Molecular serotyping and antimicrobial susceptibility profiles of Pasteurella multocida isolated from cases of hemorrhagic septicemia in cattle from selected districts of Keffa and Bench Sheko zones, South West Ethiopia

Abstract

Background

Hemorrhagic septicemia is a highly fatal disease of cattle caused by the bacteria; Pasteurella multocida serotypes B and E in Asia and Africa respectively. Even though the capsular serotype E is considered to be the common cause of Hemorrhagic septicemia in Africa, there is not enough evidence that other serotypes are not involved. Furthermore, the serotypes currently circulating in South West Ethiopia have not been identified. This study was carried out to identify circulating capsular serotypes of Pasteurella multocida and assess its antimicrobial resistance on hemorrhagic Septicemic cattle through bacterial isolation, molecular identifications, and antimicrobial susceptibility tests in Keffa and Bench Sheko Zones of South West Ethiopia Peoples’ Regional State.

Results

The bacteriological analysis from 45 purposively collected nasopharyngeal swab samples of hemorrhagic Septicemic cattle revealed that 12 (26.7%) isolates were identified as Pasteurella multocida. Similarly, the molecular analysis of these isolates revealed all twelve (12) isolates were confirmed to be Pasteurella multocida. On further capsular typing, serotype B (n = 5, 41.6%) and E (n = 5, 41.6%) were the confirmed circulating strains in the area while two (n = 2, 16.6%) isolates formed non-specific bands. All the Pasteurella multocida isolates were susceptible to Gentamicin (100%), Chloramphenicol (100%), Oxytetracycline (91.7%), and Streptomycin (75%). However, all the isolates showed multidrug resistance (100%), to four antibiotics “Ampicillin, Clindamycin, Penicillin-G, and Vancomycin”.

Conclusions

Molecular analysis of the study isolates confirmed serotypes B and E as the etiology for Hemorrhagic septicemia in cattle in the study area. A multivalent vaccine comprising serotypes B and E is recommended to prevent outbreaks along with early treatment of suspected cases during the pyretic stage using antibiotics that are effective against the strains.

Introduction

Hemorrhagic septicemia (HS), is an acute, fatal, and septicemic disease of cattle and buffalo caused by the bacteria Pasteurella multocida (P. multocida) serotypes B: 2 (6:B), Asian type and E: 2 (6:E), African type [1, 2]. Hemorrhagic septicemia, classified as a List B disease by the Office International des Epizooties (OIE) [3], is a primary pasteurellosis with high mortality in endemic areas of Africa and Asia, which makes HS the most economically important bacterial disease of cattle and water buffalo in these countries [4, 5]. According to the report from [6] in India, during the past four decades, it has been found that HS accounted for 46–55% of all bovine deaths. In Africa in 2014, the Union Inter-African Bureau for Animal Resources (AU-IBAR) reported from fourteen countries that, HS with other pasteurellosis were responsible for a total of 391 outbreaks, 3916 cases, and 906 deaths in cattle [7].

Pasteurella multocida belongs to the family Pasteurellaceae under the genera, Pasteurella which is obligate and/or opportunistic commensals of vertebrate animals, colonizing mainly the mucosal surfaces of the upper respiratory tract [8, 9]. Pasteurella multocida is categorized into five capsule serotypes (A, B, D, E, and F) [10], and 16 somatic antigen serotypes (1–16) [11, 12]. Although P. multocida does not survive outside the host for long periods; moist soil, animal tissues, and pasture may prolong its survivability [6].

Pasteurella multocida infects a wide spectrum of domestic and wild animals including humans [13]. But there are no published reports of infections with P. multocida serotypes B:2 or E:2 in people, and human illnesses have not been associated with outbreaks of Hemorrhagic septicemia [14]. Infection to P. multocida is essentially triggered by stress factors such as extremely bad weather, poor management, overcrowding, transportation, or previous respiratory infections [15]. The disease can be transmitted by contact or ingestion of the pathogen from oral or nasal secretions of the infected animal. Once clinical signs appear, case fatality is nearly 100% and significant numbers of immune carriers are present in animal populations, particularly in endemic areas [16].

