MALDI TOF Mass Spectrometry and Molecular detection of Mannheimia haemolytica from sheep and goats in Holeta and Sebeta Town, Oromia Special Zone, Ethiopia

doi:10.21203/rs.3.rs-3119485/v1

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MALDI TOF Mass Spectrometry and Molecular detection of Mannheimia haemolytica from sheep and goats in Holeta and Sebeta Town, Oromia Special Zone, Ethiopia

https://doi.org/10.21203/rs.3.rs-3119485/v1

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Background:Mannhemia haemolytica is one of the most important bacteria among causative agent of pneumonic pasteurellosis in small ruminants throughout worldwide and it is also economically devastating pathogen in Ethiopia.

Methods: A cross sectional study was carried out from November 2021 to May 2022 with the aim to identify Mannheimia haemolytica from sheep and goat in Sebeta and Holeta town, Oromia special zone, Ethiopia. A total of 235 samples (213 nasal swabs and 22 whole blood) were collected. Sheep and goat with clinical signs suggestive of pneumonic pasteurollosis were purposively sampling. Bacterial identification was conducted using biochemical, Biolog, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI TOF MS) and Real time PCR detection. Moreover, antimicrobials susceptibility test was also conducted on the identified bacterial isolates using disc diffusion method.

Results: The result showed that from a total of 235 samples, only two nasal swab samples were positive for M. haemolytica (0.85%). The two isolates were confirmed by all the tests and similar result was obtained by; biochemical, Biolog, MALDI TOF MS and real time PCR. Up on antimicrobial susceptibility testing, the two isolates were resistant to Streptomycin, Erythromycin and Clindamycin whereas they were susceptible to Tetracycline, Chloramphenicol, Trimethoprim/sulfonamides and Penicillin. Generally, this study revealed that M. haemolytica is among the causative agent of pneumonic pasteurellosis in sheep and goat in the study area. Although, the other remaining bacteria responsible for the disease.

Conclusion: The research suggests that a combination of diagnostic methods such as MALDI TOF MS, Biolog, and real-time PCR should be used, as well as for a more in-depth investigation to identify the strain or serotype of M. haemolytica using advanced molecular sequencing and also analysis of the remaining causal agent from various species and locations in countries are significant to address the present vaccination and antibiotic resistance issues.

MALDI TOF MS

M. haemolytica

Real time PCR

Sebeta

Holeta

Sheep

Goat

Mannheimia haemolytica is gram-negative rods or coccobacilli that are non-motile, non-spore forming, facultative anaerobic, oxidase and catalase-positive, bipolar that are classified as belonging to the genus Mannheimia[1]. Mannheimia haemolytica is among causative agent of pneumonic pasteurellosis in sheep and goats. Infection by Mannheimia haemolytica is often associated with insufficient health services, poor management, and climate changes [2]. It is one most serious pathogen that cause significant economic loss due to morbidity and mortality[3]. M. haemolytica is an opportunistic pathogen that residing in the respiratory tract of healthy small ruminants. This pathogen identified by an acute infection with high fever, coughing, dyspnea, muco-purulent nasal discharge, anorexia and depression and can cause infection when the body's defenses are impaired by a number of stress[4]

P.haemolytica was first identified in 1932, while P.multocida was first discovered in 1878 from fowl cholera-infected birds. Louis Pasteur is the man whom Pasteurella named in his honor in 1880 [5]. P.haemolytica biotype A was allocated to a new genus Mannhaemia in 1999 [6]. The name Mannhaemia was given in tribute to the German scientist Walter Mannheim for his significant contributions in the recent taxonomy of the family Pasteurellaceae. This new genus now contains several species based on phenotypic and genomic characteristics [7]. P. haemolytica, formerly known as Mannhaeimia haemolytica, is divided into two biotypes, A and T, based on the fermentation of arabinose and trehalose. Trehalose-negative P.haemolytica classified into five new species M. haemolytica, M.glucosidal, M.ruminalis, M.granulomatis and M.varigena while Trehalose positive classified as four serotype of P.trehalosi. The 17 serotypes of M. haemolytica and P. trehalosi are identified based on extractable surface antigen. Biotype A, is divided into 13 serotypes (A1, A2, A5, A6, A7, A8, A9, A11, A12, A13, A14, A16, and A17) and remaining four T Serotype 3, 4, 10, and 15 are classified as P. trehalosi [8]. There are two forms of pasteurellosis in small ruminants, namely pneumonic and septicemic. The septicemic form is caused by B. trehalosi and the pneumonic form by M. haemolytica type A and P. multocida type A and D [9][10]. Pneumonic form is the most important respiratory disease affecting small ruminants and develops disease when the immune system of the animal is compromised by stress factors [11]

