Detection of IgM-rheumatoid factor and anti-citrullinated protein antibodies in healthy horses and their comparison.

Rheumatoid factor (RF) is used in human and veterinary medicine in the form of IgM RF traditionally to support the diagnosis of rheumatoid arthritis (RA). In the latest diagnostic criteria, presence of anti - citrullinated protein antibodies (ACPA) was added to the grading system for the diagnosis of RA in humans. A change which is not integrated or routinely used in veterinary medicine. The criteria changed partly because of RF's diagnostic shortcomings, which include its increased titer detection in humans with non-rheumatoid diseases, inability to predict the disease and increased titers over the limit in the older population. Clinical signs similar to human RA were reported in horses in a condition known as idiopathic polysynovitis. Similarities in the clinical presentation to RA raised a question to the usability of RF and ACPA in horses. In our study, sixty clinically healthy horses, ranging from 3 days to 30 years of age, were evaluated for their serum levels of IgM RF. 55 of these horses were tested for ACPA, using methods of ELISA measuring Anti - CFG (Anti citrullinated fibrinogen antibody). The results of the study demonstrated the existence of an age-dependent increase in the level of IgM RF up to the age of about 9 years and ACPA's independence of the horse's age as well as both markers independence of the horse sex.

Chemotactic droplet swimmers in complex geometries.

Chemotaxis1 and auto-chemotaxis are key mechanisms in the dynamics of micro-organisms, e.g. in the acquisition of nutrients and in the communication between individuals, influencing the collective behaviour. However, chemical signalling and the natural environment of biological swimmers are generally complex, making them hard to access analytically. We present a well-controlled, tunable artificial model to study chemotaxis and autochemotaxis in complex geometries, using microfluidic assays of self-propelling oil droplets in an aqueous surfactant solution (Herminghaus et al 2014 Soft Matter 10 7008-22; Krüger et al 2016 Phys. Rev. Lett. 117). Droplets propel via interfacial Marangoni stresses powered by micellar solubilisation. Moreover, filled micelles act as a chemical repellent by diffusive phoretic gradient forces. We have studied these chemotactic effects in a series of microfluidic geometries, as published in Jin et al (2017 Proc. Natl Acad. Sci. 114 5089-94): first, droplets are guided along the shortest path through a maze by surfactant diffusing into the maze from the exit. Second, we let auto-chemotactic droplet swimmers pass through bifurcating microfluidic channels and record anticorrelations between the branch choices of consecutive droplets. We present an analytical Langevin model matching the experimental data. In a previously unpublished experiment, pillar arrays of variable sizes and shapes provide a convex wall interacting with the swimmer and, in the case of attachment, bending its trajectory and forcing it to revert to its own trail. We observe different behaviours based on the interplay of wall curvature and negative autochemotaxis, i.e. no attachment for highly curved interfaces, stable trapping at large pillars, and a narrow transition region where negative autochemotaxis makes the swimmers detach after a single orbit.

Epidemiology of intramammary infections with Staphylococcus aureus and mastitis streptococci in a dairy cattle herd with a history of recurrent clinical mastitis.

The aim of the present work was to examine a dairy herd with an anamnesis of recurrent clinical mastitis and decreased milk production. A total of 239 individual cow milk samples originating from asymptomatic cows were collected at four-month intervals and examined mainly for the presence of Staphylococcus aureus and mastitis streptococci using standard cultivation methods. In total, 29.7% and 9.2% samples were positive for S. aureus and mastitis streptococci, respectively. Unlike for mastitis streptococci, the prevalence of animals positive for S. aureus had an increasing trend (p<0.05; Chi-squared test for trend) with rising parity. Despite in vitro susceptibility of S. aureus to potentiated penicillins and cephalosporins, the persistence of S. aureus was observed in cows undergoing intramammary treatment with amoxicillin/clavulanic acid (a potentiated penicillin antibiotic). All isolates of S. aureus were biofilm-positive and had the same macrorestriction pattern. Furthermore, no dependence was observed between the occurrence of S. aureus in milk and previous cases of clinical mastitis, reproductive and periparturient disorders and administration of antibiotics. In contrast to S. aureus, the occurrence of mastitis streptococci in milk was linked with previous cases of clinical mastitis and intramammary administration of antibiotics.

