Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: Comparison with other isolates and genotyping methods

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.     

Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California

Corynebacterium pseudotuberculosis biovar Equi is an important pathogen of horses. It is increasing in frequency in the United States, and is responsible for various clinical forms of infection, including external abscesses, internal abscesses of the abdominal or thoracic cavities, and ulcerative lymphangitis. The host/pathogen factors dictating the form or severity of infection are currently unknown. Our recent investigations have shown that genotyping C. pseudotuberculosis isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR is useful for understanding the evolutionary genetics of the species as well for molecular epidemiology studies. The aims of the present study were to assess (i) the genetic diversity of C. pseudotuberculosis strains isolated from horses in California, United States and (ii) the epidemiologic relationships among isolates. One hundred and seven C. pseudotuberculosis biovar Equi isolates from ninety-five horses, and two C. pseudotuberculosis biovar Ovis strains, C. pseudotuberculosis ATCC 19410^T type strain and C. pseudotuberculosis 1002 vaccine strain, were fingerprinted using the ERIC 1 + 2-PCR. C. pseudotuberculosis isolated from horses showed a high genetic diversity, clustering in twenty-seven genotypes with a diversity index of 0.91. Minimal spanning tree showed four major clonal complexes with a pattern of temporal clustering. Strains isolated from the same horse showed identical ERIC 1 + 2-PCR genotype, with the exception of two strains isolated from the same animal that showed distinct genotypes, suggesting a co-infection. We found no strong genetic signals related to clinical form (including internal versus external infections). However, temporal clustering of genotypes was observed.     

Genomic characterization of Italian Clostridium botulinum group I strains

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.     

Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D

Cattle botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D resulting in economic losses. Vaccination is the most effective way to control botulism. However, the commercially available vaccines are difficult and hazardous to produce. Neutralizing antibodies against the C-terminal fragment of the BoNT heavy chain (H_C) are known to protect against lethal doses of BoNTs. We report the vaccination of cattle with a previously tested recombinant chimera consisting of Escherichia coli heat-labile enterotoxin B subunit and the H_C of BoNTs C and D. Vaccinated animals produced neutralizing antibodies against serotypes C and D averaging 5 ± 0 and 6.14 ± 1.06 IU/mL, respectively. For BoNT D, the titers were greater than those measured for the commercial vaccine, which induced titers of 5 ± 0 and 2.85 ± 1.35 against the respective serotypes, suggesting that this chimera is effective against cattle botulism.     

中国炭疽芽胞杆菌多位点可变数目串联重复序列分析基因型多态性

目的 分析中国炭疽芽胞杆菌多位点可变数目串联重复序列分析（MLVA）基因型多态性,建立中国炭疽芽胞杆菌MLVA基因型数据库。方法 收集1947年以来中国不同来源的炭疽芽胞杆菌分离菌株,采用MLVA15分型策略进行基因型鉴定,对基因型进行统一编号和命名,分析不同来源菌株MLVA基因型的分布特征,同时通过Bionumerics软件绘制聚类图,分析不同基因型间的遗传关系。结果 MLVA15分型可将533株中国炭疽芽胞杆菌分为4群91个基因型,其中54个基因型为中国独有的基因型。新命名的基因型MLVA15-CHN1~MLVA15-CHN6广泛分布于中国不同地区、不同年代和宿主,其余基因型具有地区和年代特征性。结论 本研究建立了中国炭疽芽胞杆菌MLVA基因型数据库,掌握了MLVA基因型分布特征及其遗传多态性,为中国炭疽疫情防控及分子溯源技术提供了参考依据。     

A case of infant botulism associated with honey feeding in Italy

A case of infant botulism in a 9 week-old female is described. A strain of C. botulinum type B was isolated from the feces of the baby. The epidemiologic study detected in a sample of home canned honey Clostridium botulinum spores of the same serotype that was isolated from the patient. The honey had been used only to sweeten the pacifier of the baby. This is the first case of infant botulism in Europe linked conclusively to honey.Corresponding author.     

