Characterization of bacteriophages and their carriage in Staphylococcus aureus isolated from broilers in Poland.

1. The objective of this study was the isolation and morphological characterization of temperate bacteriophages induced from Staphylococcus aureus strains isolated from clinical samples from broiler chickens and turkeys. 2. Eighty-five S. aureus strains were tested for susceptibility to oxacillin in order to determine which were methicillin resistant (MRSA). A total of 24 strains showed resistance to methicillin. 3. Thirty-one bacteriophages that were lytic against S. aureus strains were isolated and the host range of the bacteriophages was evaluated. Based on the presence of a specific nucleotide sequence, molecular identification of bacteriophages was performed and the presence of genes responsible for the production of classical enterotoxins (A-E) was also analysed. 4. All the isolated bacteriophages had an icosahedral head and a long, thin, non-contractile flexible tail, characteristic of the family Siphoviridae of the order Caudovirales. Based on multiplex PCR results, the phages were found to belong to serogroups A, B and F (Fa, Fb subgroup), which include mostly temperate phages infecting S. aureus. 5. The titre of the phages ranged from 10-4 to 10-9 PFU/ml. The bacteriophages exhibited strong lytic properties against some of the strains of Staphylococcus. The broadest spectrum of activity against the strains was observed in the case of phages sa2, sa3, sa6, sa12, sa15 and sa21. 6. The PCR results showed that of the 31 bacteriophage DNA samples, 4 (12.9%) appeared to have enterotoxigenic genes.

Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1.

Bovine herpesvirus-1 (BoHV-1), a causative agent of Infectious Bovine Rhinotracheitis (IBR), is responsible for high economic losses in cattle farming industry. The use of testing methods that allow early detection of BoHV-1-infected animals is a key element of each program of IBR eradication. The aim of the study was to design and evaluate two variants of LAMP isothermal tests with SYBR Green fluorescence probes, specific to the genes encoding gD and gE glycoproteins of BoHV-1. LAMP gE BoHV-1 assay was able to distinguish between gE- and gE+ strains of the virus. Both LAMP gD and gE assays were specific to BoHV-1 and did not react with other related to BoHV-1 alphaherpesviruses. Sensitivity of LAMP gD was 2x104 copies of the viral genome whereas for LAMP gE it was 2x105. Diagnostic sensitivity calculated for LAMP gD was 64.7% whereas for LAMP gE it was 80%. Diagnostic specificity for LAMP gD and LAMP gE was 78.9% and 89.3%, respectively. LAMP assay can be a rapid and simple method of diagnosis of acute BoHV-1 infections and discrimination of gE- strains. However, relatively low diagnostic sensitivity of the method can limit its use in routine diagnostics.

Isolation, identification and antibiotic resistance of Campylobacter strains isolated from domestic and free-living pigeons.

1. The aim of this study was to evaluate the occurrence of Campylobacter spp. in domestic and free-living pigeons and to evaluate the antibiotic resistance profiles. 2. The material consisted of cloacal swabs obtained from 108 homing pigeons and fresh faeces from 72 wild birds from Lublin and its vicinity. The identification of strains isolated on differential/selective media for Campylobacter spp. was carried out by MALDI-TOF and PCR. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in Mueller-Hinton broth. 3. A total of 35 strains of Campylobacter spp. were isolated; 27 were identified as Campylobacter jejuni and 8 as Campylobacter coli. Over half of the isolates were resistant to erythromycin and streptomycin, 40% of strains were resistant to tetracycline and ampicillin and 37% isolates were resistant to amoxicillin. Resistance to two or more antibiotics was observed in all strains tested. 4. The results indicate that both domestic and free-living pigeons are reservoirs for bacteria of the genus Campylobacter, which are characterised by varied and growing resistance to commonly used antibiotics.

Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese.

The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method. All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 µg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 µg/ml), 23.6% to tetracycline (MIC ≥ 32 µg/ml), 15% to lincomycin (MIC ≥ 64 µg/ml), 18.3% to doxycycline (MIC ≥ 32 µg/ml), 9.7% to tylosin (MIC ≥ 32 µg/ml), 56% to flumequine (MIC ≥ 256 µg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 µg/ml). Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics. Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.

Detection of the bovine viral diarrhoea virus (BVDV) in young beef cattle in eastern and south-eastern regions of Poland.