The Presumptive diagnosis of M.haemolytica was based on the presence of clinical symptoms, bacterial isolation, and identification using Biolog, MALDI TOF MS and real time PCR. Bacterial culture colonies produced by M.haemolytica are odorless, moist, smooth, grayish, form hemolysis on blood agar and ferment mannitol, glucose, maltose[12]. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry identification is a reliable alternative method for detecting protein, particularly ribosomal protein, which is abundant in M.haemolytica [13]. M.haemolytica identification using cultural, and biochemical characters is a time consuming and labor intensive process. However, molecular technologies, particularly polymerase chain reaction, allow for quick detection and identification of bacteria in mixed or pure cultures [14]. PCR approaches have been employed in nucleic acid detection of M. haemolytica for a long time to address some of the limitations of biochemical and serological methods while also improving sensitivity and speed. When compared to conventional PCR, real-time PCR is faster and may detect lower DNA quantities [15].

In most situations, M. haemolytica identified by culture and biochemical test but they cannot be confirmatory by MALDI TOF MS, Biolog and Real Time PCR. In Ethiopia M. haemolytica has been identified by[11] who reported (32.62% of M.haemolytica) from ovine pneumonic pasteurellosis case in Selected Areas of Amhara Region. However, there is a limited information that identified M. haemolytica in Sebeta and Holeta towns from sheep and goat using Biolog, MALDI Biotyper, Real time PCR and determine antimicrobial susceptibility.

2.1. Description of the Study area

The study was conducted from November 2021 to May 2022 in Sebeta and Holeta town of Oromia special zone, Ethiopia (Fig. 1). Sebeta town is located at 25 km south west from Addis Ababa at an altitude of 1800–3385m above sea level and at 8°55–8.917°N and 38°37–38.617°E, latitude and longitude respectively. It receives an average annual rainfall of 1073 ml and annual temperature that ranges from 11.3°C to 28°C. It has a total area of 102,758km²[16].

Holeta town is located in the central part of the country, 31 km west of Addis Ababa in Oromia Special Zone, Ethiopia. Surrounded between latitude 8°53′ to 9°14′ N and longitude 38°21′ to 38°36′ E. Holeta town has an area of 5550 hectares and 2449m above sea level. Annual mean temperatures are 25.9 and 7.2°C, respectively. The minimum and maximum annual rainfall of the area ranges from 834mm and 1300mm and with an average 1067mm [16]. Mixed farming where a main farming system in the area. Traditional housing and natural grazing pasture were the largest husbandry practice.

2.2. Study Population

The study populations were clinical suspected sheep and goats suggestive of pneumonic pasteurollosis or other respiratory problems that brought to veterinary clinics for treatment. Sheep and goat with clinical sign of nasal discharge, respiratory difficulty, fast respirations, salivation, and coughing with regardless of age and sex were considered for sampling.

2.3. Study design

Cross sectional study was carried out in Sebeta and Holeta town of the Oromia special zone from November 2021 to May 2022 with the aim of M.haemolytica identification. Nasal swab and whole blood samples were collected from clinically suspected sheep and goat. Clinical information of sheep and goats were collected including the name of the clinic, date of sampling, type of sample, age, sex, and animals’ identification number were recorded for each animal on the sample collection format.

2.4. Sampling method

Before the beginning of the study, primary data were obtained from veterinary clinics about the disease. Individual animals were selected purposively based on the suggestive clinical signs of respiratory infection of sheep and goat that brought to veterinary clinics. Study site were selected based on presence of veterinary clinic. Clinically suspected animals’ nasal swab and whole blood were sampled for identification of M. haemolytica.

2.5. Sample Size Determination

Purposive sampling techniques were used based on the presence of clinical signs of pneumonia suggestive of pneumonic pasteurellosis in sheep and goats brought to the respective veterinary clinics of Sebeta and Holeta towns.

