Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Characterization of atypical Mycoplasma anserisalpingitidis strains.

Mycoplasma anserisalpingitidis is a waterfowl colonizing mycoplasma, mainly found in geese. In this study, we compared the whole genomes of five atypical M. anserisalpingitidis strains originating from China, Vietnam and Hungary, with the rest of the collection. Common methods used in the description of species are genomic analyses like the analysis of 16 S - intergenic transcribed spacer (ITS) - 23 S rRNA, of housekeeping genes, of the average nucleotide identity (ANI) and average amino acid identity (AAI) and phenotypic analyses like testing the growth inhibition and the growth parameters of the strains. The atypical strains showed notable genomic differences in all of the genetic analyses: on average ANI and AAI 95% (M. anserisalpingitidis ANI Minimum: 92.45, Maximum: 95.10; AAI Minimum: 93.34, Maximum: 96.37). The atypical strains formed a separate branch among the M. anserisalpingitidis strains in all phylogenetic studies. The small genome size and possibly higher mutation rate of the M. anserisalpingitidis species likely contributed to the observed genetic difference. Based on genetic analyses, the studied strains clearly represent a new genotype of M. anserisalpingitidis. The atypical strains showed slower growth in the medium containing fructose and three of the atypical strains showed diminished growth in the inhibition test. However, no definitive geno-phenotype associations were found regarding the fructose metabolism pathway in the atypical strains. The atypical strains are potentially at an early stage of speciation.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada.

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog's condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal's disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Genetic diversity of Mycobacterium bovis evaluated by spoligotyping and MIRU-VNTR in an intensive dairy cattle breeding area in Mexico.

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis. In Mexico, dairy cattle play an important role in the persistence and spread of the bacillus. In order to describe M. bovis genetic diversity, we genotyped a total of 132 strains isolated from slaughtered cattle with bTB suggestive lesions between 2009 and 2010 in Hidalgo, Mexico, using a panel of 9-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) and spoligotyping. We found 21 spoligotypes, and 124 isolates were grouped in 13 clusters. The most frequent spoligotypes were SB0121 (49, 37.1%) and SB0673 (27, 20.5%); three new spoligotypes were reported SB02703, SB02704 and SB02705. We observed 37 MIRU-VNTR patterns, 107 isolates were grouped in 12 clusters and 25 isolates were unique. Spoligotypes SB0121, SB0673, SB0140, SB0145 and SB0120 showed marked subdivision applying MIRU-VNTR method; meanwhile, spoligotypes SB0971 and SB0327 showed single MIRU-VNTR profiles. The Hunter-Gaston discriminatory index (HGDI) was 0.88, 0.78 and 0.90 for 9-loci MIRU-VNTR, spoligotyping and both methods, respectively. Additionally, allelic diversity (h) analysis showed high diversity for QUB3232, QUB26 and QUB11b with h = 0.79, 0.66 and 0.63, respectively. Overall, high genetic variability was observed among M. bovis isolates. Thus, the use of 9-loci MIRU-VNTR panel is enough to describe genetic diversity, evolution and distribution of M. bovis. This study supports the use of these tools for subsequent epidemiological studies in high incidence areas.

Proposal of a subtype of serovar 4, K4b:O3, of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.

The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14-760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14-760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14-760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14-760, but not with antigens prepared from serovars 1-18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called "K4b:O3″, and we propose isolate 14-760 as the reference strain.

Survival patterns of Streptococcus suis serotypes 1 and 14 in porcine blood indicate cross-reactive bactericidal antibodies in naturally infected pigs.

