Molecular characterization of Mannheimia haemolytica isolates associated with pneumonic cases of sheep in selected areas of Central Ethiopia

Abstract

BackgroundMannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area.ResultsBacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin.ConclusionThe study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.

Biological and molecular characterization of a sheep pathogen isolate of Mannheimia haemolytica and leukotoxin production kinetics

“…Phylogenetic analyses comparing partial Rpt2 gene sequences of the Moroccan field isolate with reference and variant strain sequences revealed that the Moroccan isolate was homologous with the American genotype, especially Mha (USDA/ ARS/USMARC/184) isolated in the USA in 2013. This homology in genotype distribution among the Mha isolates observed agrees with the findings of Legesse et al [15], which revealed the existence of three genotypes of Mha circulating in the study areas consistent with the site of isolation. The growth kinetics of the Mha isolate in broth media showed a latency phase of 5 h, followed by an exponential phase of 3.5 h and a stationary phase of 1.5 h. The comparative growth kinetics between the local isolate and the reference strain of bovine origin (ATCC) revealed a higher bacterial concentration with the isolate than with the referred ATCC strain with 1 log difference.…”

“…Amplification of the Rpt2 gene sequence by PCR was performed using the Rpt2 forward (5′-GTT TGT AAG ATA TCC CAT TT-3′) and reverse (5′-CGT TTT CCA CTT GCG TGA-3′) primers [15]. PCR was performed using the Applied Biosystems kit protocol, in a 20-μL reaction mixture containing 2 μL buffer (10×), 2.5 μL MgCl 2 ( 25 mmol/l), 2.5 μL dNTP (10 mmol/l), 0.75 μL of each primer (10 μmol/l), 10.2 μL of sterilized water, 0.5 μL of RNase inhibitor (20 U/μL), 0.3 μL RT (50 U/μL), and 0.5 μL of Gold Taq polymerase (5 U/μL).…”

Bacteria isolated from the lower respiratory tract of sheep and their relationship to clinical signs of sheep respiratory disease

“…Respiratory rate, rectal temperature, nasal discharge, cough, dyspnea, percussion and altered sounds in pulmonary auscultation were increased in pneumonic sheep (P<0.05). These findings confirm the importance of the observed clinical signs of sheep to determine the respiratory health during physical examination (Viana et al 2007, Legesse et al 2018.…”

Serotyping, Genotyping and Virulence Genes Characterization of Pasteurella multocida and Mannheimia haemolytica Isolates Recovered from Pneumonic Cattle Calves in North Upper Egypt

“…P. haemolytica biotype A was later allocated to a new genus Mannheimia and renamed as M. haemolytica , while the four T serotypes were named Bibersteinia trehalosi . Later, serotype A:11 was classified into a new taxon as M. glucosida , due to its different biochemical profile, leaving 12 serotypes of M. haemolytica [ 9 ].…”