MLVA Genotyping Research Articles

Molecular Detection and Characterisation of Coxiella burnetii in Koala (Phascolarctos cinereus) Urogenital Tract Swabs.

Q fever is a zoonosis caused by Coxiella burnetii, primarily affecting those in close contact with domestic ruminants, the main source of human infection. Coxiella burnetii has also been detected in various wildlife species globally. In Australia, serological and molecular studies have shown exposure to and infection by C. burnetii in macropods, bandicoots, and koalas. However, the extent to which these species contribute to human infection remains unclear. An unpublished public health investigation into a Q fever case in a person involved in koala care could not conclusively link the infection to koalas due to the patient's broad animal exposure. This study aimed to explore the potential role of koalas in transmitting C. burnetii to humans by investigating the presence of C. burnetii DNA in urogenital tract (UGT) swabs from koalas. DNA was extracted from UGT swabs from koalas in three regions in New South Wales, Australia. An optimised multiplex qPCR assay detected C. burnetii DNA in 2 out of 225 samples (0.89%) at approximately 10 genome equivalents per reaction. Both positive samples amplified all three gene targets. MLVA genotyping identified two distinct C. burnetii genotypes previously isolated from Australian Q fever cases. These findings highlight the need for vaccination against Q fever for those in close contact with koalas.

Comparative Genomics of Legionella pneumophila Isolates from the West Bank and Germany Support Molecular Epidemiology of Legionnaires’ Disease

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires' disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Genetic diversity of Brucella melitensis isolates from sheep and goat milk in Iran.

The genetic diversity of Brucella strains has not been fully understood. To investigate this, the genetic characteristics of 64 isolates of Brucella melitensis from sheep and goats' milk were studied using random fragment length polymorphism (RFLP) and multiple locus variable-number tandem repeat analysis (MLVA-16) methods developed in Orsay, France (MLVA-16Orsay). The RFLP analysis revealed that all 64 isolates were of biovar one. The MLVA-typing showed that one sample was simultaneously infected with two strains of B. melitensis and the genotype of 65 isolate was analyzed. Four genotypes (47, 42, 43, and 63) were identified using MLVA-8 (panel 1), whereas six genotypes (138, 125, 116, 108, and two unknown genotypes) were identified using MLVA11 (panels 1 and 2A). From the review of MLVA-16 (panels 1, 2A, and 2B), panel 2B showed a very high discriminatory power. Two loci of Bruc04 and Bruc30 from this panel had diversity index values higher than 0.71 and the average diversity index was 0.619. So MLVA-16Orsay 34 showed the genotype indicating a low genetic homogeneity among the isolates. The findings of MLVA genotyping of the isolates suggest that strains of B. melitensis isolated from the milk of small ruminants in Iran are most closely related to the isolates from neighboring countries of the Eastern Mediterranean group. To the best of our knowledge, this is the first study to indicate the potential use of MLVA genotyping for simultaneous detection of specimen contamination using two different B. melitensis biovars.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Tracking the diversity and Mediterranean lineage of Brucella melitensis isolates from different animal species in Turkey using MLVA-16 genotyping

Brucellosis is a zoonotic disease with a high prevalence in humans and farm animals in Turkey. However, data on the genetic diversity of Brucella spp. circulating in Turkey and parts of the Mediterranean region are limited. In the present study, the genetic diversity of 50 B. melitensis isolates from seven regions of Turkey was investigated using multi-locus variable number tandem repeats analysis (MLVA-16). The profiles were compared with 163 B. melitensis isolates recovered from the Mediterranean basin. B. melitensis strains from Turkey contain 46 different genotypes and consist of two main clusters. B. melitensis isolates from Turkey were closely related to isolates from Greece and some Portuguese strains. The same genotypes isolated from different sites show the spread between sites. Therefore, uncontrolled animal movements and the trade of imported animals can be important factors for the spread of brucellosis. The endemic occurrence of B. melitensis in the Mediterranean basin is a result of socio-historical links between Mediterranean countries. Turkish strains belong to the Eastern Mediterranean line. Eradicating brucellosis in countries of the Mediterranean basin with high prevalence is a demanding need to reduce trade barriers and, more importantly, prevent human suffering