Hemorrhagic septicemia in cattle is characterized by fever, depression, multiple haemorrhages, signs of pneumonia, and subcutaneous swelling (pharyngeal region, ventral neck) [12, 17]. Hemorrhagic septicemia can be diagnosed by observing clinical signs, isolation and identification of the pathogen, using immunological tests [6] and molecular techniques [18, 19]. The clinical cases of HS are effectively treated with antibiotics at earlier stages of the disease. However, in recent times, shifts in the antibiotic sensitivity spectrum of P. multocida are evident [20] and there has been an increase in incidences of morbidity and mortality [21, 22]. The control and prevention of HS has centered on reducing the predisposing factors in combination with vaccination where herds are at high risk [6, 23].

A PCR assay built on primer pair targeting the KMT1 gene a transmembrane protein; is a rapid, and sensitive method used for identifying the field strains of P. multocida regardless of capsular serotypes [18]. A capsulated strain of P. multocida can be organized into one of the groups A, B, D, E, and F based on their distinct capsular polysaccharides. A serotype-specific primers amplifying a gene bcbD and ecbJ encoding glycosyl transferase for capsular group B and E respectively [10] were applicable for detecting the capsular serotypes of P. multocida that cause HS in cattle.

In Ethiopia, published studies regarding the status of HS are very rare. However, according to the Animal Production and Health Regulatory Directorate (APHRD) from major animal diseases reported from 2007 to 2011, HS is one of the most important animal diseases with 64,011 cases in 3,857 outbreaks [24]. The only study conducted in Ethiopia, Benishangul Gumuz indicated the prevalence of 13 (3.39%) using biochemical tests, and molecularly, the presence of B:2 was detected using conventional primers [19], while both serotypes B:2 and E: 2 were identified in Egypt [20].

Therefore, these inadequate reports regarding serotypes indicate that there is a lack of research in this area and has led to several unresolved questions. For example, besides the so-called African serotype or strain (E:2), to what extent this serotype or other new serotypes of P. multocida are involved in HS outbreaks in Africa including Ethiopia [25]. In addition, attempts have not been made enough to relate the field strains and reference vaccine strains for effective control of the disease [25]. By the same token, in Ethiopia, the vaccine is produced from the local serotype B:2 which is isolated from Southern Ethiopia specifically from Sidama (unpublished). However, its distribution and whether it is the dominant serotype is yet to be determined. Other strains, if any, circulating in the country are not well documented either. Until recently, despite the presence of HS outbreaks, cases and deaths which have been frequently reported to AU-IBAR, the serotype responsible for such cases in the country is not reported. Thus, the objectives of this study are designed; to identify the capsular serogroups of P. multocida causing Hemorrhagic septicemia and determine antimicrobial susceptibility profiles of the isolates in the study area.

Materials and methods

Description of the study area

The study was conducted in selected districts of Bench Sheko and Keffa Zones; South West Ethiopia Peoples’ Regional State (SWEPRS). The Bench Sheko Zone (BSZ) comprises of 6 districts; Debub Bench, Semien Bench, Guraferda, She Bench, Sheko and Maji and 2 city administrations Mizan Teferi and Mizan Aman Twon. Mizan Teferi Town, the capital of BSZ is 561 km away from Addis Ababa, in South West direction with a longitude of 35⁰ 3’ 5” to 35⁰ 5’ 8” West and latitude 7⁰ 2’ 1” to 7⁰ 4’ 9” North. Kaffa Zone is located in the South Western part of Ethiopia between 60 24’ to 7070’ N and 350 69’ to 36078’ E, about 460 km from Addis Ababa. Administratively, this zone comprises 12 districts; Chena, Decha, Gimbo, Gewata, Menjiwo, Bita, Gesha, Saylem, Shisho Ende, Guba, Cheta, Tello and one administrative town; Bonga Town. Mixed crop and livestock production is the major economic backbone of the areas and comprises a livestock population of 32,230 cattle, 23,091 sheep, 21,603 goats, 1,061 equines, and 46,896 Poultry [26]. This study comprises four selected districts; Aman and Wacha Maji from BSZ, and Chena and Shisho Ende from Keffa Zone [27] (See Fig. 1).

