Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis

Isolates of Pseudomonas aeruginosa from clinical infections in mink were subjected to serotyping and pulsed-field gel electrophoresis (PFGE) using SpeI. A total of 212 isolates of P. aeruginosa from the year 1998 to 2001 were included in this study: 168 isolates from mink obtained from 74 farm outbreaks of haemorrhagic pneumonia. Isolates from mink were separated into 34 distinct clones by PFGE subtyping. All isolates from mink infected during the same farm outbreak were identical, except in one case where two different strains were isolated from mink obtained from the same farm outbreak. P. aeruginosa of specific PFGE types were found to cause clusters of outbreaks on several farms within a few weeks of each other. However, PFGE types of strains causing clusters of farm outbreaks changed from year to year. These results suggest that some outbreaks of haemorrhagic pneumonia are caused by pathogenic strains of P. aeruginosa spread between farms and animals either mechanically, or through feed or water from a common source, rather than by random nosocomial infections with strains from the farm environment.     

Mastitis in mink due to Staphylococcus aureus and Escherichia coli

An epizootic of mastitis in mink due to Staphylococcus aureus and Escherichia coli associated with food poisoning was studied on a Connecticut ranch with 3,500 mink. In the course of the epizootic, approximately 2,000 mink kits and 480 adult mink, mostly nursing females, died within 10 days. Affected females had swollen mammary glands due to acute mastitis; S. aureus was isolated in pure culture from 2 mink and E. coli in pure culture from a 3rd. Staphylococcus aureus was isolated from the organs of 1 mink kit, S. aureus and E. coli from a 2nd kit, and E. coli from a 3rd. The organs of the remaining 7 kits examined did not contain bacteria. Both isolates were pathogenic when inoculated intraperitoneally into mice and mink, causing fatal septicemia within 16 to 24 hours. The meat from a septicemic bovine carcass fed prior to the epizootic was considered a possible source of infection, since it was found to be heavily contaminated with E. coli, S. aureus, and Streptococcus spp.     

Characterization of Escherichia coli Isolates Incriminated in Colisepticaemia in Mink

Colisepticaemia is a major health and economic concern for the mink industry, yet little information is available about the Escherichia coli that cause this disease. In this study, 40 E. coli, isolated from mink clinically diagnosed with colisepticaemia that had been submitted to the North Dakota State University Veterinary Diagnostic Laboratory, were randomly selected for characterization. These isolates were serotyped and screened for resistance to 18 antimicrobials, possession of transmissible R plasmids, and the presence of several virulence traits or genes using bioassays or the polymerase chain reaction. Several serotypes were identified that have previously been associated with septicaemia in other animal species. The majority of the isolates exhibited multiple antimicrobial resistance phenotypes. Common resistance phenotypes observed included those to tetracycline, sulfamethoxazole, streptomycin, ampicillin and kanamycin. Several of the isolates that could be studied by conjugation contained transmissible R plasmids coding for multiple antimicrobial resistance phenotypes. About half of the isolates produced colicin; all produced enterobactin; and all but one-quarter produced aerobactin. None of the isolates tested produced enterohaemolysin, and one-fifth were considered to be beta haemolytic. About half appeared to contain the gene encoding cytotoxic necrotizing factor-I; three contained the gene encoding EAE, but none appeared to contain the genes coding for LT, Sta/b, SLT-I/II or CNF-II toxins or K99 antigen. Approximately one-third of the isolates elaborated capsule. The results show that the E. coli isolates implicated in mink colisepticaemia possess similar virulence traits and antimicrobial resistance phenotypes to those associated with diarrhoeal diseases in food animals.     

Usage of antimicrobials and occurrence of antimicrobial resistance among bacteria from mink

The usage of antimicrobials for treatment of mink and the occurrence of antimicrobial resistance among the most important bacterial pathogens in mink was investigated. The aim of the study was to provide data, which may serve as a basis for the formulation of recommendations for prudent use of antimicrobials for mink. A total of 164 haemolytic staphylococci, 49 haemolytic streptococci, 39 Pseudomonas aeruginosa, 13 Pasteurella multocida, and 1093 Escherichia coli isolates from Danish mink were included in the study. A high frequency of resistance among S. intermedius was found for tetracyclines (54.7%), followed by penicillin (21.7%), lincosamides (20.4%), macrolides (19.1%), and spectinomycin (18.5%). Very low frequencies of resistance were recorded for other antimicrobials. The highest frequency among the E. coli isolates was recorded for ampicillin, streptomycin, sulphonamides, and tetracyclines, whereas resistance to other antimicrobials was rare. All P. aeruginosa were sensitive to gentamicin and colistin and sensitive or intermediate to enrofloxacin, whereas most isolates were resistant to all other antimicrobials. All P. multocida and haemolytic streptococci were sensitive to penicillin. There was a steady increase in the use of antimicrobials during the period 2001-2006, the majority of the prescribed amount being extended spectrum penicillins followed by aminoglycosides, sulphonamides with trimethoprim, and macrolides.     

