First case of canine visceral leishmaniasis in the midwestern of Santa Catarina State, Brazil / Primeiros casos de leishmaniose visceral canina no Meio-oeste do Estado de Santa Catarina, Brasil

Canine visceral leishmaniasis (CVL) caused by Leishmania (Leishmania) infantum is transmitted by phlebotomine sandflies and a major zoonotic disease in Brazil. Due to the southward expansion of the disease within the country and the central role of dogs as urban reservoirs of the parasite, we have investigated the occurrence of CVL in two municipalities Erval Velho and Herval d'Oeste in the Midwest region of Santa Catarina state. Peripheral blood samples from 126 dogs were collected in both cities and tested for anti-L. infantum antibodies by indirect enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence reaction (IIF) and for the presence of parasite DNA by polymerase chain reaction (PCR) in peripheral blood. From examined dogs, 35.71% (45/126) were positive for at least one of the three tests and two (1.6%) were positive in all performed tests. Twelve dogs (9.5%) were positive for both ELISA and IIF, while 21 dogs were exclusively positive for ELISA (16.7%), and 15 (11.9%) for IIF. L. infantum k-DNA was detected by PCR in 9 out of 126 dogs (7.1%) and clinical symptoms compatible with CVL were observed for 6 dogs. Taken together, these results indicate the transmission of CVL in this region, highlighting the needs for epidemiological surveillance and implementation of control measures for CVL transmission in this region.

A Leishmaniose Visceral Canina (LVC) causada pela Leishmania (Leishmania) infantum e transmitida por flebotomíneos e é uma das principais zoonoses do Brasil que se encontra em expansão em estados da região sul do país, sendo os cães o principal reservatório urbano do parasito. O presente estudo investigou a ocorrência de LVC em dois municípios, Erval Velho e Herval d’Oeste localizados no meio-oeste de Santa Catarina. Para tanto, amostras de sangue periférico de 126 cães foram coletadas em ambas as cidades e submetidas à detecção de anticorpos anti-L. infantum por meio de testes de ELISA e imunofluorescência indireta (IFI), bem com a detecção de k-DNA pela reação em cadeia de polimerase (PCR). Além disso, também foram observados os sintomas clínicos e as condições ambientais associadas a esses animais. Dos cães examinados, 35,7% (45/126) foram positivos para pelo menos um dos três testes, dois cães (1,6%) foram positivos em todos os três testes, 12 cães (9,5%) foram positivos tanto no ELISA quanto na IFI, enquanto 21 cães (16,7%) foram positivos para ELISA e 15 (11,9%) para o IFI. A amplificação do k-DNA de L. infantum foi positiva em 9 dos 126 cães (7,1%). Entre os cães positivos seis apresentaram um ou mais sintomas clínicos correlacionados com a LVC. Esses resultados confirmaram a ocorrência de LVC na região e destacaram a importância do monitoramento e implementação de medidas de controle para a LVC nessa região.

First case of canine visceral leishmaniasis in the midwestern of Santa Catarina State, Brazil

Abstract Canine visceral leishmaniasis (CVL) caused by Leishmania (Leishmania) infantum is transmitted by phlebotomine sandflies and a major zoonotic disease in Brazil. Due to the southward expansion of the disease within the country and the central role of dogs as urban reservoirs of the parasite, we have investigated the occurrence of CVL in two municipalities Erval Velho and Herval dOeste in the Midwest region of Santa Catarina state. Peripheral blood samples from 126 dogs were collected in both cities and tested for anti-L. infantum antibodies by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence reaction (IIF) and for the presence of parasite DNA by polymerase chain reaction (PCR) in peripheral blood. From examined dogs, 35.71% (45/126) were positive for at least one of the three tests and two (1.6%) were positive in all performed tests. Twelve dogs (9.5%) were positive for both ELISA and IIF, while 21 dogs were exclusively positive for ELISA (16.7%), and 15 (11.9%) for IIF. L. infantum k-DNA was detected by PCR in 9 out of 126 dogs (7.1%) and clinical symptoms compatible with CVL were observed for 6 dogs. Taken together, these results indicate the transmission of CVL in this region, highlighting the needs for epidemiological surveillance and implementation of control measures for CVL transmission in this region.