2.6. Sample collection and transportation

Nasal swab samples were collected from Sebeta and Holeta veterinary clinic after disinfecting external part of the nose with 70% alcohol. Samples were collected from clinically pneumonic sheep and goat with respiratory signs such as an irregular breathing, coughing and nasal discharge [17]. Sterile cotton tipped swabs moistened in Stuart transport medium (Oxoid, England) and the swabs are carefully inserted into nostril and rotated against the wall of the nasal cavity. Then swab were placed in labeled sterile screw capped test tube that contains 5 ml of Stuart transport medium [14]. Also, whole blood samples were collected from Jugular vein of sheep and goat by aseptic procedure in anticoagulant green stopper vacutainer tube. Then, kept in ice box and transported to Animal Health Institute (AHI), Sebeta for bacteriological investigation.

2.7. Isolation, identification and molecular detection

2.7.1. Clinical Investigation

Clinical investigation was conducted at the veterinary clinics of Sebeta and Holeta to collect samples from sheep and goats that with difficulty breathing, depression, high fever, fast respirations, salivation, coughing, and nasal discharge.

2.7.2. Isolation and Biochemically methods

Nasal swabs were enriched in Brain heart infusion broth (CRITERION, USA) and incubated at 37˚c for 24 hours. Then, a loop full of broth cultures were streaked on to blood agar (TY MEDIA, India) supplemented with 7% sheep blood and incubated at 37ºC for 24 to 48 hours and the colony of M. haemolytica checked for β-hemolytic, white-grayish, medium-sized, round, and mucoid. Typical colonies were sub cultured on MacConkey agar (CRITERION, USA) and subsequently subcultured on BHIA for further testing. A single colony from BHIA was tested for Gram reaction, Geimsa stain, and pin point colony on Mac Conkey agar, Motility, oxidase, MALDI TOF MS, Biolog, and Real time PCR confirmation [18].

Gram's staining was used to figure out the size, shape, and arrangement of bacteria. Gram negative M.haemolytica isolates were small, rod or cocci bacilli, single or pairs chain, and pink hue [19] and Giemsa staining was used to reveal M.haemolytica's bipolar appearance of coccobacilli arranged in pairs with purple color [20].

Biochemical Tests

All of the retrieved isolates were identified using the techniques proposed by [21]. M.haemolytica was tested for oxidase test to detect cytochrome oxidase activity on the (OxiStrips; Hardy Diagnostics, Santa Maria, CA 93455) [21]. Catalase test to determine bacteria produce the enzyme catalase according to [22] and for motility test M.haemolytica suspicious isolates were stab inoculated using a straight wire down the center of motility medium. Organisms were not migrated away from the stab line and grew only along the stab line, leaving the surrounding medium reasonably clear. Indole, urease, TSI, Simmon’s citrate tests and tested for sugar fermentation such as arabinose, maltose, sucrose, glucose, lactose and trehalose [23].

2.7.3. MALDI TOF MS identification system

Direct sample preparation methods was performed by using young and pure singe colony sub cultured on BHI agar (CRITERION, USA) and incubated at 37°C for 24 hours. A single colony was deposited directly on an assigned position of an MSP 96 target plate, followed by drying at ambient temperature. The dried sample was overlaid with 1 µl of HCCA matrix solution (α-cyano-4-hydroxycinnamic acid, Bruker Daltonik GmbH, Lot number- 7020120107), followed by air-drying at ambient temperature for crystallizing. Then, samples were analyzed in an MALDI-Biotyper (Bruker Daltonik, Germany) and spectra were calibrated before analysis using a standard calibration mixture including peptides and proteins obtained from E.coli Bacterial test standard(BTS) (Bruker Daltonik, Lot number:- 7030220010)[24]. Then, interpreted M. haemolytica organism with score value and National Center for Biotechnology Information (NCBI). Isolated M. haemolytica was further tested by Real time PCR.

2.7.4. Biolog identification system

Biolog bacterial identification was performed by using sinlge colony cultured on BHI agar (CRITERION, USA) at 37°C for 24 they were sub cultured on Biolog Universal growth (BUG) media (BIOLOG, USA) incubated at 37^oC for 24hrs. A pure colony was selected from BUG and emulsified into‘inoculating fluid A’ (IF-A (Catalog No. 72401) for subsequent inoculation on to the GEN III Microplate (Cat. No 1030). Prepared inoculum was measured to a specified transmittance using a turbidometer at (90–98%). For each isolate, 100 µl of the bacterial cell suspension was inoculated into each well of the Microplate, using automatic multichannel pipette and incubated at 33°C for 22 hr [25]. Microplate was read in the Micro Station reader after 22 h and results interpreted by the Biolog identification data base system software (GEN III_2.7.1.40.15G).

