Effect of Sucrose Concentration on Sucrose-Dependent Adhesion and Glucosyltransferase Expression of S. mutans in Children with Severe Early-Childhood Caries (S-ECC)

Sucrose, extracellular polysaccharide, and glucosyltransferases (GTFs) are key factors in sucrose-dependent adhesion and play important roles in the process of severe early-childhood caries (S-ECC). However, whether sucrose concentration regulates gtf expression, extracellular polysaccharide synthesis, and sucrose-dependent adhesion is related to the different genotypes of S. mutans isolated from ECC in children and still needs to be investigated. In this study, 52 strains of S. mutans were isolated from children with S-ECC and caries-free (CF) children. Water-insoluble glucan (WIG) synthesis was detected by the anthrone method, adhesion capacity by the turbidimetric method, and expression of gtf by RT-PCR in an in vitro model containing 1%–20% sucrose. The genotypes of S. mutans were analyzed by AP-PCR. The results showed that WIG synthesis, adhesion capacity, and gtf expression increased significantly when the sucrose concentration was from 1% to 10%. WIG synthesis and gtfB as well as gtfC expression of the 1% and 5% groups were significantly lower than those of the 10% and 20% groups (p < 0.05). There were no significant differences between the 10% and 20% groups. The fingerprints of S. mutans detected from individuals in the S-ECC group exhibited a significant difference in diversity compared with those from CF individuals (p < 0.05). Further, the expression of gtfB and gtfC in the S-ECC group was significantly different among the 1- to 5-genotype groups (p < 0.05). It can be concluded that sucrose-dependent adhesion might be related to the diversity of genotypes of S. mutans, and the 10% sucrose level can be seen as a “turning point” and essential factor for the prevention of S-ECC.     

Association between Vitamin D and Dental Caries in a Sample of Canadian and American Preschool-Aged Children

Background: Inadequate vitamin D levels may increase the risk of caries during childhood. The purpose of this study was to investigate the association between 25-hydroxyvitamin D (25(OH)D) status and severe early childhood caries (S-ECC) in preschool children. Methods: Data were obtained from children <72 months of age in two case–control studies in Winnipeg, Manitoba and Richmond, Virginia. Serum analysis assessed 25(OH)D, calcium and parathyroid concentrations. Data on demographics, dental history and oral hygiene were obtained via questionnaires. Bivariate and multiple logistic regression analyses were performed to assess the relationships between demographic and biological variables and S-ECC. A p-value of ≤0.05 was significant. Results: Data were available for 200 children with S-ECC and 144 caries-free controls. Children with S-ECC had significantly lower 25(OH)D levels than those who were caries-free (p < 0.001), and children with deficient 25(OH)D levels were 10 times more likely to have S-ECC (p < 0.001). Multiple logistic regression revealed that having higher 25(OH)D and calcium concentrations (p = 0.019 and p < 0.0001, respectively), as well as being breastfed in infancy (p < 0.001), were significantly and independently associated with lower odds of S-ECC, while dental insurance (p = 0.006) was associated with higher odds of S-ECC. Conclusions: This study provides additional evidence of an association between nutritional status, specifically vitamin D and calcium levels, and S-ECC.     

Vitamin D Supplementation,Characteristics of Mastication,and Parent-Supervised Toothbrushing as Crucial Factors in the Prevention of Caries in 12- to 36-Month-Old Children

