Multiple-locus Variable-number Tandem Repeat Analysis Profiles Research Articles

Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis Profiles of Enteropathogenic Yersinia spp. Obtained from Humans, Domestic Pigs, Wild Boars, Rodents, Pork and Dog Food.

The enteropathogenic Yersinia genus is commonly detected in wildlife including wild boars. Difficulties in its cultivation may hamper subsequent epidemiological studies and outbreak investigations. Multiple-locus variable-number tandem repeat analysis (MLVA) of Yersinia (Y.) enterocolitica and Y. pseudotuberculosis has proven useful in source attribution and epidemiological studies but has hitherto relied on the analysis of isolates. In the present study, MLVA profiles generated from 254 isolates of Y. enterocolitica indicated similarities between human, pig and rodent isolates. Further, MLVA analyses of 13 Y. pseudotuberculosis pure-cultured isolates were compared to MLVA analyses performed directly on the 14 PCR-positive enrichment broths from which the isolates originated, which showed matching MLVA profiles. This indicates that MLVA analysis performed directly on enrichment broths could be a useful method for molecular epidemiological investigations. In addition, 10 out of 32 samples of wild boar minced meat obtained from private hunters and from approved wild-game-handling establishments were PCR-positive for the presence of Y. enterocolitica and may indicate a risk for public health.

Rapid, simple multi-locus variable number tandem repeat analysis: a reliable tool for Klebsiella pneumoniae outbreak screening

Klebsiella pneumoniae causing nosocomial infections is increasingly multidrug-resistant. Rapid and efficient typing tools are required for monitoring. To assess a simple and rapid (<5 hours) multiplex-PCR method based on Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA) as a screening tool to determine whether K. pneumoniae strains are related or not. We assessed the global discriminatory power of our method on 72 unrelated K. pneumoniae isolates including community carriage isolates, highly virulent strains causing liver abscess and extended spectrum beta-lactamase and carbapenemase-producing strains. Suspected related strains from a suspected outbreak and relapsed meningitis case were also studied. MLVA results were compared with Whole Genome Sequencing (WGS) analysis and Multi-Locus Sequence-typing (MLST). MLVA and MLST have similar discriminatory power, each distinguishing 54 profiles among the 72 unrelated isolates (Hunter-Gaston index: 0.989). Each strain belonging to one ST or ST-complex had its own MLVA-type with few exceptions. Two strains of ST268 and ST1119 shared the same MLVA profile while two unrelated strains of ST307, ST86, ST45 and ST37 exhibited each two different MLVA-types. Moreover, investigation of seven grouped cases of K. pneumoniae neonatal sepsis pointed to strong suspicion of a common source for five isolates while two isolates with a different MLVA profile were excluded from this cluster. Our MLVA approach is a useful, rapid and reliable tool for epidemiological investigation requiring only basic molecular biology equipment and permits identification of sporadic isolates that are not part of an outbreak. However, analysis of strains sharing the same MLVA-type by a highly discriminatory technique such as WGS remains necessary.

Coxiella burnetii Shedding in Milk and Molecular Typing of Strains Infecting Dairy Cows in Greece.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.

Phylogeography of Human and Animal Coxiella burnetii Strains: Genetic Fingerprinting of Q Fever in Belgium.

Q fever is a zoonotic disease caused by the bacteria Coxiella burnetii. Domestic ruminants are the primary source for human infection, and the identification of likely contamination routes from the reservoir animals the critical point to implement control programs. This study shows that Q fever is detected in Belgium in abortion of cattle, goat and sheep at a different degree of apparent prevalence (1.93%, 9.19%, and 5.50%, respectively). In addition, and for the first time, it is detected in abortion of alpaca (Vicugna pacos), raising questions on the role of these animals as reservoirs. To determine the relationship between animal and human strains, Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) (n=146), Single-Nucleotide Polymorphism (SNP) (n=92) and Whole Genome Sequencing (WGS) (n=4) methods were used to characterize samples/strains during 2009-2019. Three MLVA clusters (A, B, C) subdivided in 23 subclusters (A1-A12, B1-B8, C1-C3) and 3 SNP types (SNP1, SNP2, SNP6) were identified. The SNP2 type/MLVA cluster A was the most abundant and dispersed genotype over the entire territory, but it seemed not responsible for human cases, as it was only present in animal samples. The SNP1/MLVA B and SNP6/MLVA C clusters were mostly found in small ruminant and human samples, with the rare possibility of spillovers in cattle. SNP1/MLVA B cluster was present in all Belgian areas, while the SNP6/MLVA C cluster appeared more concentrated in the Western provinces. A broad analysis of European MLVA profiles confirmed the host-species distribution described for Belgian samples. In silico genotyping (WGS) further identified the spacer types and the genomic groups of C. burnetii Belgian strains: cattle and goat SNP2/MLVA A isolates belonged to ST61 and genomic group III, while the goat SNP1/MLVA B strain was classified as ST33 and genomic group II. In conclusion, Q fever is widespread in all Belgian domestic ruminants and in alpaca. We determined that the public health risk in Belgium is likely linked to specific genomic groups (SNP1/MLVA B and SNP6/MLVA C) mostly found in small ruminant strains. Considering the concordance between Belgian and European results, these considerations could be extended to other European countries.