2.7.5. Real Time PCR

Deoxyribose nucleic acid (DNA) extraction

The isolate inoculated in to the Brain-Heart Infusion (BHI) broth (CRITERION, USA) incubated overnight at 37°C were used to DNA extraction. The bacterial genomic DNA was extracted using QIAamp DNA Mini kit (QIAGEN, Germany) (Lot: 166034227) according to manufacturer’s guidelines. The extracted DNA samples were stored at -20°C until PCR amplification was performed. The primers and probes from the BactoReal® Kit were used to detect specific genes 16S rDNA of M. haemolytica in real-time PCR (ingenetix, GmbH, Arsenalstra 11, A-1030 Vienna, Austria). The amplification of M. haemolytica specific DNA was revealed by a probe-specific amplification curve (FAM channel) (BactoReal® Kit- M. haemolytica Manuals). In this study, primers targeting 16s rDNA gene of M. haemolytica were used, as they have been used in previous studies [26].

The PCR was carried out in final volume of 20 µl reaction mixture containing 1µl of each primer and CR-1 Assay Mix of M.haemolytica, 10µl of DNA Reaction Mix, 3µl PCR grade water, and 5µl extracted DNA from samples. A total volume of 15 µl of master mix was added into wells of micro plate and 5 µl of extracted DNA. For negative and positive controls, nuclease free water and positive Control-DNA were used from the BactoReal® kit, respectively. The micro plate was sealed tightly by optical adhesive film. PCR was conducted on AB Applied Biosystems (7500 Fast system) using cycling conditions. The PCR cycling conditions used to amplify 16s rDNA gene of M. haemolytica included an initial denaturation at 50⁰c for 2minutes and at 95°C for 20sec, followed by 45 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 1 min and the data was analyzed based on manual in the BactoReal® kit of M. haemolytica.

2.7.6. Antimicrobial sensitivity testing

Antimicrobial susceptibility testing of M. haemolytica isolates was performed according to [27]. The bacterial isolates were tested for antibiotic susceptibility test using the Kirby-Bauer disk diffusion method. Three to five isolated colonies were transferred to 5 ml of 0.85% sterile saline water and turbidity was measured using densitometer and adjusted to 0.5 standards McFarland. After measuring the turbidity, a sterile cotton swab was dipped into the suspension and then bacteria were swabbed uniformly over the surface of Mueller-Hinton agar plate (CRITERION, USA) by rotating 60° and allowed to stand for 3–5 minutes to observe any excess moisture from the medium. Antimicrobial discs (Oxoid, England) were applied to the media using a disc dispenser gently pressed with the point of forceps to ensure complete contact with the agar surface and then incubated at 37⁰C for 16–18 hrs. Zone of inhibition was measure by using a digital caliper. 10 antimicrobials were selected according to the national AMR surveillance strategic document of Ethiopia [28].

2.8. Data analysis

The information gathered was coded and then entered into a Microsoft Excel spreadsheet 2016. The frequency and percentages were analyzed using descriptive statistics.

3.1. Clinical investigation

Nasal discharge, fever, irregular breathing patterns, fast respirations, salivation, and coughing were all observed sign in the study animals (Fig. 2).

3.2. Laboratory analysis

Two M.haemolytica positive isolate from nasal swab were found a total of 235 animals (213 nasal swabs and 22 whole blood samples) with clinical manifestation of respiratory problems. Collected samples 154 and 81 were from Sebeta and Holeta town, respectively.

3.2.1. Isolation and biochemical test results

The putative colonies of two M. haemolytica positive isolates were identified as beta -hemolysis, white to gray, medium-sized, round, and mucoid colonies on blood agar and BHI agar growth, as well as lactose fermentation and pin point pink colonies on MacConkey agar (Fig. 3). Isolates phenotypically identified as M. hemolytica were gram-negative, cocci bacilli arranged in chains with an occasional tendency of bipolar on Giemsa staining (Fig. 4).

Biochemically, the isolates were positive for catalase bubbles formation, oxidase positive showing purple color, non-motile and negative for indole, urease, Simmon’s citrate tests, and yellow slant and yellow butt on TSI tests. (Fig .5). Sugar fermentation profile indicated that the isolates were able to metabolize different sugars such as arabinose, sucrose, glucose and lactose but not trehalose (Table 1).

