Antimicrobial susceptibility testing of Spanish field isolates of Brachyspira hyodysenteriae

This study is the first conducted in Spain to evaluate antimicrobial susceptibility of field isolates of Brachyspira hyodysenteriae. One hundred and eight isolates of the bacterium, recovered from different Spanish swine farms between 2000 and 2007, were investigated. The minimum inhibitory concentrations (MIC) of erythromycin, tylosin, tiamulin, valnemulin, clindamycin and lincomycin were determined using a broth microdilution technique. Most of the isolates showed poor susceptibility to erythromycin (MIC₉₀ > 256 μg/ml), tylosin (MIC₉₀ > 256 μg/ml), clindamycin (MIC₉₀ > 4 μg/ml) and lincomycin (MIC₉₀ = 128 μg/ml). Reduced susceptibility to tiamulin and valnemulin was observed with a MIC > 2 μg/ml in 17.6% and 7.41% of the B. hyodysenteriae isolates, respectively. Moreover, a survival analysis permitted the detection of an increasing trend in the MIC values for almost all the antimicrobials used in the treatment of swine dysentery when comparing recent isolates (from 2006 to 2007) with those recovered in earlier years (between 2000 and 2004).     

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.     

Cessation of clinical disease and spirochete shedding after tiamulin treatment in pigs experimentally infected with “Brachyspira hampsonii”

With the emergence of “Brachyspira hampsonii” associated with swine dysentery in North America, identification of effective treatments and interventions is a pressing need. Denagard® (tiamulin hydrogen fumarate) Liquid Concentrate 12.5% is approved in the United States for treatment of dysentery caused by Brachyspira hyodysenteriae at 0.006% in the water. In this study, the effectiveness of tiamulin in resolving clinical disease, eliminating viable spirochete shedding, and reducing neutrophilic colitis following infection with either “B. hampsonii” or B. hyodysenteriae was evaluated. Seventy-eight 7-week-old crossbred pigs were divided into three groups [sham-inoculated (n = 18), “B. hampsonii”-inoculated (n = 30), and B. hyodysenteriae-inoculated (n = 30)]. Each inoculum group was divided into three subgroups which received either 0.006% tiamulin, 0.018% tiamulin, or no medication. Both levels of tiamulin resolved clinical disease within 24 h of treatment initiation, eliminated spirochete shedding within 72 h of treatment initiation, and resolved and/or prevented histologic lesions in pigs infected with either Brachyspira spp.     

Multiple typing for the epidemiological study of contamination of broilers with thermotolerant Campylobacter

This study aims to investigate the genetic diversity of thermotolerant Campylobacter in commercial broiler flocks and in the environment of broiler farms in Belgium. Seven out of 18 investigated flocks became colonized during rearing. Fluorescent amplified fragment length polymorphism (FAFLP), pulsed field gel electrophoresis (PFGE), restriction fragment length polymorphism of the flagellin A gene (flaA-RFLP) and antimicrobial resistance profile (ARP) were used for typing of the isolates. By the combination of FAFLP and PFGE, 22 Campylobacter genotypes could be distinguished. Colonization was almost exclusively with Campylobacter jejuni and unique genotypes were found in each flock. Multiple genotypes were detected in the broilers of 3 flocks, either simultaneously or successively. In 5 flocks, strains that were resistant to at least one antibiotic (mostly tetracycline) were found. The presence of other broiler houses on the farm did not result in a higher probability of colonization. The nipple water was contaminated with the same genotype as the broilers, illustrating its importance for transmission of Campylobacter. The same genotype was detected in a water puddle and in the broiler flock during rearing in 3 flocks. Once, the same genotype was isolated from the ditch water shortly before it was detected in the broilers.     

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

Background

The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed.

Methods

The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum.

Results

The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78^T (ATCC® 27164^T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml.

Conclusions

The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78^T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.     

