Assessment of potentially probiotic properties of Lactobacillus strains isolated from chickens.

This study was performed in order to isolate lactobacilli from chicken droppings and to select strains with the most promising probiotic properties. Lactobacillus strains were isolated from a flock of healthy laying hens. The first selection criterion was the ability to inhibit the growth of Salmonella Enteritidis. Then the tolerance to low pH and bile salt, the ability to coaggregate with pathogenic bacteria and hydrogen peroxide production were evaluated. Four isolates showing the best antagonistic activity against Salmonella Enetritidis were selected for further research. All isolates tested tolerated low pH and bile salt, likewise all produced hydrogen peroxide. They efficiently coaggregated with C. perfringens and relatively less with E. coli. Isolate 03'04 displayed above-average results in all criteria, thus it is considered as a potential probiotic for chickens, and will be further evaluated for health promoting effect in animals. The results presented in this study confirm the strain specific probiotic properties and prove the probiotic potential of isolate 03'04. Strong antagonistic properties against C. perfringens exhibited by certain Lactobacillus strains indicate the possibility to use them as a component of probiotic supplement in necrotic enteritis of poultry.

Genetic characterization of coagulase-positive staphylococci isolated from healthy pigeons.

Coagulase-positive staphylococci (CoPS) are opportunistic veterinary pathogens, of which Staphylococcus aureus, S. delphini and S. intermedius can be isolated from pigeons. The biochemical identification of S. delphini and S. intermedius isolates may be incorrect, because of their phenotypic similarity. The purpose of the present study was to isolate and identify CoPS from domestic and feral pigeons and to determine their genetic relatedness by PFGE. A total number of 31 isolates of CoPS were obtained, 15 were identified as S. delphini group B, six as S. aureus, four as S. delphini group A, three as S. intermedius and three as S. schleiferi subsp. coagulans. The results indicate that S. delphini group B is the predominant CoPS species among pigeons studied. PFGE restriction patterns of S. delphini group A and S. delphini group B form separate clusters, demonstrating their genetic heterogeneity. Indistinguishable or very similar PFGE patterns observed among S. delphini group B isolates from domestic and feral pigeons confirm the possibility of CoPS transmission between these birds.

Chicken intestinal microbiota function with a special emphasis on the role of probiotic bacteria.

Bacterial colonization of the chicken gut by environmental microbes begins immediately after hatching. Composition of the intestinal microbiota is dependent on the surrounding environment, diet variation, pathological conditions, antibiotic therapy, and others. The genomes of all these intestinal microbes form a microbiome which by far outnumbers the host's genome. As a consequence, the microbiome provides additional metabolic functions to the host, including nutrient utilization and absorption, fermentation of non-digestible dietary fiber, synthesis of some vitamins, biotransformation of bile acids, and the well-being of their chicken host. Microorganisms can also directly interact with the lining of the gastrointestinal tract, which may alter the physiology and immunological status of the bird. Since newly hatched broiler chickens demonstrate delayed commensal colonization and low bacterial diversity, the most effective and harmless method available to control the development and composition of the intestinal microbiota is a competitive exclusion treatment by applying probiotic bacteria. Additionally, recent research has shown that probiotic bacteria have a variety of beneficial effects, including counteraction of dysbiosis, promotion of gut health and homeostasis, enhancement of immune defenses and antagonization of infectious agents.

PFGE and AFLP genotyping of Staphylococcus aureus subsp. anaerobius isolated from goats with Morel's disease.

Staphylococcus aureus subsp. anaerobius is the etiological agent of the Morel's disease in sheep and goats. The disease presents with subcutaneous abscesses, located mainly in the superficial lymph nodes. Forty-one isolates of S. aureus subsp. anaerobius were collected from two outbreaks of the Morel's disease in Poland in years 2006-2008. Analysis of DNA SmaI digests by PFGE showed that 35 of 41 isolates belonged to the same PFGE type, identical to the type strain of S. aureus subsp. anaerobius ATCC 35844, confirming high level of clonality of the species. The DNA patterns of the remaining identical 6 isolates, different from the reference strain only by two bands, were found closely related. Genotyping performed with AFLP technique revealed two clonal groups including 16 and 25 isolates, respectively. The study indicated that AFLP technique might be a better discriminatory tool for genetic analysis of S. aureus subsp. anaerobius isolates, when compared to PFGE.

