Virulence Gene Characterization and Serotyping of Major Bacterial Pathogens Isolated from Bovine Respiratory Disease in Ethiopia

Akalu, Mirtneh; Abayneh, Takele; Gelaye, Esayas; Derese, Getaw; Tefera, Behailu; Degefa, Teferi; Murthy, Vemulapati Bhadra

doi:10.4236/aim.2022.121002

Cited by 3 publications

(5 citation statements)

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“…PCR was carried out for the detection of an iron-binding protein encoding gene using PmFbpA primer and amplicon size of 500bp was observed for all P. multocida serotypes which are consistent with the previous report [36]. The iron acquisition system of M. hemolytica (A: 1) allows obtaining iron from the host's iron-binding proteins under iron-deficient conditions [43].…”

Section: Discussionsupporting

confidence: 80%

“…For most PCR reactions, 5µl template DNA was added to the total 20 µl reaction mixture containing 4.5µl of nuclease-free water, 2µl of each forward and reverse primer pair (5pmol/µl), 5 µl of 10x DreamTaq™ Buffer that contains 20mM MgCl2, 5µl of dNTP Mix (2mM each) and 1.5µl of Dream Taq™ polymerase Amplification protocols for the capsular biosynthesis gene of P. multocida, PHSSA-1, and methylene transferase encoding gene(Rpt2) of M. hemolytica and manganese-dependent superoxide dismutase encoding gene of B. trehalosi (BtsodA) were used as previously described by respectively [29, 33 and 34]. PCR thermal cycling conditions for the iron acquisition TbpA2 and FbpA genes were also prepared as previously stated by respectively [35,36]. PCR instructions provided by the University of Toronto were used as a PCR protocol for surface lipoproteins (PmSLP) locus [37].…”

Section: Primers and Pcr Conditionsmentioning

confidence: 99%

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Molecular Detection and Genotyping of P. Multocida, M. Hemolytica, and B. Trehalosi Isolates Targeting the Virulence Associated Genes from Ethiopian Cattle and Sheep

2022

JGEBR

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Pasteurellaceae families are usually considered opportunistic pathogens which inhabit normal flora on the mucosal membranes of the upper respiratory and the lower genital tracts of mammals and birds. The majority of P. multocida, M. hemolytica, and B. trehalosi isolates are opportunistic animal pathogens and cause disease only under certain conditions. Some of the target genes are shared by isolates found in the same or different genera.. The alpha / beta hydrolase kmt1 genes part of OMP and SLP are shared by all P.multocida species and FbpA is common gene for both P.multocida and M. hemolytica isolates. Other virulence related genes are used for genotyping of P.multocida, M.hemolytica and B.trehalosi isolates. The objective of the current research was focused on the detection and genotyping of different target genes that can be found in the isolates of P. multocida, M. hemolytica, and B. trehalosi using various PCR primers. The distributions of virulence-associated genes were assessed in the isolates of the three genera. Materials and methods A total of eight isolates from P. multocida, M. hemolytica, and B. trehalosi isolated from clinical cases of cattle and sheep and stored at the Bacteriology laboratory of NVI were used as source of samples for the present study. DNeasy® Blood & Tissue Kit (Qiagen, Germany) was used for the extraction of DNA from bacterial isolates. Different primers were used for genotyping of P. multocida, P.hemolytica and B.trehalosi isolates. Results P.multocida was detected using species-specific kmt1 primer with an amplicon product of 460bp. Four serogroups of P.multocida (A, B, D, &E) were detected using serotype-specific primers with amplification products of 1044bp, 760bp, 657bp, and 511bp respectively. PCR was conducted to HS causing P.multocida type B and detected 620 bp amplicons. SLP genes were detected in all P.multocida serotypes with an amplification product size 1400-1553bp. In addition to that, the four P. multocida serotypes were found to be positive for BRD-PmSLP with an amplification product of 460bp but using recombinant HS-SLP primers resulted in the double band at 460 bp and 541bp for serotypes A and D. However, HS causing type B& E serotypes were detected with an amplified product of 541bp. TbpA2 has been detected with an amplicon size of approximately 750 bp for types (A and D) and 1300bp for type B using TbpA-F2/Rev primers. All of the P.multocida serotypes (A, B, D&E) and M. hemolytica (A: 1) were strongly positive for the iron acquisition FbpA gene with amplification bands at 500bp and 1000bp, respectively. A multiplex PCR as-say was carried out for detection of M. haemolytica A: 1 using PHSSA and Rpt2 primers with amplification products of 327pb and 1022 bp respectively. The three isolates of B. trehalosi were identified using BtsodA primers with the result of 144bp amplicon size. Conclusion From this work, we understood that P.multocida and M. haemolytica shared genetic materials like iron binding proteins (FbpA) and some of the target genes (kmt1 and SLP) were commonly found in all isolates of P. multocida. However, TbpA2 was found to be in most but not in all Pasteurella isolates. We also observed that PHSSA and Rpt2 genes were exclusively found in M. hemolytica isolates whereas Bt-sodA was prominent in all B. trehalosi isolates (T3, T4&T15).