Severe early childhood caries (S-ECC), defined as any sign of smooth-surface caries in a child younger than three years of age, remains a serious health issue. The aim of this study was to indicate oral health behaviours related to S-ECC. The study surveyed parents (socio-economic and medical factors, oral health behaviours) and clinically examined children including non-cavitated d1,2/cavitated d ≥ 3 lesions. %S-ECC, and caries indices (d1,2 d ≥ 3 mft and d1,2 d ≥ 3 mfs) were calculated. Spearman’s correlation and simple and multiple logistic regression were used to assess the relationships between various factors and S-ECC. A total of 496 children were examined. S-ECC occurred in 44.8%: d1,2 d ≥ 3 mft = 2.62 ± 3.88, d1,2 d ≥ 3 mfs = 4.46 ± 8.42. S-ECC was correlated with socio-economic factors, vitamin D supplementation, breastfeeding and using formula after the 18th month, and toothbrushing. Supplementation of vitamin D and toothbrushing tended to decrease the odds of S-ECC (OR = 0.49 (0.27–0.87); p = 0.016, OR = 0.46 (0.24–0.86) p = 0.015, respectively). Feeding exclusively with formula was observed to increase the odds of S-ECC (OR = 2.20 (1.29–3.76); p = 0.004). Consuming > three snacks daily (OR = 1.39 (0.97–1.98); p = 0.072) and the reluctance to eat resilient foods (OR = 1.63 (1.05–2.51); p = 0.028) were nullified by the confounding factors. Vitamin D supplementation, mastication of resilient food, breastfeeding in the first six months of a baby’s life, and parent-supervised toothbrushing are factors in the prevention of caries in toddlers. Breast- and bottle-feeding after the 18th month of life, the reluctance to eat solids, lack of vitamin D supplementation, hygienic neglect, and delay in introducing oral health behaviours may contribute to the development of caries in toddlers.     

Evolution und Infektionsbiologie der mit dem hämolytisch-urämischen Syndrom (HUS) assoziierten E. coli (HUSEC)

Shiga toxin (Stx)-producing Escherichia coli (STEC), which cause hemolytic-uremic syndrome (HUS), are designated as HUSEC. Their exceptional genome variability driven by evolutionary diversification permits fast adaptation to changed environmental conditions. The HUSEC collection (http://www.ehec.org), which has been established at the Institute for Hygiene in Münster, contains 42 EHEC reference strains (HUSEC001–HUSEC042). It represents a unique repository collection of pathogens and is extremely helpful for the analysis of evolutionary changes and fixed properties in the STEC that cause the most severe host injury. Such genomic attributes include slowly evolving loci, mobile genetic elements that often encode virulence factors and are assimilated via horizontal gene transfer. Current evolutionary models indicate that numerous outbreak strains evolved recently and that highly pathogenic HUSEC descend from less pathogenic progenitors. However, additional data suggest that HUSEC have small effective population sizes. The HUSEC collection is also a valuable resource with which to study important non-Shiga toxin virulence factors.     

Identification of Brucella melitensis Rev.1 vaccine-strain genetic markers: Towards understanding the molecular mechanism behind virulence attenuation

Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers.The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains.We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings.     

Association of long-duration breastfeeding and dental caries estimated with marginal structural models

PurposeTo estimate the association between breastfeeding 24 months or beyond and severe early childhood caries (S-ECC).MethodsWithin a birth cohort (n = 715) from low-income families in Porto Alegre, Brazil, the age 38-month prevalence of S-ECC (≥4 affected tooth surfaces or ≥1 affected maxillary anterior teeth) was compared over breastfeeding duration categories using marginal structural models to account for time-dependent confounding by other feeding habits and child growth. Additional analyses assessed whether daily breastfeeding frequency modified the association of breastfeeding duration and S-ECC. Multiple imputation and censoring weights were used to address incomplete covariate information and missing outcomes, respectively. Confidence intervals (CIs) were estimated using bootstrap resampling.ResultsBreastfeeding 24 months or beyond was associated with the highest adjusted population-average S-ECC prevalence (0.45; 95% CI, 0.36 to 0.54) compared with breastfeeding less than 6 months (0.22; 95% CI, 0.15 to 0.28), 6–11 months (0.38; 95% CI, 0.25 to 0.53), or 12–23 months (0.39; 95% CI, 0.20 to 0.56). High-frequency breastfeeding enhanced the association between long-duration breastfeeding and caries (excess prevalence due to interaction: 0.13; 80% CI, −0.03 to 0.30).ConclusionsIn this population, breastfeeding 24 months or beyond, particularly if frequent, was associated with S-ECC. Dental health should be one consideration, among many, in evaluating health outcomes associated with breastfeeding 24 months or beyond.     