Fig. 1

Map of the study areas

Study design

A cross-sectional study was carried out from September 2021 to February 2022 for the isolation of P. multocida and molecular identification of the serotypes from the isolates obtained from cases of Hemorrhagic septicemia in cattle presented to veterinary clinics of Aman, Wacha Maji /Semien Bench/, Shisho Ende and Chena. Molecularly confirmed isolates were subjected to antimicrobial susceptibility tests to assess resistance or susceptibility for commonly used antibiotics in the selected districts and drugs which are effective for P. multocida were evaluated.

Sample size determination and sampling methods

Since the purposive sampling technique was used, samples suspected for only HS cases were collected from cattle presented at selected clinics. A total of 45 cattle were sampled from the study districts. Antibiotics for susceptibility tests were selected based on information obtained from clinicians about the common drugs used to treat HS cases and previous studies of antimicrobial susceptibility tests. Hence, a total of 12 antibiotics were used for susceptibility testing.

Four clinics in the districts; Aman, Wacha Maji /Semien Bench/, Shisho Ende, and Chena were selected purposively based on having repeated reports of HS cases, information obtained from clinicians and Mizan regional laboratory reports, accessibility of cold chain and transportation. All cattle presented at these clinics in the study period were examined for typical signs of HS; high fever, depression, coughing, hypersalivation, lacrimation, serous to mucopurulent nasal discharge, subcutaneous swelling on the pharyngeal region, ventral neck and brisket [17], were sampled for bacteria isolation and identification.

Study population

The study included clinically diseased indigenous cattle presented to Aman, Wacha Maji, Shisho Ende, and Chena veterinary clinics, regardless of breed, sex, age groups, body conditions, vaccination history, and management systems. Cattle having characteristic signs for HS; high fever, depression, coughing, hypersalivation, lacrimation, serous to mucopurulent nasal discharge, subcutaneous swelling on the pharyngeal region, ventral neck and brisket, and diarrhea were sampled and all information from the sampled animal were recorded. The age grouping /young, adult, and old/ of sampled cattle was done based on [19] after age information was obtained from the owners.

Ethical considerations

All the study activities in this work were conducted based on ethical standards after ethical approval from the Jigjiga University Research Ethics Review Committee Reg.No. (JJU-RERC 08/2021). Investigators were taking owners’ verbal consent and handling animals as the established animal welfare recommendations for the handling of animals in scientific research.

Inclusion and exclusion criteria of the study

All cattle with clinical signs of high fever, depression, coughing, hypersalivation, lacrimation, serous to mucopurulent nasal discharge, subcutaneous swelling on the pharyngeal region, ventral neck, and brisket and diarrhea, were included despite varying with sex, age, breed, body condition, vaccination history, and management system. Apparently healthy cattle were excluded from this study.

Study methods

Sample collection and transportation

In this study, a total of 45 nasopharyngeal swab samples were collected from cattle clinically suspected of HS cases. Swab samples were collected by disinfecting the external part of the nose with 70% alcohol, and a sterile cotton-tipped 20–25 cm long swab (Tamil Nadu, India) moistened with sterile brain heart infusion broth was directed via the ventral nasal meatus into the nasopharynx. Then the swabs were rolled gently on the mucosa surface and put back into the test tube and put back into the test tube containing a broth with transport enriched medium; brain heart infusion (BHI) broth, kept in an ice box as recommended by [28]. Then collected specimens were transported to the Microbiology Laboratory, Mizan Regional Veterinary Diagnostic Laboratory for bacteriological analysis. Simultaneously, the Sex, age, breed, management system, and vaccination history of the sampled animals were recorded.

Bacteriological analysis

Bacterial isolation

Bacterial isolation and identification of P. multocida were performed at Mizan-Aman Regional Veterinary Diagnostic Laboratory by using bacteriological standard procedures recommended by Quinn et al. [23]. Nasopharyngeal swab specimens were incubated on Brain heart infusion/BHI/ broth at 37 °C for 24 h. Then, a loopful of the broth cultures were taken and streaked on blood agar base supplemented with 5% sheep blood and incubated aerobically at 37 °C for 24 h.