Resumo A Leishmaniose Visceral Canina (LVC) causada pela Leishmania (Leishmania) infantum e transmitida por flebotomíneos e é uma das principais zoonoses do Brasil que se encontra em expansão em estados da região sul do país, sendo os cães o principal reservatório urbano do parasito. O presente estudo investigou a ocorrência de LVC em dois municípios, Erval Velho e Herval dOeste localizados no meio-oeste de Santa Catarina. Para tanto, amostras de sangue periférico de 126 cães foram coletadas em ambas as cidades e submetidas à detecção de anticorpos anti-L. infantum por meio de testes de ELISA e imunofluorescência indireta (IFI), bem com a detecção de k-DNA pela reação em cadeia de polimerase (PCR). Além disso, também foram observados os sintomas clínicos e as condições ambientais associadas a esses animais. Dos cães examinados, 35,7% (45/126) foram positivos para pelo menos um dos três testes, dois cães (1,6%) foram positivos em todos os três testes, 12 cães (9,5%) foram positivos tanto no ELISA quanto na IFI, enquanto 21 cães (16,7%) foram positivos para ELISA e 15 (11,9%) para o IFI. A amplificação do k-DNA de L. infantum foi positiva em 9 dos 126 cães (7,1%). Entre os cães positivos seis apresentaram um ou mais sintomas clínicos correlacionados com a LVC. Esses resultados confirmaram a ocorrência de LVC na região e destacaram a importância do monitoramento e implementação de medidas de controle para a LVC nessa região

First case of canine visceral leishmaniasis in the midwestern of Santa Catarina State, Brazil / Primeiros casos de leishmaniose visceral canina no Meio-oeste do Estado de Santa Catarina, Brasil

Canine visceral leishmaniasis (CVL) caused by Leishmania (Leishmania) infantum is transmitted by phlebotomine sandflies and a major zoonotic disease in Brazil. Due to the southward expansion of the disease within the country and the central role of dogs as urban reservoirs of the parasite, we have investigated the occurrence of CVL in two municipalities Erval Velho and Herval d'Oeste in the Midwest region of Santa Catarina state. Peripheral blood samples from 126 dogs were collected in both cities and tested for anti-L. infantum antibodies by indirect enzymelinked immunosorbent assay (ELISA) and indirect immunofluorescence reaction (IIF) and for the presence of parasite DNA by polymerase chain reaction (PCR) in peripheral blood. From examined dogs, 35.71% (45/126) were positive for at least one of the three tests and two (1.6%) were positive in all performed tests. Twelve dogs (9.5%) were positive for both ELISA and IIF, while 21 dogs were exclusively positive for ELISA (16.7%), and 15 (11.9%) for IIF. L. infantum k-DNA was detected by PCR in 9 out of 126 dogs (7.1%) and clinical symptoms compatible with CVL were observed for 6 dogs. Taken together, these results indicate the transmission of CVL in this region, highlighting the needs for epidemiological surveillance and implementation of control measures for CVL transmission in this region.