Molecular characterization of Staphylococcus pseudintermedius strains isolated from clinical samples of animal origin.

The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable.

Antibiotic resistance of canine Staphylococcus intermedius group (SIG)--practical implications.

A total of 221 SIG strains were isolated from clinical samples of canine origin submitted to the Diagnostic Laboratory of the Division of Bacteriology and Molecular Biology at the Warsaw University of Life Sciences in Warsaw during the period 2006-2010. The aim of the study was to investigate the frequency of prevalence of methicillin-resistant SIG strains and to determine the MIC values of cephalotin, amoxicillin/clavulanic acid, ciprofloxacin, clindamycin, gentamicin, chloramphenicol, mupirocin for a collection of randomly selected 79 strains belonging to Staphylococcus intermedius group (SIG), including 23 mecA-positive and 56 mecA-negative strains. All isolates were identified as belonging to SIG based on their phenotypic properties and PCR amplification of S. intermedius-specific fragment of the 16S rRNA gene. The mecA gene was detected in 26 (12%) of 221 SIG strains. All tested mecA-negative SIG strains were susceptible to amoxicillin/clavulanic acid and cephalotin. One of the 56 mecA-negative SIG strains was resistant to ciprofloxacin, six (11%) to gentamicin. It was found that sixteen (29%) of 56 mecA-negative SIG strains were resistant to clindamycin. Most of the mecA-positive SIG strains were resistant to ciprofloxacin (96%), clindamycin (96%), and gentamicin (96%). Only one MRSIG strain was resistant to chloramphenicol. All examined mecA-positive SIG strains were found to be susceptible to mupirocin. Our results imply that staphylococcal multidrug resistance has become more prevalent, which could lead to difficulties in effective treatment. With some resistant strains the only therapeutic possibility are antimicrobial agents important in human medicine. New regulations for veterinary medicine concerning appropriate therapy of infections caused by multidrug-resistat staphylococci are needed.

Survival of Corynebacterium pseudotuberculosis within macrophages and induction of phagocytes death.

Since C. pseudotuberculosis is a facultative intracellular pathogen the aim of this study was focused on evaluating mechanisms that allowed these bacteria to survive in macrophages and determining their influence on induction of cell death. The influence of Corynebacteria on the programmed cell death of macrophages was determined on the basis of induction the autophagy and apoptosis in the cultures of murine macrophage cell lines J774 infected with bacteria. Corynebacterium pseudotuberculosis strains could survive within macrophages more than 48 hours. During that time bacteria were released as a result of the process that lead to death of phagocytes. This property varied among studied strains. There was no increase of microtubule-associated protein I light chain 3 (MAP I LC3) activity in macrophages infected with examined strains comparing with uninfected cultures and cultures treated with autophagy inducer (rapamycin) that served as negative and positive controls, respectively. The study with confocal microscopy did not show the increasing of caspase-3 activity in the infected macrophages and their nucleus did not reveal the fragmentation.

Epidemiological features of Morel's disease in goats.