Then, plates were examined for bacterial growth, and colonies were inspected for colony characteristics (color, shape, size, consistency, and odor). Consequently, from culture-positive plates P. multocida suspected; grayish white, small to medium-sized circular, none -hemolytic smooth, and mucoid colonies were picked and sub-cultured on a nutrient agar plate at 37 °C for 24 h. After that, sub-cultured pure colonies were subjected to gram staining to study staining reactions and cellular morphology under the light microscope at 100x magnifications. Those gram-negative, coccobacilli, or short rod bacteria were further sub-cultured, on both blood agar containing 5% sheep blood plate for examining hemolysis and MacConkey agar for identification. Those typical colony isolates passing through and satisfying P. multocida characteristics (non-hemolytic, smooth round and mucoid, gram-negative, coccobacillary rods) proceeded for primary biochemical tests; oxidase, catalase [23].

Biochemical tests

Furthermore, the characteristics of the bacterial isolates were determined using a variety of secondary biochemical tests. Suspected isolates were characterized for H₂S production and sugar fermentation on triple sugar iron agar (TSI) test, indole production, urea utilization, methyl red /MR/, Voges-Proskauer/VP/ and sugar fermentation tests (lactose, maltose, trehalose, and arabinose) conducted according to the procedure described by [23, 29]. All the data obtained were recorded and compared for confirmation of the isolates to P. multocida. Finally, isolates identified as P. multocida were preserved at room temperature using mineral oil after the slant was cultured on a test tube containing BHI agar for 24 h. and mineral oil was poured on cultured media possibly 1 cm above the end of the slant as recommended by [30].

Molecular identification

Molecular confirmation of presumptively identified P. multocida isolates were done at NVI (National Veterinary Institute) using species-specific primer sets. Further typing of the isolates were also done using capsular type-specific primers to identify the capsular type of the strain causing the disease in the study area.

Bacterial culture Preparation for PCR

Presumptive isolates of P. multocida were streaked on brain heart infusion (BHI) agar (Hi media, India) enriched with 10% horse serum. Plates were incubated at 37ºC for 24 h. aerobically. Loopful colonies of each of the pure isolates were suspended into sterile 1.5 ml Eppendorf tubes and washed twice in nuclease-free water. Cells were harvested on centrifuging at 8000 rpm for 1 min by removing the supernatant and 1 ml of cell suspension was transferred to the extraction step.

DNA extraction

Bacterial genomic DNA was extracted using DNeasy^® Blood and Tissue kit (QIAGEN GmbH, Germany) following the manufacturer’s instructions.

Polymerase chain reaction for detection of pasteurella multocida

Polymerase chain reaction (PCR) assay of P. multocida was performed using species-specific amplification gene primer (KMT1) (Eurofins Genomics, Austria) [18] as indicated in Table 1. Polymerase chain reaction amplification was carried out having a total of 20 µl reaction mixture containing 2 µl 5pmol/ µl of each primer (Fow and Rev), 10 µl IQ super mix (Bio-Rad), 3 µl RNase free water and 3 µl DNA template using thermal cycler (PCR max™ Alpha Cycler 2, AC296, UK) based on the following protocol. Initial denaturation at 95℃ for 5 min for 1 cycle, followed by 40 cycles of denaturation at 95℃ for 45 s, annealing at 55℃ for 1 min, extension at 72℃ for 1 min, and final extension of 1 cycle at 72℃ for 7 min [18]. Also, the PCR reaction has included both negative and positive (P. multocida capsular type B) controls taking all components of the reaction mixture except the DNA template.

Polymerase chain reaction for capsular typing of Pasteurella multocida

Further capsular types of the isolates were confirmed through a PCR assay using capsular antigen gene primers “capB and capE” (Eurofins Genomics, Austria) specific for each serotype B and E [10], respectively (Table 1). The PCR reaction was passed through a thermal cycler (PCR max™ Alpha Cycler 2, AC296, UK) having a total of 24 µl reaction mixture containing 2 µl 5pmol/ µl of each two primers, 10 µl IQ super mix (Bio-Rad), 3 µl RNase free water and 3 µl DNA template based on the following protocol. Initial denaturation of 1 cycle at 95℃ for 5 min, followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 1 min, and final extension of 1 cycle at 72℃ for 7 min [10]. Also, the PCR reaction has included both negative and positive controls taking all components of the reaction mixture except the DNA template. Finally, the amplified PCR products were preserved at -20℃ until proceeded to separation by agarose gel electrophoresis.