A Leishmaniose Visceral Canina (LVC) causada pela Leishmania (Leishmania) infantum e transmitida por flebotomíneos e é uma das principais zoonoses do Brasil que se encontra em expansão em estados da região sul do país, sendo os cães o principal reservatório urbano do parasito. O presente estudo investigou a ocorrência de LVC em dois municípios, Erval Velho e Herval d'Oeste localizados no meio-oeste de Santa Catarina. Para tanto, amostras de sangue periférico de 126 cães foram coletadas em ambas as cidades e submetidas à detecção de anticorpos anti-L. infantum por meio de testes de ELISA e imunofluorescência indireta (IFI), bem com a detecção de k-DNA pela reação em cadeia de polimerase (PCR). Além disso, também foram observados os sintomas clínicos e as condições ambientais associadas a esses animais. Dos cães examinados, 35,7% (45/126) foram positivos para pelo menos um dos três testes, dois cães (1,6%) foram positivos em todos os três testes, 12 cães (9,5%) foram positivos tanto no ELISA quanto na IFI, enquanto 21 cães (16,7%) foram positivos para ELISA e 15 (11,9%) para o IFI. A amplificação do k-DNA de L. infantum foi positiva em 9 dos 126 cães (7,1%). Entre os cães positivos seis apresentaram um ou mais sintomas clínicos correlacionados com a LVC. Esses resultados confirmaram a ocorrência de LVC na região e destacaram a importância do monitoramento e implementação de medidas de controle para a LVC nessa região.

First case of canine visceral leishmaniasis in the midwestern of Santa Catarina State, Brazil.

Canine visceral leishmaniasis (CVL) caused by Leishmania (Leishmania) infantum is transmitted by phlebotomine sandflies and a major zoonotic disease in Brazil. Due to the southward expansion of the disease within the country and the central role of dogs as urban reservoirs of the parasite, we have investigated the occurrence of CVL in two municipalities Erval Velho and Herval d'Oeste in the Midwest region of Santa Catarina state. Peripheral blood samples from 126 dogs were collected in both cities and tested for anti-L. infantum antibodies by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence reaction (IIF) and for the presence of parasite DNA by polymerase chain reaction (PCR) in peripheral blood. From examined dogs, 35.71% (45/126) were positive for at least one of the three tests and two (1.6%) were positive in all performed tests. Twelve dogs (9.5%) were positive for both ELISA and IIF, while 21 dogs were exclusively positive for ELISA (16.7%), and 15 (11.9%) for IIF. L. infantum k-DNA was detected by PCR in 9 out of 126 dogs (7.1%) and clinical symptoms compatible with CVL were observed for 6 dogs. Taken together, these results indicate the transmission of CVL in this region, highlighting the needs for epidemiological surveillance and implementation of control measures for CVL transmission in this region.

Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation.

Calcineurin B, the regulatory subunit of calcineurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leishmania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non-virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide polymorphism (314A>C) were obtained in spite of a single-copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79% of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsimony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth.

Internal transcribed spacers (ITS) of Trypanosoma rangeli ribosomal DNA (rDNA): a useful marker for inter-specific differentiation.

The internal transcribed spacers (ITS) flanking the 5.8S subunit of the ribosomal RNA genes (rDNA) of Trypanosoma rangeli strains isolated from distinct geographical regions and hosts were studied. The results revealed the sequence variability of the ITS spacers showing the presence of microsatellite repeats and single nucleotide polymorphisms (SNP), which were also observed within the 5.8S rDNA sequence. ITS-2 spacer was the most phylogenetically informative region due the presence of a higher number of parsimonious sites in both inter- and intra-specific analysis. Sequence analysis of both ITS spacers plus the 5.8S rDNA of T. rangeli strains allowed a clear inter-specific differentiation from Trypanosoma cruzi strains representative of the parasite zymodemes.

Detection and characterization of rabies virus in Southern Brazil by PCR amplification and sequencing of the nucleoprotein gene.

Due to the medical and socio-economical importance of both human and animal rabies infection, several studies have suggested the use of molecular techniques such as RT-PCR and DNA sequencing for diagnosis and phylogenetic studies of the rabies virus. Considering the conservancy of the nucleoprotein (N) gene of the virus, we herein describe a RT-PCR assay for rabies diagnosis and characterization. A total of 75 samples obtained from a variety of animal species in the state of Santa Catarina (SC), Southern Brazil, were comparatively studied by fluorescence antibody test (FAT), mouse inoculation test (MIT), cell infection assay and RT-PCR, which revealed itself to be as sensitive as FAT and MIT and less time-consuming than MIT. Direct sequencing of the 5' end of the N gene allowed the clustering of the SC samples with samples from the vampire bat-related or sylvatic cycle through comparative sequence analysis.

