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.

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.