Autochthonous Leishmania siamensis in Horse, Florida, USA

To the Editor: Leishmania siamensis, a recently described species, was identified as the cause of autochthonous visceral leishmaniasis in 2 men in southern Thailand (1,2). Cutaneous leishmaniasis has been reported in horses in Europe and South America. Lesions in horses are solitary or multiple nodules that are often ulcerated and most commonly occur on the head, pinnae, legs, and neck. Other clinical signs are usually absent. In South America, biochemical characterization has identified L. braziliensis in horses (3). Leishmaniasis has been reported in horses in Puerto Rico (4), and equine leishmaniasis has been described, but no reports have been published, in the United States. L. infantum has been reported in equine cutaneous leishmaniasis in Europe (5). A report from central Europe recently identified an organism with 98% nucleotide identity over the ITS (internal transcribed spacer) 1 region to L. siamensis as the cause of cutaneous leishmaniasis in 4 horses (6). L. siamensis was also identified in a case of cutaneous bovine leishmaniasis in Switzerland (7). 
 
In August 2011, a 10-year-old, 505-kg Morgan horse mare in Florida, USA, with no history of travel outside the eastern United States was evaluated at the University of Florida for an ulcerated mass in the left pinna. When, 6 months earlier, the owner had noticed the mass, it was ≈1 cm in diameter, firm, raised, and covered with hair. Three months later, the ear was unchanged, and the mare was successfully impregnated. Over the subsequent 2 months, the mass began to grow and ulcerate. At that time, veterinary consultation was obtained and a biopsy performed. Histologic study showed that the dermis was hyperplastic and diffusely infiltrated with neutrophils, macrophages, and lymphocytes (Technical Appendix Figure 1). Numerous intracytoplasmic protozoal organisms with a small nucleoid and smaller kinetoplast most consistent with Leishmania sp. were observed in macrophages. No treatment was pursued. After 45 days, the mare was seen at the University of Florida because of progression of the lesions. The mass on the internal aspect of the pinna was 6 cm × 3 cm and ulcerated, and 3 new firm 1 cm–diameter nodules were observed on the outer pinna of the same ear. Multiple soft, less-defined, 1 cm to 3 cm–diameter nodules were observed along both sides of the neck, shoulders, and withers. No other abnormalities were observed on physical examination or thoracic and abdominal ultrasound, and lymph nodes were not enlarged. Ultrasound confirmed a ≈90 day viable pregnancy. Complete blood count and plasma chemistry were within normal limits. 
 
Tissue aspirates were taken of the multiple ear lesions and of the nodules along the neck and shoulder. From the ulcerated lesion, marked mixed, predominantly neutrophilic inflammation was seen, and rare neutrophils and macrophages contained intracellular protozoal organisms consistent with Leishmania sp. amastigotes (Technical Appendix Figure 2). These organisms were 4–5 µm in diameter and round with pale basophilic cytoplasm. They had an eccentrically placed, basophilic, oval nucleus and a small, basophilic, rod-shaped kinetoplast oriented perpendicular to the long axis of the oval nucleus. No organisms were seen in any other aspirates. 
 
Fresh tissue was submitted for PCR, which has been determined suitable for detecting Old World leishmaniasis in dogs (8). Results were negative. Given the clear clinical, cytologic, and histologic evidence for cutaneous leishmaniasis, additional consensus PCR was performed as described (9), targeting the ITS1 region. Direct sequencing was performed by using the BigDye Terminator Kit (Applied Biosystems, Foster City, CA, USA) and analyzed on ABI 3130 automated DNA sequencers (Applied Biosystems) at the University of Florida Interdisciplinary Center for Biotechnology Research Sequencing Facilities (Gainesville, FL, USA). The resultant sequence was 310 bp after primers were edited out. Sequence alignment yielded a genotype with 99% identity to the first L. siamensis isolate (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF200012","term_id":"147907566","term_text":"EF200012"}}EF200012) and 100% identity to 2 more recently submitted sequences from human visceral leishmaniasis isolates from Thailand (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JQ001751","term_id":"367461079","term_text":"JQ001751"}}JQ001751 and {"type":"entrez-nucleotide","attrs":{"text":"JQ001752","term_id":"367461080","term_text":"JQ001752"}}JQ001752) (Technical Appendix). The sequence was submitted to GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"JQ617283","term_id":"386762987","term_text":"JQ617283"}}JQ617283). 
 