Interictal serum S100B levels in chronic neurocysticercosis and idiopathic epilepsy.

OBJECTIVE: To assess whether serum S100B levels could reflect a glial response in patients with epilepsy secondary to neurocysticercosis (NCC) and with idiopathic epilepsy. SUBJECTS AND METHODS: Serum S100B levels were measured using an immunoluminometric assay in 20 patients with focal epilepsy related to chronic NCC (NCC group), and 19 patients with focal epilepsy (EPI group), matched by epidemiological and clinical data. Epileptic patients were compared with 20 healthy controls (CON group) matched by age and sex. RESULTS: No difference was observed in S100B levels among NCC, EPI and CON groups (P>0.39). Serum S100B levels were not affected by antiepileptic drugs, frequency and type of seizures. Preliminarily, significantly higher levels of S100B were observed in patients with bilateral electroencephalographic (EEG) findings than in patients with unilateral and normal EEG findings (P<0.05). CONCLUSION: Serum S100B is normal in patients with focal epilepsy related or not to chronic NCC.

A new species of trypanosome, Trypanosoma desterrensis sp. n., isolated from South American bats.

Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from hats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.

Trypanocidal activity of extracts from Brazilian Atlantic Rain Forest plant species.

The trypanocidal activity of crude hydro alcoholic extracts and several fractions of 13 plants from Brazilian Atlantic Rain Forest were tested in vitro against epimastigote and trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. Crude ethanol extracts with promising in vitro activity (DL50 between 5-10 microg/ml) against epimastigotes were fractionated by solvent partition and further tested against bloodstream form of the parasite. Activity against bloodstream parasites was observed in both dichloromethane and hexane fractions of Polygala sabulosa and P. paniculata.

Biological and molecular characterization of Besnoitia akodoni n.sp. (Protozoa: Apicomplexa) from the rodent Akodon montensis in Brazil.

The diversity among coccidian parasites of the genus Besnoitia is incompletely known. Of the eight currently described members of the genus, only B. jellisoni is known to parasitize a rodent host. Here, we propose a new name, Besnoitia akodoni, for the species initially isolated form the rodent Akodon montensis in Brazil. The tissue cysts of B. akodoni were up to 442 microm in diameter and bradyzoites were 8.4 x 1.4 microm in size. The bradyzoites contained enigmatic bodies, micronemes and rhoptries. Tachyzoites were 5.8 x 1.5 microm in size and they could be grown in vitro in bovine monocytes and African Green monkey cells where they divided by endodyogeny. Besnoitia akodoni was infective to laboratory-raised mice (Mus musculus) and gerbils (Meriones unguiculatus) but not to cats (Felis catus). Comparison of the conserved sequences of the small subunit rDNA clearly established the close relationship of B. akodoni with other members of the genus. However, sequences of the more variable first internal transcribed spacer portion of the ribosomal DNA repeat support its differentiation from the other species of the genus.

Diplonema spp. possess spliced leader RNA genes similar to the Kinetoplastida.

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

Interaction of Trypanosoma rangeli Tejera, 1920 with different cell lines in vitro.

Intracellular multiplication of Trypanosoma rangeli was evaluated in vitro using experimental infection of Vero cells, murine macrophages, and promonocytes with T. rangeli Choachi, Macias, and SC-58 clone B1 strains. Our results revealed a low infectivity of all T. rangeli strains to these cell lines. Macrophages showed the highest infection rate; however, intracellular forms were no longer observed 48 h post infection Despite the observation of intracellular parasites up to 144 h post infection, the infection rates of Vero cells and J774G.8 promonocytes with these parasite strains were always below 5%. Pre-incubation of parasites with normal mouse serum increased the initial infectivity but not the time course of the infection. Under our experimental conditions, we did not observe any evidence of intracellular multiplication of T. rangeli within these cell lines.

