Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.     

Comparison of seven nucleic acid amplification tests for detection of Taylorella equigenitalis

Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the use of sensitive molecular assays is important for the efficient detection of T. equigenitalis in clinical samples.     

Comparison of culture versus quantitative real-time polymerase chain reaction for the detection of Taylorella equigenitalis in field samples from naturally infected horses in Canada and Germany

A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.     

Structural characterization and serological specificities of lipopolysaccharides from Salmonella enterica serovar Gallinarum biovar Pullorum standard, intermediate and variant antigenic type strains

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.     

An investigation into the suitability of a commercial real‐time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs

Reasons for performing study: Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real‐time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real‐time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under ‘field trial’ conditions. Materials and methods: Routine prebreeding genital swabs (n = 2072) collected from Thoroughbred mares and stallions during 2009 were examined together with stored T. equigenitalis positive material. Swabs were cultured for T. equigenitalis using standard microbiological techniques. Bacterial lysates were isolated from the swabs and examined for the presence of a 16S DNA fragment of T. equigenitalis, using a commercial multiplex real‐time PCR assay system. Results: There was complete concordance between positive and negative results obtained by the 2 methods. Real‐time PCR also detected T. equigenitalis DNA from swabs that were negative using standard microbiological culture after 6 months' storage at +4°C but from which T. equigenitalis had been isolated following collection. The sensitivities of realtime PCR and bacterial culture were both 10^?3 (equivalent to 3 colony‐forming units). Conclusion and clinical relevance: Routine bacterial culture of T. equigenitalis requires an incubation period of not less than 7 days before a conclusive negative result can be obtained, whereas bacterial extraction and real‐time PCR assay can be completed in less than 6 h. The commercially‐available PCR assay tested provided a rapid and reliable method for the identification of T. equigenitalis from equine genital swabs and could be usefully employed for the screening of mares and stallions for preseason Horserace Betting Levy Board (HBLB) Code of Practice and in other situations such as for bloodstock sales screening requirements, overcoming the current delays imposed by bacterial culture requirements. Its use could be quality assured by the existing HBLB biannual testing scheme for designated laboratories.     

Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation

Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol’s anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein.     

Analysis of chromosome-sized DNA and genome typing of isolated strains ofTaylorella equigenitalis

Analysis of chromosome-sized DNA and genome typing ofTaylorella equigenitalis NCTC11184, Kentucky 188, and five strains ofT. equigenitalis isolated in Japan were carried out. The three restriction enzymes used,ApaI,NaeI andNotI, cleaved the genomic DNAs of five Japanese strains ofT. equigenitalis into relatively limited numbers of restriction fragments, which were well resolved on crossed-field gel electrophoresis (CFGE). The respective profiles after CFGE of the restriction fragments from all five strains were essentially identical to each other after digestion byApaI,NaeI orNotI. Hence it appears that these strains have a common genome type with respect to these three restriction enzymes. It was also shown that the respective profiles from these strains were essentially different from those ofT. equigenitalis NCTC11184 and those of Kentucky 188 after digestion withApaI,NaeI orNotI.Abbreviations CFGE crossed-field gel electrophoresis - FIGE field inversion gel electrophoresis - PFGE pulsed-field gel electrophoresis     

Associations between maternal milk protein genotypes with preweaning calf growth traits in beef cattle

The objectives of this study were to investigate milk casein polymorphisms in dams and to determine the impacts of maternal casein genotypes on growth traits of their sucking calves. Milk samples from 433 dams of the breeds German Angus (GA) and German Simmental (GS) were typed at the milk protein loci α _s1-casein (α_s1-CN), β-casein (β-CN), α _s2-casein (α_s2-CN), and κ-casein (κ-CN) via isoelectric focusing. Associations between casein genotypes in maternal milk with growth traits of their 1,872 calves were analyzed until the age of weaning using linear mixed models, considering either genotypes of individual casein loci (model 1) or composite α _s1-β-α _s2-κ-CN genotypes within the casein cluster (model 2). Besides environmental effects such as sex, age of the dam, and calving year-season, genetic effects (breed group and maternal and paternal effects) were considered in statistical models. The composite casein genotype BBǀA2A2ǀAAǀAB (order of genes on bovine chromosome 6: α _s1-ǀβ-ǀα _s2-ǀκ-CN) was associated with greater average daily weight gains (ADG) and heavier age-adjusted weaning weights (WW) of calves (P < 0.05). The effects of composite genotypes on birth weight of calves were similar (P > 0.05; model 2). With regard to individual casein loci, greater ADG and WW were observed for calves from dams with the genotypes κ-CN BB and α _s1-CN BB, respectively (P < 0.05; model 1). Age-adjusted WW was largest for calves from dams carrying the κ-CN genotype BB (215 kg) compared with calves representing the maternal AB and AA genotypes (both 204 kg). Results from the present study suggested selectable casein genotypes due to their nutritional value of milk (value in terms of offspring performances), offering new perspectives for breeding strategies in beef cattle to improve preweaning calf performance.     