In view of the scarcity of information concerning viral diarrhoea virus (BVDV) infections in beef cattle in Poland, the aim of this study was to evaluate the presence of the BVDV in young beef cattle from selected herds in eastern and south-eastern regions of Poland. The material consisted of 78 sera obtained from beef cattle from 15 farms, aged 6-12 months. The anti-BVDV antibody level in the sera was estimated with an ELISA kit, and detection of the BVDV was carried out by standard PCR and one step Real-Time RT-PCR. The ELISA results showed a high degree (80%) of positivity in 5 of the 78 samples. In 7 samples the degree of positivity was in the very low range: < 40%. Of the 78 cDNA samples, the presence of genetic material with a length of 288 bp was found by standard PCR in 3 sera. The genetic material of BVDV was also found in the sera of the same three calves by Real-Time HRM PCR. BVDV infection in young beef cattle in south-eastern Poland is not a significant problem. This was confirmed by the positive ELISA results for 6.4% of the animals and the positive PCR results for 3.9%. The percentage of positive beef herds was about 8.6%. However, due to the severe nature of the disease and rapid transmission of the virus, regular monitoring of BVDV should be carried out.

Assessment of lipid and protein peroxidation markers in non-pregnant and pregnant female dogs.

The aim of the study was to investigate oxidative stress during normal pregnancy in female dogs based on an evaluation of plasma markers for lipid and protein peroxidation. Twenty clinically healthy female dogs (10 non-pregnant and 10 pregnant) were used in the study. Blood samples from the pregnant animals were collected at 19-21, 38-40, and 56-58 days of pregnancy. Blood samples from non-pregnant female dogs were obtained between 20 and 35 days after ineffective breeding. As indicators of oxidative stress, we measured the following using spectrophotometric and spectrof- luorimetric methods: thiobarbituric acid reactive substances (TBARS), radical cations of N,N, diethylparaphenylene diamine (RC-DEPPD), sulfhydryl groups (SH groups), bityrosine and formylkynurenine. The mean plasma TBARS concentration in the pregnant dogs (0.486 ± 0.071-0.581 ± 0.191 µmol/g protein) was significantly higher (p < 0.05) than that found in the non-pregnant animals (0.274 ± 0.111 µmol/g protein). A marked, although not significant, decrease in SH group content, as well as an increase in bityrosine and formylkynurenine concentration were concurrently observed in the pregnant dogs. No significant differences were found in terms of the studied markers in the pregnant animals when comparing the values obtained during the investigated periods of pregnancy, although there was a progressive decrease in TBARS concentration and a progressive increase in RC-DEPPD, bityrosine and formylkynurenine contents. Our findings suggest that normal pregnancy in female dogs is associated with oxidative stress. Further studies are necessary to establish the physiological ranges of antioxidative/oxidative profiles in pregnant dogs and to explain if and how the intensity of oxidative stress might contribute to the risk of the complications of pregnancy.

Rapid Detection of Bovine Respiratory Syncytial Virus in Poland Using a Human Patient-Side Diagnostic Assay.

Bovine respiratory syncytial virus (BRSV) plays a significant role in the etiopathogenesis of the respiratory syndrome in young cattle during their first year of life. Development of rapid and accurate BRSV diagnostic tools would aid in the appropriate control of this important pathogen. The objective of this study was to characterize infections induced by BRSV by means of rapid patient-side immunomigration assays used for diagnosis of human respiratory syncytial virus (hRSV) in humans. Nasal and tracheal swabs were obtained from healthy calves of various beef and dairy breeds - Holstein-Friesian, Simmental, Charolais, Belgian Blue and Limousin, between the ages of 5 and 12 months, from 26 farms. BRSV was identified using two rapid immunomigration assays, TruRSV® and Clearview® RSV, and compared with RT-PCR as a reference technique. BRSV was found in 73.1% of all the herds tested. High agreement with RT-PCR was obtained for TruRSV® (κ = 0.824), while in the case of the Clearview® RSV test, agreement with PCR was moderate (κ = 0.420). The results demonstrate that rapid patient-side immunomigration assays designed to detect hRSV can be used to accurately detect BRSV in field samples collected from cattle.

Oxidative parameters and expression of 70kDa heat shock proteins in pig heart tissue after transport and slaughter.

In view of the significant role of Hsp70 in protecting the organism against the destructive effects of stress, and the possibility of using this protein as a marker of the infarction process in the heart, the aim of this study was to conduct an evaluation of the expression of 70kDa heat shock proteins (Hsp70) and the concentration of TBARS (thiobarbituric acid reactive substances) and nitric oxide ions (NO), determined as nitrite ions, as markers of oxidative stress in hearts obtained from healthy pigs following slaughter and pigs which had died during or immediately after transport with symptoms of sudden cardiac death. The material consisted of hearts obtained from 90 pigs following slaughter and from pigs which had died. Oxidative stress was determined in heart lysates based on the concentration of TBARS and nitrite ions. Expression and concentration of Hsp70 were determined using SDS-PAGE, Western blotting, and ELISA. Expression of Hsp70 was observed in hearts lysates obtained from slaughtered pigs and from those which had died with symptoms of sudden death. The strongest reaction in the Western Blotting was noted in hearts lysates from pigs with no pathological changes. The highest TBARS concentration was observed in lysates from hearts in pigs which had died during or immediately after transport. The highest concentration of NO ions, determined as nitrite ions, was noted in hearts from pigs with myocardial infarction lesions. The significant decrease observed in Hsp70 concentration in heart tissue obtained from the pigs which had died in comparison to the hearts from healthy pigs indicates the important role of this protein in protecting the heart muscle against the destructive effects of stress, which limits the occurrence of post-stress cardiomyopathy in pigs following transport.

