Molecular and Serological Characteristics of Avian Pathogenic Escherichia coli Isolated from Various Clinical Cases of Poultry Colibacillosis in Poland.

Escherichia coli infections are a major problem in modern poultry production. Avian pathogenic E. coli (APEC) strains have several mechanisms that enable them to colonize various ecosystems. In this study, 290 E. coli isolates were recovered from clinical cases of colibacillosis in chicken and turkey broilers and from laying and breeding hens. The samples were taken from organs with pathological changes suggesting colibacillosis. The lesions were assigned to three groups depending on their advancement, of which the largest (60% of the isolates) was group 3, with the most extensive changes. The most common serotype was shown to be O78 (14%). The most frequently detected gene among those tested was iss, while papC was the least prevalent. An analysis of the number of genes present per isolate revealed that the presence of four genes was the most common (22%), while only 1% of the strains tested had all eight genes. The most frequently detected genes for each serotype were iss and iucD for O78; irp2 and cvi/cva for O1; irp2, iucD, and iss for O2, and iss and iucD for O8, for which the least frequent was papC. All O18 serotype strains had the iss gene, while none had the vat gene.

Therapeutic and Prophylactic Effect of the Experimental Bacteriophage Treatment to Control Diarrhea Caused by E. coli in Newborn Calves.

The prevalence of antibiotic-resistant bacteria causing neonatal diarrhea in calves has become a serious problem in the control of infection. Due to increasing antibiotic resistance, bacteriophages with probiotics are considered the best alternative. The aim of the study was to evaluate the use of a suppository containing probiotic strains of Lactobacillus spp. and bacteriophages specific for pathogenic E. coli in young calves with diarrhea. The study evaluated therapeutic and prophylactic effects (specific and nonspecific humoral response). The study was carried out on 24 female HF calves, aged 2 to 7 days and weighing from 35 to 46 kg. The calves were divided into four groups (n = 6) as follows: Group 1, healthy control that received no medicine; Group 2, positive control with diarrhea; Group 3, healthy calves that received medicine; Group 4, calves with diarrhea that received medicine. The animals received suppositories containing Lactobacillus spp. and bacteriophages specific for pathogenic E. coli for 5 days. On the first day, the calves received the suppositories twice-in the morning and 12 h later; subsequently they were administered once a day. The health status of the calves was observed for 11 days after the first application of suppositories. A protective and preventive effect of the experimental therapy was obtained in the research. The probiotic-phage suppositories reduced the duration of diarrhea in calves, completely eliminating it within 24-48 h after use. The therapy stimulated the activation of immune mechanisms in calves, which translated into an enhanced specific and nonspecific response and increased resistance to infection.

The in vitro efficacy of eye drops containing a bacteriophage solution specific for Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.

BACKGROUND: The purpose of the study was to evaluate the in vitro antibacterial effect of experimental eye drops with bacteriophages in elimination of Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.. The bacterial material was collected from dogs with independent clinical signs of bacterial conjunctivitis. Staphylococcus spp. were identified by phenotypic and genotypic methods (MALDI-TOF MS mass spectrometry). Antibiotic resistance was determined by the disc-diffusion method. Phage activity (Plaque forming units, PFU) was determined on double-layer agar plates. Phages with lytic titres > 108 PFU were used to prepare eye drops. The stability of the antibacterial titre was evaluated for preparations stored in sealed bottles as well as after opening and reclosing. RESULTS: The tests confirmed the occurrence of Staphylococcus spp. strains as etiological agents of bacterial conjunctivitis in dogs. A high percentage of strains were resistant to more than three antibiotics. The experimental phage eye drops used in the study exhibited 100% efficacy in vitro against the tested Staphylococcus isolates. Particularly noteworthy is the long duration of activity and constant antibacterial lytic titre of ≥108 PFU/mL of two eye drop solutions, nos. 7 and 12, after the bottle had been opened (21 days) and after hermetically sealed packaging (28 days) at 4-8 °C. CONCLUSIONS: The results represent the first stage of research and require continuation in vivo. If positive effects are obtained in animals, the results can be used in applied research in humans and animals.

Efficacy of experimental phage therapies in livestock.