A Pilot Study to Investigate the Feasibility of a Multiple Locus Variable Number Tandem Repeat Analysis to Understand the Epidemiology of Dichelobacter nodosus in Ovine Footrot.

Dichelobacter nodosus is the essential pathogen in ovine footrot, an important cause of lameness in sheep that reduces productivity and welfare. The aim of this study was to investigate the feasibility of using multiple locus variable number tandem repeat analysis (MLVA) developed to investigate isolates to understand the molecular epidemiology of Dichelobacter nodosus in ovine footrot by investigation of communities of strains. MLVA sensitivity was improved by optimizing PCR conditions to 100% specificity for D. nodosus. The improved MLVA scheme was used to investigate non-cultured DNA purified from swabs (swab DNA) and cultured DNA from isolates (isolate DNA) from 152 foot and 38 gingival swab samples from 10 sheep sampled on four occasions in a longitudinal study. Isolate DNA was obtained from 6/152 (3.9%) feet and 5/6 yielded complete MLVA profiles, three strains were detected. Two of the three isolate strains were also detected in isolate DNA from 2 gingival crevice cultures. Complete MLVA profiles were obtained from swab DNA from 39 (25.7%) feet. There were 22 D. nodosus community types that were comprised of 7 single strain and 15 multi-strain communities. Six community types were detected more than once and three of these were detected on the same four sheep and the same two feet over time. There were a minimum of 17 and a maximum of 25 strain types of D. nodosus in the study. The three isolate strain types were also the most frequently detected strain types in swab DNA. We conclude that the MLVA from swab DNA detects the same strains as culture, is much more sensitive and can be used to describe and differentiate communities and strains on sheep, feet and over time. It is therefore a sensitive molecular tool to study D. nodosus strains directly from DNA without culture.

IN SILICO DEVELOPMENT OF HIGH-RESOLUTION MLVA TYPING SCHEME FOR ENTEROCOCCUS FAECIUM

Emergence of enterococci as nosocomial pathogens is frequently associated with hospital outbreaks. Vancomycin resistance is especially perturbing as it limits the possible therapeutic options. Based on vast in silico analysis, we introduce a new multiple-locus variable-number tandem repeat analysis (MLVA) scheme for genotyping of Enterococcus faecium isolates, parameters of which are comparable to these of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The scheme was tested in silico on all available at this time E. faecium genomes in NCBI Genbank. Searching for suitable variable number of tandem repeats (VNTR) loci was conducted with a set of free access applications. Ten VNTR loci were selected according to their polymorphic structure and stability using the Primer-BLAST utility of NCBI. Primers were designed to be compatible in a multiplex reaction and the method was adapted for high resolution separation techniques. As a result, a total of 60 MLVA profiles and 35 MLST profiles were generated from the analysis of 114 sequenced genomes. Minimum spanning trees were created for both MLVA and MLST in order to analyze the genetic relatedness between isolates. Hunter Gaston discriminatory index was measured for both MLVA (0,959) and MLST (0,926). Typeability was also measured for both methods (MLVA – 85.9%; MLST – 89.4%). These results suggest that the new MLVA scheme is suitable for epidemiological studies of hospital-adapted E. faecium isolates.

Network properties of salmonella epidemics

We examine non-typhoidal Salmonella (S. Typhimurium or STM) epidemics as complex systems, driven by evolution and interactions of diverse microbial strains, and focus on emergence of successful strains. Our findings challenge the established view that seasonal epidemics are associated with random sets of co-circulating STM genotypes. We use high-resolution molecular genotyping data comprising 17,107 STM isolates representing nine consecutive seasonal epidemics in Australia, genotyped by multiple-locus variable-number tandem-repeats analysis (MLVA). From these data, we infer weighted undirected networks based on distances between the MLVA profiles, depicting epidemics as networks of individual bacterial strains. The network analysis demonstrated dichotomy in STM populations which split into two distinct genetic branches, with markedly different prevalences. This distinction revealed the emergence of dominant STM strains defined by their local network topological properties, such as centrality, while correlating the development of new epidemics with global network features, such as small-world propensity.