Streptococcus suis serotype (cps) 1 and cps14 have been detected in association with severe diseases such as meningitis and polyarthritis in pigs. Though these two cps are very similar, only cps14 is an important zoonotic agent in Asia and only cps1 is described to be associated with diseases in suckling piglets rather than weaning piglets. The main objective of this study was to assess restriction of survival of cps14 and cps1 in porcine blood by IgG and IgM putatively cross-reacting with these two cps. Furthermore, we differentiate recent European cps1/14 strains by agglutination, cpsK sequencing, MLST and virulence-associated gene profiling. Our data confirmed cps1 of clonal complex 1 as an important pathotype causing polyarthritis in suckling piglets in Europe. The experimental design included also bactericidal assays with blood samples drawn at different ages of piglets naturally infected with different S. suis cps types including cps1 but not cps14. We report survival of a cps1 and a cps14 strain (both of sequence type 1) in blood of suckling piglets with high levels of maternal IgG binding to the bacterial surface. In contrast, killing of cps1 and cps14 was recorded in older piglets due to an increase of IgM as demonstrated by specific cleavage of IgM. Heterologous absorption of antibodies with cps1 or cps14 is sufficient to significantly increase the survival of the other cps. In conclusion, IgM elicited by natural S. suis infection is crucial for killing of S. suis cps1 and cps14 in older weaning piglets and has most likely the potential to cross-react between cps1 and cps14.

Phylogeny of Mycoplasma bovis isolates from cattle and bison based on multi locus sequence typing and multiple-locus variable-number tandem repeats.

Multiple outbreaks of Mycoplasma bovis (M. bovis) have been reported in North American bison (Bison bison) in Alberta, Manitoba, Saskatchewan, Nebraska, New Mexico, Montana, North Dakota, and Kansas. M. bovis is mainly spread through direct contact and disseminated via animal movements thus, reliable genotyping is crucial for epidemiological investigations. The present study describes the genotyping of sixty-one M. bovis strains from cattle and bison isolated from different provinces of Canada by multi locus sequence typing (MLST), and multiple-locus variable-number tandem repeat analysis (MLVA). The sixty M. bovis clinical isolates together with the reference strain PG45 were divided into ten sequence types by MLST. Three novel sequence types were identified. Two isolates, one from cattle and one from bison shared the same sequence type, whereas one strain had the same sequence type as PG45. The cattle isolates could be further subdivided in Clade A with two subclades and bison isolates were grouped in Clade B with two subclades. With the exception of one animal, isolates originating from the same animal had the same sequence type. The sixty-one isolates also formed three main clades with several subclades when analyzed by MLVA. A total of 20 VNTR (Variable number tandem repeats) types were distinguished, 8 in cattle and 12 in bison isolates. The results showed multiple sequence types and genotype populations of M. bovis in bison and cattle. The results may further help to understand the evolution of M. bovis and develop strain specific or sequence type diagnostic tools.

Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis.

BACKGROUND: Although the pathogenic effect of members of the Mycobacterium tuberculosis complex in susceptible hosts is well known, differences in clinical signs and pathological findings observed in infected animals have been reported, likely due to a combination of host and pathogen-related factors. Here, we investigated whether Mycobacterium bovis strains belonging to different spoligotypes were associated with a higher risk of occurrence of visible/more severe lesions in target organs (lungs and/or lymph nodes) from infected animals. A large collection of 8889 samples belonging to cattle were classified depending on the presence/absence of tuberculosis-like lesions and its degree of severity. All samples were subjected to culture irrespective of the presence of lesions, and isolates retrieved were identified and subjected to spoligotyping. The association between the presence/severity of the lesions and the isolation of strains from a given spoligotype was assessed using non-parametric tests and Bayesian mixed multivariable logistic regression models that accounted for origin (region and herd) effects. RESULTS: Results suggested a difference in severity in lesioned samples depending on the strain's spoligotype. An association between specific spoligotypes and presence of lesions was observed, with a higher risk of finding lesions in animals infected with strains with spoligotypes SB0120, SB0295 and SB1142 compared with SB0121, and in those coming from certain regions in Spain. CONCLUSIONS: Our results suggest that strains belonging to certain spoligotypes may be associated with a higher probability in the occurrence of gross/macroscopic lesions in infected cattle, although these observational findings should be confirmed in further studies that allow accounting for the effect of other possible confounders not considered here, and ultimately through experimental studies.

Blood cultures and blood microbiota analysis as surrogates for bronchoalveolar lavage fluid analysis in dogs with bacterial pneumonia.

BACKGROUND: Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. RESULTS: Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). CONCLUSIONS: Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001-2018.

Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

Assessment of Campylobacter fetus subsp. venerealis molecular diagnosis using clinical samples of bulls.

