Prevalence and Molecular Detection of Pasteurella multocida, Mannheimia hemolytica, and Bibersteinia trehalosi in Sheep, Western Oromia, Ethiopia

A cross-sectional study conducted in Western Oromia, Ethiopia, between January and December 2022, revealed a 21.1% overall prevalence of pneumonic pasteurellosis-causing bacteria (*Pasteurella multocida*, *Mannheimia haemolytica*, and *Bibersteinia trehalosi*) in sheep, with significantly higher infection rates observed in clinically ill and younger animals, underscoring the need for targeted management and molecular surveillance.

The Gist

Imagine a vast, bustling farm in Ethiopia, home to millions of sheep. These animals are the lifeblood of the local economy, providing meat, skins, and income for families. But there's a silent thief lurking in the air, stealing the health of these flocks and the money of their owners. This thief is a group of invisible bacteria that cause a nasty lung infection called pneumonic pasteurellosis.

Think of these bacteria as uninvited guests who usually hang out quietly in the back of the sheep's nose and throat, like harmless neighbors. But when the sheep get stressed—maybe from bad weather, a long journey, or being crowded together—these "neighbors" turn into burglars. They break into the lungs, cause pneumonia, and can kill the sheep or make them too sick to sell.

This study is like a detective investigation carried out by scientists in the Western Oromia region of Ethiopia. Their mission was to catch these burglars, figure out exactly who they are, and see how often they show up in both healthy sheep and sick ones.

The Investigation: How They Caught the Culprits

The detectives (the researchers) went to three different neighborhoods (zones) and checked 384 sheep. They split the group into two teams:

1. The "Fit and Healthy" Team: Sheep that looked and acted normal.
2. The "Sick" Team: Sheep that were coughing, wheezing, and looked miserable.

Using special swabs (like Q-tips for the nose), they collected samples and took them to the lab. There, they played a game of "Guess Who" using two methods:

• The Microscope Game: They grew the bacteria on special plates to see what they looked like under a microscope.
• The DNA Fingerprint Game (PCR): This is the high-tech part. They looked for specific genetic "barcodes" (genes) that prove exactly which species of bacteria they were dealing with.

The Suspects: The Three "Burglars"

The study focused on three main bacterial species:

1. Pasteurella multocida (The Mastermind): This was the most common culprit found.
2. Mannheimia haemolytica (The Heavy Hitter): The second most common.
3. Bibersteinia trehalosi (The Wildcard): Found less often.

What the Clues Revealed

Here is what the detectives discovered, translated into plain English:

1. The "Sick" Sheep Were the Hotspot
It wasn't a surprise, but it was important to prove: The bacteria were found much more often in the sick sheep than the healthy ones.

• Analogy: If you find a lot of broken windows in a house, you know someone broke in. Similarly, finding these bacteria in sick sheep confirms they are the ones causing the trouble.
• The Big Finding: A sick sheep was 8 times more likely to have Pasteurella multocida than a healthy one. This proves this specific bacteria is a major villain in causing pneumonia.

2. The "Young" Sheep Are the Most Vulnerable
The study found that young sheep (lambs) were getting sick much more often than the adults.

• Analogy: Think of a young sheep's immune system like a newly built castle wall that hasn't been reinforced yet. It's easier for the bacterial "burglars" to climb over. Adult sheep have stronger, thicker walls (immunity) that keep the intruders out better.
• The Stat: Young sheep were more than twice as likely to be infected as adults.

3. Location and Gender Didn't Matter Much
The detectives checked if the bacteria were more common in specific towns or if they targeted male vs. female sheep.

• The Result: It didn't matter if the sheep were in the north or south of the study area, or if they were a boy or a girl. The risk was pretty much the same everywhere. The bacteria are everywhere, waiting for the right opportunity (stress) to strike.

4. The "DNA Fingerprint" Check
The scientists took the bacteria they found and ran a DNA test to be 100% sure of their identity.

• They found that the bacteria causing the pneumonia were indeed the specific "bad" versions (serotypes) known to attack sheep lungs.
• Interestingly, while they found a lot of bacteria in the sick sheep, the DNA test confirmed that the specific "burglar" genes were present in about 70% of the Mannheimia samples and 30% of the Pasteurella samples they tested. This tells us that while we can see the bacteria, we need high-tech tools to be absolutely sure of which ones are the dangerous ones.

Why This Matters (The Takeaway)

Imagine trying to fix a leaky roof. If you don't know where the leak is or what is causing it, you might just paint the ceiling, which won't help.

This study is like finding the exact spot of the leak.

• For Farmers: It tells them that young sheep and sick sheep need the most protection. They should be extra careful with these groups during stressful times (like moving them or bad weather).
• For Doctors/Vets: It shows that the current vaccines might need to be updated. Since Pasteurella multocida is the main villain, vaccines need to be perfect against that specific type, not just a generic one.
• For the Economy: By understanding exactly who the enemy is, farmers can stop losing money to dead or sick sheep.

The Final Verdict

The study concludes that pneumonic pasteurellosis is a persistent threat in Ethiopia. It's not just a random illness; it's a predictable pattern where stress turns harmless nose bacteria into deadly lung killers, especially for young sheep.

The scientists are saying: "We know who the bad guys are now. We need to use this knowledge to build better defenses (vaccines and management) so that the sheep stay healthy, and the farmers keep their income."

In short: The bacteria are everywhere, but they mostly attack the young and the stressed. By knowing this, we can stop the "burglars" before they break into the lungs.