Comparisons of feline panleukopenia virus, canine parvovirus, raccoon parvovirus, and mink enteritis virus and their pathogenicity for mink and ferrets

Parvoviruses from mink (mink enteritis virus [MEV]), cats (feline panleukopenia virus [FPV]), raccoons (raccoon parvovirus [RPV]), and dogs (canine parvovirus [CPV]) were compared. Restriction enzyme analysis of the viral replicative-form DNA revealed no consistent differences between FPV and RPV isolates, but CPV and MEV isolates could be distinguished readily from other virus types. Feline panleukopenia virus, RPV, and MEV, but not CPV, replicated to high titers in mink. However, on the first passage, disease and microscopic lesions were observed only in mink inoculated with MEV. Feline panleukopenia virus and RPV isolates replicated in ferrets, but disease or microscopic lesions were not observed. Feline panleukopenia virus and RPV isolates could be passaged repeatedly in mink and ferrets. Virulence of FPV and RPV isolates was low compared with that of MEV, and only a single mink inoculated with FPV or with RPV developed clinical disease on the sixth passage of virus.     

阿留申病细小病毒的分离及VP2基因遗传衍生分析

水貂阿留申病（Aleutian disease of mink，AD）是水貂的一种慢性传染病，病原为阿留申病细小病毒（Aleutian mink disease parvovirus，AMDV），属细小病毒科、细小病毒属。AD自1940年发现以来至今60年里，已经普遍存在于世界各地人工养殖的水貂种群中。对水貂养殖业造成了不可估量的经济损失。     

Staphylococcus spp., Streptococcus canis,and Arcanobacterium phocae of healthy Canadian farmed mink and mink with pododermatitis

Pododermatitis is a disease of concern for mink breeders in Canada and worldwide, as it causes discomfort and lowers the breeding rates on farms affected by the disease. Unfortunately, the etiology and pathogenesis of pododermatitis are still unknown. In this study, we compared Staphylococcus spp. and Streptococcus canis isolates from healthy mink with isolates from animals with pododermatitis on 2 farms in Ontario. Almost all hemolytic Staphylococcus spp. isolated were shown to be Staphylococcus delphini Group A by 16S ribosomal ribonucleic acid (rRNA) sequence analysis and polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) did not reveal any S. delphini or S. canis clonal lineages specifically associated with pododermatitis, which suggests that these bacteria do not act as primary pathogens, but does not dismiss their potential roles as opportunistic pathogens. While S. delphini and S. canis were the most prevalent bacterial pathogens in mink pododermatitis, they were also present in samples from healthy mink. Arcanobacterium phocae is occasionally isolated from pododermatitis cases, but is difficult to recover with conventional culture methods due to its slow growth. A quantitative real-time PCR was developed for the detection of A. phocae and was tested on 138 samples of footpad tissues from 14 farms. The bacterium was detected only in pododermatitis-endemic farms in Canada and was at higher concentrations in tissues from infected footpads than in healthy tissues. This finding suggests that A. phocae is involved in the pathogenesis of pododermatitis.     

水貂绿脓杆菌分离株的血清型分析和Eric-PCR指纹图谱多样性研究

为了掌握山东地区水貂出血性肺炎流行情况,从2012年山东省内多个养貂社区进行水貂出血性肺炎流行病学调查。从病貂中分离获得48株绿脓杆菌,分别测定了分离株的生化特性、血清型以及部分菌株对小鼠与水貂的半数致死量（LD50）,同时运用Eric-PCR方法对分离进行了进行分型。结果显示,48株分离菌株与标准菌株生化特性基本一致,血清型G型为36株,血清型Ⅰ型为7株,血清型C型为4株;48株分离株大多数对恩诺沙星最敏感;动物试验显示,48株分离株对小鼠与水貂均能致病,所选4株分离株对小鼠与水貂的LD50有差异,且水貂对所选菌株显著敏感于小鼠;分子分型分为13个基因型。结果表明,该地区水貂出血性肺炎病原呈现遗传多样性。本试验为水貂出血性肺炎的防治提供了理论依据。     

Diversity and stability of Aleutian mink disease virus during bottleneck transitions resulting from eradication in domestic mink in Denmark