BACKGROUND: Campylobacter fetus subsp. venerealis (Cfv) is the pathogen responsible for Bovine Genital Campylobacteriosis (BGC), a venereal disease of cattle associated with impaired reproductive performance. Although several PCR assays were developed to identify this pathogen, most of them are still poorly evaluated in clinical samples. This study evaluated real-time PCR assays for Cfv detection in preputial samples of bulls (n = 308). RESULTS: The detection at the subspecies level (Cfv) compared four assays: two targeting ISCfe1 and two targeting parA gene. The detection at the species level (C. fetus) considered an assay targeting the nahE gene and a commercial kit for C. fetus identification. At the subspecies level, assays directed either to different targets (parA and ISCfe1), or to the same target (ISCfe1 or parA), showed a high percentage of disagreeing results. All samples positive at the subspecies level (n = 169) were negative in C. fetus detection assays, which strongly suggests the horizontal gene transfer of ISCfe1 and parA to other bacterial species. This was confirmed by microbiological isolation of three Campylobacter portucalensis strains responsible for false positive results. Sequences with a high level of identity with ISCfe1 and parA gene of Cfv were identified in C. portucalensis genome. CONCLUSIONS: Overall, this study reveals that PCR assays solely directed to a subspecies target originate a high rate of false positive results, due to the presence of parA and ISCfe1 homologous sequences in other bacterial species, namely of the genus Campylobacter. Although the specificity of these methods may be higher if applied to bulls from herds with clinical features of BGC or in other geographical regions, current PCR diagnosis should couple subspecies and species targets, and further research must be envisaged to identify Cfv specific molecular targets.

Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany.

AIMS: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. METHODS AND RESULTS: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. CONCLUSIONS: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing.

Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp-1) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys.

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil.

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.

Streptococcus canis multilocus sequence typing in a case series of dogs with ulcerative keratitis.

OBJECTIVE: To determine whether four isolates of Streptococcus canis (S canis) recovered from dogs diagnosed with ulcerative keratitis at the Animal Health Trust (AHT) were genetically related to other ocular isolates that are registered in the online database. ANIMAL STUDIED: Four S canis corneal isolates. PROCEDURES: Clinical and laboratory records between 2016 and 2017 were searched for dogs with ulcerative keratitis for which microbiology analysis was consistent with the growth of S canis. Genomic DNA was extracted for sequencing (Illumina MiSeq), and multilocus sequence types (STs) were determined using MLST 1.8 relative to the 44 sequence types of S canis available. A neighbor-joining tree was constructed in MEGA v4.0. A two-sided Fisher's exact test was used to determine any associations between the isolated strains and ocular infections of dogs. RESULTS: Four strains were isolated from pugs (cases 1-4) with ulcerative keratitis. Genome sequencing identified ST-27 (case 1), ST-9 (case 3), and ST-13 (cases 2 and 4). STs 13 and 27 are members of Clonal Complex (CC)-13. Analysis of the multilocus sequence typing database revealed that CC-13 strains accounted for six of the twelve isolates recovered from the eye exudates of dogs (P = .0078). CONCLUSIONS: There is early evidence that the CC-13 group of S canis is associated with ocular infections in dogs. We provide draft genome sequences toward the future identification of virulence mechanisms associated with streptococcal keratitis in dogs.

Molecular epidemiology of Leptospira noguchii reveals important insights into a One Health context.

Leptospirosis presents a complex and dynamic epidemiology. Bovine leptospirosis has been described as a major infectious disease impairing reproductive efficiency. Although infections by Leptospira interrogans, L. santarosai and L. borgpetersenii are frequently reported in cattle, the presence of L. noguchii in these animals should not be neglected. In this study, we describe serological (MAT) and molecular characterization (rrs and secY gene sequencing, multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]) of eight L. noguchii strains obtained from slaughtered cows. Intraspecific genetic diversity was evaluated, and haplotype networks were constructed based on hosts and geographical localizations. Strains were characterized as belonging to serogroups Australis, Autumnalis and Panama, and molecular characterization showed a high heterogeneity of these strains. Ten different STs were found (including nine new STs and 39 novel alleles) as well as nine different pulsotypes. Two clonal complexes were found. Phylogenetic trees based on secY locus and concatenated MLST loci showed two main clusters, with sequences from the present study included in the first. In general, there was no relationship between the geographical origin and the secY phylogenetic clusters, as well as between secY phylogenetic clusters and serogroups. Molecular diversity indexes confirmed a high variability (H > 0.8). This high intraspecific variation observed may be related to differences in virulence, pathogenicity and antigenicity or even adaptability of the strains. In addition, haplotype networks clearly demonstrated the circulation of genotypes between humans and animals, confirming the zoonotic potential. The present study provides relevant data for the study of leptospirosis in the One Health context, where human, animal and environmental health is closely connected.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Reverse zoonotic tuberculosis transmission from an emerging Uganda I strain between pastoralists and cattle in South-Eastern Nigeria.

