Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A–L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.     

ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis

The ermB gene was identified in 111 erythromycin resistant isolates of Streptococcus uberis from cases of bovine mastitis associated either with a constitutive (47/111) or an inducible (64/111) phenotype, as well as a phenotypic resistance to all macrolides tested. Resistance to lincosamides was identified in 14 other isolates of S. uberis from bovine mastitis cases and was mainly mediated by the linB gene; resistance conferred by a combination of two genes (linB–lnuD, ermB–linB) was also detected.     

Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States

Streptococcus suis is an important swine pathogen and a zoonotic agent. Differences in virulence have been noted among the 33 described serotypes, serotype 2 being considered the most virulent. In this study, we aimed at assessing the serotype distribution and the production of virulence-associated markers by strains recovered from diseased pigs in the United States (U.S.). Results showed that among the 100 strains evaluated, serotype 3 (20% of the isolates) and serotype 2 (17%) were the most prevalent. We then investigated the presence in these isolates of the genes sly, epf and mrp, encoding the virulence-associated markers suilysin (SLY), extracellular factor (EF) and muramidase-released (MRP) protein, respectively. The effective production of the markers by the strains was also verified. Results showed that the presence of the gene did not always correlate with actual expression of the respective protein. In the case of MRP, this was due, in most cases, to frameshift mutations at the 5′ end of the gene resulting in premature stop codons. The most prevalent phenotypes among U.S. strains were MRP⁺EF⁻SLY⁻ (40%) and MRP⁻EF⁻SLY⁺ (35%). Serotype distribution greatly differed from that reported in several European countries, as did the production of virulence markers, particularly for serotype 2. On the other hand, our results for the U.S. S. suis isolates are similar to those reported for Canadian strains, suggesting a common status in North America.     

Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates

Nineteen Streptococcus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5′ end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/sly+ isolates.     

Effect of bacitracin on tetracycline resistance in Escherichia coli and Salmonella

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.     

Application of the California mastitis test in intramammary Streptococcus agalactiae and Staphylococcus aureus infections of camels (Camelus dromedarius) in Kenya

A study was conducted on 207 lactating camels in six herds in Kenya to evaluate the California mastitis test (CMT) for the detection of intramammary infections (IMIs) caused by Streptococcus agalactiae and Staphylococcus aureus and to investigate the prevalence of both the pathogens in the camel udder. IMI with S. agalactiae was found in 12% of all camels sampled. IMI with S. aureus was present in 11% of all camels sampled. The herd-level prevalence of IMI varied between 0 and 50% for S. agalactiae and between 0 and 13% for S. aureus. Longitudinal observations over 10–12 months confirmed persistent infections for both pathogens. Observations in one herd suggested that camel pox was a contributing factor in spreading and exacerbating S. agalactiae udder infections.The CMT had quarter-level sensitivities of 77 and 68% for S. agalactiae and S. aureus in camels, respectively. The CMT specificities were 91% for both the pathogens.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Identification of a Streptococcus equi Strain Responsible for Four Outbreaks of Strangles in Colorado

The objective of this study was to use a new subtyping technique to determine the identity of five Streptococcus equi isolates from four outbreaks of strangles in Colorado during 2005−2006. All five of the isolates from the four strangles outbreaks in Colorado contained SeM allele 28. This SeM allele is typical of American isolates of S. equi and has previously been linked with strangles outbreaks in Minnesota in 1994 and Kentucky in 1995. Use of the new S. equi strain subtyping method allowed for characterization of recent S. equi isolates associated with outbreaks of strangles in Colorado. To our knowledge, this is the first report of typing of more recent isolates from North America. This approach, however, has been used in situations in the United Kingdom to differentiate the vaccine strain of S. equi from that of the wild type of the bacteria.     