Agarose gel electrophoresis

PCR amplified products were analyzed by agarose gel electrophoresis prepared, 1.5% agarose gel in Tris/Acetate/EDTA (TAE) buffer. Four µl loading buffers containing loading dye were mixed with each PCR product and, a total of 10 µl ladder (100 bp), dye mixed amplicon, negative control, positive control for capsular type B-2 and ladder were loaded to wells of the gel correspondingly and run at 120 volts for 1 h. DNA bands were visualized under a gel documentation system (UVItec, UK) and interpreted as becoming in align with the ladder for each serotype [10]. Positive control for serotype E was not run to the gel as there was no capsular serotype E isolate in the laboratory.

Table 1 Oligonucleotide sequences /primers/ used in PCR assay

Antimicrobial susceptibility tests

All Pasteurella multocida isolates were tested for antimicrobial susceptibility against twelve types of antibiotics (Oxoid, UK), (Table 2) using the disc diffusion method as described by Hudzicki [31]. Isolates of 18–24 h. old colonies were suspended in 4 ml of sterile saline (0.9%) adjusted at a turbidity absorbance of (0.08–0.13) at the wavelength of 625 nm which is equivalent to a 0.5 McFarland standard using a Microprocessor Photocolorimeter (Vendor, India). Then the bacterial suspension was inoculated using a swab covering the entire surface of the Mueller Hinton agar (Hi media, India). Subsequently, antimicrobial discs were fixed individually on the surface of the inoculated agar plate and plates were allowed to incubate at 37℃ for 18–24 h. Finally, after its zone of inhibitions were measured results were recorded and interpreted based on Clinical and Laboratory Standard Institute breakpoints (CLSI) guideline for each antimicrobial, indicated on the recording sheet (Table 2) [32].

Table 2 Zone of interpretation chart for antimicrobials (inhibition zone diameter in mm) in antimicrobial susceptibility test

Data management and analysis

Data (districts, animal ages and sexes) obtained from the sampled animals were recorded and analyzed using SPSS sversion 20 software. Frequency measurements for field investigated data and compound bar chart for AST results were employed for presentation.

Results

Clinical investigation of hemorrhagic septicemia

In this study a total of 45 clinically sicked cattle were sampled in Veterinary clinics of; Aman (n = 6), Wacha Maji (n = 13), Chana (n = 16) and Shisho Ende (n = 10), found in the study districts. Sampled cattle exhibited signs of high fever, copious salivation and froth from the mouth, dullness with lethargy, a serous nasal discharge and edematous swellings in the submandibular region. Additionally, cattle showed inappetence and roughed hair coats (Fig. 2).

Based on this study, the highest number of P. multocida were identified from chena (n = 6, 37.5%) followed by Wacha Maji (n = 4, 30.8%) and Shisho Ende (n = 2, 20%), and none of the isolates were identified from Aman. Cattle with the age of less than or equal to 2 years (Young) and adults with the age greater than two and less than or equal to five years had a high rate of isolates (n = 7, 35.0%) and (n = 4, 23.5%) respectively. In addition, higher numbers of isolates were identified from male (n = 8, 28.6%) than female (n = 4, 23.5%) cattle (Table 3).

Table 3 Summary of P. multocida isolated sampled from HS cases in cattle in the study area

On the other hand, P. Multocida (n = 5, 41.7%) isolates of both capsular serotypes; B and E were identified, and those were isolated in all study districts except in Aman (Table 4).

Table 4 Frequency of isolated P. Multocida capsular serotypes in the study areas

Cultural and biochemical characteristics of the isolates

Out of 45 nasopharyngeal swab samples, 12 (26.7%) isolates had revealed cultural characteristics of P. multocida; non-hemolytic, grayish-white shiny, medium-sized circular and mucoid colonies on blood agar (Fig. 3). Surprisingly, isolates were growing and appreciated with small to medium-sized, circular and pale or opaque colorless colonies on MacConkey agar (Fig. 4). On Gram’s reaction, isolates were gram-negative and coccobacillary-short rods. The biochemical tests showed that isolates were positive for oxidase, catalase, and indole tests (Fig. 5, A, B, C) respectively. Conversely, they were negative for Voges-Proskauer /VP/, Methyl Red/MR/, and urease tests and were unable to ferment lactose, maltose, trehalose, and arabinose. Additionally, on triple sugar iron agar test /TSI/; yellow slant yellow butt without gas and H₂S production were the recorded features of all isolates (Fig. 5, D).