The mare delivered a stillborn foal at 350 days’ gestation. Histopathology did not reveal any infectious organisms in the fetal tissues; however, the chorioallantois showed moderate villous atrophy, which was presumed to be the cause of fetal death. One month after foaling, the mare’s cutaneous lesions were 90% resolved. 
 
Because the mare in this report was born in the United States and had never left the country, this case appears to be autochthonous. Mode of transmission is unknown. Phlebotomine sand flies found in Florida include Lutzomyia shannoni, Lu. cubensis, Lu. vexator, and Lu. cruciata. Lutzomyia sp. are competent vectors of Leishmania spp. in other areas of the world. However, the vector for reported cases of L. siamensis in other regions has not been identified. Although leishmaniasis is infrequently diagnosed in any species in Florida, models have shown that with climate change, the range of sand flies and accompanying leishmaniasis in North America is expected to expand substantially (10). This report raises many avenues for further investigation: the prevalence of leishmaniasis in horses in the United States, understanding of the life cycle and vectors, and the zoonotic potential of this Leishmania species. 
 
Technical Appendix: 
Multiple alignment with Fast Fourier Transform of Leishmania siamensis ITS1 sequences; histologic section and fine needle aspirate of ulcerated mass from a horse with cutaneous leishmaniasis. 
 