Physiological properties and classification of strains of Treponema sp. isolated from pigs in poland

Fifty-one treponemas were isolated from pigs. Twenty-three isolates with typical morphology and growth characteristic were beta hemolytic, enteropathogenic, produced indole and with exception of three strains did not ferment fructose. These strains were classified as typical T. hyodysenteriae and were usually isolated from pigs with symptoms of mucohemorrhagic diarrhoea. The seventeen other isolates were weakly beta hemolytic after 48 h incubation, enteropathogenic, 12 out of 17 produced indole, 10 out 17 fermented fructose. These strains were usually isolated from pigs with symptoms of gray-green diarrhoea and classified as T. hyodysenteriae 2 biotype or intermediate type. They may be compared with Treponema sp. isolated by Taylor et al. Eleven non enteropathogenic strains showed typical characteristic for T. innocens. Gas chromatography analysis of the fatty acids production from glucose, showed that all isolated treponemas produced acetate and butyrate. Typical T. hyodysenteriae produced additionally propionate. Strains of T. hyodysenteriae biotype 2 produced propionate or isobutyrate as well.     

Blood concentrations of amino acids, glucose and lactate during experimental swine dysentery

The aim of this study was to examine blood concentrations of amino acids, glucose and lactate in association with experimental swine dysentery. Ten pigs (approximately 23kg) were orally inoculated with Brachyspira hyodysenteriae. Eight animals developed muco-haemorrhagic diarrhoea with impaired general appearance, changes in white blood cell counts and increased levels of the acute phase protein Serum Amyolid A. Blood samples were taken before inoculation, during the incubation period, during clinical signs of dysentery and during recovery. Neither plasma glucose nor lactate concentrations changed during the course of swine dysentery, but the serum concentrations of gluconeogenic non-essential amino acids decreased during dysentery. This was mainly due to decreases in alanine, glutamine, serine and tyrosine. Lysine increased during dysentery and at the beginning of the recovery period, and leucine increased during recovery. Glutamine, alanine and tyrosine levels show negative correlations with the numbers of neutrophils and monocytes. In conclusion, swine dysentery altered the blood concentrations of amino acids, but not of glucose or lactate.     

Spatial epidemiology of the Asian honey bee mite (Varroa destructor) in the North Island of New Zealand

We describe the spatial epidemiology of Varroa destructor infestation among honey bee apiaries in the greater Auckland area of the North Island of New Zealand. The study population was comprised of 641 apiaries located within the boundaries of the study area on 11 April 2000. Cases were those members of the study population declared Varroa-infested on the basis of testing conducted between April and June 2000. The odds of Varroa was highest in apiaries in the area surrounding transport and storage facilities in the vicinity of Auckland International Airport. A mixed-effects geostatistical model, accounting for spatial extra-binomial variation in Varroa prevalence, showed a 17% reduction in the odds of an apiary being Varroa infested for each kilometre increase in the squared distance from the likely site of incursion (95% Bayesian credible interval 7–28%). The pattern of spatially autocorrelated risk that remained after controlling for the effect of distance from the likely incursion site identified areas thought to be ‘secondary’ foci of Varroa infestation initiated by beekeeper-assisted movement of infested bees. Targeted investigations within these identified areas indicated that the maximum rate of local spread of Varroa was in the order of 12 km/year (interquartile range 10–15 km/year).     

Molecular epidemiology of Cryptosporidium subtypes in cattle in England

Samples of Cryptosporidium spp., collected in a cross-sectional study of calves (median age 26 days) from 41 farms in Cheshire, UK, underwent molecular typing. The aim was to determine naturally occurring species/genotypes and to investigate transmission pathways within a local area. Of 60 positive samples, 54 were sequenced at an 18S rRNA locus and 51 were typed at a GP60 locus. C. parvum was identified in 50 samples, three cases were C. bovis and one was Cryptosporidium deer-like genotype. Six GP60 subgenotypes were identified. One subgenotype (IIaA15G2R1) was highly prevalent throughout the study area. A single subgenotype was identified on 20/22 farms. Two subgenotypes were found on 2/22 farms. The spatial scan statistic detected a cluster of subgenotype IIaA15G2R1 comprising seven farms. This suggests local transmission of the parasite between farms. As most of the isolates detected were the potentially zoonotic C. parvum allele IIa, intervention strategies should be considered to reduce the threat to public health. Biosecurity measures may reduce transmission between farms and result in lower levels of environmental contamination.     

Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province, Iran

The aim of this study was to determine the prevalence, variability with host age, and the genotypes of species of Cryptosporidium in cattle from 15 dairy farms in Qazvin province, Iran. Fecal samples, collected from 272 cattle during May 2006 to December 2007, were characterized microscopically. Oocysts from 51 positive samples were analyzed using PCR assay of 18S SSU rRNA, restriction fragment length polymorphism (RFLP) and sequencing. We identified 72.6% of the positive samples as Cryptosporidium parvum, 17.7% as Cryptosporidium andersoni, 7.8% as Cryptosporidium bovis and 1.9% as a novel genotype of C. parvum possessing a single mutation on MboII restriction. An infection rate of 19.5% of C. parvum among 174 pre-weaned calves was significantly higher than the 3.1% among 98 post-weaned calves (P < 0.0006). This is the first report of C. bovis and the new subgenotype of C. parvum in Iranian cattle.     

Molecular epidemiology of Streptococcus zooepidemicus infection in naturally occurring equine respiratory disease

The objective of the study was to characterise the molecular epidemiology of Streptococcus zooepidemicus infection among isolates collected sequentially from recently weaned, pasture maintained Welsh mountain ponies with naturally occurring respiratory disease. Weekly nasopharyngeal and tracheal lavage samplings over a 10-week period were conducted in 29 ponies. Two PCR typing methods based on characterisation of the M-protein hypervariable (HV) region and the 16S-23S rRNA gene intergenic spacer were then applied to isolates of S. zooepidemicus recovered from nasopharyngeal swab and tracheal wash samples. S. zooepidemicus infection was highly prevalent during the study, being isolated from 94% of tracheal washes and 88% of nasopharyngeal swabs. Among 39 different S. zooepidemicus types isolated, more were isolated from the trachea (n=33) than the nasopharynx (n=27). There was evidence from temporal patterns of infection for clonal succession over time by the more prevalent S. zooepidemicus types. Novel S. zooepidemicus types were identified, including previously untyped HV regions and intra-strain multiples of both the HV region and intergenic spacer types.     

Rapid virulence typing of Pasteurella multocida by multiplex PCR

The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.     

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

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.     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.     

家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析

本试验旨在通过细菌分离鉴定,明确家养观赏地图鱼的死因,筛选敏感药物。采用常规方法分离纯化细菌后,进行细菌形态学观察,并通过小白鼠致病性试验、细菌主要生化鉴定、16SrDNA序列测定分析、药敏试验及耐药基因检测等方法对分离的细菌进行鉴定及耐药分析。分离出3株革兰氏阴性短杆菌,根据细菌形态特征及理化特性,结合16SrDNA序列测定与系统发育分析结果,判定其分别为肺炎克雷伯氏菌(Klebsiella pneumoniae)、维氏气单胞菌(Aeromonas veronii)和黏质沙雷氏菌(Serratia marcescens),其中肺炎克雷伯氏菌具有较强致病性。3株细菌均对洛美沙星、氧氟沙星、阿米卡星、卡那霉素敏感,对阿莫西林、氟苯尼考、克林霉素、甲硝唑等具有较强耐药性。结果表明,肺炎克雷伯氏菌、维氏气单胞菌、黏质沙雷氏菌的混合感染是家养观赏地图鱼的死亡原因。     

Molecular epidemiology of Mycobacterium bovis: usefulness in international trade

Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.     

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).     

Molecular characterisation of Cryptosporidium isolates from pet reptiles

In this study, we investigated the presence of Cryptosporidium in 171 faecal samples from reptiles commonly used as pet animals. These include lizards belonging to the genera Eublepharis, Pogona, Chlamydosaurus, Hemiteconyx, Teratoscincus, Tiliqua, Iguana, and Chamaeleo, snakes of the genera Lampropeltis, Elaphe, Python, Boa and Corallus, and tortoises belonging to the genera Testudo and Kinixys. Cryptosporidium oocysts were detected by immunofluorescence using a commercially available kit and cryptosporidial DNA by amplification of a polymorphic fragment of the 18S rDNA and the HSP70 locus.Cryptosporidium was detected in 38.6% and 25.1% of the samples analysed by immunofluorescence and PCR, respectively. Molecular characterisation of the isolates confirmed that C. serpentis and C. varanii (syn. C. saurophilum) are the main species involved in infection in pet reptiles but also showed the presence of C. parvum and C. muris, as well as other species or genotypes of this parasite including the Cryptosporidium mouse genotype and Cryptosporidium tortoise genotype previously described in reptiles. In addition, a Cryptosporidium sp. was isolated from a chameleon and a python.