Fig. 2

Cattle showing clinical signs of HS

Fig. 3

Colonies of P. multocida grown on nutrient (A) and blood agar (B) (5% sheep blood)

Fig. 4

Colonies of P. multocida grown on MacConkey agar

Fig. 5

Some of the biochemical test results A: Oxidase and, B: Catalase, C; indole, D; TSI test for isolated P. multocida

Polymerase chain reaction (PCR) assay

On PCR assay, all presumptive isolates (n = 12, 100%), were conformed for P. multocida having an amplified band size of 460 bp (Fig. 6). On further capsular typing, five (41.7%) isolates (lane 2, 3, 5, 7 & 9) revealed P. multocida serotype B and five (41.7%) isolates (lane 1, 4, 6, 10 & 11) for P. multocida serotype E having a band size around 760 and 511 bp respectively. Whereas, 2 (16.7%) isolates (lanes 8 &12), were recorded for non-specific bands between serotypes B and E (Fig. 7).

Fig. 6

A PCR assay showing the amplified DNA fragments for P. multocida isolated from HS cases in cattle. key: Lane M: molecular markers, Lane 1–12: samples, Lane 13: negative control, Lane 14: positive control; P. multocida

Fig. 7

A PCR assay showing the amplified DNA fragments for P. multocida serotypes; B and E isolated from HS cases in cattle. key: Lane M: molecular markers, Lane 1–12: samples, Lane 13: negative control, Lane 14: positive control; capsular type B-2

Antimicrobial susceptibility tests

All molecularly confirmed isolates of P. multocida were subjected to in-vitro antimicrobial susceptibility tests (Fig. 8). Consequently, all P. multocida isolates were susceptible to Gentamicin (100%) and Chloramphenicol (100%), similarly, almost all isolates were susceptible to Oxytetracycline (91.7%) and Streptomycin (75%). On the contrary, all the isolates were multidrug-resistant (100%), to four antibiotic discs of Ampicillin (AMP), Clindamycin (DA), Penicillin-G (P), and Vancomycin (VA). Isolates showed intermediate resistance to Erythromycin (66.7%) and Kanamycin (50%) (Fig. 9).

Fig. 8

Antimicrobial Susceptibility tests on Mueller Hinton Agar plates for P. multocida isolated from the study area. Keys: 1: Trimethoprim-Sulfamethoxazole, 2: Vancomycin, 3: Chloramphenicol, 4: Erythromycin, 5: Clindamycin, 6: Streptomycin, 7: Oxytetracycline, 8: Tetracycline, 9: Gentamicin, 10: Ampicillin, 11: Kanamycin, 12: Penicillin-G

Fig. 9

Invitro antimicrobial susceptibility profiles of P. multocida isolates; N: number of isolates

Discussion

Pasteurella multocida is a pathogen causing a significant number of diseases in various domestic and wild animals and avian species. Among these, Hemorrhagic septicemia is one of the most important diseases in cattle [33]. Hence this study was intended for the identification of P. multocida serotypes causing HS in the study areas and their antimicrobial resistance pattern.

In the current study, P. multocida (n = 12) was isolated and identified in cattle suspected of HS cases in the Wacha Maji, Chena, and Shisho Ende districts. Bacteriological characterization of the isolates was held following the standards of microbiological investigation for the identification of Pasteurella species [23]. Similar characteristics of the isolates were reported by Bote et al. [19]. and Sugun et al. [25]. who identified P. multocida from HS cases in cattle in Benishangul Gumuz, Ethiopia, and Central Nigeria. However, in this study isolates were able to grow on MacConkey agar exhibiting pale or colorless opaque colonies is uncommon except for the recent finding of Desem et al. [34], where two P. multocida isolates were recovered forming pinkish colonies. In fact, similar records are found in old findings that, several strains of this bacteria were occasionally recovered on MacConkey agar [35,36,37].