First report of Salmonella 1,4,[5],12:i:- in free-ranging striped dolphins (Stenella coeruleoalba), Italy

Between 2015 and the beginning of 2018 (January-March), 30 cetaceans were found stranded along the Ligurian Sea coast of Italy. Necropsies were performed in 22 cases and infectious diseases resulted the most common cause of death. Three striped dolphins, showed a severe coinfection involving the monophasic variant of Salmonella Typhimurium (Salmonella 1,4,[5],12:i:-). The isolates were characterized based on antimicrobial resistance, Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) and whole-genome sequencing (WGS). All isolates demonstrated the same multidrug resistant genotype (ASSuT isolates), showed three different MLVA profiles, two of which closely related, and were identified as Sequence Type 34. Moreover, Single nucleotide polymorphisms (SNP) analysis confirmed strong correlations between two out of the three isolates. To our knowledge, S. 1,4,[5],12:i:-, one of the most common serovars in cases of human infection and food sources worldwide, has not previously been described in marine mammals, and reports of Salmonella-associated disease in free-ranging cetaceans are rare. These results highlight the role of cetaceans as sentinel species for zoonotic and terrestrial pathogens in the marine environment, suggest a potential risk for cetaceans and public health along the North Western Italian coastline and indicate cetaceans as a novel potential reservoir for one of the most widespread Salmonella serovars.

Salmonella spp. transmission in a vertically integrated poultry operation: Clustering and diversity analysis using phenotyping (serotyping, phage typing) and genotyping (MLVA).

The transmission of Salmonella enterica within a vertically integrated poultry operation was investigated longitudinally over an 18-month period (2013–2014). Thirty six percent of all samples collected (1503 of 4219) were positive for salmonellae with seven Salmonella enterica subsp. enterica serovars, and one Salmonella enterica subsp. salamae serovar detected. Both Salmonella enterica subsp. enterica serovars Infantis and Typhimurium were detected in all locations sampled. Salmonella Typhimurium was the most frequently detected serovar (63% of serotyped samples) with 8 phage types (PT) and 41 multiple-locus variable-number tandem-repeats analysis (MLVA) profiles identified. The most frequently identified phage types were PT135a and DT135. A total of 62 PT/MLVA combinations were observed. MLVA profiles 03-14-10-09-525 and 03-15-11-11-525 were the most frequently identified and 83% of the isolates shared at least one MLVA profile with an isolate from another phage type. The use of phage typing and MLVA profiling, on their own or in combination, were insufficient to understand the complexity of the epidemiological relationships between locations within this production system. Despite the high level of apparent diversity, cluster analysis was unable to differentiate the transmission pathways of all S. Typhimurium variants detected within the integrated enterprise. Using additional epidemiological information, the parent breeder rearing site was identified as the most likely point of introduction of two S. Typhimurium isolates into the production system with subsequent dissemination to the broiler flocks via the hatchery. This complexity is unable to be resolved in the absence of intensive sampling programs at all generations of the production system.

Molecular Investigation of the Transmission Dynamics of Brucellosis Observed Among Children in the Province of South - East Anatolia, Turkey

Background: Brucellosis is a zoonotic disease caused by Brucella species. Although brucellosis is considered as an occupational disease in adults, recently it has become an infectious disease affecting all age groups, including children. Molecular epidemiological studies are crucial for control and treatment of disease in children. Objectives: This study aimed at identifying Brucella species, to detect antibiotic susceptibilities and define transmission dynamics between the Brucella isolates in children. Methods: A total of 77 Brucella isolates were identified by conventional and polymerase chain reaction methods. Anti - biotic susceptibilities were investigated by E - test strips. The isolates were genotyped by using multiple locus variable number tandem repeat analysis (MLVA) (MLVA - 16 Orsay), including 8 mini - satellite (panel 1) and 8 microsatellite (panel 2A and 2B) markers. Results: The mean age was 9.14 ± 3.4 years. All patients had been consuming unpasteurized milk. All isolates were Brucella melitensis biovar 3. Only 2 isolates were resistant to ceftriaxone, while the other isolates were susceptible to other antimicrobial agents. The MLVA - 16 typing revealed 42 MLVA profiles. Eighteen profiles included 2 or more isolates, indicating a clustering rate of 66.7%. Twenty - four isolates showed a unique profile. Single locus, double locus, and 3 locus variants were detected in 32, 26, and 15 isolates, respectively. Bruce 30, Bruce 16, Bruce 9, Bruce 7, and Bruce 4 were highly discriminatory loci, respectively. All strains were defined as genotype 122, according to MLVA - 11, and genotype 43 according to MLVA - 8, and were in the Eastern Mediterranean genotype. Conclusions: High clustering rate revealed that brucellosis among the children mainly resulted from common sources. Controlling animal movements and avoiding contaminated milk products have an importance to interrupt spread of brucellosis in children.