Bacteriophages are the most abundant form of life on earth and are present everywhere. The total number of bacteriophages has been estimated to be 1032 virions. The main division of bacteriophages is based on the type of nucleic acid (DNA or RNA) and on the structure of the capsid. Due to the significant increase in the number of multi-drug-resistant bacteria, bacteriophages could be a useful tool as an alternative to antibiotics in experimental therapies to prevent and to control bacterial infections in people and animals. The aim of this review was to discuss the history of phage therapy as a replacement for antibiotics, in response to EU regulations prohibiting the use of antibiotics in livestock, and to present current examples and results of experimental phage treatments in comparison to antibiotics. The use of bacteriophages to control human infections has had a high success rate, especially in mixed infections caused mainly by Staphylococcus, Pseudomonas, Enterobacter, and Enterococcus. Bacteriophages have also proven to be an effective tool in experimental treatments for combating diseases in livestock.

Evaluation of the ability of colistin, amoxicillin (components of Potencil® ), and fluoroquinolones to attenuate bacterial endotoxin- and Shiga exotoxin-mediated cytotoxicity-In vitro studies.

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS- and Stx-mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS-mediated cytotoxicity in an experiment mimicking "natural infection," (c) the effect of antibiotics to decrease Stx2e-mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS- and Stx2e-induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS- and Stx2e-mediated cytotoxicity.

Probiotic potential of Lactobacillus isolates of chicken origin with anti-Campylobacter activity.

Campylobacteriosis is currently the most frequent zoonosis in humans and the main source of infection is contaminated poultry meat. As chickens are a natural host for Campylobacter species, one strategy to prevent infection in humans is to eliminate these bacteria on poultry farms. A study was conducted to evaluate the probiotic potential of 46 Lactobacillus isolates from chickens faeces or cloacae. All lactobacilli were able to produce active compounds on solid media with antagonistic properties against C. jejuni and C. coli, with L. salivarius and L. reuteri exhibiting particularly strong antagonism. The cell-free culture supernatants had a much weaker inhibitory effect on the growth of Campylobacter, and the neutralization of organic acids caused them to completely lose their inhibitory properties. The ability to produce H2O2 was exhibited by 93% of isolates; most of isolates had a hydrophobic surface, showed excellent survival at pH 2.0 or 1.5, and displayed tolerance to bile; 50% isolates displayed the ability to biofilm formation. Determination of MICs of various antibiotics showed that as much as 80.4% of Lactobacillus isolates were resistant to at least one antimicrobial agent. Seven ultimately selected isolates that met all the basic criteria for probiotics may have potential application in reducing Camylobacter spp. in chickens and thus prevent infections in both birds and humans.

Protein profiles of bacteriophages of the family Myoviridae-like induced on M. haemolytica.

The aim of study was to isolate, characterize and analyse the protein profiles of Myoviridae-like bacteriophages obtained from M. haemolytica using MALDI TOF mass spectrometry. The material consisted of the M. haemolytica reference strain ATCC® BAA410, reference serotypes A1, A2, A5, A6, A7, A9, and A11, and wild-type isolates of serotype A1. Bacteriophage morphology was examined with a transmission electron microscope. The proteins were separated in SDS-PAGE and two-dimensional electrophoresis and characterized by MALDI-TOF. Among the phages obtained, seven were specific for strains A1, A2, A5, A6, A7 and 25, and PHL-1 was specific for the BAA410 strain. The protein profiles for the phages were very similar to one another, but differed from the reference phage in that they lacked protein fractions with molecular weights of 22.9, 56.3 and 73.1 kDa. 2D electrophoresis revealed significant differences in the size of proteins and their localization in the pH gradient. The most similar profiles were observed in phages specific for strains BAA-410 and A6. In all profiles two main spots were observed in the molecular weight range from 44 to 70 kDa at pH < 4. The results indicate that 2D electrophoresis is a very useful tool for characterization of phage protein profiles. An important objective was to determine the molecular differences between morphologically similar phages belonging to one family and to find similarities to phages specific for other pathogens. The study also assessed the suitability of the methods used to characterize phages.

Surface properties of Enterococcus faecalis cells isolated from chicken hearts determine their low ability to form biofilms.