GENETIC DIVERSITY OF B. MELITENSIS STRAINS ISOLATED IN THE ZHAMBYL PROVINCE OF KAZAKHSTAN DURING 2016-2019

Brucellosis is a dangerous zoonotic infection which also infects humans. Brucellosis-causing pathogen is prevalent throughout the world and causes damage to animal husbandry and public health. Genetic typing methods have become an integral component of the epidemiological/epidemiological surveillance to track the spread of the brucellosis pathogen, because genetic methods allow identifying circulating strains by their unique characteristics. The positive effect of the use of highly discriminatory genotyping methods was demonstrated on various samples of pathogen’s isolates. However, genetic diversity can change over time, and data on the temporal dynamics of the genetic diversity are of interest to study pathogen evolution. In this study, we performed MLVA16 genotyping of B. melitensis strains isolated in 2016 and 2019 in the Zhambyl province of Kazakhstan. The results obtained indicate a significant population dynamics of the pathogen, because only 19% of the strains from 2016 had homoplastic analogues among the strains from 2019. Expanding the sample size and observation time are needed to discern reliable picture of the population dynamics and changes in genetic diversity of Brucella strains.

Analysis of cases of brucellosis in humans and molecular-biological characteristics of <i>Brucella melitensis</i> strains in regions of South European Russia with a high brucellosis incidence

Introduction. To improve the epidemiological surveillance of brucellosis in Russia, a detailed analysis of incidence and characteristics of Brucella melitensis strains circulating in the territories of the country where there is a persistent epizootic and epidemiological problem with brucellosis is required.Purpose of the study. To study current trends in the brucellosis epidemic process and perform molecular analysis of Brucella isolates from the territories of Southern and North Caucasian Federal Districts with long lasting unfavorable situation with brucellosis — the Republics of Dagestan (RD), Kalmykia (RK) and Stavropol Krai (SK).Materials and methods. The analysis of the brucellosis incidence in the North Caucasus Federal District and the Southern Federal District was carried out on the basis of data from the Rospotrebnadzor Departments for the period 2011–2020. Total 56 cultures of Brucella melitensis, isolated in 1999–2019 from patients with brucellosis that permanently resided in RD, RK and SK were studied.Results. The key factor contributing to the epidemic manifestations of brucellosis in the south of the European part of Russia is the persistence of epizootic foci in cattle and small ruminants. The leading concomitant factors are as follows: the presence in the region of "hidden" epizootic foci, uncontrolled production of food products of animal husbandry and its illegal sale to customers, unauthorized movement of animals and livestock products, untimely delivery (concealment) of sick cattle for stamping out. Results of molecular analysis of B. melitensis strains indicate the circulation of a mixed population of brucella, which is generally characteristic of the entire region, without a pronounced confinement of isolates to separate administrative territories. At the same time, the entire studied sample of strains can be divided into two groups (72% and 28% of strains) based on MLVA-16 genotypes by the variability in the Bruce 19 locus, which makes it possible to associate a part of the studied strains with the territory of their isolation.Discussion. The data obtained make it possible to scientifically substantiate the possibility of considering the affected territories of the Southern Federal District and the North Caucasian Federal District as a single common persistent active anthropurgic epizootic focus with circulation of a mixed, but typical for the region population of Brucella strains.

Diversity identification of KPC- producing Klebsiella pneumoniae using multilocus variable number tandem repeat analysis

