同一组群，66.7%和Brescia株（意大利）是同一组群。13株猪瘟流行毒株中7株和C株在同一组群，其中70～80年代的1株，90年代的6株。其余的6株猪瘟流行毒株均在另一组群。不同地区及不同材料C-株疫苗的E2基因的核苷酸序列有一定的差异，GPE株与中国的C株有较近的遗传进化关系，而法国的Thiverval株则与中国C株的遗传进化关系较远。本研究的结果对了解我国猪瘟分子流行病学的特点具有重要的意义。     

Lleida Bat Lyssavirus isolation in Miniopterus schreibersii in France

Bat rabies cases are attributed in Europe to five different Lyssavirus species of 16 recognized Lyssavirus species causing rabies. One of the most genetically divergent Lyssavirus spp. has been detected in a dead Miniopterus schreibersii bat in France. Brain samples were found positive for the presence of antigen, infectious virus and viral RNA by classical virological methods and molecular methods respectively. The complete genome sequence was determined by next‐generation sequencing. The analysis of the complete genome sequence confirmed the presence of Lleida bat lyssavirus (LLEBV) in bats in France with 99.7% of nucleotide identity with the Spanish LLEBV strain (KY006983).     

Prevalence and molecular typing of Giardia spp. in captive mammals at the zoo of Zagreb, Croatia

A total of 131 faecal samples from 57 mammalian species housed at the zoo of Zagreb, Croatia, were tested for the presence of Giardia spp. cysts using epifluorescence microscopy. The overall prevalence (29%) was high, yet all animals were asymptomatic at the time of sampling. Positive samples were characterized by PCR and sequence analysis of both conserved and variable loci, for the identification of Giardia species and G. duodenalis assemblages and genotypes. Assemblages A and C were identified in Artiodactyla, assemblage B in Primates, Rodentia and Hyracoidea, and assemblages A, B, C and D, as well as Giardia microti, in Carnivora. Genotyping at the ITS1-5.8S-ITS2 region, at the triose phosphate isomerase, glutamate dehydrogenase and beta-giardin genes revealed extensive polymorphisms, particularly among assemblage B isolates. A phylogenetic analysis of concatenated sequences showed that isolates from captive mammals housed at the zoo are genetically different from isolates of human and domestic animal origin. This is the first survey in a zoological garden to include a molecular characterization of the parasite, and provides novel sequence data of G. duodenalis from many previously uncharacterized hosts.     

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.     

Anaplasmosis in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum

Two dogs in France were diagnosed with Anaplasma phagocytophilum infection by real-time PCR. The most remarkable hematologic and biochemical findings were severe thrombocytopenia, mild neutrophilia, morulae in neutrophils, and increased serum concentration of the α2-globulin fraction detected by agarose gel electrophoresis of serum proteins. Using sequencing of the partial 16S rRNA and ankA genes, molecular characterization of the A. phagocytophilum strains showed that the organisms from both dogs were identical to the European strains isolated from horses and people. Based on phylogenetic analysis, the ankA gene was more discriminating than the 16S rRNA gene in distinguishing the majority of European and American strains of A. phagocytophilum infecting people and animals. Three isolates of A. phagocytophilum, 1 from Spain (cow) and 2 from Norway (sheep and deer), were external to the European and American clades.     

猪脑心肌炎病毒GXLC株的分离及其3D基因分子特征的分析

采集临床疑似脑心肌炎死亡仔猪的组织作为接种材料,接种于BHK-21细胞系,观察细胞病变（CPE）,并用RT-PCR和间接荧光抗体试验（IFA）进行鉴定,证实分离到1株脑心肌炎病毒（encephalomyocarditis virus,EMCV）,命名为GXLC株。应用RT-PCR方法扩增GXLC株的3D基因,扩增产物克隆入pMD18-T载体后进行测序,对获得的3D基因序列进行分析。序列分析结果表明,GXLC株3D基因全长1380 nt,编码460个氨基酸,含有7个抗原表位。同源性分析结果表明,GXLC株与国内外其它EMCV分离株3D基因核苷酸序列的同源性在84.7%～99.7%之间,氨基酸序列的同源性在96.1%～99.6%之间。遗传进化分析结果表明,基于3D基因核苷酸序列绘制的系统进化树可将所有EMCV分离株分成2个群：Ⅰ群和Ⅱ群,Ⅰ群可再细分为Ⅰa亚群和Ⅰb亚群,其中猪源EMCV在Ⅰ群和Ⅱ群中均有分布,而鼠源EMCV分布在Ⅰ群,人源EMCV分布在Ⅱ群;GXLC株与其它中国分离株均属于Ⅰa亚群。     