First characterization in Italy of clinical isolates of mutans streptococci by using specific monoclonal antibodies

The aim of this investigation was to gain further insight into the prevalence of different serotypes of mutans streptococci in the Italian population by using specific monoclonal antibodies in an enzyme immunoassay. Isolates from dental plaque samples, collected from an adult population living in Pisa (Italy), were identified as mutans streptococci on the basis of their morphological and biochemical properties, and were then serotyped. The results show that 77.5% of the strains isolated belonged to serotype c or f (i.e., S. mutans), 15.9% were serotype e (i.e., S. mutans) and only two strains (1.4%) belonged to serotype g (i.e., S. sobrinus). These data are partially in agreement with other studies in Europe and in the U.S.A. The distribution pattern of the various serotypes turned out to be substantially similar among the different groups of patients, subdivided on the basis of their caries status, indicating that none of the serotypes was particularly associated with dental caries.     

鼠疫噬菌体基因组研究进展

随着不同来源鼠疫噬菌体基因组测序的完成,为从基因水平研究鼠疫噬菌体提供了机遇。本文从全基因组的水平描述了鼠疫噬菌体的基本特征,对已测序的鼠疫噬菌体基因组进行分析比较,并探讨其遗传进化关系,以及从基因预测和蛋白注释方面描述鼠疫噬菌体基因组的功能,为进一步研究鼠疫噬菌体提供数据。     

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

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

山东省聊城市2020-2021年小肠结肠炎耶尔森菌食品分离株分子特征分析

目的了解 2020-2021年山东省聊城市小肠结肠炎耶尔森菌(Y.e)食品分离株分子特征。方法对聊城市市售生肉与肉制品中Y.e开展分离培养、药敏试验、毒力基因检测、PFGE和全基因组测序(WGS)。利用微生物基因组注释系统对全基因组数据进行拼接组装, 对组装基因组开展多位点序列分型(MLST)和核心基因组多位点序列分型(cgMLST), 并应用基于WGS的单核苷酸多态性(wg-SNPs)分型方法与美国生物技术信息中心上获取的14株国内外Y.e的基因组进行遗传进化分析。结果 165份样品中共检出21株Y.e, 检出率为12.73%, 20株Y.e测序成功。Y.e携带多种耐药基因和毒力基因, 出现多重耐药现象, 毒力基因PCR检测显示21株Y.e呈现2种毒力基因特征;PFGE、MLST和cgMLST聚类分析显示, 21株Y.e呈高度多样性;wg-SNPs遗传进化分析显示20株Y.e可分为2个进化主分支。结论聊城市生肉与肉制品中分离的Y.e携带多种耐药基因和毒力基因, 且分子分型呈现高度多样性, 应加强生肉与肉制品中Y.e分子生物学监测, 开展基因组测序及分子分型检测工作, 为预防由Y.e引...     

Population analysis of clinical and environmental Vibrio parahaemolyticus isolated from eastern provinces in China by removing the recombinant SNPs in the MLST loci

Vibrio parahaemolyticus is a common seafood-borne pathogenic bacterium which causes gastroenteritis in humans. Continuous surveillance on the molecular characters of the clinical and environmental V. parahaemolyticus strains needs to be conducted for the epidemiological and genetic purposes. To generate a picture of the population distribution of V. parahaemolyticus in eastern China isolated from clinical cases of gastroenteritis and environmental samples, we investigated the genetic and evolutionary relationships of the strains using the commonly used multi-locus sequence typing (MLST, in which seven house-keeping genes are used in the protocol). A highly genetic diversity within the V. parahaemolyticus population was observed but ST3 was still dominant in the clinical strains, and 103 new sequence types (ST) were found in the clinical strains by searching in the global V. parahaemolyticus MLST database. With these genetically diverse strains, we estimated the recombination rates of the loci in MLST analysis. The locus recA was found to be subject to exceptionally high rate of recombination, and the recombinant single nucleotide polymorphisms (SNPs) were also identified within the seven loci. The phylogenetic tree of the strains was re-constructed using the maximum likelihood method by removing the recombination SNPs of the seven loci, and the minimum spanning tree was re-constructed with the six loci without recA. Some changes were observed in comparison with the previously used methods, suggesting that the homologous recombination has roles in shaping the clonal structure of V. parahaemolyticus. We propose the recombination-free SNPs strategy in the clonality analysis of V. parahaemolyticus, especially when using the maximum likelihood method.     

