Study Areas
The study was conducted in selected districts of Borana pastoral zone (southern Ethiopia) and Arsi zone (central Ethiopia), which were selected purposively (Fig. 1). Borana zone is a typical pastoral zone featured by livestock production in dominantly semi-arid and arid climate. Arsi zone, on the other hand, is one of the typical highland areas in central Ethiopia where mixed crop-livestock production is practiced.
Three localities namely Yabello, Gomole, and Dire were selected from Borana pastoral zone. The study areas are located in the southern part of Ethiopia between 3°36’- 6°38’ North latitude and 3°43’- 39°30’ East longitude. Yabello, which is the center of the zone, is situated at 570 km from the capital, Addis Ababa. The study districts receive an annual average rainfall ranging from 300 mm to 700 mm. The annual mean temperature of the areas varies from 19 ºC to 24 ºC. Livestock production (pastoralism) is the dominant economic activity in the areas. The mean number of sheep and goats per household in the areas is 9.5 and 13.8. The incidence of disease and mortality in sheep and goats is known to be high in this zone. The reports of the Central Statistical Agency12 for instance showed that 88,689 sheep and 88,502 goats were diseased during 2020/21 of which 49,987 and 38,797, respectively died.
Arsi zone is located in Oromia Regional State at about 175 km away from the capital Addis Ababa. Two districts (Tiyo and D/Tijjo), which are found surrounding Asella town, the capital of the zone, were selected for this study. The districts have elevations ranging from 2400 to 3000 meters above sea level. The average annual rainfall of the districts is 1658 mm whereas the mean annual humidity ranges from 43–60%. The annual mean temperature of the area ranges from 10 to 22.6 ºC. The daily maximum temperature can reach up to 28 ºC and the minimum temperature is as low as 10 ºC. In both districts sheep and goat production play an important role in the livelihood of the farmers. This zone is also one of the zones in central Ethiopia where high incidence of disease and mortality has been recorded. During the 2020/21 survey of the Central Statistical Authority 151,693 sheep and 104,965 goats succumb to diseases and 79,635 and 46,709 of them died12.
Figure 1.
Study Animals And Sampling
The study population consists of sheep and goats owned by pastoral community in the southern lowlands of Ethiopia (Borana zone) and smallholder mixed crop-livestock farmers in the central highland (Arsi zone). Borana pastoral zone and Arsi zone were selected purposively due to their agro-ecologies, the presence of a high number of sheep and goats, and the higher incidence of diseases and mortality as shown above. The study districts were also selected purposively for the same reason. Empirical evidence from the field showed that there has been frequent occurrence of respiratory diseases suggestive of pneumonic pasteurellosis in sheep and goats in the selected districts. Those sheep and goats brought to each district veterinary clinic were examined for the clinical evidence of pneumonia and those which were observed with anorexia, coughing, dyspnea, lethargy, bilateral nasal discharge, and abnormal lung sounds were considered to have pneumonia. Those pneumonic sheep and goats were considered in this study for bacteriologic and molecular characterization. In addition, sera samples were collected from apparently healthy sheep and goats for identification of the serotypes of M. haemolytica and P. multocida circulating in the study areas.
Collection Of Nasal Swabs
Nasal swabs were collected from those sheep and goats clinically diagnosed with pneumonia. Collection of the swab samples was conducted after appropriately restraining the animals with the assistance of veterinary personnel. The external part of the nostril was disinfected with 70% ethanol and sterile cotton-tipped, 20–25 cm long swabs were inserted into the nasopharynx via the ventral nasal meatus, rolled gently and pulled out. After sampling, the tips of each nasal swab were placed into individual tubes containing Amies transport medium (Oxoid, UK) and immediately transported on ice to the Yabello and Asella Regional Veterinary Laboratories.
Isolation And Phenotypic Identification
The swabs were removed from the transport media and placed individually into brain heart infusion (BHI) broth (Oxoid, UK) and incubated aerobically at 37°C for 6–8 hours. A loop full of broth was streaked onto blood agar supplemented with 5% defibrinated sheep blood and MacConkey agar (Oxoid, UK) and incubated aerobically for 24 hours at 37°C. Growth of bacteria was monitored for Pasteurella/Mannheimia-like features such as colony color, motility, morphology after staining with Gram’s stain and haemolysis13,14. Those suspected colonies were sub-cultured onto nutrient agar and nutrient broth to perform primary (catalase and oxidase) and secondary biochemical tests as described by Quinn and colleague15. Presumptive identification was made on the basis of colony morphology, haemolysis, and Gram’s staining and biochemical tests. The biochemical characteristics examined include TSI, oxidase, indol, urease, citrate, catalase, and fermentation of glucose, lactose, sucrose, maltose, xylose and arabinose.
From the Phenotypically identified M. haemolytica and P. multocida, pure colonies were cultured onto slant nutrient agar in a screw cupped tube for 24 h at 37 ºC and covered with sterile paraffin and stored at -20°C until transported to Holota Institute of Biotechnology for molecular characterization.
Molecular Detection And Characterization
DNA extraction
For DNA extraction few colonies were taken from the pure cultures presumptively identified as M. haemolytica and P. multocida grown on blood agar for 24–48 hours and transferred into 1.5 mL Eppendorf tubes. The genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue Kit as per the manufacturer’s instructions (Qiagen, Germantown, MD, USA). The DNA purity was checked on 0.8% agarose gel electrophoresis and stored at − 20°C until used.
