Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil

Background: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The ""Folmer region"" detects a single taxon using a 3% divergence threshold. Methods: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2). Results: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (< 100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (similar to 798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapa state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage. Conclusions: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

A longitudinal study on the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection in Amazonian Brazil, with special reference to its prevalence and incidence

This was a longitudinal study carried out during a period over 2 years with a cohort of 946 individuals of both sexes, aged 1 year and older, from an endemic area of American visceral leishmaniasis (AVL) in Para State, Brazil. The object was to analyze the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection based principally on the prevalence and incidence. For diagnosis of the infection, the indirect fluorescent antibody test (IFAT) and leishmanin skin test (LST) were performed with amastigote and promastigote antigens of the parasite, respectively. The prevalence by LST (11.2%) was higher (p < 0.0001) than that (3.4%) by IFAT, and the combined prevalence by both tests was 12.6%. The incidences by LST were also higher (p < 0.05) than those by IFAT at 6 (4.7% A- 0.6%), 12 (4.7% A- 2.7%), and 24 months (2.9% A- 0.3%). Moreover, there were no differences (p > 0.05) between the combined incidences by both tests on the same point surveys, 5.2%, 6.3%, and 3.6%. During the study, 12 infected persons showed high IFAT IgG titers with no LST reactions: five children and two adults developed AVL (2,560-10,120), and two children and three adults developed subclinical oligosymptomatic infection (1,280-2,560). The combined tests diagnosed a total of 231 cases of infection leading to an accumulated prevalence of 24.4%.

Further evidences on a new diagnostic approach for monitoring human Leishmania (L.) infantum chagasi infection in Amazonian Brazil

This was a prospective study carried out during a period over 2 years (May/2006-September/2008) with a cohort of 1,099 individuals of both genders, aged 1 year old and older, from an endemic area of American visceral leishmaniasis (AVL) in Para state, Brazil. The object was to analyze the prevalence and incidence of human Leishmania (L.) infantum chagasi infection as well as the dynamics evolution of its clinical-immunological profiles prior identified: (1) asymptomatic infection (AI); (2) symptomatic infection (SI = AVL); (3) sub-clinical oligosymptomatic infection (SOI); (4) sub-clinical resistant infection (SRI) and; (5) indeterminate initial infection (III). The infection diagnosis was performed by using both the indirect fluorescent antibody test and leishmanin skin test with amastigotes and promastigotes antigens of L. (L.) i. chagasi, respectively. A total of 187 cases of infection were recorded in the prevalence (17%), 117 in the final incidence (6.9%), and 304 in the accumulated prevalence (26.7%), which provided the following distribution into the clinical-immunological profiles: AI, 51.6%; III, 22.4%; SRI, 20.1%; SOI, 4.3%; and SI (=AVL), 1.6%. The major finding regarding the dynamics evolution of infection was concerned to III profile, from which the cases of infection evolved to either the resistant profiles, SRI (21 cases, 30.8%) and AI (30 cases, 44.1%), or the susceptible SI (=AVL; 1 case, 1.5%); the latter 16 cases remained as III till the end of the study. These results provided the conclusion that this diagnostic approach may be useful for monitoring human L. (L.) i. chagasi infection in endemic area and preventing the high morbidity of severe AVL cases.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

Phlebotominae sand flies (Diptera: Psychodidae): potential vectors of American cutaneous leishmaniasis agents in the area associated with the Santo Antônio Hydroelectric System in Western Amazonian Brazil

INTRODUCTION: An entomological study was conducted as part of a vector-monitoring program in the area associated with the Santo Antônio hydroelectric system in State of Rondônia, Western Amazonian Brazil. METHODS: Fourteen sampling sites were surveyed to obtain data on the potential vectors of Leishmania spp. in the area. Sand flies were collected from 2011 to 2014 during the months of January/February (rainy season), May/June (dry season), and September/October (intermediary season) using light traps arranged in three vertical strata (0.5, 1, and 20m). RESULTS : A total of 7,575 individuals belonging to 62 species/subspecies were collected. The five most frequently collected sand flies were Psychodopygus davisi (Root) (36.67%), Trichophoromyia ubiquitalis (Mangabeira) (8.51%), Nyssomyia umbratilis (Ward & Fraiha) (6.14%), Bichromomyia flaviscutellata (Mangabeira) (5.74%), and Psychodopygus complexus (Mangabeira) (5.25%). These species have been implicated in the transmission of American cutaneous leishmaniasis agents in the Brazilian Amazon region and described as potential vectors of this disease in the study area. CONCLUSIONS: Additional surveillance is needed, especially in areas where these five species of sand fly are found.