Table 1

Summary of *M. haemolytica* biochemical test results
Biochemical	M. haemolytica
Haemolysis on Blood agar	+ (beta hemolysis)
MacConkey agar	Pink Pin point
Gram stain	G-ve (Pink color)
Giemsa stain	Bipolar
Oxidase	+ (Purple color)
Indole	-
Motility	Non motile
Glucose	+
Trehalose	-
Arabinose	+

3.2.2. MALDI Biotyper identification results

The two Mannheimia haemolytica presumptive isolates were confirmed by MALDI Biotyper from a total of 213 nasal swab samples tested by culture and biochemical tests (Fig. 6) and (Table 2)

Table 2

Isolates confirmed by MALDI TOF MS
Town	Typeof samples	Score value	Matched Pattern	NCBI Code
Sebeta	Nasal swab	2.05	Mannheimia haemolytica ISU 29772 ISUV	75985
Sebeta	Nasal swab	1.83	Mannheimia haemolytica DSM 10531T DSM	75985

3.2.3. Biolog identification results

Biolog confirmed phenotypically two Mannheimia haemolytica isolates that had been confirmed by MALDI TOF MS too (Fig. 7) and (Table 3).

Table 3

Isolates confirmed phenotypically by Biolog
Town	SPP	Age	Sex	Type of samples	ID %	species ID
Sebeta	Caprine	Young	M	Nasal swab	81	Mannheimia haemolytica
Sebeta	Ovine	Adult	M	Nasal swab	78	Mannheimia haemolytica

3.2.4. Real time PCR results

Out of 213 nasal swab samples, two bacterial isolates confirmed by MALDI Biotyper were found positive in real-time PCR for M.haemolytica specific 16S rDNA genes (Fig. 8) and (Table 4).

Table 4

Real time PCR detection of *M. haemolytica* and CT value
Kit used	Gene detected	Type of samples	Ct Value		Result
Kit used	Gene detected	Type of samples	IPC	Sample	Result
Bactorealkit@ Mannheimia haemolytica	16S rDNA gene of M. haemolytica	Nasal swab	33.29	9.82	Mannhemia haemolytica
		Nasal swab	33.17	9.71	Mannhemia haemolytica
		NTC	34.67	Undet	Negative
		Positive control	34.07	25.81	Positive

3.2.5. Antimicrobial susceptibility testing results

The antibiotic susceptibility testing of M. haemolytica isolates were subjected to a panel of 10 antimicrobials. The isolates' antimicrobial susceptibility pattern shown on (Table 5) and (Fig. 9).

Table 5

Antimicrobial sensitivity test results
Antimicrobial agent	Disk Potency (µ g)	Number of isolates
Antimicrobial agent	Disk Potency (µ g)	S	I	R
Amoxicillin/clavulanate	AMC-25 µ g	2	-	-
Ampicillin	AMP-10 µ g	2	-	-
Penicillin	P-10 units	2	-	-
Ceftriaxone	DA-30 µg	2	-	-
Tetracycline	TE-30 µg	2	-	-
Erythromycin	E-15 µg	-	-	2
Chloramphenicol	C-30 µg	2	-	-
Trimethoprim/ Sulfamethoxazole	SXT-25µg	2	-	-
Streptomycin	S-10 µg	-	-	2
Clindamycin	DA-10 µg	-	-	2
S = Susceptible, I = Intermediate, R = Resistance

Mannheimia haemolytica is one of the most economically significant etiological agents troubling sheep and goats globally, and it is the primary causal bacterium associated to pneumonic pasteurollosis [29]. In this study, M. haemolytica was identified from sheep k2and goats in Sebeta and Holeta town using culture, biochemical, and Biolog, MALDI TOF MS and Real time PCR detection. Clinical finding of pneumonic pasteurollosis caused by Mannheimia haemolytica agent reveals are nasal discharge, fever, respiratory distress, fast respirations, salivation, in appetence and coughing. These clinical manifestations resembled to those described by [30]. The identification of M. haemolytica with biochemical methods is often challenging in some conditions due to antibiotic treatment, vaccination, transportation and storages [31]. Of the 213 cultures two (2) nasal swabs were tentatively identified as M.haemolytica. This finding is lower than the findings of [32], [33], [34] whose reports were 2.22% in sheep and goat, 11.2% in sheep and 14.10% and 11.80% in sheep and goats respectively.