PurposeThe current study aimed to determine blaKPC, blaGES, blaVIM, blaNDM, blaOXA-23, and blaOXA-48 genes in clinical strains of Klebsiella pneumoniae isolated in Tehran, Iran to assess genetic diversity using MLVA as a typing method. MethodsA total of 181 K. pneumoniae isolates were obtained from various clinical samples. CLSI 2018 (clinical and laboratory standards institute) guidelines were used to determine antibiotic susceptibility and the Modified Hodge Test (MHT).To detect blaKPC, blaGES, blaVIM, blaNDM, blaOXA-23, and blaOXA-48, the polymerase chain reaction (PCR) method was used. The MLVA method was used to type K. pneumoniae isolates by using PCR for 8 Variable Number Tandem Repeats (VNTRs). ResultsImipenem was the most effective antibiotic against K. pneumoniae, with 36.5% susceptibility. 100 (55.24%) of the isolates tested positive for KPC, and 30 (30%) tested positive for six genes. Thirty MLVA genotypes were distinguished, and an examination of diversity indexes (DIs) for eight loci revealed that seven different alleles were the most polymorphic, with the highest DI of 0.780. ConclusionsThe present study showed that MLVA could be helpful for typing clinical strains of K. pneumoniae. Our K. pneumoniae isolates are thought to be derived from a small number of clones that have undergone minor genetic changes over time. The results also showed that this method had great potential to differentiate those strains with high phenotypic similarity. The current study has revealed some intriguing facts about K. pneumoniae genetic relatedness in Tehran, Iran.

MLVA GENOTYPING OF B. ANTHRACIS IN THE FORMATION OF A COLLECTION OF REPRESENTATIVE STRAINS

Anthrax is an infectious disease that affects many species of mammals and humans. Bacteriological isolation of the pathogen and its identification by phenotypic and PCR methods remains the gold standard. However, these methods do not allow determining the cause of the outbreak, namely, their origin from one or more sources. In addition, the causative agents of especially dangerous infections are deposited in the National Collections, and genotyping with the selection of a single representative strain is a prerequisite to prevent the loss of strains with unique properties, as well as the deposition of identical strains. In this study, we performed MLVA genotyping of 10 isolates from 5 outbreaks of anthrax in animals in 2021. The obtained MLVA profiles allowed differentiation of isolates in different outbreaks and confirmed the identity of genotypes in each outbreak. MLVA genotyping of B. anthracis can be recommended as the primary method for genotyping and selection of unique strains for replenishment of the collection.

Seroprevalence trend of human brucellosis and MLVA genotyping characteristics of Brucella melitensis in Shaanxi Province, China, during 2008-2020.

In this study, a total of 179,907 blood samples from populations with suspected Brucella spp. infections were collected between 2008 and 2020 and analyzed by the Rose Bengal plate test (RBPT) and serum agglutination test (SAT). Moreover, conventional biotyping, B. abortus-melitensis-ovis-suis polymerase chain reaction (AMOS-PCR), and multiple-locus variable-number tandem repeat analysis (MLVA) was applied to characterize the isolated strains. A total of 8103 (4.50%) samples were positive in RBPT, while 7705 (4.28%, 95% confidence interval (CI) 4.19-4.37) samples were positive in SAT. There was a significant difference in seroprevalence for human brucellosis over time, in different areas and different cities (districts) (χ2 = 2 = 32.23, 1984.14, and 3749.51, p < .05). The highest seropositivity (8.22% (4, 965/60393; 95% CI 8.00-8.44) was observed in Yulin City, which borders Inner Mongolia, Ningxia, and Gansu Province, China, regions that have a high incidence of human brucellosis. Moreover, 174 Brucella strains were obtained, including nine with B. melitensis bv. 1, 145 with B. melitensis bv. 3, and 20 with B. melitensis variants. After random selection, 132 B. melitensis were further genotyped using MLVA-16. The 132 strains were sorted into 100 MLVA-16 genotypes (GTs) (GT 1-100), 81 of which were single GTs represented by singular independent strains. The remaining 19 shared GTs involved 51 strains, and each GT included two to seven isolates from the Shaan northern and Guanzhong areas. These data indicated that although sporadic cases were a dominant epidemic characteristic of human brucellosis in this province, more than 38.6% (51/132) outbreaks were also found in the Shaan northern area and Guanzhong areas. The 47 shared MLVA-16 GTs were observed in strains (n = 71) from this study and strains (n = 337) from 19 other provinces of China. These data suggest that strains from the northern provinces are a potential source of human brucellosis cases in Shaanxi Province. It is urgent to strengthen the surveillance and control of the trade and transfer of infected sheep among regions.

Identification of Tick-Borne Pathogens and Genotyping of Coxiella burnetii in Rhipicephalus microplus in Yunnan Province, China.