BACKGROUND: Tuberculosis remains a major public health challenge globally with increasing risks for inter-transmission between pastoralists and cattle in Nigeria. This study was aimed at using molecular tools to establish zoonotic transmission of tuberculosis between pastoralists and their cattle in Ebonyi State, Nigeria. Sputum (n = 149) and milk (n = 144) samples from pastoralists and cattle, respectively were screened on the assumption of subclinical infections considering unguarded human-livestock interactions. Isolates obtained were analysed using deletion typing, spoligotyping and 24-Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR). RESULTS: Fifty-four MTC were confirmed by deletion typing and were differentiated accordingly (M. tuberculosis: pastoralists =42, cattle = 2; M. bovis: pastoralists =1; M. africanum: pastoralists =9). Spoligotyping indicated 59.2% Uganda I/SIT46 (pastoralists =28; cattle = 1), 16.3% Latin American Mediterranean/SIT61 (pastoralists =8), 2.0% T/SIT53 (pastoralists =1) strains of M. tuberculosis and new strains of M. bovis and M. africanum. The 24-MIRU-VNTR of selected predominant cluster isolates shared by cattle and pastoralists (Uganda I/SIT46: pastoralists =9; cattle = 1) showed the same number of copies at each of the repetitive loci. CONCLUSIONS: Mycobacterium bovis was confirmed in humans and a reverse zoonotic tuberculosis transmission from an emerging Uganda I M. tuberculosis strain between pastoralists and cattle in Nigeria evidenced by MIRU-VNTR. Using molecular tools will help mitigate disease burden through informed epidemiological insights.

Characterization of Pasteurella multocida isolated from dead rabbits with respiratory disease in Fujian, China.

BACKGROUND: Pasteurella multocida is one of the important pathogens that infect rabbits, causing major economic losses in commercial rabbit farming. In this study, 205 P. multocida isolates recovered from lungs of dead rabbits with respiratory disease were defined by capsular serogroups, lipopolysaccharide (LPS) genotypes, multi-locus sequence types and screened virulence factors by using PCR assays, and tested antimicrobial susceptibility. RESULTS: The 205 isolates were assigned into 2 capsular types, A and D, and 2 LPS genotypes, L3 and L6. When combining capsular types with LPS genotypes, 4 serotypes were detected. A:L3 (51.22%, 105/205) was the most predominant serotype, followed by A:L6 (24.88%, 51/205), D:L6 (19.02%, 39/205) and D:L3 (4.88%, 10/205). The 205 isolates were grouped into 3 sequence types, ST10, ST11 and ST12. ST12 (56.10%, 115/205) was the most prevalent sequence type, followed by ST10 (24.88%, 51/205) and ST11 (19.02%, 39/205). In the 205 isolates, virulence associated genes ptfA, fur, hgbB, ompA, ompH and oma87 were positive in the PCR screening, whereas the toxA and tbpA genes were negative. Notably, the 156 capsular serogroup A isolates carried the pmHAS gene. All the 205 isolates were susceptible to most of the used antibiotics, except for streptomycin, gentamycin, kanamycin and ceftriaxone, and the resistance rates of which were 27.80, 15.61, 9.27 and 2.44%, respectively. CONCLUSIONS: This study, for the first time, described the prevalence and characteristics of P. multocida causing respiratory disease in rabbits in Fujian Province, which might be useful for tracking the epidemic strains and development of efficient vaccines and methods to prevent and control the pathogen.