Análisis filogenético de Neorickettsia risticii detectada en murciélagos de Argentina

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Abstract

La fiebre equina del Potomac (ehrlichiosis monocítica equina) es causada por Neorickettsia risticii (anteriormente Ehrlichia risticii), con casos clínicos notificados solamente en EEUU, Canadá, Brasil y Uruguay. Neorickettsia risticii es un endosimbionte de trematodos que presentan un complejo ciclo de vida con varios hospedadores como artrópodos, caracoles acuáticos y vertebrados insectívoros. Distintos estudios filogenéticos revelaron que se compone de diversas cepas en EEUU, pero no se cuenta con información disponible de Brasil y Uruguay. En nuestro país, N. risticii se detectó por primera vez en 2013 en murciélagos Tadarida brasiliensis procedentes de la Ciudad Autónoma de Buenos Aires (CABA). El objetivo de nuestro estudio fue caracterizar filogenéticamente a N. risticii detectada en CABA anteriormente. Se estudiaron tres muestras de ADN previamente obtenidas resultando positivas mediante una PCR anidada para un fragmento del ARNr 16S del género Neorickettsia. Las secuencias tuvieron un 100 % de similitud entre ellas y 99,5 a 99,7 % con distintos hallazgos de N. risticii en EEUU. Sólo una muestra pudo ser amplificada con la PCR anidada de un fragmento del gen groESL y el análisis filogenético reveló que N. risticii hallada en CABA se relaciona con hallazgos de la costa oeste de EEUU. Dado el potencial impacto en medicina veterinaria, la caracterización molecular de N. risticii circulante en Sudamérica es de suma importancia. Además, la circulación de este patógeno en nuestro país hace necesario realizar estudios adicionales para comprender la ecología y transmisión de N. risticii, y detectar la aparición de casos equinos.

Translated abstract

Potomac horse fever (equine monocytic ehrlichiosis) is caused by Neorickettsia risticii (formerly Ehrlichia risticii), with clinical cases reported only in the USA, Brazil and Uruguay. N. risticii is an endosymbiont of flukes with a complex life cycle utilizing various hosts such as aquatic snails and arthropods, and vertebrate insectivores. Different phylogenetic studies revealed that N. risticii consists of various strains in the USA, but no information is available from Brazil and Uruguay. In our country, N. risticii was first detected in 2013 in Tadarida brasiliensis bats from Buenos Aires city (CABA). The aim of our study was to characterize phylogenetically N. risticii detected in CABA. The three DNA samples previously obtained were positive by nested-PCR for a fragment of 16S rRNA of Neorickettsia genus and were sequenced, resulting in a 100% similarity among them and 99.5 to 99.7% with various strains of N. risticii from USA. Only one sample could be amplified by nested-PCR for a fragment of groESL gene. Phylogenetic analysis revealed that the current strain is related to strains of N. risticii along the west coast of the USA. Given the potential impact of veterinary medicine, molecular characterization of N. risticii circulating in South America is of great importance. In addition, the circulation of this pathogen in our country requires additional studies to understand the ecology and transmission of N. risticii, and detecting the occurrence of equine cases.

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Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever

Mingqun Lin, Chunbin Zhang, Kathryn Gibson … (2009)

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis.

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Neorickettsia risticii is vertically transmitted in the trematode Acanthatrium oregonense and horizontally transmitted to bats.

Chunbin Zhang, Yasuko Rikihisa, Carole Salaun-Martin … (2005)

Potomac horse fever is known to be transmitted through the ingestion of caddisflies parasitized with Neorickettsia (formerly Ehrlichia) risticii-infected metacercaria. However, the species of trematode involved and how N. risticii is maintained in nature are unknown. In this study, gravid trematodes were recovered from the intestines of 12 out of 15 Eptesicus fuscus big brown bats and eight out of nine Myotis lucifugus little brown bats from various sites in Pennsylvania, USA. Trematode specimens isolated from six E. fuscus bats contained N. risticii DNA. The trematode was identified as Acanthatrium oregonense. N. risticii was detected within individual trematode eggs by polymerase chain reaction as well as by immunofluorescence labelling with an anti-N. risticii antibody, indicating that N. risticii is vertically transmitted (from adult to egg) in A. oregonense. Furthermore, N. risticii DNA was detected in the blood, liver or spleen of 23 out of 53 E. fuscus and M. lucifugus bats, suggesting that N. risticii can also be transmitted horizontally from trematode to bat. These results indicate that A. oregonense is a natural reservoir and probably a vector of N. risticii.

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Equine monocytic Ehrlichiosis (Potomac horse fever) in horses in Uruguay and southern Brazil.

Alexson F. Dutra, L F Schuch, E Delucchi … (2001)

A disease named locally as churrío or churrido equino (i.e., equine scours) has occurred for at least 100 years in Uruguay and southern Brazil in farms along both shores of the Merín lake. This report describes cases of churrido equino and provides serologic, pathologic, and DNA-based evidence indicating that the disease is in fact equine monocytic ehrlichiosis (Potomac horse fever). Results of an epidemiological investigation conducted on an endemic farm are also presented. Clinical signs in 12 horses were fever, depression, diarrhea, dehydration, and sometimes colic and distal hind limb edema. Postmortem findings of 3 horses were of acute enterocolitis. Inclusion bodies containing ehrlichial organisms were found in the cytoplasm of macrophages of the large colon of 1 horse. Eleven of the 12 horses were serologically positive to Ehrlichia risticii (indirect fluorescent antibody assay) and, of 3 paired samples, 2 showed seroconversion. Ehrlichia risticii DNA was identified by a nested polymerase chain reaction in peripheral blood of an affected horse. A healthy horse inoculated with peripheral blood from an affected horse developed the disease and antibodies to E. risticii. The disease had a peak incidence in March (summer) and was statistically associated with a marshy ecosystem near the Merín lake, where large numbers of Pomacea spp. (Ampullariidae) snails were found. Incidence density was almost 8 times higher in nonnative horses than in native horses. It was concluded that the previous diarrheic disease of horses known in Uruguay and southern Brazil as churrido equino is equine monocytic ehrlichiosis.