Phenotypic and Molecular Characterization of the Capsular Serotypes of Pasteurella multocida Isolated from Pneumonic Cases of Cattle in Ethiopia

The genus Pasteurella is a member of the Pasteurellaceae family, which includes a large and diverse group of gram-negative Gammaproteobacteria. Pasteurella species are widely prevalent and cause several economically important endemic and epizootic diseases in a wide range of animals worldwide (Harper et al., 2006; Wilson and Ho, 2013). Characteristically, the organism is small (0.2 µm up to 2.0 µm), rods/coccobacilli, capsulated, non-spore-forming, non-motile and bipolar in a stain. The bacterium is facultatively anaerobic, fermentative and grows best with media supplemented in serum or blood (Quinn et al., 2002). Among members of this species Pasteurella multocida (P. multocida), a commensal and opportunistic pathogen, that resides in the upper respiratory tracts of cattle (Dabo et al., 2007; Taylor et al., 2010). It is a common bacterial pathogen implicated with bovine respiratory disease (BRD), or “shipping fever”, non-septicemic pneumonia (Dabo et al., 2007).
       
Strains of P. multocida are classified into five capsular serogroups (A, B, D, E and F) according to Carter (1955) using indirect hemagglutination test and into 16 somatic or LPS serotypes by Heddleston gel diffusion precipitation assay (Heddleston et al., 1972). Polymerase chain reaction (PCR) typing has been applied as a rapid and sensitive molecular method for capsular genotyping using primers designed for species and type-specific detection that was unique to all P. multocida strains (Townsend et al., 1998). Moreover, a multiplex PCR assay was employed as an alternative technique for capsular typing with primers designed following the identification, sequence determination and analysis of the capsular biosynthetic loci of each capsular serogroup (Townsend et al., 2001). Serogroups were further classified into eight LPS genotypes (L1-L8) according to Harper et al., (2015).
       
Despite the extensive research conducted over several years on the respiratory disease of cattle, it continues to result in a great economic impact on the cattle industry (Taylor et al., 2010). Previous studies revealed the extent of respiratory disease problems in the country and losses due to mortality, morbidity and associated expenses are estimated to be higher in Ethiopia. In those studies,  P. multocida was isolated at species level as one of the major bacterial pathogens from the nasal, trans-tracheal swab and pneumonic lung samples using conventional bacteriological methods (Abera et al., 2014; Musteria et al., 2017; Gebremeskel et al., 2017). However, limited information is available regarding the capsular types and genotypes of P. multocida isolates circulating in the country. Hence, molecular advances are indispensable to understand the capsular Serogroups of P. multocida representing severe threats to the cattle population in Ethiopia. Therefore, the present study was designed to determine the biotypes and capsular serotypes of P. multocida associated with pneumonic cases of cattle in Ethiopia.

Study area
 
The study was conducted in selected areas of Ethiopia. Samples were collected from Asosa (10^o04' N, 34^o31' E), Bale-Robe (7^o7' N, 40^o0' E), Bishoftu (8^o45' N, 38^o59' E), Mekele (13^o29' N, 39^o28' E) and Yabelo (4^o53' N, 38^o5' E). The areas are located in different agro-ecological zones of highland and lowland areas from 550 to 2492 meters above sea level (Fig 1). Bacteriological and molecular assays were carried out at National Veterinary Institute (NVI), Ethiopia.
 

 
Sample size and sample collection
 
A cross-sectional study with a purposive sampling method was employed to collect samples from reported pneumonic cases of cattle in the study area. Nasopharyngeal swabs and pneumonic lung tissue samples were collected from veterinary clinics and abattoir, respectively. A total of 400 samples were collected from (n=170) calves and (n=230) adult cattle, during the study period from April 2018 to January 2019.
 
Nasopharyngeal swab sample
 
Clinical cases of respiratory infection were inspected and nasopharyngeal swab samples were collected from clinically sick calves and adult cattle using a sterile laryngeal swab. Briefly, the swab was directed via the ventral nasal meatus into the nasopharynx, rotated vigorously for 30 sec at the contralateral side. The swab was retracted and inserted into a sterile screw-capped test tube containing a transport medium of modified Cary-Blair Medium (Park Scientific, UK).
 