In vitro invasion capacity of Salmonella Typhimurium DT9 isolates sourced from humans and layer hen environments.

In Australia, Salmonella Typhimurium definitive type 9 is frequently isolated during foodborne outbreaks of salmonellosis. Multiple-locus variable number tandem repeat analysis (MLVA) trace back investigations frequently identify isolate distribution patterns that may be epidemiologically linked to disease outbreaks. In this study, the in vitro virulence potential of S. Typhimurium DT9 isolates possessing different MLVA patterns (03 15 07 11 550, 03 24 11 10 523, 03 15 08 11 550 and 03 14 08 11 550) isolated from either humans or layer hens was assessed using a human colon carcinoma cell line. Four strains per MLVA from each host for a total of 32 isolates were included in these experiments. Bacteria were grown to stationary phase and added to cells at a multiplicity of infection of 100. Across all isolates, mean percent recovery ranged from 7.1±1.1 to 33.3±7.1%. The layer hen isolate, KC900 (MLVA profile 03 15 08 11 550), exhibited the greatest invasion with a mean percent recovery of 33.3±7.1%. Overall, layer hen isolates of S. Typhimurium DT9 had significantly higher invasion into Caco2 cells than human isolates (p=.0021). RAPD and enterobacterial repetitive intergenic consensus genomic fingerprinting was also performed. Irrespective of source, the SalmonellaDT9 isolates included in this study exhibited similar fingerprint patterns.

Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years

Two cross-sectional studies were carried out in 2013 and 2015 monitoring for Mycoplasma hyopneumoniae presence in a swine farm. In these studies, the genetic diversity of M. hyopneumoniae was assessed in clinical specimens using a Multiple Locus Variable-number tandem repeat Analysis (MLVA) targeting P97 R1, P146 R3 and H4 loci. The samples from August 2015 showed the MLVA profile prevalent in June 2013, therefore it can be concluded that a same genetic type of M. hyopneumoniae can persist for at least two years in a closed herd. In addition, the nested PCR reactions implemented in this study showed to be useful for MLVA typing in non-invasive clinical samples.

Molecular Epidemiology of Cholera Outbreaks during the Rainy Season in Mandalay, Myanmar.

Cholera, caused by Vibrio cholerae, remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit (ctxB), the toxin-coregulated pilus A (tcpA) of Haitian type, and repeat sequence transcriptional regulator (rstR) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

Outbreak of Salmonella enteritidis PT14b gastroenteritis at a restaurant in England: the use of molecular typing to achieve a successful prosecution

ObjectivesTo describe an outbreak of Salmonella enteritidis phage type (PT) 14b in people who had eaten at a restaurant, and the investigation and subsequent prosecution of the food business operator (FBO). Study designThe local health protection team and environmental health department formed an outbreak control team to investigate the outbreak. MethodsEpidemiological, microbiological, and environmental investigations were undertaken. Epidemiological investigations involved case finding and interviews. Microbiological investigation: stool samples from the suspected cases and environmental samples from the implicated food business were investigated. Salmonella isolates obtained were subjected to multiple locus variable-number tandem repeat analysis (MLVA) profiling and whole genome sequencing. In addition, adenosine triphosphate (ATP) hygiene swab tests were used to verify the quality of cleaning procedures and data loggers were used to determine the water temperature of the mechanical dishwasher. ResultsFifteen cases of illness where the causative agent was shown to be S. enteritidis PT14b were identified, all of whom had eaten at the same restaurant. S. enteritidis PT14b was also identified from three of the 11 food and environmental samples taken at the restaurant and found to have the same MLVA profile as the cases. A case for prosecution was built and the FBO was successfully prosecuted in July 2015. ConclusionsThis investigation highlighted that the use of molecular typing as part of thorough epidemiological, microbiological, and environmental investigations can present a robust case for prosecution against restaurants which pose a risk to public health.

