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

Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (n = 107) and pneumonic lung tissue (n = 59). Laboratory assay confirmed isolation of M. haemolytica 37 (22.29%), P. multocida 25 (15.06%), B. trehalosi 12 (7.23%), and H. somni 15 (9.04%). PCR assay of P. multocida capsular typing revealed 21 (84.0%) cap A (hyaD-hyaC) and 4 (16.0%) cap D (dcbF) strains. M. haemolytica serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. P. multocida biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%),


Introduction
Ethiopia has the largest number of livestock in Africa and home for various animal species. The country is naturally endowed with different agro-ecological zones and suitable for livestock production. Cattle are the most important livestock subsector in the country and the main providers of nutrition and income for livelihoods. The total cattle population in the country is estimated to be 59.5 million and distributed evenly throughout the country, with higher density in the highland areas [1] [2]. However, the productivity potential remained very low to meet the growing demand of the population. This is due to various constraints such as high prevalence of veterinary disease, low animal health service coverage, and shortage of access to inputs. Moreover, animal health issues are the major barriers to productivity. Prevalence of veterinary important diseases like respiratory illness exert negative impact on productivity and contributes to economic losses in the country [3] [4].
BRD is considered as one of the potential disease in all age of cattle [5] [6].
Moreover, M. haemolytica and H. somni do not constantly involve stressors (environmental and management) or other concomitant infections to cause fatal pneumonia [7].

Isolation of Bacterial Pathogens
Bacterial strains were isolated using the standard bacteriological assay. Briefly, pneumonic lung tissue samples were minced, reconstituted in 4 ml sterile physiological saline (pH 7.0 ± 0.2), centrifuged at 3000 × g for 3 min, and the supernatant was discarded. The remaining sediment was reconstituted with 100 µl sterile physiological saline. Ten µl of pneumonic lung sample suspension and nasopharyngeal swabs were streaked comparably onto blood agar base (HiMedia, India) with 5% sheep blood and MacConkey agar (HiMedia, India). Plates were incubated at 37˚C for 24 -48 hrs aerobically.
Colonies characteristics of Pasteurellaceae were further assayed as per the standard bacteriological method. Presumptive isolates were further analyzed using biochemical test (catalase and oxidase reaction, ornithine decarboxylase (ODC), indole production, nitrate reduction and urease). Identification of BRD-associated bacteria pathogens to species level was conducted using carbohydrate fermentation reaction (glucose, lactose, sucrose, arabinose, trehalose, dulcitol, mannitol, sorbitol, and D-xylose).

Molecular Assay
A PCR assay was conducted on presumptive bacterial pathogens of BRD as a confirmatory assay. Species-specific universal genes Rpt2 and Kmt1 were used for molecular detection of M. haemolytica and P. multocida, respectively. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (QIAGEN GmbH, Germany) following the manufacturer's instruction.

Serotyping
Rabbit antisera were produced against M. haemolytica reference strains which were kindly provided by the NVI, Ethiopia. The assay was conducted using a rapid plate agglutination test against rabbit serum and classified to their respective serotypes [27]. Biotypes of P. multocida were assigned based on their sugar fermentation profiles [28].

Electrophoresis
Amplified PCR products were electrophoresed in 1.5% agarose gels. PCR products (10 µl) were mixed with loading buffer (6×) and loaded into each separate

Data Analysis
Data were coded and stored in Excel and analyzed using statistical software (STATA 11). Descriptive statistics were used and statistical result was considered at p < 0.05.

Outbreak Investigation
Diseased animals showed high fever (>40.0˚C), coughing, anorexia, severe respiratory distress, marked depression, salivation, and absence of rales when the ventral lung auscultated. Infected animals appeared dull and respiratory grunts were observed in advanced cases. Cattle slaughtered at the abattoir were inspected for typical gross pathological lesions. The affected parts of the lung showed firm, friable, irregularity in shape, consolidation, and dark red coloration were frequently observed in pneumonic cases. In some advanced cases, pulmonary parenchymal consolidation and interstitial edema were evidenced.

Biochemical Characterization
Bacterial pathogens isolated from nasopharyngeal and pneumonic lung samples of cattle revealed the identification of M. haemolytica, P. multocida, B. trehalosi, and H. somni. Isolates were Gram-negative, coccobacilli, and pleomorphic (Table 3).

Isolation of Bacterial Pathogens
Bacteriological assay showed that 37 (

Discussion
BRD is a multiplex illness that causes a severe form of pneumonia in all age groups of cattle. It is caused by range of factors such as multiple bacterial and viral pathogens together with environmental stressors are responsible for the outbreak of this disease [29]. BRD infected animals are detected late or not detected    [31]. Therefore, outbreak monitoring, investigation, and identification of bacterial pathogens are significant to design effective controlling strategies. BRD outbreak investigation aids in defining the frequency of the major infectious pathogens. The clinical signs observed in the current study include coughing, depression, loss of appetite, respiratory distress, and elevated temperature (DART). These findings are consistent with previous studies [7] [8]. Pneumonic lung gross pathological lesion inspection showed irregularity in shape, dark red coloration, marbling, non-friable foci, or fibrinous pleuritis, pulmonary parenchymal consolidation, and interstitial edema in advanced cases were consistent with previous findings [7] [30] [32]. However, diagnosis of BRD infection based on clinical symptoms is difficult. Consequently, diagnosis has to be supported with the identification of the exact pathogens.
In the current outbreak investigation, bacteriological assay revealed higher prevalence of M. haemolytica 37 (22.29%) followed by P. multocida 25 [21]. Besides, primers targeting fragment of the kmt1 gene of P. multocida produce unique amplification product to all strains [12] [14]. The PCR result of M. haemolytica and P. multocida isolates using the rpt2 and kmt1 universal genes confirmed 37 (100%) and 25 (96.15%) of M. haemolytica and P. multocida isolates, respectively. PCR results of M. haemolytica and P. multocida using the universal primers rpt2 and kmt1 are in agreement with the previous findings in Ethiopia from pneumonic cattle and sheep [3] [12]. Besides, the PCR assay designed to detect P. multocida capsular type A and D strains using specific cap loci (hyaD-hyaC) and (dcbF) genes, respectively [13].

Conclusion
In conclusion, remarkable evidence was identified in the distribution of capsular type, serotypes, and virulence-associated genes of M. haemolytica and P. multocida. PCR assay indicated that M. haemolytica (A: 1) and P. multocida (A: 3) were the most prevalent isolates to cause BRD. Detection of virulence genes of M. haemolytica (ssa, sodA, FbpA, TbpA, and lktC) and P. multocida (toxA, FbpA, TbpA, and pmSLP) in most of the strains implies the pathogenic potential of both pathogens to cause disease outbreak. Hence, the current findings provide relevant information to understand the capsular types, serotypes, and virulence gene incidence associated with M. haemolytica and P. multocida. Therefore, continuous outbreak surveillance from wider areas of the country and molecular epidemiology are indispensable in designing efficient prevention strategies.
search. Great appreciation was forwarded to Veterinary clinics located in the study areas, animal owners, and abattoir staff for their cooperation during sample collection. The authors also thank the Department of Biotechnology, Koneru Lakshmaiah Education Foundation (KLEF) for supporting the study.

Data Availability
All data supporting the findings of this study can be obtained from the corresponding author upon formal request.

Ethical Consideration
Consent was first obtained from animal owners. Samples collection followed scientific protocols and animal handling employed with basic animal welfare ethics. Laboratory assay was executed following the standard bacteriological and molecular methods.

Conflicts of Interest
Authors declare that they have no conflict of interests.