M. haemolytica in present study discovered on blood agar as, white to grey colonies, smooth round and β-type haemolysis and on MacConkey agar appeared as pink pin point colonies as described by [34]. The isolate of M.haemolytica was found to be Gram-negative, coccobacilli, non-motile, bipolar, non-spore-forming, catalase and oxidase positive. These findings were consistent with [35]. Biochemically isolates fermented mannitol, arabinose and glucose while negative for indole, citrate, urease, and, trehalose this finding in line with observations of (Legesse et al., 2018).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) identification method uses ribosomal proteins, which are commonly found in proteins. MALDI TOF MS is faster, reduced labor and reagent expenses for confirming M. haemolytica [36]. In present study two (2) M. haemolytica isolate were tentatively identified by biochemical and confirmed by MALDI TOF MS similar results were obtained by those two methods. This study supports the agreement between biochemical and MALDI TOF (Urban-chmiel et al., 2016) who state that degree of agreement between the two methods for identifying M. haemolytica was found 100%. In addition to biochemical and MALDI TOF MS similar results of M. haemolytica were found by Biolog in recent study. Association of this method consistent with those of [25] who used MALDI-TOF MS and Biolog GEN III. The effectiveness of these methods in identifying bacteria to species was found to be similar with[37]. Also, MALDI-TOF MS has the possiblity to replace these time-consuming [38].

Polymerase Chain Reaction is one of the most rapid and sensitive molecular tools for bacterial identification. PCR-based identifications rely on the amplification of nucleic acid[39]. The PCR results revealed that only two (2) M.haemolytica isolate were positive, which was consistent with the findings by [40]. The current detection rate is lower than previously study by [14],[41], [26] and [3] whose report 9.09%, 20.2%, 6% and 34.21% respectively. To our knowledge, this is the first study in Holeta and Sebeta town described by multi approaches such as biochemical, MALDI Biotyper, Biolog, and Real time PCR detection of M.haemolytica.

Several factors, including identification methods, misidentification, seasonal variation and veterinary service, are likely to be responsible for the disparity of the result [34]. Holeta and Sebeta towns are in the same agro ecology zone. However, result from Holeta town were the negative for M. haemolytica 0 (0%). Finding of this study is in agreement with [35] who found results of one hundred nasopharyngeal swabs of apparently healthy field goats (0.00%) were negative for M. haemolytica and stated that seasonal variation is one factor for the agent, in Baghdad, Iraq. Predisposing factors such as stress in the region's current climatic conditions and changing weather patterns may have led to stress formation in the form of natural incidences of pneumonic pasteurollosis caused by M.haemolytica [42]. They also coincide with [34], who discovered a higher prevalence of M. haemolytica in the spring and early summer.

Antimicrobial susceptibility testing of M.haemolytica is necessary to determine resistance development. Antibiotic resistance has been documented in M. haemolytica isolates [43]. The most susceptible antibiotic disc in our investigation were Amoxicillin/clavulanicacid,Chloramphenicol,Oxytetracycline,PenicillinG,Trimethoprim/sulfamethoxazole, Ceftriaxone, Tetracycline, and Ampicillin. The current findings contrasted with those of [44] who found 100% resistance to ampicillin, penicillin-G, 83.3% resistance to tetracycline and 58.3% to chloramphenicol, respectively. Furthermore, M. haemolytica isolates have been found to be extremely resistant to tetracycline according to [45]. This study was also in line with the findings of [46], who claimed that chloramphenicol is 66% effective and well tolerated against a broad range of bacteria. In addition [44], found that trimethoprim-sulfamethoxazole was 83.3% effective against M. haemolytica isolates.

Another obtained finding indicates that, M. haemolytica isolates were resistant to streptomycin, erythromycin, and clindamycin. This resistance may be linked to the widespread use of these drugs for disease prevention and treatment [47] This finding agrees with that of [48], who discovered that M. haemolytica isolates were 75% resistant to streptomycin. Furthermore, this outcome was consistent with a previous study from a Chinese veterinary clinic, in which the sick goat was given streptomycin on a regular basis, resulting in significant bacterial resistance. However, studies from Australia imply that streptomycin which are not available to food-producing animals, are responsible for the sensitivity.