Prevalence of Streptococcus mutans and dental decay in schoolchildren living in Genoa (Italy)

In this study population (105 schoolchildren aged 5.5 – 11.5 yrs), dental decay was detected in 75.2% and S. mutans in 55.2% of the subjects. The presence of S. mutans was assessed - using the selective GSTB medium - in unstimulated saliva and in pooled occlusal and pooled buccal plaques from the four most posterior teeth. All three samples showed association between S. mutans presence and caries prevalence. Of the two types of plaque, the occlusal not only had a higher frequency of isolation but also a significantly higher proportion of S. mutans.The presence of S. mutans was significantly associated with both caries prevalence and extent of caries experience. Both S. mutans prevalence and S. mutans proportion in plaque increased with the number of decayed teeth present among those sampled.Sucrose consumption between meals appeared to be more correlated with the degree of caries experience rather than with caries or S. mutans prevalence.A second clinical examination was scheduled six months after the first for S. mutans-positive children who either were free of active carious lesions, or were caries-active but without signs of dental decay in the sampled teeth. Caries-active subjects proved to be more prone to new carious lesions than caries-free subjects, who tended to remain caries-free even when they had a high proportion of S. mutans in plaque, thus indicating the basic importance of the host factor in the caries process.     

Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.     

Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats

Early life vaccination is necessary to protect young children from dental caries. Our group had previously reported that a plasmid DNA vaccine pGJA-P/VAX against the glucosyltransferase (GTF) enzyme and cell surface antigen AgI/II (PAc) of Streptococcus mutans (S. mutans) elicited a specific and protective immunity in adult experimental animal models. In this report, early life immunization with the same plasmid was studied following intranasal (i.n.) and intramuscular (i.m.) delivery in murine models. The potential of inducing mucosal and systemic immune responses to special antigens was measured by ELISA. In addition, cytokine production and protection effectiveness against dental caries formation were also investigated. In the i.n. route, rats were primed when they were 5 days old, and boosted after 10 and 20 days with either plasmid pGJA-P/VAX–bupivacaine complexes, or pGJA-P/VAX alone, or empty vector. The pGJA-P/VAX–bupivacaine combination was able to mount the immune responses characterized by increased antibody levels of specific salivary IgA and serum IgG, preferential IFN-γ production and significant reduction in the dental caries lesions. In the i.m. route, rats were vaccinated with either pGJA-P/VAX alone or empty vector with the same immunization schedule as the i.n. route. Plasmid pGJA-P/VAX alone induced a significant increase in the serum IgG and IFN-γ production. However, it was not effective in eliciting specific salivary IgA and in decreasing the dental caries formation. All these findings indicate the feasibility of immunity with a targeted fusion DNA vaccine to a young immune system.     

Phylogenomics of Brazilian epidemic isolates of Mycobacterium abscessus subsp. bolletii reveals relationships of global outbreak strains