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Section: Discussionsupporting

confidence: 80%

Section: Primers and Pcr Conditionsmentioning

confidence: 99%

Molecular Detection and Genotyping of P. Multocida, M. Hemolytica, and B. Trehalosi Isolates Targeting the Virulence Associated Genes from Ethiopian Cattle and Sheep

2022

JGEBR

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show abstract

Section: Discussionsupporting

confidence: 80%

“…For most PCR reactions, 5µl template DNA was added to the total 20 µl reaction mixture containing 4.5µl of nucleasefree water, 2µl of each forward and reverse primer pair (5pmol/µl), 5 µl of 10x DreamTaq™ Buffer that contains 20mM MgCl 2 , 5µl of dNTP Mix (2mM each) and 1.5µl of Dream Taq™ polymerase Ampli cation protocols for the capsular biosynthesis gene of P. multocida, PHSSA-1, and methylene transferase encoding gene (Rpt2) of M. hemolytica and manganese-dependent superoxide dismutase encoding gene of B. trehalosi (BtsodA) were used as previously described by Townsend et al [33], Kumar et al [28] and Ewers et al [34], respectively. PCR thermal cycling conditions for the iron acquisition TbpA2 and FbpA genes were also prepared as previously stated by Sujatha et al [35] and Akalu [36], respectively. PCR instructions provided by the University of Calgary were used as a PCR protocol for surface lipoproteins (PmSLP) locus [37].…”

Section: Bacterial Isolatesmentioning

confidence: 99%

Molecular detection and genotyping of P. multocida, M. hemolytica, and B. trehalosi isolates targeting the virulence associated genes from Ethiopian cattle and sheep

Deresse¹,

Gelaye²,

Abayneh³

et al. 2022

Preprint

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BackgroundPasteurellaceae families are usually considered opportunistic pathogens which inhabit normal flora on the mucosal membranes of the upper respiratory and the lower genital tracts of mammals and birds. The majority of P. multocida, M. hemolytica, and B. trehalosi isolates are opportunistic animal pathogens and cause disease only under certain conditions. Some of the target genes are shared by isolates found in the same or different genuses. The alpha / beta hydrolase kmt1 genes part of OMP and SLP are shared by all P.multocida species and FbpA is common gene for both P.multocida and M. hemolytica isolates. Other virulence related genes are used for genotyping of P.multocida, M.hemolytica and B.trehalosi isolates. The objective of the current research was focused on the detection and genotyping of different target genes that can be found in the isolates of P. multocida, M. hemolytica, and B. trehalosi using various PCR primers. The distributions of virulence-associated genes were assessed in the isolates of the three genera.Materials and methodsA total of eight isolates from P. multocida, M. hemolytica, and B. trehalosi isolated from clinical cases of cattle and sheep and stored at the Bacteriology laboratory of NVI were used as source of samples for the present study. DNeasy® Blood & Tissue Kit (Qiagen, Germany) was used for the extraction of DNA from bacterial isolates. Different primers were used for genotyping of P. multocida, P.hemolytica and B.trehalosi isolates.ResultsP.multocida was detected using species-specific kmt1 primer with an amplicon product of 460bp. Four serogroups of P.multocida (A, B, D, &E) were detected using serotype-specific primers with amplification products of 1044bp, 760bp, 657bp, and 511bp respectively. PCR was conducted to HS causing P.multocida type B and detected 620 bp amplicons. SLP genes were detected in all P.multocida serotypes with an amplification product size 1400-1553bp. In addition to that, the four P. multocida serotypes were found to be positive for BRD-PmSLP with an amplification product of 460bp but using recombinant HS-SLP primers resulted in the double band at 460 bp and 541bp for serotypes A and D. However, HS causing type B& E serotypes were detected with an amplified product of 541bp. TbpA2 has been detected with an amplicon size of approximately 750 bp for types (A and D) and 1300bp for type B using TbpA-F2/Rev primers. All of the P.multocida serotypes (A, B, D&E) and M. hemolytica (A: 1) were strongly positive for the iron acquisition FbpA gene with amplification bands at 500bp and 1000bp, respectively. A multiplex PCR assay was carried out for detection of M. haemolytica A: 1 using PHSSA and Rpt2 primers with amplification products of 327pb and 1022 bp respectively. The three isolates of B. trehalosi were identified using BtsodA primers with the result of 144bp amplicon size.ConclusionFrom this work, we understood that P.multocida and M. haemolytica shared genetic materials like iron binding proteins (FbpA) and some of the target genes (kmt1 and SLP) were commonly found in all isolates of P. multocida. However, TbpA2 was found to be in most but not in all Pasteurella isolates. We also observed that PHSSA and Rpt2 genes were exclusively found in M. hemolytica isolates whereas Bt-sodA was prominent in all B. trehalosi isolates (T3, T4&T15).

