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

Abstract

Background: Mannheimia 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.

Results: Bacteriological 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.

Conclusion: The 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.

Keywords: Central Ethiopia; Mannheimia haemolytica; Molecular characterization; Sheep.

Fig. 1

Agarose gel electrophoresis showing PCR products of Rpt2 and PHSSA genes approximately 1022 and 325 bp, respectively. Lanes: M = 1 kb plus DNA molecular markers; 1–16: Isolates (lane 10 and 14: B.trehalosi isolates, please describe the other M. haemolytica positive lanes). N: Negative control-; P: positive control

Fig. 2

Agarose gel electrophoresis showing PCR products (approximately 1044 bp) using primer pairs targeting capsular biosynthesis gene of P. multocida. Lanes: M = 1 kb plus DNA molecular marker, E: Extraction control, Lane 1–9:M.haemolytica isolates. N: Negative control, V: NVI vaccine strain (P. multocida type A) positive around 1044 bp. P: positive control

Fig. 3

Agarose gel electrophoresis showing PCR products (approximately 1044 bp) using primer pairs targeting capsular biosynthesis gene of P. multocida and B trehalosi as template. Lanes: M = 1 kb plus DNA molecular marker, Lane VC: NVI reference strain (M. haemolytica serotype A2 isolate), Lane 1–2: B. trehalosi isolates, N: Negative control; E: extraction control, P: positive control

Fig. 4

Evolutionary relationships of 19 Mannheimia haemolytica isolates including three isolates from Ethiopia with respect to PHSSA gene partial sequence. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 301 positions in the final dataset

Fig. 5

Phylogenetic analysis of 18 Mannheimia haemolytica strains including four isolates from Ethiopia using Rpt2 gene sequence. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 780 positions in the final dataset