Necrotizing Infectious Enteritis in Piglets,Caused by Clostridium Perfringens Type C: I. Biochemical and Toxigenic Properties of the Clostridium

Twenty Danish and 3 foreign strains of the porcine subtype of Cl. perfringens type G have been studied with regard to biochemical activity and toxin formation.The strains examined showed no deviation from the biochemical features characteristic of Gl. perfringens. Two of the strains showed delayed fermentation of saccharose, 1 of them also of salicin. Only 3 of the strains gave a positive nitrite reaction. The rest of the strains (20) presumably reduced nitrate to NH₃ within 3 days.All of the strains examined produced the major antigens α and β, none of them ε and ι. Of the minor antigens, θ and v were produced by all the strains, x by 2 of the foreign strains and 5 of the Danish strains. None of the strains gave reaction for the δ, λ and μ antigens.Some of the Danish strains were hemolytic in the presence of α, δ, and θ antitoxins, others not, while a few varied. The foreign strains were non-αδθ hemolytic.     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

Properties of macrophages and lymphocytes appearing in rat renal fibrosis followed by repeated injection of cisplatin

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68⁺ M1-macrophages and major histocompatibility complex (MHC) class II⁺ macrophages remarkably increased persistently, whereas CD163⁺ M2-macrophages slightly increased. MHC class II⁺/CD68⁺ and MHC class II⁺/CD163⁺ macrophages were present, indicating that MHC class II⁺ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-β1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4⁺ and CD8⁺ T cells appeared in close relation with MHC class II⁺ macrophages, and mainly CD4⁺ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.     

Effect of Antibiotic-containing Extenders on Taylorella equigenitalis Contaminated Semen

Taylorella equigenitalis is a gram-negative coccobacillus and the causative agent of a transmissible venereal disease in horses known as contagious equine metritis. Outbreaks of contagious equine metritis have been documented in various countries since 1977, with the most recent discovery in the United States in December 2008. During disease occurrences, culturing semen samples for T equigenitalis before breeding may help to prevent transmission of this disease; however, little is known about the antimicrobial activity of equine semen extenders against the organism. The purpose of this study was to investigate the infectivity levels of T equigenitalis in three equine semen extenders inoculated with known concentrations of the organism. The semen extenders used for this study included INRA 96, E-Z Mixin BF, and VMDZ. In addition, Timentin was added to INRA 96 at three different concentrations (0.5, 1.0, and 1.5 mg/mL) to investigate possible synergistic effects of antibiotic supplementation of extenders. Results were based on the visual counting of the colonies on chocolate Eugon agar plates. Both INRA 96 (with added Timentin) and VMDZ (as supplied by the manufacturer) significantly reduced the numbers of T equigenitalis isolated from semen extenders as compared with INRA 96 (as supplied by the manufacturer) or the antibiotic free E-Z Mixin BF. Our findings indicate that INRA 96 (with added Timentin) or VMDZ may significantly decrease the growth of T equigenitalis in extended semen; however, it is also important to consider the possible effects of antibiotic supplemented extenders on sperm longevity and fertility in addition to eliminating specific pathogens in semen.     

Resistance phenotypes and genotypes among multiple-antimicrobial-resistant Salmonella enterica subspecies enterica serovar Choleraesuis strains isolated between 2008 and 2012 from slaughter pigs in Okinawa Prefecture,Japan

A total of 349 Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) strains, which were isolated between 2008 and 2012 from 349 pigs at two slaughterhouses in Okinawa Prefecture, Japan, were investigated for antimicrobial susceptibility and the presence of antimicrobial resistance genes. All isolates were resistant to at least four antimicrobial agents. The antimicrobial agents for which isolates showed a high incidence of resistance were as follows: ampicillin (100%) and streptomycin (100%), followed by gentamicin (99.7%), oxytetracycline (99.7%), sulfamethoxazole/trimethoprim (99.4%), nalidixic acid (40.1%) and oxolinic acid (40.1%). All isolates were sensitive to cefuroxime, ceftiofur, colistin, fosfomycin, enrofloxacin, orbifloxacin and danofloxacin. The predominant resistance phenotypes and genotypes were: resistance to ampicillin, streptomycin, gentamicin, oxytetracycline and sulfamethoxazole/trimethoprim (58.5%, 204/349) and blaTEM-strA-strB-aadA1-aadA2-aacC2-tet (B)-sul1-sul2-dhfrXII-dhfrXIII (36.1%, 126/349). The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of the quinolone-resistant isolates (n=12) showed amino acid substitutions of Ser-83→Phe or Asp-87→Tyr in GyrA and Ser-107→Ala in ParC. To our knowledge, this is the first report on the molecular characterization of antimicrobial resistance among S. Choleraesuis strains in Japan.     

High Prevalence of BlaCTX-M Group Genes in Aeromonas dhakensis Isolated from Aquaculture Fish Species in South Korea

The prevalence of resistant genes against β-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more β- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all β-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect β-lactamase-encoding genes, bla_TEM, bla_OXA-B and bla_CTX-M. In the results, the bla_TEM-1 gene was harbored in all strains, whereas only 3 strains harbored bla_OXA gene. In the case of bla_CTX-M gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the bla_CTX-M positive strains showed simultaneous resistance to all β-lactams (18 out of 30 strains). In sequence analysis for bla_CTX-M genes detected, they were CTX-M group 1-encoding genes including bla_CTX-M-33 from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.     