An electrophoretic characterization of iron-transporting proteins in Mannheimia haemolytica A1.

Iron-regulated outer membrane proteins (IROMPs) in Mannheimia haemolytica A1, which function as a receptor for complexes containing iron ions, are induced by iron deficiency in the growth environment of the bacteria. Densitometric analysis of SDS-PAGE separation showed expression of IROMPs of 71, 77, and 100 kDa in the case of bacteria grown in a medium with 2,2-dipyridyl. The electrophoregrams obtained in 2-DE separations confirmed the presence of protein fractions with these molecular weights and isoelectric points ranging from 5.4 to 6.4. The results of the study also confirmed the ability of M. haemolytica A1 proteins involved in iron uptake to induce a protective immune response. In Western blot with serum from convalescent calves naturally infected with M. haemolytica A1, distinct reactions were obtained for IROMPs of 71, 77, and 100 kDa.

Effect of conglutinin on phagocytic activity of bovine granulocytes.

In the present study we investigated the effect of bovine conglutinin on the phagocytic activity of leukocytes. We measured both the chemotactic activity of conglutinin and its effect on the internalization of zymosan particles and E. coli by granulocytes. We also assessed the binding of conglutinin to various microorganisms isolated from clinical cases in cattle. We showed that conglutinin binds strongly to the surface of yeast cells and to mannan-rich zymosan particles, while weak binding was observed in the case of the bacterial strains tested, including those whose O antigen is composed of mannan. Conglutinin (1-10 microg/ml) neither acts as a chemotactic factor for peripheral blood leukocytes nor affects ingestion of E. coli by granulocytes. However, as flow cytometry based assay showed, conglutinin (0.1-1 microg/ml) increased ingestion of zymosan expressed as mean fluorescence intensity (MFI) of positive cells.

The influence of selected techniques of bovine leukocyte isolation on their viability and metabolism.

The aim of the study was to assess the effect of selected isolation methods on the viability and metabolism of bovine leukocytes. The cells were isolated using a Ficoll 1077, Histopaque 1083 gradient and osmotic shock method, and Ficoll or Histopaque with osmotic shock. Evaluation were made of the total number of cells, viability after isolation and in 24h culture on RPMI 1640 medium and metabolism with NBT reduction assay. Microscopic and cytometric evaluation of the leukocytes revealed that the isolation methods applied had an influence on their number and viability. Based on the results it can be concluded that isolation methods of cells in a Histopaque or Ficoll yield highly pure cell fractions with high viability.

Characterization of outer membrane proteins participating in iron transport in Pasteurella multocida serotype A3.

Iron-regulated outer membrane proteins (IROMPs) of P. multocida serotype A3, which function as receptors for complexes containing iron ions, are induced by iron deficiency in the bacterial growth environment. Analysis of an electrophoresis image of proteins isolated from bacteria grown on medium supplemented with 2,2'-dipyridyl revealed expression of 16 new proteins that were not noted in the case of the bacteria grown in standard conditions, with molecular weights from 30 to 160 kDa. Induction of IROMP expression occurred within 30 minutes after restricted iron conditions were established. In immunoblotting, distinct reactions were noted for proteins of molecular weight ranges of 25-49 kDa, 61-95 kDa, and 108-214 kDa. Proteins of the molecular weight of 68, 75 and 86 kDa were analysed using mass spectrometry and matched with the highest probability to proteins in the NCBI data base. Several dozen different proteins with similar amino acid sequences were matched to each sample.

Characterization of Hsp70 proteins in bovine leukocytes induced by the temperature 41 degrees C.

Heat shock proteins (Hsp) are the group of proteins observed in both prokaryotic and eukaryotic cell types. Hsp synthesis takes place in response to many environmental conditions, including ultraviolet radiation, heavy metal ions, hypoxia and toxic agents. Many authors have suggested that Hsp can be used in immunoprophylaxis, yet Hsp70 proteins expressed in bovine leukocytes have not been fully characterized. Hence the aim of this study was to evaluate the expression of Hsp70 proteins in bovine leukocytes exposed to temp. 41degrees C. The material for the study consisted of bovine white blood cells incubated at 41 degrees C for 2 hours. SDS-Page electrophoresis, Western blotting, and two-dimensional electrophoresis (2D) were performed to estimate the proteins obtained. The results of the study confirmed the influence of the temperature of 41 degrees C on induction of Hsp70 in bovine leukocytes. These proteins were mainly localized within molecular mass 70kDa. Some of the proteins with molecular mass from 20 to 50 kDa also showed positive reactions in Western blotting with anti-Hsp70 antibodies. Analysis of 2D electrophoresis showed a change in the localization of these proteins in the pH gradient. It can be postulated that analysis of Hsp70 expression in bovine leukocytes can be a very helpful marker for evaluating an organism's adaptation to environmental heat stress. The proteins obtained also showed immunological reactivity with rabbit antibodies in Western blotting reactions, indicating that they can be used as protective factors in the pathogenesis of many diseases.

