Decoding Leishmania in equines: A comparative analysis of molecular targets.

Overview

• This study investigates the prevalence of Leishmania infection in equines (horses, mules, and donkeys) in Southern Punjab, Pakistan, using molecular techniques to identify the parasite and compare the effectiveness of two genetic markers.
• The research highlights the molecular detection of Leishmania, analyzes risk factors, and performs genetic characterization of the parasite strains found in the equine population.

Background and Importance

• Leishmania spp. are parasitic protozoa that cause leishmaniasis, affecting various animals and resulting in health problems and economic losses.
• In equines, Leishmania infection can cause complex clinical symptoms, including poor weight gain and damage to skin, liver, and spleen.
• There was limited information about Leishmania prevalence in equines specifically in Southern Punjab, Pakistan, creating a knowledge gap.
• Addressing this gap is important for veterinary health monitoring and disease control in the region’s animal populations.

Study Design and Methods

• Location: District Rahim Yar Khan, Southern Punjab, Pakistan.
• Sample: 384 equines (horses, mules, donkeys) were selected using a calculated sampling formula to ensure representative data.
• Sample Collection: Blood samples were drawn from animals for both microscopic examination and molecular analysis.
• Microscopic Detection:
  • Microhematocrit method was used to concentrate the parasite by examining the buffy coat layer after centrifugation.
  • Parasites were identified under a microscope.
• Molecular Detection:
  • DNA was extracted from blood samples using standardized commercial kits.
  • PCR amplification targeted two genetic markers: mitochondrial Cytochrome b and nuclear 18S rRNA genes.
  • These genes were chosen to compare sensitivity and confirm presence of Leishmania DNA.
• Data on potential risk factors such as sex, age, body condition, pregnancy status, deworming, and clinical symptoms were collected via questionnaires.
• Phylogenetic and sequence analyses were performed to classify the identified Leishmania strains.

Results

• Prevalence Rates:
  • Microscopic examination identified Leishmania in 2.1% of animals.
  • PCR targeting 18S rRNA gene detected infection in 7.3% of samples.
  • PCR targeting Cytochrome b gene detected infection in 8.8% of samples.
• Risk Factors:
  • Infection rates were significantly higher in female equines compared to males.
  • Older and underweight animals showed higher infection rates than younger and healthier individuals.
  • Non-significant trends indicated higher infection in:
    • Gestating females
    • Non-dewormed animals
    • Symptomatic animals
    • Animals in poor body condition
• Phylogenetic Analysis:
  • The sequences clustered with Leishmania (Leishmania) infantum species known from previous reports.
  • This suggests the circulating parasite strain in equines is similar to those infecting other hosts in different regions.
• Marker Sensitivity:
  • The nuclear 18S rRNA gene was found to be a more sensitive molecular marker than the mitochondrial Cytochrome b gene for detecting Leishmania infection in equine blood samples.

Conclusions and Implications

• This work provides the first molecular epidemiological evidence of Leishmania infection in equines from Southern Punjab, highlighting a previously underreported animal health issue.
• Molecular methods are more effective than microscopy in detecting the parasite, especially using the 18S rRNA gene as a sensitive target.
• Female, older, and underweight animals are at higher risk, indicating that health and demographic factors influence infection rates.
• Identification of Leishmania infantum helps in understanding the transmission dynamics and potential zoonotic risk.
• This research supports the need for continued surveillance and targeted control measures to manage Leishmania infections in equine populations.