Morel's disease caused by Staphylococcus aureus subsp. anaerobius was diagnosed for the first time in Poland in October 2006 in a goat flock. A second infected flock was found two months later. The course of the disease in both flocks was observed for 15-17 months. Clinical manifestation was confined to abscesses located near major superficial lymph nodes, mostly: superficial cervical, subiliac, parotid and mandibular. At necropsy no other lesions were found. The incubation period was estimated at 3 weeks. Clinical signs were seen both in young and adult goats and up to 7 abscesses in one animal were noted. Abscesses tended to persist for 1 to 5 months, then rupture and heal completely. The initial high in-flock point prevalence in both flocks (93.6% and 84.4%) dropped to approximately 10-30% during next 3-4 months. Until the end of the observation period the in-flock point prevalence remained at this level and only single abscesses were observed, mainly in young animals. No influence of the concurrent caprine arthritis encephalitis virus (CAEV) infection on the clinical course of Morel's disease was noticed. It is to be concluded that the clinical course of Morel's disease in a goat flock resembles caseous lymphadenitis (CLA). However, in Morel's disease abscesses occur more frequently in young goats and are located near, not inside, the lymph nodes, as in the case with CLA. Also, the incubation period of Morel's disease seems to be shorter (3 weeks versus 2-6 months in CLA).

Antibiotic resistance patterns and occurrence of mecA gene in Staphylococcus intermedius strains of canine origin.

We have evaluated 102 Staphylococcus intermedius isolates of canine origin for susceptibility to antimicrobial primary agents, i.e. penicillin, amoxicillin, amoxicillin with clavulanic acid, cefuroxime, trimethoprim/sulfonamides, neomycin, streptomycin, gentamicin, norfloxacin, tetracycline, vancomycin, erythromycin and secondary agents, i.e., chloramphenicol, ciprofloxacin, lincomycin, teicoplanin, rifampicin, imipenem, mupirocin. Antimicrobial sensitivity was examined using the disk diffusion method and performed according to NCCLS quidelines. Methicillin resistance was detected using the disk diffusion method with oxacillin, and the occurrence of mecA gene was detected by PCR. Resistance to streptomycin, penicillin, amoxicillin, neomycin, followed by tetracycline was predominant. From 14 mecA-positive strains, 12 were multidrug-resitant, and the remaining two showed atypical susceptibility. One strain resistant to oxacillin in the disc diffusion method was mecA-negative, suggesting a different mechanism of resistance. Our results indicate that the emergence of S. intermedius resistance to methicillin may be underestimated. In case of clinical multidrug-resitant S. intermedius isolates, resistance to methicillin should be considered.

Protective effect of potentially probiotic Lactobacillus strain on infection with pathogenic bacteria in chickens.

The probiotic potential of a Lactobacillus salivarius 3d strain isolated from chicken faeces was assessed in one day old chickens. Lactobacillus salivarius 3d was administered per os at a concentration of 10(8) cfu in 100 microl of PBS. The chickens were then challenged with pathogenic bacteria: Salmonella Enteritidis, Campylobacter jejuni and Clostridium perfringens. Samples of caecal contents and livers were collected after 1, 2, 3, 7 and 14 days after infection. Lactobacilli and pathogenic bacterial cell counts were determined in the samples. This study showed that L. salivarius 3d reduced the number of Salmonella Enteritidis and Clostridium perfringens in the group of chickens treated with Lactobacillus. Therefore it may be concluded that L. salivarius 3d may be used as a potential probiotic for chickens.

Resistance to antimicrobial agents of Campylobacter spp. strains isolated from animals in Poland.