Differential susceptibility of triatomines of the genus Rhodnius to Trypanosoma rangeli strains from different geographical origins.

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.

Trypanosoma cruzi infection in Didelphis marsupialis in Santa Catarina and Arvoredo Islands, southern Brazil.

Between 1984 and 1993 the prevalence of the Trypanosoma cruzi infection in opossums (Didelphis marsupialis) was studied in Santa Catarina and Arvoredo Islands, State of Santa Catarina, Brazil. The association of the triatomine bug Panstrongylus megistus with opossums nests and the infection rate of these triatomines by T. cruzi was also studied. Thirteen different locations were studied in Santa Catarina Island (SCI), in which 137 D. marsupialis were collected. Sixty two opossums were collected at the Arvoredo Island (AI), located 12 miles north from SCI. All captured animals were submitted to parasitological examinations that revealed the presence of T. cruzi in 21.9% of the opossums captured in SCI and 45.2% among opossums captured in the AI. The presence of P. megistus was detected in most of the D. marsupialis nests collected in the SCI, however, in the non-inhabited AI only eight triatomines were collected during the whole study. The presence of T. cruzi-infected D. marsupialis associated with P. megistus in human dwellings in the SCI, and the high infection rate of D. marsupilais by T. cruzi in the absence of a high vector density are discussed.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors.

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil.

Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Elimination of bancroftian filariasis (Wuchereria bancrofti) in Santa Catarina state, Brazil.

During the 1950s, three foci of Wuchereria bancrofti transmission were identified in the State of Santa Catarina, Brazil. In Florianópolis, São José da Ponta Grossa and Barra da Laguna community treatment of bancroftian filariasis with diethylcarbamazine (DEC) was performed using two distinct approaches, without vector control or improvements in sanitation. In two of the three communities only microfilaraemic individuals were treated, while in Barra da Laguna the entire population received DEC treatment regardless of their infection status. In both cases, transmission of the parasite was blocked and no new cases were detected in all localities for up to 10 years. Recently, a new survey in São José da Ponta Grossa and Barra communities revealed no microfilaria-positive individuals, including residents that were positive in the 1950s. These data confirm that transmission of W. bancrofti was interrupted in Santa Catarina, and mass treatment appears to be more effective than treatment of microfilaraemic individuals only.

Characterization of Trypanosoma rangeli strains isolated in Central and South America: an overview.

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.

Mini-exon gene sequence polymorphism among Trypanosoma rangeli strains isolated from distinct geographical regions.

Trypanosoma rangeli can infect humans and the same domestic and wild animals and triatomine vectors infected by T. cruzi in Central and South America. This overlapping distribution complicates the epidemiology of Chagas disease because of the cross-reactivity between T. rangeli and T. cruzi antigens. We have studied T. rangeli strains isolated from different geographical regions using the mini-exon gene as a genetic marker. Two pairs of oligonucleotides directed to this gene were designed in order to detect specifically T. rangeli DNA by PCR assays. This assay was highly sensitive, able to amplify the target sequence using the equivalent DNA content of a single parasite as template, and demonstrated no cross-reactivity with T. cruzi DNA. T. rangeli SC-58 strain, isolated in southern Brazil, showed a distinct electrophoretic pattern from the other T. rangeli strains tested. Low stringency single specific primer-PCR (LSSP) assays were able to detect sequence polymorphisms at the mini-exon gene among T. rangeli strains. Sequence comparisons of this gene revealed that the SC-58 strain was genetically distinct from strains isolated in Central America and northern South America. In addition to insertion/deletion events, the presence of microsatellite repeats in the non-transcribed region of the gene contribute to the intra-species variability.