Rhipicephalus microplus, a vector that can transmit many pathogens to humans and domestic animals, is widely distributed in Yunnan province, China. However, few reports on the prevalence of tick-borne pathogens (TBPs) in Rh. microplus in Yunnan are available. The aim of this study was to detect TBPs in Rh. microplus in Yunnan and to analyze the phylogenetic characterization of TBPs detected in these ticks. The adult Rh. microplus (n = 516) feeding on cattle were collected. The pooled DNA samples of these ticks were evaluated using metagenomic next-generation sequencing (mNGS) and then TBPs in individual ticks were identified using genus- or group-specific nested polymerase chain reaction (PCR) combined with DNA sequencing assay. As a result, Candidatus Rickettsia jingxinensis (24.61%, 127/516), Anaplasma marginale (13.18%, 68/516), Coxiella burnetii (3.10%, 16/516), and Coxiella-like endosymbiont (CLE) (8.33%, 43/516) were detected. The dual coinfection with Ca. R. jingxinensis and A. marginale and the triple coinfection with Ca. R. jingxinensis, A. marginale, and CLE were most frequent and detected in 3.68% (19/516) and 3.10% (16/516) of these ticks, respectively. The results provide insight into the diversity of TBPs and their coinfections in Rh. microplus in Yunnan province of China, reporting for the first time that C. burnetii had been found in Rh. microplus in China. Multilocus variable number tandem repeat analysis with 6 loci (MLVA-6) discriminated the C. burnetii detected in Rh. microplus in Yunnan into MLVA genotype 1, which is closely related to previously described genotypes found primarily in tick and human samples from different regions of the globe, indicating a potential public health threat posed by C. burnetii in Rh. microplus in Yunnan.

Genotyping of Brucella strains isolated from humans and cattle of different geographical regions of Pakistan using MLVA‐15

BackgroundPrevalence of brucellosis and MLVA genotyping in animals and humans, isolated from different regions of Pakistan was performed. Animals having history of brucellosis from the field and local farms of Bannu, Mardan, Peshawar, Swat, Lahore and Islamabad were selected for blood collection. Humans that work with them were also selected for sampling in this study. Total of 600 samples were taken from cattle and humans and subjected to Rose Bengal plate Test (RBPT) for the initial screening of positive samples. Designed primers of B.abortus for cattle and B.melitensis for humans were utilised to perform PCR. Culturing and isolation was carried to further to perform MLVA genotyping assay through the selection of two panels of primer markers.ResultsRBPT showed more number of cases of brucellosis in animals and humans compared to the PCR findings. Genotyping findings based upon MLVA‐15 set of markers demonstrated that the isolated strains of B.abortus fall in the same clade with strain A1, P8 and A2 from Pakistan and also similar with BCCN#02‐45 strain from India. On the other hand, B.melitensis isolated from different districts of Pakistan shared the same clade with BwIM‐AFG 63, BwIM‐AFG 44 strains from Afghanistan and BwIM IRN 37 strain from Iran. Selected VNTR alleles were sequenced for calibration purposes.ConclusionIt is concluded that B rucella is prevalent in animals and humans in studied districts of Pakistan. Moreover, A1, P8, BwIM‐AFG 63, BwIM‐AFG 44 and A2 were found the common genotypes in Pakistan.

Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan.

Tularemia is a highly dangerous zoonotic infection due to the bacteria Francisella tularensis. Low genetic diversity promoted the use of polymorphic tandem repeats (MLVA) as first-line assay for genetic description. Whole genome sequencing (WGS) is becoming increasingly accessible, opening the perspective of a time when WGS might become the universal genotyping assay. The main goal of this study was to describe F. tularensis strains circulating in Kazakhstan based on WGS data and develop a MLVA assay compatible with in vitro and in silico analysis. In vitro MLVA genotyping and WGS were performed for the vaccine strain and for 38 strains isolated in Kazakhstan from natural water bodies, ticks, rodents, carnivores, and from one migratory bird, an Isabellina wheatear captured in a rodent burrow. The two genotyping approaches were congruent and allowed to attribute all strains to two F. tularensis holarctica lineages, B.4 and B.12. The seven tandem repeats polymorphic in the investigated strain collection could be typed in a single multiplex PCR assay. Identical MLVA genotypes were produced by in vitro and in silico analysis, demonstrating full compatibility between the two approaches. The strains from Kazakhstan were compared to all publicly available WGS data of worldwide origin by whole genome SNP (wgSNP) analysis. Genotypes differing at a single SNP position were collected within a time interval of more than fifty years, from locations separated from each other by more than one thousand kilometers, supporting a role for migratory birds in the worldwide spread of the bacteria.