Enterococcus faecalis is one of the most significant bacterial pathogens associated with the first-week mortality of chickens. Here, the surface properties of bacterial cells and the selected virulence factors of E. faecalis strains isolated from the hearts of clinically healthy broiler chickens were studied. Investigations were carried out on live and autoclaved cells. E. faecalis (ATCC 29212) was used as a reference strain. The bacterial cells revealed different haemolytic activities. Their surface free energy was dominated by the hydrophobic component. The cell walls of the bird isolates showed slightly weaker acidic characteristics than those of E. faecalis (ATCC 29212). Moreover, the bacterial cells from the chicken hearts showed higher electrophoretic mobility and surface electrical charge than the reference strain, and consequently demonstrated a low ability to form biofilms.

Assessment of antibiotic susceptibility in Lactobacillus isolates from chickens.

BACKGROUND: The aim of this study was to determine the susceptibility of 88 Lactobacillus isolates derived from chickens to antibiotic substances and to detect drug-resistance genes. RESULTS: The minimal inhibitory concentration of 13 antimicrobial substances was determined by the broth microdilution method, and resistance genes were detected by PCR. We recorded a high prevalence of resistance to tiamulin (90% resistant isolates), tetracyclines (74%) and lincomycin (70%), and a moderately high frequency of resistance to enrofloxacin (48%), macrolides (42%), aminoglycosides (12.5-31%), ampicillin (26%) and chloramphenicol (23%). Multi-drug resistance was observed in 79.5% of isolates. The presence of resistance genes was generally correlated with phenotypic resistance, but some molecular determinants were also recorded in susceptible isolates. Among tetracycline resistance genes, the most frequently identified was tetW (45% isolates), followed by tetM (26%) and tetL (24%). The ermB, ermC and lnuA genes, associated with resistance to macrolides and lincosamides, were observed in 39, 12 and 39% of isolates, respectively. Among genes determining resistance to aminoglycoside antibiotics, we identified ant(6)-Ia (10% of isolates), aac(6')-Ie-aph(2')-Ia (8%), aph(2″)-Ic (6%) and aadE (4.5%). The cat gene was present in 32 isolates, including 8 of 20 found to be resistant to chloramphenicol. Two genes encoding efflux pumps were identified-the acrA gene was present in all isolates tested, and 10 of 79 lactobacilli determined to be phenotypically resistant to tiamulin contained the lsaE gene. We were unable to explain the resistance mechanism of Lactobacillus isolates to ampicillin, but showed that it did not involve the production of ß-lactamases. CONCLUSIONS: Our findings indicate that intestinal lactobacilli should be considered a reservoir of resistance genes and that antibiotics must be used prudently in poultry production. The data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorization of susceptible and resistant strains within the genus Lactobacillus.

Bacteriophage therapy to combat bacterial infections in poultry.

Infections in poultry are an economic and health problem in Europe and worldwide. The most common infections are associated with salmonellosis, colibacillosis, campylobacteriosis, and others. The prevalence of Campylobacter-positive poultry flocks in European countries varies from 18% to 90%. In the United States, the prevalence of infected flocks is nearly 90%. A similar percentage of infection has been noted for salmonellosis (about 75-90%) and E. coli (90-95%). The occurence of Clostridium perfringens is a major problem for the poultry industry, with some estimates suggesting colonization of as many as 95% of chickens, resulting in clinical or subclinical infections. In the US, annual economic losses due to Salmonella infections run from $1.188 billion to over $11.588 billion, based on an estimated 1.92 million cases. Similar costs are observed in the case of other types of infections. In 2005 economic losses in the the poultry industry due to mortalities reached 1,000,000 USD.Infections caused by these pathogens, often through poultry products, are also a serious public health issue.The progressive increase in the number of multi-drug resistant bacteria and the complete ban on the use of antibiotics in livestock feed in the EU, as well as the partial ban in the US, have led to the growth of research on the use of bacteriophages to combat bacterial infections in humans and animals.The high success rate and safety of phage therapy in comparison with antibiotics are partly due to their specificity for selected bacteria and the ability to infect only one species, serotype or strain. This mechanism does not cause the destruction of commensal bacterial flora. Phages are currently being used with success in humans and animals in targeted therapies for slow-healing infections. They have also found application in the US in eliminating pathogens from the surface of foods of animal and plant origin. At a time of growing antibiotic resistance in bacteria and the resulting restrictions on the use of antibiotics, bacteriophages can provide an alternative means of eliminating pathogens.