The family Trypetheliaceae (Loculoascomycetes: lichenized Melanommatales) in Amazonian Brazil.

The family Trypetheliaceae is redefined including a key to the genera. Exiliseptum gen. nov. is described. Keys to the species are provided for the six genera which occur in Amazonian Brazil along with brief comments on the three other genera. Thirty-five species are included, tem new species described and ten new combinations proposed.

A new species of Abarema Pittier (Mimosaceae ingeae) from Amazonian Brazil.

Abarema piresii Barneby & Grimes is described from the Brazilian states of Amazonas and Pará. The species is illustrated an tis relationships are discussed. Two kindred species are incidentally transferred from Pithecellobium to Abarema as A. curvicarpa (Irwin) and A. moniliformis (Ducke) Barneby & Grimes.

Eimeria species (Apicomplexa: Eimeriidae) of podocnemis expansa (Schweigger) and geochelone denticulata (LINN.) from Amazonian Brazil (Reptilia: Chelonia)

Eimeria lagunculata, Eimeria mammiformis and Eimeria podocnemis n. spp., are described from the faeces of the fresh-water turtle Podocnemis expansa, in Pará State, north Brasil. Oocysts of E. lagunculata are ellipsoidal, 19.2 x 12.8 (17.0-20.7 x 11.8-14.1) mum, shape-index (= length/ width) 1.5 (1.4-1.7). Oocyst wall about 0.5-0.7 mum thick, with a prominent stopper-like micropyle at one pole. No oocyst residuum and no polar body. Sporocysts elongate ellipsoidal, 11.0 x 5.4 (10.4-11.8 x 5.2-6.0) mum, shape-index 2.0 (1.8-2.1): no Stieda body. A compact, ellipsoidal sporocyst residuum lies between the two sporozoites, which possess a posterior and an anterior refractile body. Oocysts of E. mammiformis broadly ellipsoidal, 30.0 x 19.4 (23.0-37.0 x 16.3-21.5) mum, shape-index 1.5 (1.1-1.9). Oocyst wall about 0.7 mum thick, with a prominent micropyle: no oocyst residuum and rarely a single polar body. Sporocysts ellipsoidal, 15.3 x 7.9 (14.8-17.0 x 7.4-9.6) mum, shape-index 2.0 (1.8-2.2), with a tiny Stieda body. Sporocyst residuum bulky, ellipsoidal: sporozoites with two conspicuous refractile bodies. E. podocnemis has broadly ellipsoidal oocysts, 17.0 x 12.8 (14.8-19.2 x 11.8-14.1) mum, shape-index 1.3 (1.1-1.4). Oocyst wall about 0.5-0.7 mum thick, with no micropyle. No oocyst residuum, but always a single polar body. Sporocysts ellipsoidal, 9.7 x 5.2 (8.9-10.4 x 4.4-6.0) mum, shape-index 1.9 (1.6-2.0), with no Stieda body. Sporocyst residuum bulky, ellipsoidal: sporocysts with 2 refractile bodies. Eimeria carinii n. sp., is recorded from the tortoise Geochelone denticulata, also from Pará. Oocyst wall about 1.2 mum thicl. No micropyle. Oocyst residuum limited to a number (about 10-20) of scattered granules: no polar body. Sporocysts broadly ellipsoidal, and with no Stieda body: they measure 8,8 x 7.3 (8.0-9.0 x 7.0-7.5) mum, shape-index 1.2 (1.1-1.3). Sporocyst residuum bulky, spherical to ellipsoidal: sporozoites possess both posterior and anterior refractile bodies.