Rapidly growing, non-tuberculous mycobacteria (NTM) in the Mycobacterium abscessus (MAB) species are emerging pathogens that cause various diseases including skin and respiratory infections. The species has undergone recent taxonomic nomenclature refinement, and is currently recognized as two subspecies, M. abscessus subsp. abscessus (MAB-A) and M. abscessus subsp. bolletii (MAB-B). The recently reported outbreaks of MAB-B in surgical patients in Brazil from 2004 to 2009 and in cystic fibrosis patients in the United Kingdom (UK) in 2006 to 2012 underscore the need to investigate the genetic diversity of clinical MAB strains. To this end, we sequenced the genomes of two Brazilian MAB-B epidemic isolates (CRM-0019 and CRM-0020) derived from an outbreak of skin infections in Rio de Janeiro, two unrelated MAB strains from patients with pulmonary infections in the United States (US) (NJH8 and NJH11) and one type MAB-B strain (CCUG 48898) and compared them to 25 publically available genomes of globally diverse MAB strains. Genome-wide analyses of 27,598 core genome single nucleotide polymorphisms (SNPs) revealed that the two Brazilian derived CRM strains are nearly indistinguishable from one another and are more closely related to UK outbreak isolates infecting CF patients than to strains from the US, Malaysia or France. Comparative genomic analyses of six closely related outbreak strains revealed geographic-specific large-scale insertion/deletion variation that corresponds to bacteriophage insertions and recombination hotspots. Our study integrates new genome sequence data with existing genomic information to explore the global diversity of infectious M. abscessus isolates and to compare clinically relevant outbreak strains from different continents.     

Lactobacilli Infection Case Reports in the Last Three Years and Safety Implications

Lactobacilli constitute the dominant microbiota in many fermented foods and comprise widely used probiotics. However, these bacteria cause rare infections mostly in diabetic and immunocompromised subjects in presence of risk factors such as prosthetic hearth valves and dental procedures or caries. The scope of this survey was re-assessing the pathogenic potential of lactobacilli based on the infection case reports published in the last three years. In 2019, 2020, and 2021, total of 17, 15, and 16 cases, respectively, including endocarditis, bacteremia, and other infections, were reported. These annual numbers are higher than those observed previously. Lacticaseibacillus rhamnosus (13 cases), comprising strain GG (ATCC 53103) with established applications in healthcare, L. paracasei (7 cases), Lactobacillus acidophilus (5 cases), L. jensenii (5 cases), Lactiplantibacillus plantarum (3 cases), L. paraplantarum, L. delbrueckii subsp. delbrueckii, L. gasseri, L. paragasseri, Limosilactobacillus fermentum, and L. reuteri (1 case each) were involved. Virulence characterization of two strains that caused infections, a derivative of L. rhamnosus GG and L. paracasei LP10266, indicated that increased biofilm-forming capacity favors pathogenicity and it is determined by variable genetic traits. This survey highlights that the strains of lactobacilli that cause infections are little characterized genetically. Instead, to avoid that these bacteria become a hazard, genetic stability should be periodically re-evaluated by whole genome sequencing (WGS) to ensure that only non-pathogenic variants are administered to vulnerable individuals.     

Genomics of Streptococcus salivarius,a major human commensal

The salivarius group of streptococci is of particular importance for humans. This group consists of three genetically similar species, Streptococcus salivarius, Streptococcus vestibularis and Streptococcus thermophilus. S. salivarius and S. vestibularis are commensal organisms that may occasionally cause opportunistic infections in humans, whereas S. thermophilus is a food bacterium widely used in dairy production. We developed Multilocus sequence typing (MLST) and comparative genomic analysis to confirm the clear separation of these three species. These analyses also identified a subgroup of four strains, with a core genome diverging by about 10%, in terms of its nucleotide sequence, from that of S. salivarius sensu stricto. S. thermophilus species displays a low level of nucleotide variability, due to its recent emergence with the development of agriculture. By contrast, nucleotide variability is high in the other two species of the salivarius group, reflecting their long-standing association with humans. The species of the salivarius group have genome sizes ranging from the smallest (∼1.7 Mb for S. thermophilus) to the largest (∼2.3 Mb for S. salivarius) among streptococci, reflecting genome reduction linked to a narrow, nutritionally rich environment for S. thermophilus, and natural, more competitive niches for the other two species. Analyses of genomic content have indicated that the core genes of S. salivarius account for about two thirds of the genome, indicating considerable variability of gene content and differences in potential adaptive features. Furthermore, we showed that the genome of this species is exceptionally rich in genes encoding surface factors, glycosyltransferases and response regulators. Evidence of widespread genetic exchanges was obtained, probably involving a natural competence system and the presence of diverse mobile elements. However, although the S. salivarius strains studied were isolated from several human body-related sites (all levels of the digestive tract, skin, breast milk, and body fluids) and included clinical strains, no genetic or genomic niche-specific features could be identified to discriminate specific group.     