Molecular epidemiology of Coxiella burnetii in French livestock reveals the existence of three main genotype clusters and suggests species-specific associations as well as regional stability

Q fever is a worldwide zoonosis caused by the bacterium Coxiella burnetii. In domestic ruminants, Q fever main clinical manifestations are abortions. Although the clinical signs may differ between ruminant species, C. burnetii's genetic diversity remains understudied in enzootic areas. Here, we focused on France, where Q fever is enzootic, with the aims to (a) identify potential associations between C. burnetii genotypes and ruminant host species; (b) assess the distribution of C. burnetii genotypes both within French farms and across France's major livestock-farming regions; and (c) suggest a subset of markers for future genotypic studies. We used DNA samples collected between 2006 and 2015 from 301 females (160 cows, 76 ewes, 65 goats) aborted of Q fever within 7 different farming regions. C. burnetii diversity was determined using a multiple-locus variable-number of tandem repeat analysis (MLVA) considering 17 markers. Using a phylogenetic approach, we identified 3 main genotypic clusters divided into 12 sub-clusters. These clusters were significantly associated with ruminant species: almost all the cattle genotypes were found in a “cattle-specific” cluster whereas small ruminants genotypes essentially grouped into the two other clusters. The clusters also proved stable over space and time, some genotypes being more specifically observed in certain farming regions. We also observed some within-farm diversity but this diversity was restricted to a same genotypic cluster. Finally, we identified 6 MLVA markers that maximized the representativeness of the diversity described. Overall, we highlighted that molecular epidemiology is a relevant approach to assess C. burnetii's genetic diversity and to reveal the existence of species-specific associations and regional stability. These results will be valuable in the field to trace genotype circulation among ruminants and from ruminants to humans. Ultimately, the potential links between genotypes and virulence traits need to be investigated to adapt control measures in livestock farms.     

A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes

Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA₁₃ scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential.     

Phylogenetic Analysis of Enterohemorrhagic Escherichia coli O157, Germany, 1987�C2008

Multilocus variable number tandem repeat analysis (MLVA) is a subtyping technique for characterizing human pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157. We determined the phylogeny of 202 epidemiologically unrelated EHEC O157:H7/H^– clinical isolates through 8 MLVA loci obtained in Germany during 1987–2008. Biodiversity in the loci ranged from 0.66 to 0.90. Four of 8 loci showed null alleles and a frequency <44.1%. These loci were distributed among 48.5% of all strains. Overall, 141 MLVA profiles were identified. Phylogenetic analysis assigned 67.3% of the strains to 19 MLVA clusters. Specific MLVA profiles with an evolutionary persistence were identified, particularly within sorbitol-fermenting EHEC O157:H^–.These pathogens belonged to the same MLVA cluster. Our findings indicate successful persistence of this clone.     

Multi-locus variable-number tandem repeat analysis (MLVA) reveals heterogeneity of Mycobacterium bovis strains and multiple genotype infections of cattle in Ethiopia

Bovine tuberculosis (BTB) remains a major threat to animal and human health, and obstructs international and inter-regional livestock trade in Ethiopia. Many aspects of epidemiology of BTB and its causative agent, Mycobacterium bovis (M. bovis) are not well known. Aims of the study were to elucidate molecular characteristics of M. bovis strains using MLVA typing method. Further aim was to determine infection pressure associated with occurrence of multiple genotypes in individual infected cattle. Data and samples were collected in the period July 2006–January 2007 in cattle slaughtered at five representative abattoirs across the country. Molecular investigation of the isolates was carried out using multilocus variable-number tandem-repeat analysis (MLVA) of 28 variable numbers of tandem repeat (VNTR) loci, and the results were compared to spoligotyping. This study is believed to contribute to the knowledge of molecular genetics and epidemiology of M. bovis in Ethiopia and elsewhere with similar BTB epidemic situation and livestock production settings. Four-hundred and six tissue samples from 337 carcasses revealing gross pathologic lesions compatible with BTB were collected from five abattoirs. Fifty-eight isolates obtained from cultured samples were subjected to region of difference (RD) analysis and MLVA typing. RD confirmed all isolates as being M. bovis. MLVA revealed a high heterogeneity of M. bovis (19 genotypes) and the discriminatory power of MLVA was higher than for spoligotyping (Hunter–Gaston Diversity Index (HGDI) 0.92 vs. 0.82). Adoption of the nine VNTR loci with ⩾3 alleles provided good differentiation between the isolates. However, differentiation was optimized when MLVA was combined with spoligotyping (HGDI = 0.99). MLVA confirmed infections with multiple genotypes in 38.5% (10/26) of individual animals. In conclusion, the usefulness of MLVA for genotyping M. bovis in high prevalence settings was demonstrated. BTB in Ethiopia is caused by heterogeneous populations of M. bovis and individual carcasses were often infected with different genotypes, indicating a high infection pressure perhaps related to the absence of protective immunity conferred by infection.     

Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia

Leptospira spp. are the causative agents of a worldwide zoonosis, leptospirosis, maintained by various mammals. Each Leptospira serovar is frequently associated with a particular maintenance host, and recently, Leptospira genotype–host association has also been suggested to limit serovars to restricted areas. We investigated the molecular characteristics of L. interrogans and L. borgpetersenii which were isolated from small feral and wild animals in four East Asian states using multiple-locus variable-number tandem repeat analysis (MLVA).MLVA using 11 loci was performed on 110 L. interrogans serogroups from Japan (79 strains of 5 serogroups from 3 animal species), Philippines (21; 3; 2), Taiwan (7; 2; 3), and Vietnam (3; 1; 1). A MLVA method using 4 loci for L. borgpetersenii was established and performed on 52 isolates from Japan (26; 3; 7), Philippines (13; 1; 2), and Taiwan (13; 1; 3). In L. interrogans, serogroups Autumnalis and Hebdomadis appeared more genetically diverse than serogroups Bataviae, Grippotyphosa, Icterohaemorrhagiae, Pomona, or Pyrogenes. The former serogroup strains with the exception of one Hebdomadis strain were isolated from Apodemus speciosus while all the latter serogroup strains with the exception of Grippotyphosa were isolated from Rattus norvegicus. L. borgpetersenii was isolated from at least 11 animal species while L. interrogans was isolated from five species, which might suggest a wider host range for L. borgpetersenii. Broad host preference in a single genotype was also observed, which colonized not only different species of the same genera but also multiple animal genera.This study demonstrates that there may be variability in the range of genetic diversity among different Leptospira serogroups, which may be attributed to maintenance host animals and environmental factors.     

Genetic diversity of Francisella tularensis in Poland with comments on MLVA genotyping and a proposition of a novel rapid v4-genotyping

We investigated the genotypes of Francisella tularensis (F. tularensis) strains isolated in Poland during the period 1953–2013 and studied their genetic relationship to F. tularensis strains isolated in other countries using MLVA. We examined the mosquito and tick samples collected in Poland for the presence of F. tularensis DNA using PCR. Our results revealed a high genetic diversity among the strains of F. tularensis collected from Poland, suggesting that the bacterium is commonly found in the environment. However, we did not detect F. tularensis DNA in ticks and mosquitoes, showing that the arthropod bites might not be the main source of infection.We also propose the application of a practical assay called v4-genotyping that can be directly performed on the clinical and environmental samples.In addition, we discovered genetic variations among Schu S4 reference strains used in various laboratories and showed that MLVA analysis should not be based on amplicon sizes only because point mutations occurring within the MLVA loci might not always be manifested by a change in the amplicon size.     