Observations on some Avian Coccidia (Apicomplexa: Eimeriidae) in Amazonian Brazil

Oocysts of Eimeria porphyrulae n. sp. are described in faeces of Porphyrula martinica (Aves: Gruiformes: Rallidae). They are ellipsoidal to oval, 22.4 x 17.7 (20.0-23.7 x 16.2-18.7) µm, shape-index (length/width) 1.3. Oocyst wall about 1.25 µm thick, colourless, with two layers: inner one prominently striated. Micropyle and sub-micropylar granule present: no oocyst residuum. Sporocysts 17.5 x 9.0 (17.0-19.0 x 8.0-10.0) µm, shape-index 1.9, with inconspicuous Stieda/sub-Stieda bodies. Sporocyst residuum of scattered granules, sometimes a compact mass: sporozoites with two refractile bodies. Eimeria crypturelli n. sp. is described in faeces of Crypturellus soiu (Tinamiformes: Tinamidae). Oocysts ellipsoidal-oval, 20.75 x 14.5 (17.5-25.0 x 11.25-21.25) µm, shape-index 1.4. Oocysts wall about 1.25 µm thick and bi-layered: inner layer faintly striated. Micropyle present, with oocyst residuum immediately below: single polar body rarely present. Sporocysts 13.0 x 7.5 (12.5-13.75 x 7,5-8.1) µm, shape-index 1.7, with a Stieda body but seemingly no sub-Stieda. Sporocyst residuum compact: sporozoites with two refractile bodies. Isospora cacici n. sp. is recorded from faeces of Cacicus cela cela (Passeriformes: Icteridae). Oocysts subspherical-spherical, 26.5 x 23.7 (22.5-27.5 x 20.0-26.2) µm, shape-index 1.1. Wall a single, colourless layer about 1.5 µm thick. No micropyle or oocyst residuum: 1-2 polar bodies. Sporocysts ellipsoidal, 17.7 x 12.5 (17.5-18.75 x 11.25-13.75) µm, shape-index 1.4, with pronounced Stieda/sub-Stieda bodies: residuum compact and sporozoites with two refractile bodies. Isospora thraupis n. sp. is described from faeces of Thraupis palmarum melanoptera (Passeriformes: Thraupidae). Oocysts subspherial-spherical, 19.9 x 19.0 (18.7-21.2 x 18.75-20.0) µm, shape-index 1.0. Wall about 0.6 µm thick, smooth, colourless and a single layer: no micropyle, oocyst residuum or polar bodies. Sporocysts 14.2 x 9.2 (13.7-16.2 x 8.7-10.0) µm, shape-index 1.5: Stied/sub-Stieda bodies inconspicuous. Residuum compact: sporozoites with two refractile bodies.

Schellackia (Apicomplexa: Eimeriidae) of the brazilian tree-frog, Phrynohyas venulosa (Amphibia: Anura) from Amazonian Brazil

Endogenous stages of a Schellackia species are described in histological sections of the intestine of the tree-frog, Phrynohyas venulosa, from North Brazil. Most oocysts sporulate within the epithelial cells of the gut, but a few were detected in the lamina propria.

Haematozoan parasites of the lizard Ameiva ameiva (Teiidae) from Amazonian Brazil: a preliminary note

Three different haematozoan parasites are described in the blood of the teiid lizard Ameiva ameiva Linn. from North Brazil: one in the monocytes and the other two in erythrocytes. The leucocytic parasite is probably a species of Lainsonia Landau, 1973 (Lankesterellidae) as suggested by the presence of sporogonic stages in the internal organs, morphology of the blood forms (sporozoites), and their survival and accumulation in macrophages of the liver. One of the erythrocytic parasites produces encapsulated, stain-resistant forms in the peripheral blood, very similar to gametocytes of Hemolivia Petit et al., 1990. The other is morphologically very different and characteristically adheres to the host-cell nucleus. None of the parasites underwent development in the mosquitoes Culex quinquefasciatus and Aedes aegypti and their behaviour in other haematophagous hosts is under investigation. Mixed infections of the parasites commonly occur and this often creates difficulties in relating the tissue stages in the internal organs to the forms seen in the blood. Concomitant infections with a Plasmodium tropiduri-like malaria parasite were seen and were sometimes extremely heavy.

Clinical and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian Brazil: a review

The wide variety of Leishmania species responsible for human American cutaneous leishmaniasis combined with the immune mechanisms of the host results in a large spectrum of clinical, histopathological, and immunopathological manifestations. At the middle of this spectrum are the most frequent cases of localized cutaneous leishmaniasis (LCL) caused by members of the subgenera Leishmania and Viannia, which respond well to conventional therapy. The two pathogenicity extremes of the spectrum generally recognized are represented at the hypersensitivity pole by mucocutaneous leishmaniasis (MCL) and at the hyposensitivity pole by anergic diffuse cutaneous leishmaniasis (ADCL). Following the present study on the clinical, histopathological and immunopathological features of cutaneous leishmaniasis in Amazonian Brazil, we propose the use of the term "borderline disseminated cutaneous leishmaniasis" for the disseminated form of the disease, due to parasites of the subgenera Leishmania and Viannia, which might be regarded as intermediate between LCL and the extreme pathogenicity poles MCL and ADCL.