A total of 69 Campylobacter jejuni and 16 Campylobacter coli strains isolated from chicken, dog and pig stool samples were characterized based on their resistance to five antimicrobial agents and on plasmid pTet profiles. Antimicrobials used in this study were: amoxicillin/clavulanic acid, ciprofloxacin, erythromycin, tetracycline and trimethoprim/sulfamethoxazole. Among the isolates studied, 91.7% were resistant to one or more antimicrobial agent. The highest level of resistance for the whole test group was to trimethoprim/sulfamethoxazole (57.6%), followed by ciprofloxacin (44.2%) and tetracycline (20%). All isolates were susceptible to amoxicillin/clavulanic acid. Strains isolated from chickens were susceptible to erythromycin. Few erythromycin-resistant strains were isolated from dogs and pigs (5.8%). C. coli strains exhibited a higher antibiotic resistance than C. jejuni strains, excluding resistance to trimethoprim/sulfamethoxazole. The pTet plasmid harboring the tet(O) gene was detected in 14 Campylobacter spp. strains. Our studies demonstrate that the majority (71.4%) of tetracycline-resistant isolates carry a plasmid-borne tet(O) gene, particularly strains for which the minimum inhibitory concentration (MIC) are > or = 256 microg/ml. In conclusion, we have found high-level trimethoprim/sulfamethoxazole, ciprofloxacin and tetracycline resistance in Polish strains isolated from different sources. This study has demonstrated that resistance of Campylobacter species differs depending on both the bacterial species and animal origins. All strains that displayed resistance to four antimicrobial agents were isolated from pigs. Localization of the tet(O) gene on either plasmid or chromosome was not found to be correlated with tetracycline resistance.

Prevalence of the suilysin gene in Streptococcus suis strains isolated from diseased and healthy carrier pigs.

Knowledge of virulence factors of Streptococcus suis is limited. Several virulence factor candidates have been proposed, among them suilysin, which is responsible for a toxic effect on epithelial cells. The aim of this study was to detect the suilysin gene sequence in Streptococcus suis strains of various origin. In total 63 Streptococcus suis isolates were investigated. Forty four of them originated from tissues of streptococcosis affected animals. The remaining 19 strains were isolated from tonsils of healthy carrier pigs. Suilysin gene specific sequence was detected in 79% of the strains tested. In isolates obtained from pigs with signs of streptococcosis this gene sequence was recorded in 85% of cases. In Streptococcus suis strains isolated from healthy carrier pigs the suilysin gene was detected in 63% of the isolates. It seems that suilysin toxic activity is only one of the many steps involved in the pathogenesis of Streptococcus suis infection and that strain's virulence cannot be stated only on the basis of suilysin gene sequence presence.

Occurrence of Lawsonia Intracellularis and Brachyspira spp. infection in swine suffering from diarrhoea.

Principal aim of this study was to examine fecal samples from pigs suffering from diarrhea for the presence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli. The molecular techniques such as PCR and nested PCR were employed to detect the presence of p78 fragment of genomic DNA specific for Lawsonia intracellularis as well as fragment of tlyA gene specific for Brachyspira hyodysenteriae and 16S rDNA gene of Brachyspira pilosicoli. We assumed that about 25% of pigs were infected with Lawsonia intracellularis, about 10% with Brachyspira hyodysenteriae and only 0,8% with Brachyspira pilosicoli. In about 3% mixed infection with L. intracellularis and B. hyodysenteriae was observed. Results were comparable in herds that differed in quantity, breeding technology, hygienic standards and preventive treatment with different chemotherapeutics.

The cytotoxic properties of Serpulina hyodysenteriae.

Examination of colonic enterocytes inoculate with pure culture of S. hyodysenteriae by phase-contrast microscopy revealed that only few spirochaetes adhere to epithelial cells. S. hyodysenteriae was observed to be highly motile, showed corkscrew-like movement which might suggest that bacteria were trying to penetrate and damaged the host cells. The pattern of motility provide evidence of a chemotaxis. Supernatant of S.hyodysenteriae lysate were found to cause CTE in CHO, Vero and PK-15 culture. This support the hypothesis that damage is consistent with the presence of toxin. Inhibition activity of serpulinas hemolysin preparation with streptolysin S inhibitors confirms the suggestion that the mechanism by which S. hyodysenteriae toxin effects the cells seems to be similar to the action of streptococcal toxin S.

Localisation of the mitogenic epitope of staphylococcal enterotoxin B.

