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Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

ABSTRACT: AIM:This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. INTRODUCTION:M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. MATERIALS AND METHODS:A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. RESULTS:All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database. CONCLUSION:The present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.

SUBMITTER: Kumar J

PROVIDER: S-EPMC4774775 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

Kumar Jyoti J Dixit Shivendra Kumar SK Kumar Rajiv R

Veterinary world 20150915 9

<h4>Aim</h4>This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture.<h4>Introduction</h4>M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica de ...[more]

PMID: 27047201

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Project description:Mastitis affects both dairy and meat/wool sheep industries with losses due to reductions in milk quality and quantity, increased treatment costs and restricted lamb growth. Effective vaccines would be important tools for mastitis control. However, the development of vaccines against mastitis has proved challenging due to the failure to target protective immunity to the mammary gland. In order to target responses to the mammary gland, this study tested whether local administration directly into the gland through the teat canal or in the udder skin confers protection against an intramammary infection. In this study, we tested a vaccine that confers protection against respiratory disease caused by Mannheimia haemolytica to determine if it also protects against intramammary infection by the same organism. No evidence of protection was observed in animals that received a subcutaneous immunisation in the udder skin, however, intramammary immunisation provided almost complete protection against an experimental challenge administered 7 days post immunisation but not if the challenge was delivered 14 days post immunisation. To investigate further the nature of this variation in response, the somatic cell count and concentration of cytokines Interleukin-1β, Interleukin-10 and Interleukin-17A was determined in milk over the course of each study. Intramammary immunisation induced an inflammatory response within the mammary gland, characterised by increases in SCC and in the production of cytokines IL-1β, IL-10, and IL-17A. This response was similar to that observed in un-vaccinated control animals post challenge. The SCC and cytokine levels had returned to levels comparable with un-vaccinated controls prior to challenge at both 7 and 14 days post immunisation. The transient nature of the protective effect is consistent with the priming of an innate antibacterial response within the mammary gland which provides protection against challenge at 7 days but is diminished by 14 days post-vaccination. Further studies are planned to determine the nature of the innate immune mechanisms associated with the protective effect described here to determine whether it may be exploited to improve ruminant udder health.

Project description:Six Lactobacillus strains originating from the nasopharyngeal microbiota of cattle were previously characterized in vitro and identified as candidate bacterial therapeutics (BTs) for mitigating the bovine respiratory pathogen Mannheimia haemolytica In the present study, these BT strains were evaluated for their potential to (i) reduce nasal colonization by M. haemolytica, (ii) modulate the nasal microbiota, and (iii) stimulate an immune response in calves experimentally challenged with M. haemolytica. Twenty-four Holstein bull calves (1 to 3 weeks old) received either an intranasal BT cocktail containing 6 Lactobacillus strains (3?×?109 CFU per strain; BT + Mh group) 24?h prior to intranasal M. haemolytica challenge (3?×?108 CFU) or no BTs prior to challenge (Mh, control group). Nasal swab, blood, and transtracheal aspiration samples were collected over the course of 16?days after BT inoculation. Counts of M. haemolytica were determined by culturing, and the nasal and tracheal microbiotas were evaluated using 16S rRNA gene sequencing. Serum cytokines (interleukin-6 [IL-6], IL-8, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA). Administration of BT reduced nasal colonization by M. haemolytica (P?=?0.02), modified the composition and diversity of the nasal microbiota, and altered interbacterial relationships among the 10 most relatively abundant genera. The BT + Mh calves also had a lower relative abundance of Mannheimia in the trachea (P?<?0.01) but similar cytokine levels as Mh calves. This study demonstrated that intranasal BTs developed from the bovine nasopharyngeal Lactobacillus spp. were effective in reducing nasal colonization by M. haemolytica in dairy calves.IMPORTANCE Bovine respiratory disease (BRD) is one of the significant challenges for the modern dairy industry in North America, accounting for 23 to 47% of the total mortality among pre- and postweaned dairy heifers. Mass medication with antibiotics is a common practice to control BRD in dairy cattle. However, the emergence of multidrug-resistant BRD pathogens highlights the importance of developing alternatives to antibiotics for BRD mitigation. Using a targeted approach, we recently identified 6 Lactobacillus strains originating from the bovine respiratory microbiota as candidates to be used as bacterial therapeutics (BTs) for the mitigation of the BRD pathogen Mannheimia haemolytica Here, we demonstrated that intranasal inoculation of the BT strains reduced nasal colonization by M. haemolytica in dairy calves experimentally challenged with this pathogen. This study, for the first time, shows the potential use of intranasal BTs as an alternative to mitigate BRD pathogens in cattle.

Project description: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.

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Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

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Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

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