Technical Summary

Title

Prevalence and Molecular Detection of Pasteurella multocida, Mannheimia hemolytica, and Bibersteinia trehalosi in Sheep, Western Oromia, Ethiopia

1. Problem Statement

Ethiopia possesses the largest livestock population in Africa, with small ruminants (sheep and goats) contributing significantly to national GDP, meat consumption, and foreign exchange earnings. However, productivity is severely constrained by infectious diseases, particularly pneumonic pasteurellosis. This disease causes high morbidity and mortality, especially in stressed animals, leading to substantial economic losses through mortality, reduced growth, and treatment costs.

Despite annual vaccination with monovalent inactivated P. multocida vaccines, high mortality rates persist. A critical gap exists in the epidemiological understanding of the specific pathogen strains and species circulating in Western Oromia. There is a lack of published scientific evidence regarding the prevalence and molecular characterization of Pasteurella multocida, Mannheimia haemolytica, and Bibersteinia trehalosi in this region, hindering the development of targeted, cost-effective control strategies.

2. Methodology

• Study Design: A cross-sectional study conducted between January and December 2022.
• Study Area: Three zones in Western Oromia: Horro Guduru Wollega, East Wollega, and West Shawa.
• Sampling:
  • Sample Size: 384 sheep (220 clinically healthy, 164 clinically ill).
  • Selection: Multi-stage sampling was used. Woredas (districts) and Kebeles were selected based on security and production potential. Animals were selected via simple random sampling (healthy) and purposive sampling (diseased animals presenting with respiratory distress at veterinary clinics).
  • Clinical Criteria: Diseased animals exhibited erratic breathing, grunting, coughing, dyspnea, lethargy, and muco-purulent nasal discharge.
• Sample Collection: Bilateral nasal swabs were collected, stored in peptone water, and transported to the laboratory under cold chain conditions.
• Laboratory Analysis:
  • Bacteriological Isolation: Samples were cultured on Tryptose Soy Broth, Blood Agar, and MacConkey agar. Isolates were identified based on colony morphology, hemolysis patterns, and biochemical tests (Catalase, Indole, Urease, TSI, MR-VP).
  • Molecular Detection (PCR):
    • DNA Extraction: Performed using the boiling method.
    • Target Genes:
      • M. haemolytica: PHSSA (serotype-specific antigen) and Rpt2 (methyl transferase).
      • P. multocida: CapA (capsular gene).
    • Controls: Positive controls (reference strains from NVI) and negative controls (no DNA template) were included.
  • Data Analysis: Descriptive statistics, Chi-square tests, and Logistic Regression (univariate and multivariable) were performed using R software to determine Odds Ratios (OR) and associations with risk factors.

3. Key Contributions

• First Molecular Survey in the Region: This study provides the first comprehensive data on the prevalence and molecular detection of these three specific pathogens in the Western Oromia zones of Ethiopia.
• Differentiation of Pathogens: It moves beyond general "pasteurellosis" diagnosis to distinguish between P. multocida, M. haemolytica, and B. trehalosi, which is crucial for vaccine matching and treatment protocols.
• Risk Factor Identification: The study quantitatively identifies age and health status as primary risk factors, offering data-driven insights for targeted interventions.
• Validation of Diagnostic Methods: It compares phenotypic (biochemical) identification with genotypic (PCR) confirmation, highlighting the necessity of molecular tools for accurate species-level identification.

4. Key Results

• Overall Prevalence: The total prevalence of Pasteurellaceae was 21.1% (81/384).
  • P. multocida was the most prevalent species, followed by M. haemolytica and B. trehalosi.
• Association with Health Status:
  • P. multocida: Significantly associated with diseased status. Prevalence was 18.9% in diseased sheep vs. 2.7% in healthy sheep (OR = 8.31, p < 0.001).
  • M. haemolytica: Higher in diseased sheep (9.1%) than healthy (5.0%), but the association was not statistically significant (p = 0.110).
  • B. trehalosi: Detected in 3.7% of diseased and 5.5% of healthy sheep (no significant association).
• Risk Factors:
  • Age: Young sheep (<1 year) had a significantly higher prevalence (26.9%) compared to adults (15.0%). Young animals were 2.41 times more likely to be infected (OR = 2.41, p = 0.001).
  • Sex: Females showed a slightly higher prevalence (23.5%) than males (18.3%), but this was not statistically significant in the final multivariable model (p = 0.213).
  • Location: No significant difference in prevalence was found across the different zones or districts (p > 0.05).
• Molecular Confirmation:
  • Of 26 M. haemolytica isolates identified biochemically, 71.4% (5/7 tested) were PCR positive for PHSSA/Rpt2.
  • Of 37 P. multocida isolates, 31.3% (5/16 tested) were PCR positive for CapA.
  • B. trehalosi could not be molecularly confirmed due to a lack of specific primers.

5. Significance and Recommendations

• Epidemiological Insight: The study confirms that pneumonic pasteurellosis remains a persistent threat in Western Oromia, with P. multocida being the primary driver of clinical disease.
• Targeted Interventions: The strong correlation between age (young sheep) and disease suggests that management and vaccination strategies should prioritize young stock.
• Vaccine Efficacy: The high prevalence of P. multocida despite existing vaccination programs suggests a potential mismatch between circulating serotypes and the current monovalent vaccine, or the involvement of other species (M. haemolytica) not covered by the vaccine.
• Future Directions:
  • Molecular characterization (serotyping and phylogenetics) is essential for all isolates to guide vaccine updates.
  • Further research is needed to elucidate the specific pathogenic roles of M. haemolytica and B. trehalosi in both healthy and diseased animals.
  • Development of multivalent vaccines covering the specific serotypes and species identified in this region is recommended.

Conclusion: The study establishes a baseline for the epidemiology of pasteurellosis in Western Oromia, highlighting the dominance of P. multocida and the critical need for species-specific molecular diagnostics to improve disease control and economic productivity in Ethiopian sheep farming.