Prevalence and age-related infection of Cryptosporidium suis, C. muris and Cryptosporidium pig genotype II in pigs on a farm complex in the Czech Republic

A total of 413 pig faecal samples were collected from pre-weaners (119), starters (131), pre-growers (123) and sows (40) from a farm with a closed breeding system segmented into two breeding complexes and a growing complex in the region of Vysočina, Czech Republic and screened for the presence of Cryptosporidium using staining methods and genotyping (SSU rRNA). Cryptosporidium oocysts were detected by microscopy in the faeces of 21.1% of the samples (87/413). Sequence analyses and RFLP identified C. suis in 44, Cryptosporidium pig genotype II in 23 and C. muris in 2 samples. No mixed infections were found.Pigs under 7 weeks of age were infected with C. suis only. Cryptosporidium pig genotype II was found in animals from 7 weeks of age. No relationship was found between diarrhoea and any Cryptosporidium infection in any of the different age groups (P < 0.05). The pre-weaned pigs shed significantly more Cryptosporidium oocysts than older pigs and it was associated with C. suis infection.     

Streptococcus zooepidemicus Myocardial Abscessation in a 1-Month-Old Thoroughbred Foal

A 1-month-old male Thoroughbred foal was presented at necropsy. A focal purulent lesion was observed in the right ventricular free wall of the myocardium. To the authors' knowledge, this is the first report of Streptococcus zooepidemicus myocardial abscessation in an equine.     

The two-component regulatory system CiaRH contributes to the virulence of Streptococcus suis 2

Two-component regulatory systems (TCSs) are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. In this work, we constructed a mutant strain of the TCS CiaRH and measured its virulence in vitro and in vivo. Compared with the wild type strain, the mutant strain exhibited a significant decrease in adherence to epithelial cells Hep-2 and PIEC. Furthermore, the deletion of CiaRH not only enhanced the bactericidal activity of RAW264.7 macrophage against Streptococcus suis 2, but also increased blood clearance of S. suis 2 in vivo. More importantly, the mutant was attenuated in vivo in CD1 mice and pigs, with reduced mortality, morbidity and impaired bacterial growth observed in specific organs. These results suggest that the CiaRH is required for S. suis 2 virulence.     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

Relationship between level of antibiotic use and resistance among Escherichia coli isolates from integrated multi-site cohorts of humans and swine

The objective of this longitudinal ecological study was to examine the relationship between the prevalence of antibiotic-resistant (AR) commensal Escherichia coli isolates from both monthly human wastewater and composite swine fecal samples and the concurrent aggregated monthly antibiotic use recorded within each host species in multi-site vertically integrated swine and human populations. In addition, human vocation (swine worker versus non-swine worker), swine production group, and season were examined as potential confounding variables. Human and swine E. coli isolates (n = 2469 human and 2310 swine, respectively) were tested for antimicrobial susceptibility using a commercial broth microdilution system. In the human population, among swine workers the relative odds of tetracycline resistance were increased significantly for tetracycline (class) drug use at the third quartile and above of mean monthly dosage (MMD) (OR = 1.8) as compared to the referent category (non-use). The relative odds of ciprofloxacin resistance were significantly increased for ciprofloxacin use in non-swine workers (OR = 5.5) as compared to the referent (non-use). The relative odds of tetracycline resistance were increased significantly for chlortetracycline use in medicated feed for the upper tertile of MMD category (OR = 2.9) as compared to the referent category (no use) across all swine production groups. While high variability among seasonal samples over the 3-year period was observed, no common seasonal trends relating to antibiotic use and prevalence of resistance over the 3-year period were apparent. The overall effects of concurrent human and swine antibiotic use on AR E. coli levels were inconsistent and modest in this study.     

The bovine annexin 9 gene (ANXA9) is significantly associated with milk-fat yield in a Spanish Holstein–Friesian population

On the basis of QTL studies for milk-fat yield trait on BTA3, annexin 9 protein (ANXA9), fatty acid transport protein type 3 (SLC27A3) and diacylglycerol O-acyltransferase 1 (DGAT1) were selected as candidate genes. Three different single nucleotide polymorphisms (SNPs) of bovine ANXA9, SLC27A3 and DGAT1 genes have been tested in a selective genotyping design for milk-fat yield. Significant allele frequency differences were found for ANXA9 (p = 0.02), in Holstein–Friesian animals with high and low breeding values for milk-fat yield. Regression analysis also showed a significant effect (p = 0.0207) between estimated breeding values (EBVs) for fat milk content and ANXA9 polymorphism. So ANXA9 gene falls into a significant quantitative trait loci interval for milk-fat yield that was previously reported on bovine chromosome 3 in other dairy populations. Our results suggest that the ANXA9 gene polymorphism or a linked segregating QTL contributes to variation in milk-fat yield.     