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only risk factor identifi ed among the persons reported here was older age. Unlike persons in other reports, persons in our report were all men, and 2 reported GI symptoms. The mechanism for UTI in these cases is unclear but could have included ascending and hematogeneous spread.
We calculated incubation periods for GI symptoms for 6 persons as the time between onset of GI symptoms and the May 14 wedding (5 persons) or last travel date (1 person). The incubation period was 5-7 days (average 5.5 days). The incubation period for UTI, which could be calculated for 2 persons, was an average of 25.5 days. Long incubation periods for Salmonella spp. infections have been reported (6)(7)(8)(9); reasons include exposure to a low dose of bacteria, specifi c populations (e.g., young children, child day care attendees), and method of food preparation (6)(7)(8)(9). The age of persons in our investigation did not affect the length of the incubation period. The amount of food eaten was not collected during the interview; however, most persons in our investigation reported eating a wide variety of foods, and 1 reported eating small portions. All food was prepared during the week before the wedding and served cold. This length of time and the potential for temperature abuse could have increased the infectious dose and decreased the incubation period (6). In addition, the 1 person with travel-related infection was not exposed to these food items. We found no literature on the incubation period for S. enterica ser. Agbeni. The reason for the long incubation period in this investigation is unclear and could be due to host-specifi c factors, the implicated serotype, or the food source.
The Feces from both diarrheal and surveillance specimens were collected from a cohort of children living in Mirpur (3). A total of 2,039 fecal samples were examined microscopically (0.9% saline smear) and/or by fecal culture for amebic trophozoites and cysts (4). One hundred forty-nine (7%) of the samples were positive by microscopy and/or culture for an Entamoeba parasite with both cysts and trophozoites that closely resembled those of E. histolytica, E. moshkovskii, and E. dispar.
DNA was extracted directly from fecal samples by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. DNA from positive fecal cultures was isolated by using the cetyl-trimethylammonium bromide extraction method (5). PCR was conducted to detect E. histolytica, E. dispar, and E. moshkovskii, all of which are morphologically indistinguishable by microscopy and are endemic to Bangladesh (Table) (6)(7)(8)(9). An antigen detection test (TechLab Inc., Blacksburg, VA, USA) was also used to identify fecal samples positive for E. histolytica.
Fecal samples (129) and cultures derived from fecal material (20) were tested by PCR. Forty-four fecal samples were positive for E. histolytica, 42 for E. dispar, and 7 for E. moshkovskii. PCR results for 48 samples were negative for all 3 parasites (mixed infections account for the total being >129); 5 cultures also were negative for all 3 parasites.
ENTAGEN-F and ENTAGEN-R primers, which exhibit a broad specifi city for the small subunit ribosomal RNA (SSU rRNA) gene sequences of Entamoeba, were used in PCR to amplify DNA fragments from 43 of the samples that were negative by PCR for the 3 Entamoeba species; amplifi cation conditions were adapted from Stensvold et al. (10). The amplifi ed DNA was separated by electrophoresis by using a 2% agarose gel. Bands of the size predicted for the Entamoeba spp. SSU rRNA gene amplicon were detected in 15 samples (online Technical Appendix Table, wwwnc.cdc.gov/ EID/pdfs/12-0122-Techapp.pdf). The PCR products were extracted by using the QIAquick Gel Extraction Kit (QIAGEN) and cloned by using the Zero Blunt TOPO Cloning Kit (Invitrogen, Carlsbad, CA, USA). The sequenced clones from 2 different isolates, 1 diarrheal and 1 surveillance specimen, were completely novel when compared with the SSU rRNA gene sequences from other organisms and did not match any previously sequenced Entamoeba species. These isolates represent a new species of Entamoeba (GenBank accession nos. JQ412861 and JQ412862), here named E. bangladeshi (online Technical Appendix) We examined the phylogenetic relationship between E. bangladeshi and other Entamoeba parasites by using maximum-likelihood analysis as implemented in MEGA 5 (online Technical Appendix Figure, panel  A). E. bangladeshi, although distinct, clearly grouped with the clade of Entamoeba infecting humans, including E. histolytica. E. bangladeshi, however, appeared more distantly related than the noninvasive E. dispar, but closer than E. moshkovskii, to E. histolytica.
To further characterize E. bangladeshi, we established it in xenic culture, and it displayed the ability to grow at 37°C and 25°C, a characteristic shared with E. moshkovskii and E. ecuadoriensis but that distinguishes it from E. histolytica and E. dispar. Cultured trophozoites were evaluated through light and transmission electron microscopy (online Technical Appendix Figure, panel B). By light microscopy, we detected no apparent differences between E. bangladeshi and E. histolytica. The physical resemblance between E. histolytica and E. bangladeshi is notable because direct microscopic examination of fecal samples is still used as a diagnostic tool in areas to which these species are endemic to detect E. histolytica parasites.  Our fi ndings add to the diversity of Entamoeba species found in humans. The incidence and effect of infection in infants by the newly recognized species E. bangladeshi await future epidemiologic studies.

Autochthonous
Leishmania siamensis in Horse, Florida, USA To the Editor: Leishmania siamensis, a recently described species, was identifi ed as the cause of autochthonous visceral leishmaniasis in 2 men in southern Thailand (1,2). Cutaneous leishmaniasis has been reported in horses in Europe and South America. Lesions in horses are solitary or multiple nodules that are often ulcerated and most commonly occur on the head, pinnae, legs, and neck. Other clinical signs are usually absent. In South America, biochemical characterization has identifi ed L. braziliensis in horses (3). Leishmaniasis has been reported in horses in Puerto Rico (4), and equine leishmaniasis has been described, but no reports have been published, in the United States. L. infantum has been reported in equine cutaneous leishmaniasis in Europe (5). A report from central Europe recently identifi ed an organism with 98% nucleotide identity over the ITS (internal transcribed spacer) 1 region to L. siamensis as the cause of cutaneous leishmaniasis in 4 horses (6). L. siamensis was also identifi ed in a case of cutaneous bovine leishmaniasis in Switzerland (7).
In August 2011, a 10-year-old, 505-kg Morgan horse mare in Florida, USA, with no history of travel outside the eastern United States was evaluated at the University of Florida for an ulcerated mass in the left pinna. When, 6 months earlier, the owner had noticed the mass, it was ≈1 cm in diameter, fi rm, raised, and covered with hair. Three months later, the ear was unchanged, and the mare was successfully impregnated. Over the subsequent 2 months, the mass began to grow and ulcerate. At that time, veterinary consultation was obtained and a biopsy performed. Histologic