Pneumonic lung sample
 
An Abattoir survey was carried out on cattle slaughtered and lung with pneumonic cases were inspected for irregularity in shape, cranioventral reddening, marbling, non-friable foci, or fibrinous pleuritis. Approximately a 3×3 mm piece of lung tissue was taken aseptically from the edge of the lesion and placed in a sterile universal bottle. All samples were maintained in a cold chain and transported to the Research and Development laboratory of the NVI, Ethiopia.
 
Isolation and phenotypic characterization
 
P. multocida strains were isolated employing standard bacteriological assay. Briefly, pneumonic lung tissue samples were minced and suspended in 4 ml sterile physiological saline (pH 7.0±0.2) and centrifuged (3260 × g for 3 min at +4^oC) and the supernatant was discarded. The sediment was reconstituted with 100 µl sterile physiological saline. Ten µl of the 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^oC for 24-48 hrs aerobically. The cultural, morphological and biochemical assay was conducted to identify Pasteurella species according to standard procedure (Quinn et al., 2002).
 
Molecular characterization
 
DNA extraction
 
Genomic DNA was extracted using DNeasy® Blood and Tissue kit (QIAGEN GmbH, Germany) following the manufacturer’s instructions.
 
PCR detection of P. multocida
 
PCR assay for P. multocida was carried out using species-specific primers. All amplification and sequencing primers were synthesized by Eurofins Genomics, Austria. Primer for the identification of P. multocida species was based on a previous report (Townsend et al., 1998; Jonathan and Arora, 2012) and primers were depicted in (Table 1). PCR reaction mix (20 µl) consisted of 2 µl 5 pmol of each primer, 10 µl IQ supermix (Bio-Rad), 3 µl RNase free water and 3 µl DNA template. Amplification was carried out using a thermal cycler (PCRmax^™ Alpha Cycler 2, AC296, UK). Briefly, initial denaturation at 95oC for 5 min, followed by 35 cycles of denaturation at 95^oC for 1 min, annealing at 55^oC for 1 min, extension at 72^oC for 1:30 min and final extension at 72^oC for 7 min.
 

 
PCR for capsular typing
 
The capsular antigens of P. multocida were assayed in multiplex PCR using serogroup-specific primers targeting serogroups A, B, D and E as described previously (Townsend et al., 2001). The oligonucleotides used were described in (Table 1). PCR reaction mix was prepared in 40 µl final volume of 6 µl 5 pmol of each primer, 20 µl of IQ supermix, 2 µl RNase free water and 6 µl template DNA. Amplification was carried out at initial denaturation at 95oC for 5 min, followed by 35 cycles of denaturation at 95^oC for 1 min, annealing at 55^oC for 1 min, extension at 72^oC for 30 sec and final extension at 72^oC for 7 min.
 
Agarose gel electrophoresis of PCR products
 
Amplification of PCR product was carried out in 2% (w/v) agarose gels prepared in 1x Tris borate EDTA (TBE) electrophoresis buffer. 10 µl of each PCR product was mixed with 6x gel loading dye and loaded into separate wells of the gel. 10 µl of DNA ladder (100 bp or 1 kb plus, Fermentas) was added into the last lane. Gel electrophoresis was conducted at 120V for 60 min and PCR products were visualized under gel documentation system (UVI TEC, UK) stained with GelRed (Biotium, Inc).
 
Subspecies and biovar identification
 
Confirmed P. multocida isolates were assigned into subspecies based on sorbitol and dulcitol fermentation. Isolates were further classified into their respective biovars based on carbohydrate/sugar fermentation profiles including glucose, lactose, sorbitol, mannitol, trehalose, maltose, xylose and arabinose) and ornithine decarboxylase (ODC) (Kim et al., 2019).
 
Ethical statement
 
Requirement compliance for animal ethics approval from University and Institution was not required for this study. Due to this reason, samples were collected from animals without experimental intervention. Consent was first obtained from the animal owners before sampling.
 
Data analysis
 
Data collected during the study period were analyzed using STATA software version 11. Descriptive statistics were used for analysis and statistical analysis was considered at P<.05.

Pasteurella species are highly prevalent among animal populations and economically important pathogen that causes diseases in a wide range of hosts. P. multocida is opportunist, associated with acute and chronic infections that can lead to morbidity and mortality any time when lower airway defense mechanisms are compromised (Harper et al., 2006; Peek et al., 2018). The present study was conducted to identify and characterize the most prevalent capsular serotypes of P. multocida from pneumonic cases of cattle in Ethiopia using standard bacteriological and molecular methods. Clinically sick cattle exhibited coughing, high fever (>39.5.0^oC), depression, anorexia, nasal discharge, lacrimation, breathing difficulty and inappetence. Postmortem findings showed irregularity of lung shape, cranioventral reddening, marbling, non-friable foci and fibrinous pleuritis. However, suspected cases of P. multocida associated pneumonic samples were confirmed with the bacteriological assay. 
       