The detection and quantification of Bacillus thuringiensis spores from soil and swabs using quantitative PCR as a model system for routine diagnostics of Bacillus anthracis.

AIMS: To optimize the DNA isolation for the routine detection and quantification of bacillary spores in soil and swabs. The procedure is primarily intended for diagnostics of Bacillus anthracis spores, but due to its high pathogenicity, B. thuringiensis served as its surrogate organism. METHODS AND RESULTS: Various commercial kits for soils and swabs in combination with quantitative PCR were tested with different results. The PowerSoil DNA kit and the Ultra Clean Microbial DNA kit gave the best results for the extraction from soil and swabs, respectively. Extra beating led to considerably higher yields of DNA. The effectiveness of isolation reached 23% for DNA isolation from soil and 13% from swabs. The limit of detection was assessed to be 8·85 × 103 from 250 mg of soil and 2·79 × 103 from a swab inoculated with 100 µl of spore suspension. CONCLUSIONS: The optimized protocol is suitable for direct isolation and quantification of bacillary spores without any previous culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: In contrast to previous studies, the isolation and quantification of spores was performed directly from the sample, without previous culture of spores on plates. Therefore, the method is suitable for such conditions where previous culturing is not possible, such as in military installations under field conditions.

Carbonaceous materials and their advances as a counter electrode in dye-sensitized solar cells: challenges and prospects.

Dye-sensitized solar cells (DSSCs) serve as low-costing alternatives to silicon solar cells because of their low material and fabrication costs. Usually, they utilize Pt as the counter electrode (CE) to catalyze the iodine redox couple and to complete the electric circuit. Given that Pt is a rare and expensive metal, various carbon materials have been intensively investigated because of their low costs, high surface areas, excellent electrochemical stabilities, reasonable electrochemical activities, and high corrosion resistances. In this feature article, we provide an overview of recent studies on the electrochemical properties and photovoltaic performances of carbon-based CEs (e.g., activated carbon, nanosized carbon, carbon black, graphene, graphite, carbon nanotubes, and composite carbon). We focus on scientific challenges associated with each material and highlight recent advances achieved in overcoming these obstacles. Finally, we discuss possible future directions for this field of research aimed at obtaining highly efficient DSSCs.

Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains.

In vivo spread of macrolide-lincosamide-streptogramin B (MLSB) resistance--a model study in chickens.

The influence of specific and non-specific antibiotic pressure on in vivo spread of macrolide-lincosamide-streptogramin B (MLSB) resistance was evaluated in this study. Chickens repeatedly inoculated with Enterococcus faecalis harbouring the plasmid pAMß1 carrying the erm(B) gene were perorally treated for one week with tylosin, lincomycin (both specific antibiotic pressure) and chlortetracycline (non-specific antibiotic pressure). Antibiotic non-treated but E. faecalis inoculated chickens served as a control. To quantify the erm(B) gene and characterise intestinal microflora, faecal DNA was analysed by qPCR and 454-pyrosequencing. Under the pressure of antibiotics, a significant increase in erm(B) was observed by qPCR. However, at the final stage of the experiment, an increase in erm(B) was also observed in two out of five non-treated chickens. In chickens treated with tylosin and chlortetracycline, the increase in erm(B) was accompanied by an increase in enterococci. However, E. faecalis was at the limit of detection in all animals. This suggests that the erm(B) gene spread among the gut microbiota other than E. faecalis. Pyrosequencing results indicated that, depending on the particular antibiotic pressure, different bacteria could be responsible for the spread of MLSB resistance. Different species of MLSB-resistant enterococci and streptococci were isolated from cloacal swabs during and after the treatment. PFGE analysis of MLSB-resistant enterococci revealed four clones, all differing from the challenge strain. All of the MLSB-resistant isolates harboured a plasmid of the same size as pAMß1. This study has shown that MLSB resistance may spread within the gut microbiota under specific and non-specific pressure and even in the absence of any antimicrobial pressure. Finally, depending on the particular antibiotic pressure, different bacterial species seems to be involved in the spread of MLSB resistance.

Influence of Salmonella enterica serovar enteritidis infection on the development of the cecum microbiota in newly hatched chicks.

Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of the orders Enterobacteriales, Clostridiales, and Lactobacillales. Salmonella enterica serovar Enteritidis infection caused the greatest changes in the gut microbiota when 1-day-old chicks were infected, compared with the infection of 4- and 16-day-old chicks.

