Učinak odabranih makrolida na neka obilježja goveđih leukocita

The aim of the study was to evaluate the effect of tylosin, tilmicosin and roxithromycin on viability, nitro blue tetrazolium reduction (NBT) assay, chemotaxis, apoptosis and oxidative stress in bovine leukocytes in vitro conditions. The material for the study consisted of blood collected into EDTA tubes from the external jugular vein of Holstein-Friesian cattle aged 1 week to 2.5 years, during routine veterinary examinations. In leukocytes the percent of viability, nitrate ion concentration (NO), metabolic activity (NBT, nitrotetrazolium blue reduction assay), chemotactic activity and apoptosis were determined. The results indicated a slight negative effect of these macrolides on the viability of the leukocytes, and confirmed the ability of macrolides to induce apoptosis in leukocytes in vitro. These results indicate that all of the macrolides investigated exhibit a modulatory effect on the functions of leukocytes isolated from cattle of different ages. The strongest inhibitory effect on the NBT reduction assay and chemotaxis of the leukocytes was exhibited by roxithromycin, which at the same time had the least negative effect on the leukocytes.Cilj je ovog istraživanja bio procijeniti učinak tilozina, tilmikozina i roksitromicina na preživljavanje, test redukcije “nitro-tetrazol-modrog” (NBT), kemotaksiju, apoptozu i oksidacijski stres goveđih leukocita u uvjetima in vitro. Prilikom rutinskog veterinarskog pregleda, u epruvete s EDTA bila je uzeta krv iz jugularne vene goveda holštajnsko-frizijske pasmine u dobi od tjedan dana do 2,5 godine. Određen je postotak preživljavanja leukocita, koncentracija iona nitrata (NO), metabolička aktivnost (test redukcije “nitro-tetrazol-modrog”, NBT), kemotaksijska aktivnost i apoptoza. Rezultati su pokazali blagi negativni učinak spomenutih makrolida na preživljavanje leukocita i potvrdili sposobnost makrolida da potaknu njihovu apoptozu in vitro te naznačuju da svi pretraženi makrolidi imaju modulacijski učinak na funkciju leukocita goveda različite dobi. Najjači inhibicijski učinak na test redukcije NBT i kemotaksiju leukocita pokazao je roksitromicin, koji je istodobno imao najmanje negativan učinak na leukocite

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

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.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).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.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

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

Abstract 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

Restriction analysis profiles of phages from <i>M</i>. <i>haemolytica</i> isolates single-digested with TaqI (lines 1–7).

<p>Legend: φPHL-1 (from strain BAA-410); φA1; φA2; φA5; φA6; φA7; φ25; M—DNA markers (100–1000 bp, Fermentas).</p

Cluster analysis tree diagram of PCR profiles of phage DNA from <i>M</i>. <i>haemolytica</i> isolates (lanes 1–9).

<p>Legend: φPHL-1 (from strain BAA-410); φ25; φ25a; φA1; φA1a; φA6; φA5; φA2; φA7; DNA markers (100–1000 bp, Fermentas, Li) are shown as lanes (M).</p

PCR analysis profiles of phage DNA from <i>M</i>. <i>haemolytica</i> isolates (lanes 1–9).

<p>Legend: φPHL-1 (from strain BAA-410); φ25; φ25a; φA1; φA1a; φA6; φA5; φA7; φA2; DNA markers (100–1000 bp, Fermentas) are shown as lanes (M).</p

Lytic zones (plaques) of bacteriophages specific for <i>M</i>. <i>haemolytica</i> strains.

<p>Lytic zones (plaques) of bacteriophages specific for <i>M</i>. <i>haemolytica</i> strains.</p