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

Abstract

Background and Aim: Mannheimia haemolytica (Mha) is a common agent of pneumonia in ruminants globally, causing economic losses by morbidity, mortality, and treatment costs. Infection by Mha is often associated with or promoted by respiratory viral pathogens and environmental conditions. Infections due to Mha have rarely been described in small ruminants. This study reports the biological and molecular characteristics of a new Moroccan Mha isolate from small ruminants presenting typical respiratory symptoms. We also studied the cultural parameters, growth kinetics, and Lkt excretion of the isolate and its pathogenicity on laboratory animals and small ruminants. Materials and Methods: Suspected pasteurellosis cases in sheep and goat flocks in Morocco were investigated. A local strain of Mha was isolated and identified using biochemical and molecular methods. Polymerase chain reaction-targeting specific genes were used for serotyping and phylogenetic analyses; further, leukotoxin production, cytotoxicity, and pathogenicity of the isolate in mice, goats, and sheep were investigated. Results: Phylogeny analysis revealed 98.76% sequence identity with the USA isolate of 2013; the strain growth with a cycle of 9-10 h with leukotoxin secretion was detected by NETosis and quantified by cytotoxicity and mortality of mice. Goat and sheep infections cause hyperthermia, with characteristic postmortem lesions in the trachea and lung. Conclusion: A local isolate of Mha from sheep that died of pneumonia was characterized for the 1st time in North Africa using biological and molecular methods. Although growth on appropriate culture media is accompanied by intense leukotoxin secretion, experimental infections of sheep and goats cause hyperthermia and typical lesions of pneumonia.

Comparative protection of small ruminants against Mannheimia haemolytica infection by inactivated bacterin and toxoid vaccines

“…A recent study confirmed the higher sensitivity of goats to M. haemolytica than that of sheep [15]. To assess the conferred immunity in this study, we performed experimental infection only on goats.…”

“…Vaccination with M. haemolytica bacterins failed to induce effective protection, probably because this bacterium produces some virulence factors involved in clinical disease and mortality [ 19 ]. The Mha leukotoxin is known to be cytotoxic to ruminant leukocytes and has been the subject of studies on its virulence and immunogenicity in cattle [ 15 , 20 ]. The presence of this protein in the vaccine components may contribute to an increase in its efficacy.…”

“…The strain was cultured on brain heart infusion (BHI; Solabia, France) agar for 9 h at 37°C, passaged in BHI broth, and incubated for 12 h at 37°C with moderate agitation. Culture in a bioreactor was conducted at 35°C with automatic agitation, and aeration until the end of the logarithmic growth phase reached after 7–9 h of fermentation [ 15 ]. After harvesting, the culture was inactivated with 0.5% (v/v) formaldehyde.…”

“…The bacterial suspension was harvested after centrifugation at 8000× g for 30 min at 4°C. To obtain leukotoxin supernatant, the recovered supernatant was treated by ultrafiltration to concentrate the protein [ 15 , 16 ]. Three vaccines were prepared.…”

Stimulating immunoglobulin response by intramuscular delivery of exopolysaccharides-adjuvanted mannheimiosis vaccine in goats

“…Mastitis develops in camels and ewes. It has also been isolated from certain wild birds and cattle [4][5][6][7]. In particular, M. haemolytica causes bovine pneumonic pasteurellosis (older name) or mannheimiosis.…”