PCR assay for detection of virulence associated genes of M. haemolytica
Species specific primers targeting Rpt2 gene coding for methyltransferase (Forward: 5′ - GTT TGT AAG ATA TCC CAT TT- 3’ and Reverse: 5′- CGT TTT CCA CTT GCG TGA − 3’) were used in PCR assay as described in previous studies16. In brief, the PCR was carried out in a final volume of 25 µL of reaction mixture containing 10 µL of IQ Super mix (Bio Rad, USA) (DNA polymerase, dNTPs and buffer), 2 µL (5pM/µL) of each primer pairs, 3 µL of RNase free water and 4 µL of template DNA. The PCR reaction was run at an initial denaturation at 95°C for 3 min, followed by 35 cycles of each at 95°C for 1 min, annealing at 48°C for 1 min and extension at 72°C for 30s and a final extension cycle at 72°C for 5 min. One reaction tube without the DNA template and the other with DNA template from reference M. haemolytica isolate from National Veterinary Institute culture collection (MH-NVI) were included as negative and positive controls, respectively. The primers were expected to amplify 1022bp of the target gene.
PCR assay for detection of P. multocida
For detection of P. multocida conventional PCR was carried out using primers designated KMT1T7-Forward (5’- ATC-CGC-TAT-TTA-CCC-AGT-GG- 3’) and KMT1SP6-Reverse (5’- GCT-GTA-AAC-GAA-CTC-GCC-AC- 3’) amplifying 460bp of gene as described by Townsend and collegaue17. The PCR reaction was carried out in a final volume of 25µL containing 12.5 µL of master mix (Promega, USA), 1µL of primers (10pM/µL), 3µL of DNA template and 7.5µL of RNAase free water. The polymerase chain reaction was performed on 'Prime' PCR with an initial denaturation of 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 52.1°C for 1 min, extension at 72°C for 30 sec, with the final extension at 72°C for 7 min. The vaccine strain P. multocida obtained from National Veterinary Institute served as positive control and while one reaction tube without the DNA template was used as negative control.
Detection of the PCR products was done in 2% (w/v) agarose gel, prepared from 0.5X Tris borate EDTA buffer stained with Gelred. Each PCR product (5 µL) was mixed with 6X loading buffer and loaded into separate wells of the pre-prepared gel while 1 kb plus DNA molecular marker was loaded onto the first and last lane and run at 120 V for 60 min on electrophoresis apparatus (EC 2060, USA). The different band sizes of the PCR products were visualized under UV transilluminator and photographed in a gel documentation system (UVI TEC, UK).
Identification of Serotypes of M. haemolytica and P. multocida
About 5–10 mL of blood samples were collected aseptically from the jaguar vein of sheep and goats using a plain vacutainer tube and needle. The blood samples were transported to the nearby Regional Veterinary Laboratories where they were kept overnight at room temperature to allow clotting. The sera were then separated from the clot by centrifugation at 3000 g for 10 minutes and collected into labeled cryovials and stored at -20 ℃ until transported to National Veterinary Institute, Bishoftu, Ethiopia where serotyping was carried out. Serotypes of M. haemolytica and P. multocida were identified by using the indirect haemagglutination (IHA) test as described by Biberstein and colleague18,19. Each serum sample was serotyped using the reference serotypes available in National Veterinary Institute as described below.
Capsular Antigen Preparation
Briefly, the isolates of the P. multocida and M. haemolytica (PA, A1, A2, and A7) from NVI stock were allowed to grow on tryptose broth for 20 hours at 37 oC. Full bacterial growth was monitored and the culture was centrifuged at 2000 g for 20 minutes. The sediment was resuspended with an equal volume of Phosphate Buffered Saline (PBS) and the suspension was heated in water bath at 60 oC and again centrifuged at 4500 g for 20 minutes at 4 oC. The clear supernatant was harvested and used as an antigen20.
Sensitization Of The Antigen With Sheep Rbc
Blood was drawn from a jugular vein of a male sheep into a syringe containing Alsever’s solution and stored at 4 oC overnight and centrifuged at 2000 g for 10 minutes. The supernatant was discarded and 100 µL of RBC was added to 10 mL of the capsular antigens. This was followed by mixing of the RBC and capsular antigens with 50% glutaraldehyde. The mixture was homogenized by gentle agitation, incubated at 37 oC for 1 hour and centrifuged at 2000 g for 10 minutes from which the supernatant was discarded. The sediment was resuspended with an equal volume of PBS and washed two times. Finally 10 mL PBS was added to the sediment to make a 1% suspension for use.
Titration Of Antibodies
Ninety µL of PBS was dispensed into wells in the first raw of V-shaped microplate and 50 µL of PBS containing sensitized RBC was added to the rest of the wells including the negative and positive controls. Ten µL of sera were added to the wells in the first raw to get 1:10 dilution. After pipetting and mixing, 50 µL was transferred serially to the next wells and the last 50 µL was discarded. The plate was covered with a microplate, sealed and incubated at 37 oC with constant agitation for 1 hour. The IHAT antibody titres of all the samples were recorded in comparison with the positive and negative controls. Positive results were taken if the level of antibody titer was greater than 1:10.
Antimicrobial Susceptibility Tests
Antimicrobial susceptibility test was conducted on M. haemolytica isolates using the Kirby Bauer disk diffusion method21. The isolates were tested against six different antimicrobials with varied usage in veterinary practices in Ethiopia: extensively used (penicillin and tetracycline), commonly used (gentamycin and sulfamethoxazole) and rarely used (bacitracin and chloramphenicol). The interpretations were carried out according to CLSI22. Only six M. haemolytica isolates were tested against the selected antimicrobials due to the limited antimicrobial disc availability.