Limited digestion of staphylococcal enterotoxin B (SEB) with trypsin resulted in the generation of a 12-Kda amino-terminal fragment and a 17-Kda carboxy-terminal fragment which were isolated by preparative iso-electric focusing. The carboxy-terminal fragment exhibited significant mitogenic activity for murine splenocytes, whereas the isolated amino-terminal fragment possessed little detectable mitogenic activity. Monoclonal antibodies (MAbs) specific for the carboxy-terminal fragment neutralised most of the mitogenic activity of both the intact toxin and the carboxy-terminal fragment. MAbs specific for the amino-terminal fragment had no detectable neutralising activity. These results support the hypothesis that the epitope(s) responsible for mitogenic activity is located in the carboxy-terminal region of SEB.

Transfer of plasmid Hly in vivo in pigs intestine.

The results of our study suggest the in vivo transfer of Hly plasmid from native pathogenic and enterotoxigenic Escherichia coli strain to autochtonous Escherichia coli, using ileal loop test. To confirm this hypothesis pHly::Tn5 and PHly::Tn3 were obtained using an in vitro recombination method, and introduced to Escherichia coli laboratory strain. For experiments the laboratory strain, carrying pHly::Tn5 and pHly::Tn3 and pHly::Tn5 strain which acquired K88(F4) by means of conjugation, were used. In the study in which the donor Escherichia coli pHly::Tn5 strain, carrying antigen K88(F4), was injected into the ileum, pHly conjugants were isolated from faeces after 48 h in 2 out of 5 pigs, which was a low frequency. After the oral introduction of 10(9) cells of pHly::Tn5 and pHly::Tn3 Escherichia coli strains without the colonizing factor K88(F4), conjugants were not isolated from faeces of experimental animals. However when the pigs received donor CSH55 pHly::Tn5 Escherichia coli strain orally, which were also carrying plasmid K88(F4), transconjugants were obtained in a low frequency of 3 out of 9 pigs. Our experiments confirmed the suggestion of Smith that in vivo transfer of plasmid in the intestine of animals is only possible when the donor transfers the plasmid with high frequency and readily colonizes the intestine. The pHly::Tn5 plasmid acquired by in vitro recombination does not spread with time throughout the autochtonic population of Escherichia coli present in the intestine of swine. The results of our study showed the in vivo transfer in pigs intestine of Hly pathogenicity marker from both native pathogenic strains carrying antigen K88(F4) and constructed donor laboratory strain of Escherichia coli pHly::Tn5 also carrying antigen K88(F4) to autochtonous intestinal strains.

Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens.

A total of 2450 samples of feces, intestinal contents and colon mucosal scrapings were bacteriologically examined. A total of 53 strains of Treponema sp. were isolated, and 45 strains of Bacteroides sp., 30 strains of E. coli, 30 strains of Micrococcus sp. and 10 strains of Streptococcus D isolates were randomly selected. Growth promoting studies showed statistically significant stimulation of Treponema sp. growth by yeast extract, chicken egg yolk and rumen fluid. Different growth inhibitors were also tested. For selective medium the following inhibitors were selected: spectinomycin, colistin, vancomycin, brilliant green. Optimal concentrations of these inhibitors in the medium were determined. Finally TSA medium supplemented with 0.05% yeast extract, 5% bovine blood, 0.01% DTT, 400 micrograms spectinomycin, and 250 micrograms/ml vancomycin, appeared to be optimal selective medium for intestinal Treponema sp. isolation. Quantitative studies showed that the number of Treponema C.F.U. on Songers et al. medium with spectinomycin and on spectinomycin-vancomycin medium, did not differ significantly. The number of overgrowing bacteria was statistically significantly lower on spectinomycin-vancomycin medium, than Songers et al. selective medium with spectinomycin. The TSA supplemented with blood, yeast extract 50 micrograms/ml of colistin and 1 microgram/ml of brilliant green was less selective than spectinomycin-vancomycin medium and inhibited some strains of Treponema sp. In the case of spectinomycin-vancomycin resistant of overgrowing bacteria, colistin-brilliant green medium may be suitable for isolation of Treponema sp.

Production of enterotoxins by strains of Escherichia coli isolated from pigs.

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.