Epidemiological survey of Streptococcus mutans in a group of adult patients living in Pisa (Italy)

An epidemiological investigation was carried out to evaluate the prevalence of Streptococcus mutans in a group of 134 adult patients. Markedly higher frequency of isolation was observed in caries-active subjects than in caries-inactive or caries-free subjects, indicating a significant association between the prevalence of the microorganism and the caries status. Moreover, the presence of the microorganism appeared to have a significant association with the extent of caries experience evaluated by the DMF score. These findings are in agreement with those reported previously for school children in other areas of Italy.Isolation of S. mutans was compared among patient groups with different caries activity in relation to culture times of dental plaque samples in a transport medium (Colorimetric Broth Medium). S. mutans was most frequently isolated from caries-active subjects when the medium was incubated for 48 h after inoculation with dental plaque samples.     

Gene content differences across strains of Streptococcus uberis identified using oligonucleotide microarray comparative genomic hybridization

Streptococcus uberis is one of the principal causative agents of bovine mastitis. The organism is typically considered an environmental pathogen. In this study, two multilocus sequence typing (MLST) schemes and whole genome DNA microarrays were used to evaluate the degree and nature of genome flexibility between S. uberis strains. The 21 isolates examined in this study arise from a collection of 232 international isolates for which previous epidemiological and preliminary genotyping data existed. The microarray analysis resulted in an estimate of the core genome for S. uberis, consisting of 1530 ORFs, among 1855 tested, representing 82.5% of the S. uberis 0140J genome. The remaining ORFs were variable in gene content across the 21 tested strains. A total of 26 regions of difference (RDs), consisting of three or more contiguous ORFs, were identified among the variable genes. Core genes mainly encoded housekeeping functions, while the variable genes primarily fell within categories such as protection responses, degradation of small molecules, laterally acquired elements, and two component systems. Recombination detection procedures involving the MLST loci suggested S. uberis is a highly recombinant species, precluding accurate phylogenetic reconstructions involving these data. On the other hand, the microarray data did provide limited support for an association of gene content with strains found in multiple cows and/or multiple herds, suggesting the possibility of genes related to bovine transmissibility or host-adaptation.     

Evaluation of infection status in Chinese swine with porcine reproductive and respiratory syndrome virus by nested RT-PCR targeting nsp2 gene

One of the biggest obstacles in containing porcine reproductive and respiratory syndrome virus (PRRSV) results from its genetic diversity due to the high mutation rate. The nsp2 gene of PRRSV is the most hypervariable region of the genome. Since the emergence of highly pathogenic (HP)-PRRSV, many of PRRSV strains with a mutated nsp2 gene have been reported. To decipher the epidemiology of the PRRSV and identify the epidemic strains, a nested RT-PCR able to differentiate the nsp2 gene from different PRRSV strains was developed and used to test 550 clinical samples. The amplified products of 301-bp, 211-bp and 154-bp were corresponding to low pathogenic PRRSV (LP-PRRSV) infection without deletion in nsp2 gene, HP-PRRSV infection with 90-bp deletion in nsp2 gene and a variant PRRSV strain with 147-bp deletion in nsp2 gene, respectively. Of the 550 clinical samples, 192 including 108 serum samples and 84 tissue samples were tested PRRSV RNA positive. Of the 192 positive samples, 107 were infected with a single strain and 85 were infected with two strains. 84 out of 85 samples harboring two PRRSV strains of HP-PRRSV and PRRSV variant strain were detected. 97 out of 107 samples with single strain were detected with HP-PRRSV infection. The data indicated that HP-PRRSV containing a 90-bp deletion in the nsp2 gene remained the predominant strain in China.