Comparison of immunogenic properties of native and inactivated Mannheimia haemolytica Lkt in calves.

The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorbance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MTT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.

Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro.

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.

Lipid peroxidation as the additional indicator of transport stress in broilers.

Stressful conditions (transport, temperature fluctuations, infections and excessive crowding) lead to dysfunction of immunological mechanisms in birds. They influence the lipid balance what manifests itself through the increase of the malonodialdehyde (MDA) level, reacting with the 2-thiobarbituric acid (TBA), what is an indicator of lipid peroxidation. In the case of birds, the classical indicator of the stressful reaction is the heterophile/lymphocyte ratio (H/L), which changes depending on the stress intensity. The present study defined the influence of road transport on lipid peroxidation process as the additional indicator of stress in broilers. The investigation was conducted on 10 broilers Ross 308 (39 days old), which were transported on the distance of 70 km. The birds were clinically healthy, free of parasites and free of Salmonella spp. infection in the beginning of the experiment. The determination of the TBA-reactive substances (TBARS) in serum was conducted according to the method proposed by Ledwozyw et al. (1986). The analysis of the results with a Student t-test showed the statistically significant difference (P < or = 0.05) in the TBA-reactive substances level before and directly after the birds transportation.

Evaluation of Mannheimia haemolytica leukotoxin prepared in nonsupplemented and BSA or FBS supplemented RPMI 1640 medium.

Mannheimia haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Supernatant preparations containing Lkt produced by M. haemolytica serotype 1, grown in RPMI 1640 medium supplemented with BSA or FBS and without supplements were evaluated during this study. Analysis of obtained Lkt showed presence of 105 kDa antigen (SDS-PAGE electrophoresis). The obtained bacterial protein fraction estimated as Lkt was detected by Western blotting with mouse monoclonal (Mab 605 and Mab 601) anti-Lkt antibodies. No significant differences were found in obtained leukotoxin between wildtype and reference M. haemolytica strains. Our studies showed that growth in media supplemented with BSA or FBS had no significant influence on leukotoxin production. When BSA or FBS supplements were used, additional protein fractions in electrophoregrams SDS-PAGE were observed. These protein bands did not react with Mab 605 and/or Mab 601 in Western blotting analysis. Lkt immunogenicity was detected by immunoblotting with sera from Lkt immunized rabbits and calves.

The influence of transport stress on the humoral immunological response of calves induced by Mannheimia haemolytica leukotoxin.

In the contemporary systems of cattle production, transport stress is the most essential poly-etiological factor responsible for inducing unfavourable reactions in the animals. The main reason for this phenomenon is the immunosuppressive effect of steroid hormones on cellular and humoral protective mechanisms. The purpose of the present study was to establish the relationship between the cortisol concentration as an indicator of the stress reaction occuring directly after the transportation of calves and the specific humoral immune response to the leukotoxin (Lkt) antigen produced by the M. haemolytica strain. The experiment was carried out on 19 clinically healthy calves, weighing about 100 kg and transported by track for about 2 hours. After the delivery of the animals for feeding to the traditional cattle-house, the calves were immunized s.c.: group I with 1 ml Lkt (in conc.--10 microg/ml) with 1 ml of adjuvant on the 1st and 14th day after the transportation, group II with the same Lkt doses on the 3rd and 16th day after the transportation. The animals of the control group were vaccinated on the 1st and 14th day after the transportation with the twice diluted adjuvant. In examined sera the cortisol concentrations and the level of Lkt antibodies were measured by ELISA test. The cytotoxin neutralizing (CN) antibody level (cytotoxity assay) was determined with a simple visual assay. The study revealed, significant differences (P < or = 0.01) in serum cortisol levels between the control and experimental animals. The analysis of the absorbance of the sera in both groups immunized with Lkt showed substantial differences (P < or = 0.05) from the 6th through to 22nd day of the experiment compared with the control group. The analysis of the results of CN antibody titers showed no differences between the sera from group I and II. Based on the results obtained in this experiment it can be assumed, that a short transportation stress has no important influence on the level of specific humoral anti-Lkt response.