Investigation on Anthrax in Bangladesh during the Outbreaks of 2011 and Definition of the Epidemiological Correlations

In 2011, in Bangladesh, 11 anthrax outbreaks occurred in six districts of the country. Different types of samples were collected from May to September in the six districts where anthrax had occurred in order to detect and type Bacillus anthracis (B. anthracis) strains. Anthrax was detected in 46.6% of the samples analysed, in particular in soils, but also in bone samples, water, animal feed, and rumen ingesta of dead animals. Canonical single nucleotide polymorphisms (CanSNPs) analysis showed that all the isolates belonged to the major lineage A, sublineage A.Br.001/002 of China and Southeast Asia while the multi-locus variable number of tandem repeats (VNTRs) analysis (MLVA) with 15 VNTRs demonstrated the presence of five genotypes, of which two resulted to be new genotypes. The single nucleotide repeats (SNRs) analysis showed 13 SNR types; nevertheless, due to its higher discriminatory power, the presence of two isolates with different SNR-type polymorphisms was detected within two MLVA genotypes. This study assumes that soil is not the only reason for the spread of the disease in Bangladesh; contaminated feed and water can also play an important role in the epidemiology of anthrax. Possible explanations for these epidemiological relationships are discussed.

Investigation of Genetic Relatedness of Brucella Strains in Countries Along the Silk Road.

In this study, MLVA (multiple-locus variable-number tandem repeat analysis) genotype data of Brucella strains from 11 countries along the Silk Road were downloaded from the MLVAbank. MLVA data of strains were applied to the constructed Minimum Spanning Tree to explore the species/biovars distribution, geographic origins, and genetic relationships of the strains analyzed. Moreover, whole-genome sequencing–single-nucleotide polymorphism (WGS-SNP) phylogenetic analysis of the genome of Brucella melitensis strains from GenBank was performed to discriminate the relatedness of strains further and investigate the transmission pattern of B. melitensis brucellosis. A total of 1,503 Brucella strains were analyzed in this study: 431 Brucella abortus strains (29.8%), 1,009 B. melitensis strains (65.7%), and 63 Brucella suis strains (4.5%). B. melitensis biovar 3 was the dominant species and was shown to be widespread in all of the examined regions, suggesting that the prevention and surveillance of the B. melitensis population are a main challenge in these countries. A wide host spectrum was observed for this Brucella population; many animal reservoirs are a potential reason for the continuous brucellosis circulation in these countries. Although the B. abortus strains from the examined regions had common geographic origins, only a few shared genotypes were observed in different countries. These data revealed that the majority of B. abortus strains were spreading within the national borders. However, the B. melitensis strains from Italy originated from a Western Mediterranean lineage; strains from the other 10 countries originated from Eastern Mediterranean lineage, and this lineage was shared by strains from three to nine different countries, suggesting that the introduction and reintroduction of the disease in the 10 countries might have occurred in the past. Furthermore, the most shared MLVA-16 genotypes were formed in the B. melitensis strains from China, Kazakhstan, and Turkey, suggesting that the introduction and trade in sheep and goats have occurred frequently in these countries. WGS-SNP analysis showed that the B. melitensis in this study originated from the Malta (Italy) region. According to their territorial affiliation between four clade strains from these countries in genotype B, the absence of a clear differentiation suggests that strains continuously expand and spread in countries along with Silk Road. Active exchange and trade of animals (sheep and goats) among these countries are reasonable explanations. B. suis strains from different nations showed unique geographic origins and epidemiological characteristics. Therefore, there is an urgent need for the control of transfer and trade of infected sheep (goats) in countries along the Silk Road, namely, the strengthening of the entry–exit quarantine of sheep and goats and improvements in the diagnosis of animal brucellosis.