Foodborne Salmonellosis in Italy: Characterization of Salmonella enterica Serovar Typhimurium and Monophasic Variant 4,[5],12:i− Isolated from Salami and Human Patients

Salmonella enterica serovar Typhimurium (STm) and its monophasic variant 4,[5],12:i:- (VMSTm) have been responsible for an increased number of foodborne infections in humans in Europe in recent years. The aim of this study was to investigate the origin of three foodborne salmonellosis outbreaks that occurred in Pavia Province (Lombardy region, northern Italy) in 2010. Phenotypic and genetic characteristics of the STm and VMSTm isolates from patients and from food that were recovered in the framework of the three outbreaks were evaluated through serotyping, phage typing, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Salami from three artisan producers, which had all purchased meat from the same slaughterhouse, was the food source of infection in outbreak I. STm isolates were recovered from salami and patients with symptoms of gastroenteritis. These isolates had the same PFGE type and the same rare MLVA profile (3-18-9-NA-211). The same molecular profiles were found in an STm isolate from a salami, which likely was the source of another family outbreak (II). A VMSTm strain with common phenotypic and molecular profiles was isolated from three hospitalized patients and identified as the cause of another putative outbreak (III). During the following 3 years (2011 through 2013), 360 salami produced in Pavia Province were monitored for the presence of S. enterica . In 2011, no STm and VMSTm isolates were recovered from 159 salami tested. During 2012 and 2013, 13.9% of 201 tested salami harbored S. enterica , and half of the isolates were VMSTm, mainly in salami from those artisan producers involved in the previous outbreaks. These isolates were genetically variable, especially in terms of MLVA profiles. The data collected suggest that from 2012, VMSTm has replaced STm in the environments of the salami producers monitored in this study, and these data confirm the dominance of this emergent serovar along the pork supply chain.

Prevalence, antimicrobial resistance and multiple-locus variable-number tandem-repeat analysis profiles of diarrheagenic Escherichia coli isolated from different retail foods

Diarrheagenic E. coli (DEC) isolates were recovered from local retail markets and the Osaka Municipal Central Wholesale Market in Japan. Retail food samples were collected for analysis in Osaka Japan from 2005 to 2008 and consisted of 32 beef, 28 pork, 20 poultry, 136 fish, 66 fruits and vegetables and 51 ready–to-eat (RTE) food samples. A total of 82 DEC strains were recovered from 64 (19%) food samples with the highest prevalence in poultry (100%, 20/20), followed by pork (54%, 15/28), beef (28%, 9/32), fruits and vegetables (12%, 8/66), fish (6.6%, 9/136) and RTE foods (5.9%, 3/51). Most of the strains belonged to E. coli possessing the enteroaggregative E. coli (EAEC) heat-stable enterotoxin 1 (EAST1) gene (EAST1EC; n=62, P<0.0001) and enteropathogenic E. coli (EPEC; n=16, P<0.01), whereas only 1 strain belonged to Shiga toxin-producing E. coli (STEC), 1 to EAEC and 2 to enterotoxigenic E. coli (ETEC) strains. Of the 82 DEC isolates, 22 O and 13H serogroups were detected, including some specific serogroups (O91, O103, O115, O119, O126, and O157) which have been associated with human diarrheal infections. Phylogenetic group A and B1 were predominant among the DEC isolates. Antimicrobial resistance to tetracycline was most common (49%), followed by nalidixic acid (28%), ampicillin (24%), sulfamethoxazole/trimethoprim (20%), and cephalothin (18%). All isolates were susceptible to aztreonam. Of the resistant strains, 44% (22/50) demonstrated resistance to >3 antimicrobial agents. Isolates resistant to >5 antimicrobials were only found in the meat samples, while isolates from the fruits and vegetables as well as RTE foods showed resistance to only 1 or 2 antimicrobial agents. Sixty one percent of EAST1EC, 56% of EPEC and all of the EAEC and ETEC were resistant to at least 1 antimicrobial agent. Multiple-locus variable-number tandem repeat analysis (MLVA) was used in this study for genotyping of DEC. The 82 isolates collected for this study showed 77 distinct MLVA profiles located among 3 branches. The Simpson's Index of Diversity (D) was 99.9% at its highest. The high diversity of these food strains would suggest their originating from a variety of sources and environments. In conclusion, retail food samples in Japan were contaminated with DEC; EAST1EC, a putative DEC, were detected at high rates in poultry, pork and beef. Isolates resistant to >3 antimicrobials were found only in raw meat and fish. Food animals may act as the reservoir for multi-resistant bacteria. Due to the finding that nearly 1/3 of EAST1EC strains were resistant to >3 antimicrobials, additional surveillance for EAST1EC should be initiated.