Evaluation of Brucella abortus S19 vaccines commercialized in Brazil: Immunogenicity,residual virulence and MLVA15 genotyping

Live attenuated Brucella abortus S19 is the most effective vaccine against brucellosis in cattle. The assessment of the immunological parameters is essential to guarantee the biological quality of live anti-bacteria vaccines. The evaluation of genetic stability of live bacterial vaccines is also important in quality control. The aims of the present study were to compare (i) the immunogenicity and residual virulence, and (ii) the genotypic profile (MLVA15) of the eight S19 vaccines commercialized in Brazil to the USDA S19 reference strain. Two batches of each of the eight S19 commercial vaccines used in Brazil (A–H) were tested. They were submitted to the potency and residual virulence in vivo tests recommended by OIE and typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) described for Brucella spp. Our results demonstrated that all S19 vaccines commercialized in Brazil would be approved by Brazilian and OIE recommendations for potency and residual virulence. Furthermore, the S19 vaccine is genetically very homogeneous, as all but two batches (from the same manufacturer) tested showed identical MLVA15 profile. The two batches with different profiles presented six repeat units in locus Bruce07, instead of the five found in all other strains, including the USDA S19 reference strain. Although presenting a slightly different profile, this vaccine was also protective, as demonstrated by the immunogenicity and residual virulence assays performed. Therefore, the commercial Brazilian S19 vaccines were in accordance to Brazilian and international standards for immunogenicity and residual virulence tests. Moreover, our results also show that MLVA could be a useful inclusion to the list of in vitro tests required by the official control authorities to be applied to the commercial S19 vaccines, as an efficient assay to guarantee the quality and stability of the vaccine strains.     

An investigation into the cause of the 1983 whooping cough epidemic in the Netherlands

Despite more than 50 years of vaccination, whooping cough is still an endemic disease in the Netherlands with regular epidemic outbreaks. In the last 20 years, two periods of increased notifications were observed. The causes of the increased notifications in the first period, from 1983 to 1987, are contentious. At the time it was suggested to be a surveillance artifact, caused by changes in diagnostic procedures and increased awareness. An alternative explanation, a reduction in the vaccine dose, was downplayed at the time. The aim of this study was to reinvestigate the causes of the increased notifications by identifying changes in the Bordetella pertussis population. B. pertussis strains, isolated from 1965 to 1992, were characterized by means of fimbrial serotyping, multiple-locus sequence typing of virulence genes (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Shifts in fimbrial serotypes and MLVA types were associated with changes in vaccine dose and increased number of notifications. One to three years after lowering of the vaccine dose, the predominant fimbrial serotype changed from Fim3 to Fim2, and the reverse trend was observed when the vaccine dose was increased. Significantly, changes in fimbrial serotypes were evident at least seven years before the increase in notifications. Our results provide evidence that the change in vaccine dose affected host immunity and, consequently, contributed to an increase in pertussis morbidity. Further, we show that MLVA and fimbrial serotyping of strains can be used as early warning for pertussis epidemics.     

Botulism surveillance in Italy: 1992–1996

Though a relatively rare disease, botulism can be a serious problem of public health, particularly when connected with the consumption of industrial canned food; moreover, in the last years the shortage of botulism antitoxin has caused some concern in the Public Health Authorities. This work presents the results of a five-year surveillance of botulism in Italy, with the distribution of the cases by Regions (first level administrative units which, in Italy, have administrative and legislative competencies in the sanitary field) and by vehicle of transmission. All the relevant and confirmed botulism outbreaks that occurred in the period under consideration are described.     

Foodborne Botulism in Canada, 1985–2005

During 1985–2005, a total of 91 laboratory-confirmed outbreaks of foodborne botulism occurred in Canada; these outbreaks involved 205 cases and 11 deaths. Of the outbreaks, 75 (86.2%) were caused by Clostridium botulinum type E, followed by types A (7, 8.1%) and B (5, 5.7%). Approximately 85% of the outbreaks occurred in Alaska Native communities, particularly the Inuit of Nunavik in northern Quebec and the First Nations population of the Pacific coast of British Columbia. These populations were predominantly exposed to type E botulinum toxin through the consumption of traditionally prepared marine mammal and fish products. Two botulism outbreaks were attributed to commercial ready-to-eat meat products and 3 to foods served in restaurants; several cases were attributed to non-Native home-prepared foods. Three affected pregnant women delivered healthy infants. Improvements in botulism case identification and early treatment have resulted in a reduction in the case-fatality rate in Canada.     