Detection of Mycobacterium tuberculosis complex antibodies in free-ranged wild boar and wild macaques in selected districts in Selangor and reevaluation of tuberculosis serodetection in captive Asian elephants in Pahang,Peninsular Malaysia

Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, affecting several domestic animals, wildlife species and humans. The preliminary investigation was aimed to detect antibody against MTBC among indigenous wildlife which are free-ranged wild boar, free-ranged wild macaques and captive Asian elephants in selected areas of Selangor and elephant conservation centre in Pahang, respectively. The results indicate that MTBC serodetection rate in wild boar was 16.7% (7.3–33.5 at 95% confidence interval (CI)) using an in-house ELISA bPPD IgG and 10% (3.5–25.6 at 95% CI) by DPP^®VetTB assay, while the wild macaques and Asian elephant were seronegative. The univariate analysis indicates no statistically significant difference in risk factors for sex and age of wild boar but there was a significant positive correlation (P<0.05) between bovine TB in dairy cattle and wild boar seropositivity in the Sepang district.     

Full genome characterization of Iranian H5N8 highly pathogenic avian influenza virus from Hooded Crow (Corvus cornix), 2017: The first report

During 2014–2017 Clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses (HPAIVs) have spread worldwide. In 2016, an epidemic of HPAIV H5N8 in Iran caused mass deaths among wild birds, and several commercial poultry farms and captive bird holdings were affected and continue to experience problems. Several outbreaks were reported in 2017. One of them is related to Hooded crow (Corvus cornix) in a national park in Esfahan province in 2017. Whole genome sequencing and characterization have been done on the detected H5N8 sample. Based on HA sequencing results, it belongs to 2.3.4.4 clade, and the cleavage site is (PLREKRRKR/G). Phylogenetic analysis of the HA gene showed that the Iran 2017 H5N8 virus clustered within subgroup Russia 2016 2.3.4.4 b of group B in H5 clade 2.3.4.4 HPAIV.On the other hand, the NA gene of the virus is placed in group C of Eurasian lineage. Complete genome characterization of this virus revealed probable reassortment of the virus with East-Asian low-pathogenic influenza viruses. Furthermore, the virus possessed some phenotypic markers related to the increased potential for transmission and pathogenicity to mammals at internal segments. This study is the first full genome characterization H5N8 HPAIV in Iran. The data complete the puzzle of molecular epidemiology of H5N8 HPAIV in Iran and the region. Our study provides evidence for fast and continuing reassortment of H5 clade 2.3.4.4 viruses, that might lead to changes in virus structural and functional characteristics such as the route and method of transmission of the virus and virus infective, pathogenic and zoonotic potential.     

1株库蠓源版纳病毒的分离与全基因组序列分析

为分析云南省虫媒病毒的种类与遗传特征,在云南省师宗县采集库蠓进行病毒的分离与鉴定;通过全长cDNA扩增与高通量测序技术获取病毒全基因组序列,进行序列比对与系统发生树构建。结果显示,从采集的库蠓样本中分离出1株可在C6/36细胞上引起细胞病变的毒株（YNSZ043）,病毒基因组为分节段双链RNA,琼脂糖凝胶电泳呈"2-4-3"的带型特征;电镜观察可见直径为70~80 nm,呈"指环状",表面具有纤维突起的病毒粒子。全基因组测序结果显示,YNSZ043毒株为版纳病毒（Banna virus,BAV）,基因组大小为20 683 bp,由Seg-1（3 762 bp）至Seg-12（861 bp）12个基因节段组成,与中国BAV毒株各基因节段的核苷酸序列相似性在64.8%~99.6%之间,氨基酸序列相似性在58.8%~100%之间,在系统发生树上YNSZ043毒株与中国分离的BAV聚为一簇,形成独立的中国进化支系。对决定BAV基因型的Seg-12分析结果显示,YNSZ043毒株属于A2基因型,该毒株的Seg-5/VP5与越南分离BAV毒株的核苷酸和氨基酸序列相似性高达97.1%和97.6%,表明该毒株的Seg-5基因节段很可能与越南毒株之间发生了基因重配。研究结果丰富了中国BAV的基因组序列,为开展云南省BAV的流行病学研究提供了参考。     

Perfrin,a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.     

Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile

Two cases of fatal enteritis caused by Clostridium difficile in captive Asian elephants are reported from an outbreak affecting five females in the same zoo. Post mortem examination including histopathology demonstrated fibrinonecrotic enterocolitis. C. difficile was isolated by selective cultivation from two dead and a third severely affected elephant. Four isolates were obtained and found positive for toxin A and B by PCR. All isolates were positive in a toxigenic culture assay and toxin was demonstrated in the intestinal content from one of the fatal cases and in a surviving but severely affected elephant. PCR ribotyping demonstrated that the C. difficile isolates shared an identical profile, which was different from an epidemiologically unrelated strain, indicating that the outbreak was caused by the same C. difficile clone. It is speculated that the feeding of large quantities of broccoli, a rich source of sulforaphane, which has been shown to inhibit the growth of many intestinal microorganisms may have triggered a subsequent overgrowth by C. difficile. This is the first report of C. difficile as the main cause of fatal enterocolitis in elephants. The findings emphasize the need to regard this organism as potentially dangerous for elephants and caution is recommended concerning antibiotic treatment and feeding with diets containing antimicrobials, which may trigger an expansion of a C. difficile population in the gut.     

Genomic and functional characterization of fecal sample strains of Proteus cibarius carrying two floR antibiotic resistance genes and a multiresistance plasmid-encoded cfr gene

The objective of this study was to investigate the molecular characteristics and horizontal transfer of florfenicol resistance gene-related sequences in Proteus strains isolated from animals. A total of six Proteus strains isolated from three farms between 2015 and 2016 were screened by polymerase chain reaction (PCR) for known florfenicol resistance genes. Proteus cibarius G11, isolated from the fecal material of a goose, was found to harbor both cfr and floR genes. Whole genome sequencing revealed that the strain harbored two copies of the floR gene: one was located on the chromosome and the other was located on a plasmid named pG11-152. Two floR-containing fragments 4028 bp in length were identical and showed transposon-like structures. The cfr gene was found on a plasmid named pG11-51 and flanked by a pair of IS26s. Thus, mobile genetic elements played an important role in floR replication and horizontal resistance gene transfer. Therefore, increasing attention should be paid to monitoring the spread of resistance genes and resistance in real time.     

A serosurvey of swine and free-living species on Iowa farms for antibodies against encephalomyocarditis virus

Swine and free-living nonporcine species from 20 Iowa swine farms were surveyed for antibodies against encephalomyocarditis virus (EMCV). The microtitration serum neutralization test was used, and antibody titers ≥ 1:8 were considered positive. The overall prevalence in 267 sows in various groups was 37.8% (range 20-86%). The prevalence in sows maintained in total confinement was significantly lower than in sows not maintained in total confinement (p = 0.01). Prevalence in sows was not associated with other herd characteristics or with abundance estimates of rats and/or mice.

Free-living animals tested included 74 domestic cats (Felis domestica), 203 house mice (Mus musculus), 15 mice of the genus Peromyscus, nine Norway rats (Rattus norvegicus), 34 opossums (Didelphis virginiana), 14 raccoons (Procyon lotor), and seven striped skunks (Mephitis mephitis). Of these, the only seropositive animals were two domestic cats.

The results of this study failed to implicate the free-living species surveyed as important reservoirs of EMCV for swine, and suggested that swine themselves and/or some species not included in this survey are the main reservoir of EMCV for swine in Iowa.

     

New insights into the morphology, molecular characterization and identification of Baylisascaris transfuga (Ascaridida, Ascarididae)

Species ranked within the genus Baylisascaris (Ascaridida, Ascarididae) have been implicated in clinical and subclinical intestinal diseases in their natural hosts (e.g., raccoons and bears) as well as in life-threatening larva migrans syndromes in a number of incidental hosts, including humans. Following the diagnosis of Baylisascaris transfuga infestation in two captive polar bears, living in the zoo park of Pistoia (Tuscany, Italy), nematodes (n=300; both sexes) have been characterized by morphological and molecular methods by sequencing and analysing ribosomal (large ribosomal DNA (28S) and internal transcribed spacer region 1 and 2 (ITSs)) and mitochondrial (cytochrome c oxidase subunit 1 (cox1) and cytochrome c oxidase subunit 2 (cox2)) target regions. In addition, seven faecal samples were collected from the animal enclosure and submitted to copromicroscopic and molecular examination. All nematodes were morphologically identified as B. transfuga and their main distinctive features are here presented. No variation in size and nucleotide polymorphisms was detected within each target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this little known nematode infestation in order to apply appropriate anthelmintic strategies.