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

A low prevalence of mycobacteria in freshwater fish from water reservoirs, ponds and farms.

A survey of the occurrence of mycobacteria was conducted from 717 freshwater fish (25 species) in two water reservoirs, five ponds and two farms in the Czech Republic. A total of 2182 tissue samples from these fish were examined using the conventional culture method. Thirteen mycobacterial isolates were obtained from 12 (1.7%) fish belonging to nine species. Isolates were identified using sequence analysis of the 16SrRNA gene as: Mycobacterium algericum, M. fortuitum, M. gordonae, M. insubricum, M. kumamotonense, M. nonchromogenicum, two isolates of M. peregrinum, M. terrae and M. triplex. Mycobacteria were isolated more frequently from fish skin and gills than from internal organs or muscles.

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds.

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.

Distribution, characterization and genetic bases of erythromycin resistance in staphylococci and enterococci originating from livestock.

The occurrence of staphylococci and enterococci expressing increased resistance to erythromycin (ERY) and, in particular, to macrolide-lincosamide-streptogramin B (MLS(B) ) antibiotics was investigated in dairy cattle, pigs and turkeys. Three hundred rectal (cloacal) swabs of each animal species were examined. A total of 120 and 71 staphylococci and enterococci, respectively, with increased resistance to ERY were identified. These were most frequent in turkeys (42.3% of positive animals), followed by pigs and dairy cattle (6.7% and 6.0% of positive animals, respectively). Similarly, MLS(B) -resistant isolates colonized predominantly turkeys (29.7% of animals), while their occurrence in pigs and dairy cattle was only sporadic (0.8% of animals). At least one of the erm genes encoding for MLS(B) resistance was found in 56.7% and 69.0% of staphylococci and enterococci, respectively. The erm(C) gene prevailed in staphylococci while the erm(B) gene was predominant in enterococci. Macrolide efflux genes msr(A) and msr(C) were also frequent in staphylococci and enterococci, respectively. Macrolide inactivation gene mph(C) occurred mainly in staphylococci. In staphylococci, methicillin resistance was rarely detected (7.5% of isolates), but resistance to telithromycin (ketolides) was frequent in both staphylococci and enterococci (89.2% and 47.9% of isolates, respectively). This study showed that turkeys represent an important source of ERY (MLS(B) )-resistant cocci. In addition, resistance to ketolides was also frequent.

Epidemiology and characterization of Staphylococcus epidermidis isolates from humans, raw bovine milk and a dairy plant.

Geographically related Staphylococcus epidermidis isolates from human patients (n=30), dairy farms (farmers and individual raw milk from cattle, n=36) and a dairy plant (n=55) were examined for epidemiological relatedness by pulsed-field gel electrophoresis and, using in vitro methods, for the ability to produce biofilm and antimicrobial resistance. Methicillin-resistant isolates (MRSE) were also identified and characterized. Isolates from farmers and dairy cattle were found to be genetically related, while isolates from human patients were highly diverse. Some dairy plant isolates (18.2%) were closely related to those from dairy farms. Biofilm production and resistance to antimicrobial agents were most typical for isolates from human patients, of which 76.7% were MRSE. Methicillin resistance was also widespread in farm-related isolates (61.1%). This study indicates the possible transmission of S. epidermidis between cattle and farmers. Dairy products were not proven to be an important source of either human infections or methicillin-resistant strains.

Comparison of the conventional culture, the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of Mycobacterium avium ssp. avium in tissues of naturally infected hens.

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold's and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).

The biofilm-positive Staphylococcus epidermidis isolates in raw materials, foodstuffs and on contact surfaces in processing plants.

Isolates from the "farm to fork" samples (182 isolates from 2779 samples) were examined genotypically (icaAB genes) and phenotypically (in vitro biofilm formation, typical growth on Congo red agar; CRA) with the aim to assess the risk of penetration of virulent strains of Staphylococcus epidermidis into the food chain. The contamination of meat and milk products was significantly higher in comparison with raw materials. Contamination of contact surfaces in the meat-processing plants was significantly lower than that of contact surfaces in the dairy plants. The ica genes (which precondition the biofilm formation) were concurrently detected in 20 isolates that also showed a typical growth on CRA. Two ica operon-negative isolates produced biofilm in vitro but perhaps by an ica-independent mechanism. The surfaces in the dairy plants and the milk products were more frequently contaminated with ica operon-positive strains (2.3 and 1.2 % samples) than the other sample types (0-0.6 % samples).