广东地区猪链球菌的耐药性特点及四环素耐药基因携带情况

猪链球菌(Streptococcus suis,SS)是危害世界养猪业的重要细菌性病原菌之一,本研究旨在了解猪链球菌临床分离株的耐药性特点和四环素耐药基因的携带情况,为临床上该病的防控提供科学依据。本试验采用K-B法和CLSI推荐的肉汤微量稀释法检测猪链球菌广东分离株对18种抗菌药物的耐药性。结果显示,被检菌株对四环素类、磺胺类和氨基糖苷类抗菌药物表现较强的耐药性,尤其对四环素类药物的耐药率高达98.2%;对头孢菌素类药物比较敏感;菌株都耐受6种或6种以上的抗菌药物,其中以6和14重耐药菌株的数量最多,分别占20%和17%。本试验设计了4对引物检测四环素耐药基因携带情况,结果发现56株菌中以携带tetM基因为最多(85.71%)。结果表明tetM很可能是介导广东SS分离株对四环素产生极高耐药率的主要原因之一。     

动物源性沙门氏菌的耐药性分析及氟苯尼考类耐药基因的鉴定

为了解动物沙门氏菌的流行情况和药物敏感性及氟苯尼考耐药株的耐药基因分布,本试验对临床上疑似患沙门氏菌病的病料进行病原分离和细菌的多重PCR鉴定;采用K-B法测定分离株对23种抗菌药物的敏感性;选择氟苯尼考耐药菌株扩增floR、fexA、fexB、cfr和pexA基因。结果显示,共鉴定出61株沙门氏菌,其中肠炎沙门氏菌10株,鸡白痢沙门氏菌12株,鼠伤寒沙门氏菌39株。所有菌株对青霉素、红霉素、万古霉素耐药,90.16%对6种及6种以上抗菌药耐药。floR基因广泛存在于鼠伤寒沙门氏菌氟苯尼考耐药菌株中(8/12,66.67%),未发现其他耐药基因。研究结果表明鼠伤寒沙门氏菌是鹅源分离株中的优势血清型;floR基因主要介导沙门氏菌对氟苯尼考耐药性,但可能还存在其他机制。     

Prevalence of Calodium hepaticum (Syn. Capillaria hepatica) in house mice (Mus musculus) in the Azores archipelago

Calodium hepaticum (Syn. Capillaria hepatica) is a zoonotic liver nematode of mammals distributed worldwide. Rodents are believed to be the main reservoirs of this nematode. In this paper, prevalence of the parasite was analyzed in liver histological sections from 51 house mice (Mus musculus) caught in human-inhabited houses, from two localities (Furnas and Rabo de Peixe) on São Miguel island from the Azores archipelago (Portugal). Mean prevalence of infection was 19.6%, with 33.3% prevalence in Furnas and 4.1% in Rabo de Peixe (P = 0.07). No significant differences were found between the prevalence of infection and the age, body weight and the sex of mice. Hepatic lesions found were either acute and/or chronic stage and consisted of moderate to severe multifocal pyogranulomatous hepatitis with encapsulated eggs with typical bipolar plugs and moderate to severe necrotizing hepatitis consistent with larva tracks. Periportal inflammatory infiltration, hepatocyte regeneration and bile duct hyperplasia were also noted. In most cases, hepatic lesions occupied more than 50% of the liver, but despite severe lesions, in some mice, no signs of hepatic failure were noticed. The high rate of infection found in the present study suggests that house mice are an important reservoir for this parasite in the Azores and could have a role in human transmission.     

Acaricidal effects of the essential oil of Origanum minutiflorum (Lamiaceae) against Rhipicephalus turanicus (Acari: Ixodidae)

The acaricidal effects of the volatile essential oil Origanum minutiflorum O. Schwarz & P.H. Davis (Lamiaceae) against adult Rhipicephalus turanicus was evaluated at a variety of concentrations and exposure times. Generally tick mortality increased with concentration and exposure. Ticks exposed to vapors from cotton wicks containing at least 10 μl/L resulted in complete (100%) mortality at 120 min. The major constituent of essential oil obtained from the plant material of O. minutiflorum was carvacrol.