Aleutian mink disease (plasmacytosis) virus (AMDV) in domestic mink (Neovison vison) has been subject to eradication in Denmark since 1976. In 2001, approximately 5% of Danish mink farms were still infected and all were located in the northern part of the peninsula of Jutland. In the present study a total of 274 Danish isolates of AMDV collected during the two seasons of 2004 and 2005 were characterized by partial sequencing of the coding region of the non-structural (NS) proteins. Older AMDV isolates from Denmark, available, were also included. The Danish isolates represent a very homogenous cluster compared with Swedish, Finnish and Dutch isolates and seem to represent a minor fraction of the genetic diversity previously found in Denmark. Stability of nucleotide deviations reveals that the purifying selection of bottlenecks imposed on the AMDV population in Denmark by the stamping out policy for more than 6 years exceeds the rate of mutation driven diversity. Among the isolates from farms in northern Jutland two distinct types could be identified and within each of them a number of sub-types which were all useful in tracking spread of infections. Infection at a farm the preceding season was a predisposing risk parameter for disease outbreak at a farm, and strain identity substantiates the suggestion that inadequate disinfection is involved in the recurrence of outbreaks. In cases of new introductions to farms it is indicated that contact including transport between farms played a most significant role.     

Cloning and Sequencing of cnf1 from Escherichia coli Incriminated in Mink and Bovine Colibacillosis

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.     

猪链球菌精氨酸脱亚氨酸酶序列分析及其PCR检测

对9株猪链球菌2型重庆分离株的精氨酸脱亚氨酸酶基因进行克隆测序,结果表明该基因长度为1231bp,与Genbank发表的该基因序列相比,核苷酸同源性高于99%,推导的氨基酸同源性高于96%。根据精氨酸脱亚氨酸酶基因的测序结果建立扩增片段长度为237bp的PCR检测方法,35株猪链球菌致病株中,30株精氨酸脱亚氨酸酶基因的PCR检测阳性,有5株精氨酸脱亚氨酸酶基因的PCR检测阴性;14株正常猪扁桃体分离株中,11株为精氨酸脱亚氨酸酶基因的PCR检测阳性,3株为精氨酸脱亚氨酸酶基因的PCR检测阴性;猪链球菌1型、7型、9型、13型、1/2型各一株,均能扩增出精氨酸脱亚氨酸酶基因的片段。     

水貂绿脓杆菌的分离与鉴定

2011~2013年间,从山东省、河北省和江苏省等地的多个貂场疑似水貂出血性肺炎病貂的肺脏、肝脏和脾脏中分离到425株疑似绿脓杆菌。用绿脓杆菌的OprF和PEA基因的PCR引物对分离菌株的目的基因扩增,经核苷酸测序确定其均为绿脓杆菌序列。通过日本生研株式会社血清学分型系统进行分型：G型376株,占88.5%;B型34株,占8%;C型12株,占2.8%;E型3株,占0.7%。取其中6株细菌（G血清型3株,其他血清型各1株）进行详细生化试验,结果显示该6株菌均符合绿脓杆菌的生化特性,并且从这6株中选4株细菌（每血清型各1株）腹腔接种小白鼠和气管内接种水貂,结果表明4株菌对其均有致病性。     

In vitro activity of 24 antimicrobial agents against Staphylococcus and Streptococcus isolated from diseased animals in Japan

A total of 88 Staphylococcus and 61 Streptococcus isolates from diseased animals throughout Japan were examined in 2000 for the minimum inhibitory concentrations of 24 different antimicrobials by the agar dilution method standardized by the Japanese Society of Chemotherapy. The resistance rates to aminobenzylpenicillin (36.4%) and benzylpenicillin (35.2%) were high in Staphylococcus isolates, and those to oxytetracycline (45.9%) and kanamycin (21.3%) were high in Streptococcus isolates. Two isolates resistant to oxacillin harbored the mecA gene. One was Staphylococcus epidermidis derived from a pig with arthritis, and the other Staphylococcus cohnii from a head of cattle with mastitis.     

Isolation of reoviruses from mink]

In this first report of the isolation of reovirus from mink, isolates were obtained from 18 to 26 young mink with viral enteritis, by using cultures of cat kidney cells. The isolated also produced cytopathic changes in cell cultures of mink kidney, dog kidney, piglet kidney, calf kidney, bovine embryonic kidney and calf testis. A characteristic feature was the formation of eosinophilic inclusion bodies in the cytoplasma of culture cells. Haemagglutination tests were negative with erythrocytes from cat, rabbit, pig, horse and cattle. Attempts to infect old and young mink, kittens and ferrets with tissue culture material failed. It was not known to what extent this second infection with reovirus influenced the course of mink viral enteritis.     

Outbreak of Salmonella Dublin-associated abortion in Danish fur farms

Outbreaks of Salmonella Dublin infections were recorded in 25 Danish mink and fox farms. All farms suffered extensive disease problems; clinical and pathological observations included abortion, stillbirths, necrotizing endometritis, and increased mortality. By genotyping with pulsed-field gel electrophoresis and amplified fragment length polymorphism, all isolates of S. Dublin had indistinguishable patterns. The outbreaks took place during April and May, around the time of whelping. During this period, mink are particularly susceptible to Salmonella infections. All affected farms were served by the same feed factory and it was concluded that a batch of contaminated feed was responsible for the outbreaks, although repeated culture of feed samples collected during the same period were negative. No other likely source could be identified. The results emphasize the importance of strict hygiene measures at feed factories and the proper use of ingredients of known Salmonella status, in particular during the whelping season. Infected mink farms did not have a higher risk of outbreak of salmonellosis in the year following the outbreak.     