MLVA-16 Genotyping of Brucella abortus and Brucella melitensis Isolates from Different Animal Species in Egypt: Geographical Relatedness and the Mediterranean Lineage.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

First identification of a Brucella abortus biovar 4 strain from yak in Tibet, China

Brucellosis is one of the major zoonotic diseases in the world. In China, understanding on its causative agent Brucella is still limited. Recently, we isolated a Brucella strain XZ19-1 from yak in Lhasa, Tibet. Phenotypical characterization proved that it belongs to B. abortus biovar 4, a biotype that has never been reported in China. MLVA-16 genotyping revealed a novel profile (4-5-3-12-2-2-3-3-8-32-8-5-4-3-3-3) in this strain, while MLST sequence typing demonstrated that it belongs to ST 71. Furthermore, the whole genome of XZ19-1 strain was sequenced. Subsequent phylogenetic analysis demonstrated that XZ19-1was genetically more closely related to B. abortus strains originated from European countries rather than to those collected from China previously. Isolation and identification of XZ19-1 strain may thus indicate a unique Brucella lineage existing in Qing-Tibet plateau. These findings will help to improve the diagnosis and epidemiological studies of brucellosis in animals and human in this part of China.

Seroprevalence of human brucellosis and molecular characteristics of Brucella strains in Inner Mongolia Autonomous region of China, from 2012 to 2016

ABSTRACT In the present study, a total of 1102304 serum samples were collected to detected human brucellosis between the years 2012 and 2016 in Inner Mongolia. Overall, an average of 3.79% anti-Brucella positive in Inner Mongolia was presented but the range of positive rates were among 0.90 to 7.07% in 12 regions. Seroprevalence of human brucellosis increased gradually from 2012 to 2016. However, the incidence rate of human brucellosis showed a declining trend. One hundred and seven Brucella strains were isolated and identified as B. melitensis species, and B. melitensis biovar 3 was the predominant biovar. MLVA-11 genotypes 116 was predominant and had crucial epidemiology to the human population. All 107 strains tested were sorted into 75 MLVA-16 genotypes, with 54 single genotypes representing unique isolates. This result revealed that these Brucellosis cases had epidemiologically unrelated and sporadic characteristics. The remaining 21 shared genotypes among two to four strains, confirming the occurrence of cross-infection and multiple outbreaks. Extensive genotype-events were observed between strains from this study and Kazakhstan, Mongolia, and Turkey, these countries were key members of the grassland silk road. Long-time trade in small ruminants (sheep) in these countries has possibly promoted the spread of Brucella spp. in these regions.

Molecular investigation of infection sources and transmission chains of brucellosis in Zhejiang, China

ABSTRACT In the present study, a total of 7793 samples from 5 different types of hosts were collected and tested, with a seroprevalence of 2.4% (184/7793). Although the seroprevalence of human and animal brucellosis is relatively low, numbers of human brucellosis cases reported have increased continuously from 2004 to 2018. A total of 118 Brucella strains containing 4 biotypes were obtained, including Brucella melitensis bv.1 (n = 8) and bv.3 (n = 106), Brucella abortus bv.3 (n = 3) and bv.7 (n = 1). Twenty-one shared MLVA-16 genotypes, each composed of 2 to 19 strains obtained from different hosts, suggest the occurrence of a brucellosis outbreak epidemic with multiple source points and laboratory infection events. Moreover, 30 shared MLVA-16 genotypes were observed among 59.6% (68/114) B. melitensis isolates from Zhejiang and strains from other 21 different provinces, especially northern provinces, China. The analysis highlighted the imported nature of the strains from all over the northern provinces with a dominant part from the developed areas of animal husbandry. These data revealed a potential transmission pattern of brucellosis in this region, due to introduced infected sheep leading to a brucellosis outbreak epidemic, and eventually causing multiple laboratory infection events. It is urgent to strengthen the inspection and quarantine of the introduced animals.