Phenotypic and molecular characterizations of P. multocida revealed an overall incidence of 61 (15.25%) P. multocida subspecies multocida. 21 (16.80%) and 8 (10.67%) isolates were recovered from calves and adult cattle nasopharyngeal swab samples, respectively. P. multocida incidence from pneumonic lungs of calves showed 14 (31.11%) and 18 (11.61%) from adult cattle (Table 2). P. multocida incidence was higher in calves compared to adult cattle (P<0.05). Previous reports of P. multocida incidence ranges from 3.4% to 39.2% in Ethiopia (Abera et al., 2014; Musteria et al., 2017; Gebremeskel et al., 2017). In the present study higher incidence was identified from Bishoftu (25.0%) followed by Bale-Robe (17.50%), Yabelo (16.25%), Mekele (8.75%) and Asosa (8.75%). While individual cases revealed an incidence rate as high as 44.4% from calves’ lung in Bishoftu.
 

       
Despite the distribution of P. multocida species in Ethiopia, it is not much known about the capsular types and serotypes of P. multocida strains associated with pneumonic cases in cattle. Presumptive P. multocida isolates were confirmed by PCR assay targeting KMT1 gene fragment of species-specific detection. Thus, the finding revealed ~460 bp size product in all P. multocida isolates (Fig 2). Although capsular typing confirmed the presence of hyaD-hyaC gene (~1044 bp) of serogroup-A in 56 (91.80%) isolates and dcbF gene (~657 bp) of serogroup-D in 5 (8.20%) isolates (Fig 3). The current finding is in accordance with Kong et al., (2019) who identified serogroup A as a predominant isolate from cattle. Similarly, Katsuda et al., (2013) reported the isolation of capsular type A (93.7%), D (6.3%) and Ewers et al., (2006) identified capsular type A (93.2%) and D (3.3%) from cattle.
 

 

       
P. multocida biovar assay revealed 48 (78.69%) P. multocida biovar type 3 as a predominant isolate and comprised 43 (89.58%) capsular type A isolates and 5 (10.42%) capsular type D. Moreover, P. multocida A:1, A:2 and A:12 were isolated at a rate of 7 (11.47%), 4 (6.56%) and 2 (3.28%), respectively (Table 3). P. multocida capsular type A is a principal bacterial respiratory pathogen in cattle, causing morbidity and mortality with consequent social and economic cost (Dabo et al., 2007). Hence, the findings of P. multocida capsular type A:3 as the most prevalent pathogen from identified cases calls for further microbiological investigation and assessment of the economic impact of this pathogen at the national level.
 

       
The present study revealed remarkable evidence in the distribution of P. multocida capsular types in Ethiopia. However, P. multocida is not the only pathogen-associated with pneumonic cases of cattle and other respiratory disease bacteria, Mycoplasma species and viruses might involve. Hence, subsequent monitoring on emerging pathogens and serotypes of P. multocida is essential for the development of an effective control strategy in the country. The current study depicts isolation of P. multocida A:3 strain. Thus, the finding highlights the molecular epidemiology of isolates in the study areas. Therefore, microbiological investigation covering a wider area of the country, based on the outbreak report, should be carried out to assess the serotypes and genotypes of P. multocida isolates circulating in the country. Moreover, the microbial diversity of pathogens associated with respiratory infection of cattle need to be compiled to make a definite conclusion in the improvement of the existing prevention and control strategy.

Although many pathogens are responsible to cause pneumonia in cattle are yet to be determined in Ethiopia. The present study revealed that P. multocida A:3 strain is the most common serotype isolated from pneumonic samples of cattle in the study areas. Therefore, the current finding suggests further comprehensive studies on P. multocida capsular types covering wider area of the country to understand its impact and assessment of microbial diversity of respiratory infection in cattle to design an effective control strategy.

The authors highly acknowledge the animal owners and abattoir staff for allowing sample collection. Great appreciation was forwarded to the Research and Development laboratory of the National Veterinary Institute of Ethiopia for providing the required facilities and support to conduct this research. The authors also thank the Department of Biotechnology, University of Koneru Lakshmaiah Education Foundation (KLEF) for supporting the study.