Heat Shock Proteins 70kDa, Eosinophil Cationic Protein, and Nitric Oxide During Chronic Superficial Keratitis in Dogs.

The objective of the study is to determine the levels of eosinophil cationic protein (ECP), heat shock proteins 70, and nitric oxide ions measured as nitrite ions (Griess reaction) in dogs with chronic superficial keratitis (CSK). The study was conducted on 24 dogs with CSK. Blood sera from the animals were tested for concentrations of heat shock proteins 70, ECP, and nitrite ions before treatment and again 5 weeks and 6 months after treatment. Dogs with CSK were treated for 6 months with various regimes involving the use of ophthalmic drops containing dexamethasone, dimethyl sulfoxide, and cyclosporine. The control group consisted of 16 clinically healthy German Shepherds. The results obtained indicated a significant (P ≤ 0.05) elevation in the concentrations of heat shock proteins 70 and nitrite ions in dogs with CSK in comparison to healthy dogs and dogs after 5 weeks of therapy. After 6 months of treatment, concentrations of heat shock proteins 70, ECP, and nitrite ions had fallen below pretreatment values. Significant correlations were found between concentrations of heat shock proteins 70, ECP, and nitrite ions in healthy animals and animals with CSK. The elevated concentrations of heat shock proteins 70, ECP, and nitrite ions in dogs with CSK may indicate that the disease was both localized and systemic. The significant correlation between levels of heat shock proteins 70 and nitrite ions suggests that these parameters may be used as indirect indicators of CSK.

Antimicrobial activity of Lactobacillus strains of chicken origin against bacterial pathogenss.

This study was conducted to identify and evaluate the antimicrobial activity of some Lactobacillus isolates of chicken origin. Among 90 isolates 14 Lactobacillus species were distinguished using MALDI-TOF mass spectrometry and 16S-ARDRA. The dominant species was L. salivarius (34.4%), followed by L. johnsonii (23.3%), L. crispatus (13.3%) and L. reuteri (11.1%). All lactobacilli were screened for antimicrobial activity against wild-type strains of Salmonella enterica, Escherichia coli, and Clostridium perfringens. Results from the agar slab method showed that all Lactobacillus isolates were able to produce active compounds on solid media with antagonistic properties against these pathogens. The highest sensitivity to lactobacilli was observed in C. perfringens strains, and the lowest in E. coli. Lactobacillus salivarius exhibited particularly strong antagonism towards all of the indicator bacteria. Strains of L. ingluviei and L. johnsonii and one strain of L. salivarius (10d) selectively inhibited the growth of C. perfringens. No antimicrobial activity of many Lactobacillus isolates was observed when cell-free culture supernatant was used in a well diffusion assay. All Lactobacillus isolates exhibited the ability to produce H2O2 and proved to be hydrophobic (excluding one of L. salivarius). [Int Microbiol 19(1):57-67 (2016)].

16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry.

BACKGROUND: The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. RESULTS: A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70-1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. CONCLUSIONS: The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats.

The use of MALDI-TOF mass spectrometry for rapid identification of Mannheimia haemolytica.

Mannheimia haemolytica is the most important bacterial pathogen isolated from cases of Bovine Respiratory Disease (BRD). Routine identification of these bacteria is usually performed using phenotypic methods. Our study showed that MALDI-TOF MS is a reliable alternative to these methods. All of the strains analyzed were identified as M. haemolytica. The identification results were compared to those obtained using conventional methods commonly used in microbiological diagnostics, based on detection and analysis of biochemical properties of microorganisms. The degree of agreement between the two methods for identifying M. haemolytica was 100%.

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

AIM OF STUDY: The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. MATERIAL AND METHODS: The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). RESULTS: Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. CONCLUSIONS: The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