In the current study, the overall isolation rate of P. multocida was 26.7% and higher than other records from Benishangul Gumuz at 3.39% [19], Central Nigeria at 10.3% [25] and 17.5% reported from Zimbabwe [33] at which the bacteria were recovered from outbreak cases of HS in cattle. Other similar findings were reported in 20% by [13] and 15.6% by [38] from nasal swab samples of pneumonic cattle in Ethiopia. However, the highest isolation rate of 84.6% was reported from HS cases of cattle in Egypt [20].

The bacterial isolation rate in this study was higher in the districts of Chena (n = 6, 50%) and Wacha Maji (n = 4, 33.3%), and no isolates were found in Aman those sharing similar agroecological zones. This may have been due to the exposure of cattle to stress factors like draft power use as Chena and Wacha Maji are rural areas where agricultural activities have been more practiced than Aman town district. This finding was in accordance with [39] in India, dairy cattle in rural areas (47.7%) were more susceptible to HS than in urban areas (23.6%). In addition, records from the vaccination history of cattle in Aman town district were regularly vaccinated for the HS vaccine. This possibly reduces the recovery rate of the bacteria as a supporting finding was reported by [40], vaccination reduces the bacterial isolation rate of Pasteurella multocida by lowering bacterial load and bacteremia in vaccinated animals compared to unvaccinated ones.

Isolates rate based on age, P. multocida in young cattle aged less than or equal to 2 years were 7 (35.0%), adults aged greater than 2 years and less than or equal to 5 years were 4 (23.5%) and old cattle age greater than 5 years were 1 (12.5%). This result indicates an increase in cattle age and a decrease in the isolated P. multocida although there were more significant differences in the percentages of sampled cattle in old age (17%) than adults (37%) and young (44.4%). Similarly, Bote et al. [19]. reported comparable findings of 7.96% and 2.23% in young and adults respectively. Similar findings suggested that young cattle between 6 and 24 months were more susceptible to the disease than old cattle [1, 41]. This may most probably be due to old animals being more resistant; as they acquired long-lasting [42] or solid immunity [43] from previous exposures of natural infection to HS and the vaccine respectively. Sex distribution of the isolates was higher in male 8 (28.6%) than in female cattle 4 (23.5%), which is in contrast to the report of Bote et al. [19]. 4.69% and 2.25% in females and males respectively.

In this study, species and capsular-specific PCR assays were done to investigate P. multocida and circulating serotypes causing HS in South West Ethiopia. Hence, all the isolates (n = 12, 26.7%) were confirmed to be P. multocida using primers targeting kmt1 gene, and this finding is in agreement with 13 (3.39%) isolates of Bote et al. [19], 18 (10.3%) of Sugun et al. [25], and 44 (84.6%) of Elsayed et al. [20], were identified from HS cases of cattle in Ethiopia, Nigeria, and Egypt respectively. As well, 3 isolates of P. multocida were identified from HS cases of cattle in Indonesia [44].

In the case of capsular typing, ten isolates were confirmed to be P. multocida capsular type B (n = 5, 41.7%) and E (n = 5, 41.7%), indicating that both serotypes of B and E were the principal cause of HS in cattle in the study area. This finding agreed with a recent study by Elsayed et al. [20], in Egypt that both capsular serotypes B and E were identified from HS cases in cattle in Africa.

However, only capsular stain B was reported as the cause of HS in the previous study by Bote et al. [19]. in Benishangul Gumuz Ethiopia. To the best of our knowledge, this is the first finding to show that capsular serotype E was identified as causing HS in Ethiopian cattle. Supporting findings of capsular type E were reported in Nigeria [25], and Zimbabwe [33] from HS cases in cattle. The other two isolates possessing non-specified bands between B and E indicate that they reacted for either of the two serotypes with different fragment lengths, most probably because isolates may have a gene variation or carried out nonspecific amplifications. In Ethiopia alum precipitated vaccine is used for HS produced from bacterial inactivation of P. multocida capsular serotype B which is isolated from Sidama, Ethiopia. The identified field strain, capsular serotype B of this study was matched with the vaccinal reference strain used in NVI for vaccine production for HS on the capsular serotypings.