M. haemolytica is one among the most significant etiological agents of pneumonic pasteurellosis in sheep and goats all over the world, which causes financial losses due to high mortality, delayed marketing and treatment costs. M. haemolytica pathogen exists in the upper respiratory tracts of small ruminants. It can infect and predispose in animals with compromised immune systems. In this study, M. haemolytica was identified from pneumonic pasteurellosis cases using biochemical, MALDI TOF MS, Biolog, and real-time PCR. Isolation and identification of M. haemolytica through biochemical test is more time-consuming. However, Biolog, MALDI TOF MS and Real time PCR has been more successful in the identification of the M. haemolytica since it requires less time and cost. Regarding to antimicrobial susceptibility test some isolates were susceptible such as: chloramphenicol, tetracycline, amoxicillin/clavulanic acid, respectively. However, others isolates were resistant to streptomycin, Clindamycin, and Erythromycin. The occurrence of M. haemolytica in the current investigation was found to be very low when compared to the previous finding. However, this big difference was due to specific diagnostic methods used with very specific protein detection, season of study, veterinary service and animal management systems.

Based on the above information, the following points were forwarded as recommendation:

The MALDI TOF MS, Biolog, and real-time PCR are proposed as preferable methods for rapid and easy analysis of M. haemolytica
Antibiotic usage should be regulated and guided at the national level.
Before treating the animals, an antimicrobial susceptibility test should be performed.
To determine the strain of M. haemolytica more research should be conducted utilizing sophisticated molecular characterization or DNA sequencing
It is important to isolate the M. haemolytica pathogen with the rest ones from different animal species and geographical areas by considering multivalent serotype vaccine development and antibiotic resistant.

Acknowledgments

Authors in this article would like to express specially thank to Dr. Debebe Ashenafi, Mr. Abebe Olani and Mr. Abde Aliy for their wise advice, constructive comments and supervisions on the Laboratory activities throughout the period of this research. His special gratitude also goes to Animal Health Institute (AHI) and Addis Ababa University College of Veterinary Medicine and Agriculture (CVMA) for supporting me and providing facilities to attend this program, as well as all AHI staff members, General Bacteriology, Molecular and Pathology Laboratory staff. The authors thanks to Sebeta and Holeta town animal health department staff in Vet clinic especially to Dr. Dejene Getachew, Dr. Bune Tarecha, Dr. Sidise Merga, and animal owners for allowing and providing assistance during the study period.

Authors’ contributions

A.A.U drafted the proposal and the manuscript, collected sample, laboratory processing and analyzed the data. A.O.B and A.A.M supervised the research work, sample analysis and edit writing. D.A.B advice authors, data analysis, interpretation and edited the manuscript.

Funding

There is no any funding for this study

Data availability

All data generated or analyzed during this study are included in this published

article [and its supplementary information files].

A total of 235 samples (213 nasal swabs and 22 whole blood) were collected.

Ethics approval and consent to participate

The study was reviewed and approved by the animal research ethics and review committee of Addis Ababa University College of veterinary medicine and agriculture (approval number: VM/ERC/30/03/14/2022). In the study town, permission was obtained from all the relevant authorities prior to data collection. The purpose of study was explained to the owners of the animals.Animal owner’s allowing animals for sampling and providing assistance during the study period.All the methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interest

The authors declare that they have no competing interests.

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MALDI TOF Mass Spectrometry and Molecular detection of Mannheimia haemolytica from sheep and goats in Holeta and Sebeta Town, Oromia Special Zone, Ethiopia

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Abstract

Figures

1. Background

2. Materials and Methods

2.1. Description of the Study area

2.2. Study Population

2.3. Study design

2.4. Sampling method

2.5. Sample Size Determination

2.6. Sample collection and transportation

2.7. Isolation, identification and molecular detection

2.7.1. Clinical Investigation

2.7.2. Isolation and Biochemically methods

2.7.3. MALDI TOF MS identification system

2.7.4. Biolog identification system

2.7.5. Real Time PCR

2.7.6. Antimicrobial sensitivity testing

2.8. Data analysis

3. Results

3.1. Clinical investigation

3.2. Laboratory analysis

3.2.1. Isolation and biochemical test results

3.2.2. MALDI Biotyper identification results

3.2.3. Biolog identification results

3.2.4. Real time PCR results

3.2.5. Antimicrobial susceptibility testing results

4. Discussion

5. Conclusion and Recommendations

Declarations

References

Additional Declarations

Supplementary Files

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