Putative Equine Neorickettsiosis in a Mare from Southern Brazil

Abstract

Background: This report describes the occurrence of equine neorickettsiosis (EN) in the northern region of Paraná, southern Brazil. EN is a non-contagious infectious disease caused by the Gram-negative bacterium, Neorickettsia risticii. Equine neorickettsiosis was previously known as Potomac horse fever and monocytic ehrlichiosis. The disease occurs predominantly in the USA and Canada; data relative to EN in Brazil is scarce. The aim of this study was to report the first case of putative EN in the state of Paraná due to a combination of IHC and molecular testing.

Case: A 2-year-old Quarter Horse was referred to a Veterinary Hospital with episodes of abdominal discomfort, fever, anorexia, tachycardia, and tachypnea. The animal reportedly demonstrated episodes of blackened and fetid diarrhea after the ingestion of hay. A treatment was established upon arrival at the veterinary hospital, but the mare died after 12 hours of monitoring. An autopsy examination performed soon after death revealed severe hyperemia of the mucosa of the cecum and colon, with multifocal cecal erosions and ulcerations. The principal histological lesion observed was necrotizing enterocolitis. Additional significant histopathologic lesions included widespread lymphoid depletion affecting the spleen, tonsils, and lymph nodes. An IHC assay designed to identify the antigens of N. helminthoeca (NH) in formalin fixed paraffin embedded (FFPE) tissues, identified antigens of intralesional neorickettsial organisms within macrophages of the mucosa of the colon. Additionally, a PCR assay designed to amplify the 16S rRNA gene of Neorickettsia, amplified the desired amplicon, but sequencing was frustrating.

Discussion: A putative diagnosis of equine neorickettsiosis was established due to the combination of epidemiological evidence, pathologic findings, immunohistochemical identification of intralesional antigens of neorickettsial agents, and amplification of the 16S gene of Neorickettsia spp. by PCR. The gross and microscopic lesions described must be differentiated from lesions observed in equine salmonellosis and Clostridium difficile associated disease of horses. IHC was performed on FFPE intestinal tissue using an anti-NH hyperimmune serum as primary antibody due to high antigenic cross-reactivity between Neorickettsia spp. There was positive intracytoplasmic immunolabeling of macrophages in the mucosa of the large colon indicating the presence of neorickettsial antigens. The PCR amplified the desired amplicon from the colon fragment, but sequencing was frustrating due to poor quality product that resulted in an organism consistent with Methylobacterium spp., probably, a contaminant. EN show a seasonal and endemic tendency in USA, Uruguay and in southern Brazil, occurring in warmer months, as in the case reported. Evidence suggests that an ancient disease named “churrido” known for at least 100 years in southern Brazil and Uruguay is, in fact, equine neorickettsiosis and might have been introduced to North America from South America. Considering the epidemiological evidence and the worldwide distribution of NR, we suggest that EN is probably underdiagnosed in Brazil and that the etiology of equine enterocolitis should be more thoroughly investigated. Therefore, this report broadens the knowledge of EN in our region. In conclusion, considering the epidemiologic, pathologic, immunohistochemical and molecular evidence, and the specie-specificity of Neorickettsia spp., we report a putative case of EN in southern Brazil and the first report in Paraná state. IHC is a useful technique, less technically demanding and time consuming than bacterial culture and isolation, considered the gold standard; and can be used to diagnose EN when compatible pathologic evidence is present concomitantly.