Distribution of Streptococcus suis capsular types in Quebec and western Canada

Of a total of 561 isolates of Streptococcus suis recovered from various tissues of diseased pigs, 464 were from veterinary laboratories in Quebec and 97 were from western Canada, particularly Alberta (84). Almost 83% of all these isolates belonged to the 23 known S. suis capsular types. There was no marked difference between the two groups of isolates. Capsular type 2 was the most prevalent and represented 32% of all isolates. The other important capsular types were, in decreasing order, 3, 1/2, 8 and 4. Lungs, brain, and meninges were the source of the majority of isolates. More than 40% of all S. suis isolates were found in pure culture. The number of isolations of this microorganism was higher in piglets aged five to eight weeks than in any other age group.     

Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA

Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.     

Isolation,Identification and Drug Sensitivity Analysis of Pseudomonas aeruginosa from Minks

In order to provide therapeutical guidance for drug admistration,the bacteria from five dead minks suffering with typical symptoms provided by mink farms in Shandong province were isolated and identified,and the drug sensitivity was tested.The bacteria were isolated with TSA plates,and identified using biochemical methods and PCR assay.The drug sensitivity of the isolates to antimicrobial agents was investigated using K-B method.PCR was used to detect the resistance genes.A total of 5 Pseudomonas aeruginosa isolates were obtained from sick minks.The drug sensitivity results showed that 5 strains were sensitive to fluoroquinolone,the third and fourth generations cephalosporin drugs,but were resistant to aminoglycoside,tetracycline,chloramphenicol,penicillin,the first and second generations cephalosporin drugs.There were six resistance genes were detected,aadA1,aac(3')-Ⅱc,aac(6')-Ⅰb,tetA,tetK and cat2.All of the isolates were detected more than three kinds of resistance genes.The result showed that 5 strains were all Pseudomonas aeruginosa,and Pseudomonas aeruginosa was the main cause of mink hemorrhagic pneumonia.The resistance of 5 strains were very serious and mainly for multiple drug resistance phenomenon.The resistance genes detected in the mink were various,and could cause the resistance to the drugs.     

广东地区猪链球菌的耐药性特点及四环素耐药基因携带情况

猪链球菌(Streptococcus suis,SS)是危害世界养猪业的重要细菌性病原菌之一,本研究旨在了解猪链球菌临床分离株的耐药性特点和四环素耐药基因的携带情况,为临床上该病的防控提供科学依据。本试验采用K-B法和CLSI推荐的肉汤微量稀释法检测猪链球菌广东分离株对18种抗菌药物的耐药性。结果显示,被检菌株对四环素类、磺胺类和氨基糖苷类抗菌药物表现较强的耐药性,尤其对四环素类药物的耐药率高达98.2%;对头孢菌素类药物比较敏感;菌株都耐受6种或6种以上的抗菌药物,其中以6和14重耐药菌株的数量最多,分别占20%和17%。本试验设计了4对引物检测四环素耐药基因携带情况,结果发现56株菌中以携带tetM基因为最多(85.71%)。结果表明tetM很可能是介导广东SS分离株对四环素产生极高耐药率的主要原因之一。     

水貂绿脓杆菌分离鉴定及药物敏感性分析

本试验旨在通过对山东某水貂养殖场送检的5只具有典型出血性肺炎症状的病死水貂进行细菌分离鉴定及耐药情况分析,为临床用药和治疗提供依据和参考。通过细菌分离纯化、生化鉴定和PCR方法对分离菌株进行鉴定,采用K-B药敏法检测分离菌株对常用药物的敏感性,并通过PCR方法检测分离菌株耐药基因的携带情况。经鉴定共分离得到5株绿脓杆菌,药物敏感性试验结果显示,5株绿脓杆菌对氟喹诺酮类药物、第3代和第4代头孢类药物较敏感,对氨基糖苷类、四环素、氯霉素、青霉素类、第1代和第2代头孢类药物耐药。耐药基因检测结果显示,5株绿脓杆菌共检测出6种耐药基因aadA1、aac(3')-Ⅱc、aac(6')-Ⅰb、tetA、tetK、cat2。且每株分离菌均检测出3种以上耐药基因。结果表明,分离的5株菌株均为绿脓杆菌,主要引起水貂出血性肺炎。分离菌株耐药性较严重,主要表现为多重耐药现象。菌株携带的耐药基因呈多样性,可引起菌株对相应药物产生耐药现象。