The etiological treatment of HS is only effective with the parenteral administration of antibiotics (e.g., penicillin, ampicillin, tetracycline, chloramphenicol, streptomycin), at the early stages of the disease [45]. However, the misuse of antimicrobials is expected to be a significant factor in the expression of resistance encoding genes, thus increasing the emergence of antimicrobial-resistant isolates of P. multocida [46] and increases in the incidence of multidrug-resistant (MDR) this bacterium in recent years [47].

Based on the results of the current study on antimicrobial susceptibility tests, all the isolates of P. multocida (n = 12) were 100% susceptible to Chloramphenicol and Gentamicin, followed by Oxytetracycline (91.7%) and Streptomycin (75%). This was agreed with Cuevas et al. [48], who reported that cattle isolates of P. multocida were 100% susceptible to antibiotics of Gentamicin, Chloramphenicol, and Oxytetracycline from acute septicemic cases in Spain. Similar susceptibility finding was recorded by Sugun et al. [25]. chloramphenicol (100%), oxytetracycline (88.52%), streptomycin (83.61%), and Gharibi et al. [49]. oxytetracycline (100%) isolated from pneumonic cattle. On the contrary, P. multocida isolated from HS cases of cattle exhibited resistance to oxytetracycline, erythromycin, and chloramphenicol [20].

In addition, isolates showed susceptibility to Tetracycline (58.3%), Trimethoprim-Sulfamethoxazole (50%), and Kanamycin (50%). Comparable findings of isolate susceptibility of 56.2% and 75% were reported for Tetracycline and Kanamycin respectively isolated from HS cases in buffaloes [50]. Besides, P. multocida was 100% susceptible to Trimethoprim-Sulfamethoxazole isolated from HS cases in buffaloes [51], and from pneumonic cattle [52]. Erythromycin (66.7%) and Kanamycin (50%) were intermediate-reacted antibiotics, and this finding was agreed with the report of Sugun et al. [25], and Donahue and Olson [53] erythromycin (61.30%).

On the other hand, this investigation revealed that all the isolates were resistant (100%), to four antibiotic; Ampicillin, Clindamycin, Penicillin-G, and Vancomycin. This was in agreement with Penicillin-G (77.05%) and Vancomycin (100%) [25] and 70.0% in Penicillin-G and Vancomycin [54], but in contrast to Penicillin-G (72.2%) [48] and 70.0% susceptible in India [54]. Generally, in this study 38.9% of the isolates were developing resistance to Trimethoprim-Sulfamethoxazole (41.7%), Tetracycline (41.7%), and Erythromycin (33.3%), besides Ampicillin, Clindamycin, Penicillin-G, and Vancomycin; indicating that isolates were developing multidrug resistance. Similar findings of multidrug resistance on P. multocida were reported by Kumar et al. [22], Bote et al. [19]. and Jamali et al. [55].

Conclusion

In the current study, P. multocida was isolated and confirmed from nasopharyngeal swabs of hemorrhagic Septicemic cattle. The molecular detection using conventional PCR assays and capsular typing of the isolates revealed that the African serotype E was involved in the occurrence of the disease in addition to the speculated B serotype in the study area. This finding further affirmed that the identified capsular serotype B corresponds with the vaccine reference serotype B of NVI in terms of capsular differentiation. Isolates has shown variability in susceptibility for tested antimicrobials indicating that all isolates were susceptible to certain antimicrobials. Conversely, they were developing resistance for some other antimicrobials forming multidrug resistance (MDR). Based on this finding isolates were sensitive to Chloramphenicol, Gentamicin, Oxytetracycline, and Streptomycin and thus could be the respective drugs of choice. Confidently, the current finding provides useful information for further research on HS in different areas, evaluation of currently used vaccines, and the choice of drugs for the treatment of HS in Ethiopia. Since this study was conducted only in specific areas of Ethiopia coupled with the absence of further genome typing, like whole genome sequencing and analysis, makes it unable to clearly determine the actual molecular epidemiology of P. multocida capsular serogroups involved in causing HS in the country.