Beef carcass contamination in a slaughterhouse and prevalence of resistance to antimicrobial drugs in isolates of selected microbial species.

Meat contaminating bacteria may be the direct cause of foodborne diseases and represent a potential cause for the drug resistance of human pathogenic agents. The prevalence and resistance to 17 antimicrobial drugs of isolates of selected bacterial species were investigated in 70 swabs of beef carcasses and 70 subsequent samples of beef meat. Molecular techniques (coagulase gene typing Staphylococcus aureus and original gene typing Escherichia coli) were used in the differentiation of isolates. Carcasses were already contaminated after evisceration, least frequently with S. aureus strains (7.5% of samples), most frequently with coagulase-negative staphylococci strains (52.2% of samples). During carcass processing, contamination with resistant or polyresistant strains of S. aureus and E. coli significantly increased (P<0.01). Gene typing isolates of S. aureus and E. coli indicated that the strains probably originated in the processing plant.

Staphylococcus aureus isolates from dairy cows and humans on a farm differ in coagulase genotype.

Staphylococcus aureus is a frequent cause of animal and human infections. The aim of the present study was to test diversity of the populations of S. aureus colonising cattle and humans sharing an infected environment. Eighty-six S. aureus isolates obtained from dairy cows, from people coming into contact with dairy cows on the farm and the other farm personnel were characterized by restriction fragment length polymorphism of the coagulase gene. Molecular analyses identified ten polymorphism types with prevalent presentation of type II in isolates from cow's milk and type IV in isolates from people coming into contact with dairy cows on the farm (the cattlemen) and the other farm personnel. Seven further genotypes were identified among the isolates from the cattlemen. The results indicate that the strains dominating in human population did not equate to the causative agents of bovine mastitis.

Low molecular weight heparin-loaded polymeric nanoparticles: formulation, characterization, and release characteristics.

The aim of the present work was to investigate the preparation of low molecular weight heparin (LMWH) nanoparticles (NP) as potential oral heparin carriers. The NP were formulated using an ultrasound probe by water-in-oil-in-water (w/o/w) emulsification and solvent evaporation with two biodegradable polymers [poly-epsilon-caprolactone, PCL and poly(D,L-lactic-co-glycolic acid) 50/50, PLGA] and two non-biodegradable positively charged polymers (Eudragit RS and RL) used alone or in combination. The mean diameter of LMWH-loaded NP ranged from 240 to 490 nm and was dependent on the reduced viscosity of the polymeric organic solution. The surface potential of LMWH NP prepared with Eudragit polymers used alone or blended with PCL and PLGA was changed dramatically from strong positive values obtained with unloaded NP to negative values. The highest encapsulation efficiencies were observed when Eudragit polymers took part in the composition of the polymeric matrix, compared with PCL and PLGA NP exhibiting low LMWH entrapment. The in vitro LMWH release in phosphate buffer from all formulations ranged from 10 to 25% and was more important (two- to threefold) when esterase was added into the dissolution medium. The in vitro biological activity of released LMWH, determined by the anti-factor Xa activity with a chromogenic substrate, was preserved after the encapsulation process, making these NP good candidates for oral administration.

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples.

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.

Comparison of methods for the determination of antimicrobial resistance in Staphylococcus aureus from bovine mastitis.

The results of three standard methods (broth dilution, agar dilution, disk diffusion) and an experimental modification of the microdilution method for determination of resistance to ampicillin, cephalotin, cloxacillin, neomycin, novobiocin, penicillin and streptomycin were compared using 151 Staphylococcus aureus isolates obtained from cases of mastitis. The accuracy of the dilution methods was compared by determination of minimum inhibition concentrations (MIC, MIC50, MIC90 and modal MIC) and by assessment of the agreement within the tolerance of +/-1 dilution step in 2-fold dilution series. The results of the dilution methods were further compared with those of the reference disk diffusion method and the strains were classified as sensitive or resistant using the interpretation criteria for human strains. The comparisons indicated that MIC characteristics and the final classification as sensitive or resistant were method-dependent. Resistance to aminoglycoside antibiotics was observed more often when using broth dilution methods, especially when the broth was supplemented with lactose.