An outbreak in Italy of botulism associated with a dessert made with mascarpone cream cheese

In the late 1996, an outbreak of botulism affected eight young people (age of patients ranged from 6 to 23 years) in Italy. The onset of the illness was the same for all of these patients: gastrointestinal symptoms (nausea and vomiting) followed by neurologic symptoms. The most common neurologic symptoms were dysphagia, respiratory failure (100%), diplopia (87%), dysarthria, ptosis (75%) and mydriasis (50%). All patients required mechanical ventilation. Botulinum toxin was detected from two of respectively five sera and six stool samples analysed, while spores of Clostridium botulinum type A were recovered from all patient' faeces. The epidemiological investigation led to suspect a commercial cream cheese (mascarpone) as a source of botulinum toxin: indeed, it had been eaten by all the patients before onset of the symptoms, either alone or as the (uncooked) ingredient of a dessert, tiramisù. Botulinum toxin type A was found in the tiramisù leftover consumed by two patients and in some mascarpone cheese samples collected from the same retail stores where the other patients had previously bought their cheeses. A break in the cold-chain at the retail has likely caused germination of C. botulinum spores contaminating the products, with subsequent production of the toxin. One of the patients died, while the others recovered very slowly. Prompt international alerting and recall of the mascarpone cheese prevented the spread of the outbreak due to the wide range of distribution, demonstrating the importance of a rapid surveillance system. None of the people complaining of symptoms after the public alert resulted positive for botulinum spores and toxin.     

Evaluation and Selection of Multilocus Variable-Number Tandem-Repeat Analysis Primers for Genotyping Brucella abortus Biovar 1 Isolated from Human Patients

ObjectivesBrucellosis is the most common bacterial zoonosis in the world. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is a molecular method for genotyping bacterial species. Brucella abortus biovar I was isolated from most of the brucellosis-suspected patients in Korea. This study was conducted to investigate the ability of various MLVA primers that are used for molecular typing B. abortus isolates and for analyzing their epidemiological data.MethodsA total of 80 human isolates of B. abortus biovar I isolated from human patients and the reference strain were used for MLVA. Genetic diversity was determined by calculating the Simpson's diversity index (DI) of each VNTR locus. The Brucella strains were subcultured 30 times to determine the stability of each locus. The DNA of the strains cultivated in each passage was extracted and subjected to MLVA for further investigation.ResultsThe 15 VNTR loci were selected based on high DI values. The DIs of the 15 VNTR loci showed considerable discrimination power ranging from 59% for Bruce 43 to 87% for Bruce 22. Bruce 09, Bruce 11, Bruce 16, Bruce 42, and Bruce 43 were confirmed to remain stable in vitro among the 15 VNTR loci selected.ConclusionThe results of this study suggest that the five loci subsets may be a useful epidemiological tool for investigating B. abortus biovar 1 outbreak.     

Brucella suis biovar 2 multi locus sequence type ST16 in wild boars (Sus scrofa) from Abruzzi region,Italy. Introduction from Central-Eastern Europe?

Porcine brucellosis occurs in many countries where pigs are farmed, often representing an underrated problem. B. suis biovar 2 is the most common isolate in Europe, with high prevalence reported in wild boars in which it is generally isolated in the absence of gross lesions. In the last five years, we tested for Brucella spp. 389 lymph nodes of wild boars collected during hunting seasons or during necropsy procedures.In this paper, we describe the first case of isolation of B. suis biovar 2 from a wild boar aborted foetus, and we analyse the genomic relationships with B.suis biovar 2 strains isolated in the past five years in Abruzzi Region, Central Italy. The genetic fingerprint revealed that the isolates under study belong to the MLST ST16 and to the MLVA11 Gt 57, similar to the Central-Eastern European strains. Massive restocking (for hunting purpose) of wild boars from Eastern Europe have been done since 1950 in Italy contributing to the increasing of population size and distribution, as well as to the interbreeding between these foreign breeds and the local population. The contamination of pastures with infected material such as aborted wild boars foetuses can increase the risk of transmission of Brucella among wild and domestic animals. The contact of B. suis with domestic ruminants may also cause serological reactions to brucellosis serological testing, and even unapparent infection, thus hampering the efforts made in the brucellosis eradication campaign.