Mutant prevention concentration of orbifloxacin: comparison between Escherichia coli,Pseudomonas aeruginosa,and Staphylococcus pseudintermedius of canine origin

Background

The mutant prevention concentration (MPC) is an important parameter to evaluate the likelihood of growth of fluoroquinolone-resistant mutants for antimicrobial-pathogen combinations. The MPCs of fluoroquinolones for different canine pathogens have not been compared. In this study, we compared for the first time orbifloxacin MPCs between susceptible strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus pseudintermedius of canine origin.

Methods

More than 10¹⁰ CFU/ml of 10 strains of each bacterial species were inoculated onto Muller-Hinton agar supplemented with different concentrations of orbifloxacin from 1× to 64× minimum inhibitory concentration (MIC) and the MPCs were recorded. MICs of original strains and of mutants arising after exposure to sub-MPC concentrations (one per original strain) were determined in the presence or absence of efflux pump inhibitors (EPIs). The effects of quinolone resistance-determining region (QRDR) mutations were also examined.

Results

MPCs were significantly higher for P. aeruginosa (16–128 μg/ml) than for E. coli (0.5–32 μg/ml). MPCs for S. pseudintermedius varied between the low-susceptible (16–128 μg/ml) and the high-susceptible strains (4–16 μg/ml) and were the most broadly distributed among the three species. Regarding resistance mechanisms, only one QRDR mutation in gyrA was found in all of the 10 mutants of E. coli and in 4 of the 10 mutants of P. aeruginosa, whereas mutations in both grlA and gyrA were found in 3 mutants and one mutation in grlA was found in 2 mutants among the 10 mutants of S. pseudintermedius. In the presence of an EPI, the MICs of P. aeruginosa mutants decreased markedly, those of E. coli mutants decreased moderately, and those of S. pseudintermedius mutants were unaffected.

Conclusions

MPCs of orbifloxacin vary between bacterial species of canine pathogens, possibly due to the diversity of the main fluoroquinolone resistance mechanism among these species. Therefore, the type of bacterial species should be taken into consideration when using fluoroquinolone drugs such as orbifloxacin in canines.     

Demonstration of Heterogeneous Genotypes of Taylorella equigenitalis Isolated from Horses in Six European Countries by Pulsed-Field Gel Electrophoresis

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12. Two English isolates and 4 French isolates gave identical PFGE profiles to those of Kentucky 188 from the United States. A common genotype was found in 5 isolates from Belgium and England and also in 10 isolates from France and Switzerland. The analysis of genomic DNA from 12 isolates of T. equigenitalis obtained from male horses in France, Sweden and Switzerland gave no evidence of a sex-related difference in the genomic DNA. Genomic DNA from 11 streptomycin (STM)-susceptible isolates obtained in Sweden and Switzerland were classified into four genotypes by PFGE. Each of the six genotypes determined among the 17 isolates from these two countries had single phenotypes for resistance or susceptibility to STM.     

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.     

Pleiotropic effects of polysaccharide capsule loss on selected biological properties of Streptococcus suis

In this study, an unencapsulated Streptococcus suis mutant was used to investigate the pleiotropic effects resulting from capsule loss. The capsule deficient mutant of S. suis acquired a biofilm-positive phenotype, which was associated with significantly increased cell surface hydrophobicity. Cell-associated fibrinogen-binding and chymotrypsin-like activities were decreased in the unencapsulated mutant. The mutant did not differ significantly from the encapsulated parent strain for minimal inhibitory concentrations to penicillin G, ampicillin, and tetracycline. However, while the encapsulated strain was highly resistant to the bactericidal action of penicillin G and ampicillin, the unencapsulated mutant was approximately 60-fold more sensitive. Compared with the parent strain, the unencapsulated mutant induced a much higher inflammatory response in monocyte-derived macrophages resulting in an increased secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8. The capsule appears to hinder important adhesins or hydrophobic molecules that mediate biofilm formation, as well as cell wall components capable of stimulating immune cells.     

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.     

Successful Eradication of Taylorella asinigenitalis,Pseudomonas aeruginosa,and Klebsiella pneumoniae Venereal Bacterial Pathogens Using Domestic Steam Disinfection: Implications for AI Practice

Steam disinfection has become established as a trusted method of microbial decontamination; however, there have been no reports on the use of this technology to disinfect equipment used in collection of semen in artificial insemination practice. Hence, it was the aim of this study to examine the survival of Taylorella asinigenitalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae venereal bacterial pathogens using domestic steam disinfection. Sixteen bacterial pathogens from three genera Taylorella, Pseudomonas, and Klebsiella each at an inoculum density of approximately 1.5 × 10⁷ colony-forming units were subjected to a steam disinfection cycle. No bacteria were recovered after disinfection, including following recovery and nonselective cultural enrichment techniques. In the absence of full sterilization, domestic steam disinfection of equipment offers a cheap, simple, and widely available technology for the elimination of these pathogens, thereby enhancing infection control in equine breeding.