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“…Concerning Pasteurella multocida, it is known by its host range and ability to elicit the disease in different animals other than cattle (Alarawi and Saeed 2021). For example, in pigs, it causes atrophic rhinitis, rabbits snuffles and birds fowl cholera (Boyce et al 2006;Harper et al 2006;Pesapane et al 2022;Akalu et al 2022).…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of Pasteurella Multocida and Mannheimia Haemolytica Infections in Cattle Using Lateral Flow Immunochromatographic Assay

2023

Int J Vet Sci

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This study was mainly designed to advance and recover the control measures of Bovine respiratory disease (BRD) via accelerating the field identification of some BRD causing pathogens. A simple rapid field test was developed mainly to identify Pasteurella multocida and Mannheimia haemolytica, both are highly associated with BRD. A full identified Pasteurella multocida and Mannheimia haemolytica cultures were earned from Microbiology Department, Faculty of Veterinary Medicine, Cairo University, Egypt. Species-specific bacterial antigen of outer membrane protein of each bacterial strain was prepared separately for M. haemolytica and P. multocida. Specific polyclonal antibodies were prepared by amplifying each one in guinea pigs and rabbits. Pathogens-specific lgG were purified using caprylic acid technique. Pathogens-specific lgG titers were measured by ELISA. The colloidal gold nanoparticles were conjugated to the rabbit mono specific polyclonal lgG antibody and did not conjugate to guinea pig specific polyclonal lgG. The particles were then utilized to develop the Lateral flow kits (LFK) which is a rapid assay for detection for P. multocida and M. haemolytica in clinical samples when Compared to the conventional Bacteriological tests. Results: Percentage of accuracy, specificity and sensitivity of LFK kits recorded 93, 94.4 and 90.4%, respectively, for P. multocida and 91.5, 94.3and 84.7%, respectively, for Mannheimia haemolytica.

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Virulence Gene Characterization and Serotyping of Major Bacterial Pathogens Isolated from Bovine Respiratory Disease in Ethiopia

Cited by 3 publications

References 36 publications

Molecular Detection and Genotyping of P. Multocida, M. Hemolytica, and B. Trehalosi Isolates Targeting the Virulence Associated Genes from Ethiopian Cattle and Sheep

Molecular Detection and Genotyping of P. Multocida, M. Hemolytica, and B. Trehalosi Isolates Targeting the Virulence Associated Genes from Ethiopian Cattle and Sheep

Molecular detection and genotyping of P. multocida, M. hemolytica, and B. trehalosi isolates targeting the virulence associated genes from Ethiopian cattle and sheep

Diagnosis of Pasteurella Multocida and Mannheimia Haemolytica Infections in Cattle Using Lateral Flow Immunochromatographic Assay

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