Detection of bovine respiratory syncytial virus infections in young dairy and beef cattle in Poland.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is a major contributor to bovine respiratory disease complex in dairy and beef calves, especially during the first year of life. There is a lack of comprehensive information about the prevalence of infection in cattle herds in Poland as well as in European countries outside the European Union. OBJECTIVE: The aim of this study was to estimate the prevalence of BRSV infections in young beef and dairy cattle in southeastern Poland, a region that has direct contact with non-EU countries. Animals & methods: Nasal swabs and sera (n = 120) were obtained from young cattle aged 6-12 months from 45 farms in eastern and southeastern Poland. BRSV antigen detection in the nasal swabs was carried out using a rapid immunomigration assay used in diagnosing human respiratory syncytial virus (hRSV) infections in humans, while antibodies to BRSV were detected in the sera by ELISA antibody detection. RESULTS: The study confirmed the presence of BRSV infections in young cattle under 12 months of age from both dairy and beef herds. BRSV was detected in 27 of the 45 herds (60%) sampled. CONCLUSIONS: Findings from this study indicate a high prevalence of BRSV infections in cattle in Poland, which may have a significant influence on health status and animal performance. The prevalence of infection is similar to that in other parts of Poland and other countries in Europe. Development of strategies to reduce BRSV infections is needed to improve health and productivity.

Screening of Lactobacillus strains of domestic goose origin against bacterial poultry pathogens for use as probiotics.

Lactobacilli are natural inhabitants of human and animal mucous membranes, including the avian gastrointestinal tract. Recently, increasing attention has been given to their probiotic, health-promoting capacities, among which their antagonistic potential against pathogens plays a key role. A study was conducted to evaluate probiotic properties of Lactobacillus strains isolated from feces or cloacae of domestic geese. Among the 104 examined isolates, previously identified to the species level by whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analysis of 16S-23S regions of rDNA, dominated Lactobacillus salivarius (35%), followed by Lactobacillus johnsonii (18%) and Lactobacillus ingluviei (11%). All lactobacilli were screened for antimicrobial activity toward Salmonella Enteritidis, Escherichia coli, Clostridium perfringens, Staphylococcus aureus, Pasteurella multocida, and Riemerella anatipestifer using the agar slab method and the well diffusion method. Lactobacillus salivarius and Lactobacillus plantarum exhibited particularly strong antagonism toward all of the indicator strains. In the agar slab method, the highest sensitivity to Lactobacillus was observed in R. anatipestifer and P. multocida, and the lowest in E. coli and S. aureus. The ability to produce H2O2was exhibited by 92% of isolates, but there was no correlation between the rate of production of this reactive oxygen species and the antimicrobial activity of Lactobacillus sp. All lactobacilli showed resistance to pH 3.0 and 3.5 and to 2% bile. The data demonstrate that Lactobacillus isolates from geese may have probiotic potential in reducing bacterial infections. The antibacterial activity of the selected lactobacilli is mainly due to lactic acid production by these bacteria. The selected Lactobacillus strains that strongly inhibited the growth of pathogenic bacteria, and were also resistant to low pH and bile salts, can potentially restore the balance of intestinal microflora in geese and could offer an alternative to antibiotic therapy.

Use of different cell lines for in vitro cultures of bovine respiratory syncytial virus.

This study compared the use of different cell lines for in vitro cultures of bovine respiratory syncytial virus (BRSV). The BRSV 375 strain and 3 nasal swabs obtained from Simmental calves were used for this study. The culture was performed on 3 cell lines: bovine kidney cells (LLC-PK1), bovine tracheal cells (TBTR) and primary chicken embryo-related cells (CER). A comparative analysis of titres was performed using a microplate agglutination test with human group O erythrocytes and bovine erythrocytes. The presence of BRSV in all cell lines was confirmed using the reverse transcriptase polymerase chain reaction (RT-PCR) method. The first small refractile changes in the LLC-PK1 cells occurred at 48h after infection. Syncytial changes were noted 4 days after incubation. Large refractile cell changes were observed on day 3 of growth in the TBTR culture. Syncytia were observed on the second day after infection in subsequent passages. The cytopathic effect in the CER cells occurred 24h after infection, and syncytia appeared after 3 passages. Changes in syncytia indicate an adaptation of the virus for the infection of cells other than tracheal cells in primary and secondary cultures. The highest viral titre was obtained using the TBTR line. The titres obtained in the LLC-PK1 and CER cultures averaged 10(1.86)/ml. The low virus titres in all culture types suggest the need for research aimed at the optimisation of culture conditions.

Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis.

The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1-10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing.

Characterization of heat-shock proteins in Escherichia coli strains under thermal stress in vitro.

The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress conditions. The presence of these Hsps in different cell fractions indicates a very strong protective adaptation in the bacteria in unfavourable conditions, which is critical for the organism infected by them.