Detection and phylogenetic relationship of Neorickettsia risticii in horses from Southern Rio Grande do Sul, Brazil.

Overview

• This research identified and genetically characterized the bacterium Neorickettsia risticii, which causes Potomac Horse Fever, in horses from southern Rio Grande do Sul, Brazil.
• The study conducted molecular detection, sequencing, and phylogenetic analysis of the pathogen, confirming its presence and providing new genetic information about the local strains.

Introduction and Background

• Neorickettsia risticii is an intracellular bacterium responsible for Potomac Horse Fever (PHF), a severe diarrheal disease in horses.
• PHF is characterized as a systemic, acute disease which can be fatal if untreated.
• This pathogen exists as an endosymbiont within digenetic trematodes (parasitic flatworms), with a lifecycle involving freshwater snails as intermediate hosts.
• The disease shows seasonal occurrence, mostly during warmer months, and is especially prevalent in horses living near wetlands.
• Previous studies detected the presence of N. risticii in southern Brazil through serological tests and PCR but lacked genetic sequencing and detailed phylogenetic classification.

Study Objectives

• To perform molecular diagnosis of N. risticii directly from horses displaying symptoms of PHF as well as from clinically healthy horses and freshwater snails from endemic farms in southern Brazil.
• To obtain genetic sequences of the bacterium to enable phylogenetic analysis and comparison with other known strains, especially from South America.
• To enrich the genetic database of N. risticii and support future epidemiological studies by clarifying the strain diversity and relationships.

Methods

• Samples were collected from symptomatic horses, clinically healthy horses, and Heleobia spp. snails (freshwater snails involved in the pathogen’s lifecycle).
• DNA was extracted from blood samples of horses and tissues of snails.
• Nested Polymerase Chain Reaction (nested PCR) was used to amplify a target region of the N. risticii genome to detect the presence of the pathogen’s DNA.
• Positive PCR products underwent sequencing to determine the exact genetic sequence of the pathogen.
• Phylogenetic analysis was conducted to compare these sequences with those of known N. risticii strains globally and specifically from South America.

Results

• Symptomatic horses showed successful amplification of a 529-base pair PCR product specific to N. risticii, confirming molecular diagnosis in these animals.
• Clinically healthy horses and the sampled freshwater snails did not show any amplification — suggesting no detected infection in these groups at the time of sampling.
• Sequencing of the PCR products provided the first genetic sequences of N. risticii from southern Brazil.
• Phylogenetic analysis revealed that these samples shared high genetic similarity with existing South American N. risticii sequences, indicating regional strain relatedness.

Conclusions and Significance

• The study confirmed the active circulation of N. risticii in symptomatic horses within southern Rio Grande do Sul, Brazil.
• The absence of detection in healthy horses and snails suggests that infection is likely linked to clinical disease and/or may require targeted sampling to detect in intermediate hosts.
• The newly obtained genetic sequences fill a crucial gap in the molecular epidemiology of PHF in Brazil, enabling better understanding of pathogen diversity and phylogenetic relationships.
• These findings support ongoing surveillance and control efforts for Potomac Horse Fever in endemic regions.
• Further research is encouraged to explore the full transmission cycle, including the role of snails, and to develop prevention and treatment strategies based on local pathogen strains.