Abstract
Aim of StudyThe 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 MethodsThe 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).ResultsEight 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.ConclusionsThe 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.
Highlights
Mannheimia haemolytica is the primary etiological agent of bovine respiratory disease complex (BRDC) in cattle and sheep, and exists as commensal flora in the upper respiratory tract of ruminants
The most similar profiles were observed in the case of bacteriophages φA1 and φA5
Standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential
Summary
Mannheimia haemolytica is the primary etiological agent of bovine respiratory disease complex (BRDC) in cattle and sheep, and exists as commensal flora in the upper respiratory tract of ruminants It is responsible for significant economic losses to the livestock industry [1]. A positive element in contrast to other methods used in alternative therapies is the common occurrence of bacteriophages, which are present in wastewater, water bodies, soil, forest undergrowth, and food products. They are estimated to number about 1031, which is 10 times greater than the number of bacteria that have been characterized. This is a significant factor facilitating both their acquisition and characterization of their suitability for fighting bacterial infections [2, 3]
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