Investigation of a national outbreak of STEC Escherichia coli O157 using online consumer panel control methods: Great Britain, October 2014.

In October 2014, Public Health England (PHE) identified cases of Shiga toxin-producing Escherichia coli (STEC) serogroup O157 sharing a multiple locus variable-number tandem repeat analysis (MLVA) profile. We conducted a case-control study using multivariable logistic regression to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) testing a range of exposures. Cases were defined as laboratory-confirmed STEC O157 with the implicated MLVA profile, were UK residents aged ⩾18 years with symptom onset between 25 September and 30 October 2014, and had no history of travel abroad within 5 days of symptom onset. One hundred and two cases were identified. Cases were mostly female (65%; median age 49, range 2-92 years). It was the second largest outbreak seen in England, to date, and a case-control study was conducted using market research panel controls and online survey methods. These methods were instrumental in the rapid data collection and analysis necessary to allow traceback investigations for short shelf-life products. This is a new method of control recruitment and this is the first in which it was a standalone recruitment method. The case-control study suggested a strong association between consumption of a ready-to-eat food and disease (aOR 28, 95% CI 5·0-157) from one retailer. No reactive microbiological testing of food items during the outbreak was possible due to the short shelf-life of the product. Collaboration with industrial bodies is needed to ensure timely traceback exercises to identify contamination events and initiate appropriate and focused microbiological testing and implement control measures.

Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M.orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M.orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M.orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M.orygis in South Asia.

Longitudinal study of Salmonella 1,4,[5],12:i:- shedding in five Australian pig herds

The shedding patterns of Salmonella spp. and MLVA profiles of Salmonella enterica subspecies enterica (I) serotype 1,4,[5],12:i:- were monitored in a 12-month longitudinal observational study of five pig herds to inform management; provide indications of potential hazard load at slaughter; and assist evaluation of MLVA for use by animal and public health practitioners. Twenty pooled faecal samples, stratified by age group, were collected quarterly. When Salmonella was cultured, multiple colonies were characterized by serotyping and where S. Typhimurium-like serovars were confirmed, isolates were further characterized by phage typing and multiple locus variable number tandem repeat analysis (MLVA). Salmonella was detected in 43% of samples. Salmonella 1,4,[5],12:i- was one of several serovars that persisted within the herds and was found among colonies from each production stage. Virtually all Salmonella 1,4,[5],12:i:- isolates were phage type 193, but exhibited 12 different, closely-related MLVA profiles. Salmonella 1,4,[5],12:i:- diversity within herds was low and MLVA profiles were stable indicating colonization throughout the herds and suggesting each farm had an endemic strain. High prevalence of S. 1,4,[5],12:i:- specific shedding among terminal animals indicated high hazard load at slaughter, suggesting that primary production may be an important pathway of S. 1,4,[5],12:i:- into the human food chain, this has implications for on-farm management and the application and targeting control measures and further evidence of the need for effective process control procedures to be in place during slaughter and in pork boning rooms. These findings have implications for animal health and food safety risk mitigation and risk management.

A case study on Salmonella enterica serovar Typhimurium at a dairy farm associated with massive sparrow death.

Background Salmonella enterica Typhimurium (S. Typhimurium) is the most common cause of bovine salmonellosis in Japan and where it is also cause of salmonellosis in wild birds. In 2008, a postpartum cow at a dairy farm developed diarrhea caused by S. Typhimurium. The herd was extensively surveilled for Salmonella sp. and we characterized bacterial isolates from this and other cows to determine the source of infection.ResultsEight isolates of S. Typhimurium from cattle were identified as phage type DT40 and showed a 100 % similarity by pulsed-field gel electrophoresis and the same or similar multiple-locus variable-number tandem-repeat analysis profiles as those of S. Typhimurium isolated from dead sparrows (Passer montanus) collected at Asahikawa in 2006. S. Typhimurium DT40 was considered to be a major cause of high sparrow mortality in Hokkaido in 2005–2006 and 2008–2009, suggesting that DT40 maintained in sparrows was transmitted to cattle.Conclusions S. Typhimurium DT40 may be transmitted from sparrows to dairy cattle.