Blood and intestinal parasites of squirrels (Rodentia: Sciuridae) in Amazonian Brazil

We report the result of an examination for blood and intestinal protozoa in 12 specimens of the red squirrel Sciurus spadiceus (Rodentia: Sciuridae) from Birroque, municipality of Plácido de Castro, state of Acre, Brazil. No parasites were detected in thin, Giemsa-stained blood films of the animals, but culture of the blood of three in Difco B45 medium blood-agar slants gave rise to isolates of epimastigotes. Inoculation of one isolate into laboratory mice resulted in the appearance of Trypanosoma cruzi-like trypomastigotes in their peripheral blood, and the other two isolates gave rise to transient infections with a T. lewisi-like parasite in inoculated mice and hamsters. The failure of the latter parasite to develop in the triatomine bug Rhodnius robustus suggests that it is probably not T. rangeli. This appears to be the first record of a T. lewisi-like trypanosome in neotropical squirrels. Oocysts of an Eimeria sp., were detected in the faeces of 10 animals (83.3%). The parasite develops in the epithelial cells of the intestine, where it may cause severe damage and sometimes results in death of the animal. No oocysts were detected in bile.

The Genus Cyclospora (Apicomplexa: Eimeriidae), with a description of Cyclospora schneideri n.sp. in the snake Anilius scytale scytale (Aniliidae) from Amazonian Brazil: a review

A review is made of the recorded species of the coccidian genus Cyclospora and major events leading up to the discovery of C. cayetanensis, which is responsible for serious outbreaks of diarrhoea in man and is one of the aetiological agents of "traveller's diarrhoea". Humans appear to be the specific hosts, with the entire life-cycle in the intestine: to date there is no convincing evidence that the disease is a zoonosis. A description is given of oocysts and endogenous stages of C. schneideri n.sp., in the snake Anilius scytale scytale. Sporulation is exogenous and completed after about one week at 24-26º. Mature oocysts 19.8 × 16.6 (15.1 × 13.8-25.7 × 20.1), shape-index 1.2 (1.0-1.3): no oocyst residuum or polar bodies. Oocyst wall a single colourless, smooth layer with no micropyle: it is rapidly deformed or broken. Sporocysts 13.6 × 9.4 (11.3 × 8.3-15.1 × 9.9), shape-index 1.4 (1.2-1.5) with an inconspicuous Stieda body. Sporozoites 11-13 × 2.5-3. Endogenous stages are intracytoplasmic in the epithelial cells of the small intestine and with the characters of the Eimeriorina.

Eimeria lepidosirenis n.sp. (Apicomplexa:Eimeriidae) of the South American lungfish Lepidosiren paradoxa (Osteichthyes:Dipnoi) from Amazonian Brazil

The mature oocysts of Eimeria lepidosirenis n.sp. are described in faeces removed from the lower region of the intestine of a single specimen of the South American lungfish Lepidosiren paradoxa, from Belém, state of Pará, Amazonian Brazil. Oocysts with endogenous sporulation: spherical to slightly subspherical, 30.8 × 30.3 µm (28.1 × 25.9 -33.3 × 31.8), shape-index (ratio length/width) 1.0, n = 25. Oocyst wall a very thin, single layer approximately 0.74 µm thick, smooth, colourless, with no micropyle and rapidly breaking down to release the sporocysts. Oocyst residuum a bulky ovoid to spherical mass of approximately 20.0 × 15 µm, composed of fine granules and larger globules and enclosed by a very fine membrane: no polar bodies seen. Sporocysts 15.5 × 9.0 µm (14.5 × 8.0 16.0 × 9.0), shape index 1.7 (1.6-1.8), n = 30, ovoid, with one extremity rather pointed and with a very delicate Stieda body but no sub-Stieda body: sporocyst wall a single extremely thin layer with no valves. Sporocyst residuum a spherical to ovoid mass of approximately 5.0 × 4.0 µm, composed of fine granules and small globules and enclosed by a very fine membrane. Sporozoites strongly recurved at their ends and apparently with only a single refractile body. Site of development in the host uncertain: no evidence of endogenous stages was found in fresh scrapings and stained smears of the intestinal epithelium.