Exploring Leishmania infantum cathepsin as a new molecular marker for phylogenetic relationships and visceral leishmaniasis diagnosis.

BACKGROUND: Leishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil. METHODS: We used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite. RESULTS: The cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10- 11 ng/µl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%. CONCLUSIONS: The results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.

Molecular e serological detection of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in domestic dogs in Manaus city, Amazonas, Brazil.

Leishmaniasis are zoonosis widely spread in Brazil, caused by the protozoan of the genus Leishmania, which includes several species. The disease manifests itself in the visceral or tegumentary form, and the main reservoir is the dogs. Manaus is the largest city in the Brazilian Amazon region, and despite the importance of the municipality, practically nothing is known about leishmaniosis in humans and animals. The objective of this study was to evaluate the occurrence and risk factors associated with the presence of Leishmania infantum in domiciliated dogs from Manaus. Molecular (polymerase chain reaction) and serological (immunofluorescent antibody test) methods were used as an indication of the circulation of the parasite. Blood samples for 154 domiciled dogs were obtained, and prevalence ratio and analysis of the variables were performed. Serum antibodies anti-Leishmania spp. were detected in 20.8% (95% CI: 14.4%-27.2%). Access to the street and zone of residence (P < 0.01) were associated with higher seropositivity. Molecular diagnosis for L. infantum detected positivity in 60 (39%) of the 154 (95% CI: 31.3%-46.7%) animals, and the variables street access, contact with dogs, and zone of residence were associated with higher frequencies of positivity (P < 0.05). Both serology and molecular diagnosis detected positive dogs in the municipality. This is the first description of the circulation of L. infantum infecting dogs in Manaus. As the municipality is classified as nonendemic, studies of isolation and characterization of the isolate must be done urgently.

Diversity of intestinal protozoa and clinical signs associated in wild-caught Phoneutria nigriventer kept in captivity for the anti-arachnid serum production.

The phylum Arthropoda comprises approximately 85% of all described animal species. The class Arachnida includes some invertebrates of great importance as they are either involved in the transmission of diseases or poses a risk of human envenomation. Spiders belonging to the genus Phoneutria sp., are the arachnids exhibiting medical importance. These animals were quarantined and maintained in captivity at the Biotério de Artrópodes of the Instituto Butantan, São Paulo, Brazil, for the production of the anti-arachnid serum. A total 509 feces samples from different Phoneutria nigriventer were analyzed, and 131 (25.73%) samples were found to be positive for flagellates and ciliates. All positive samples were subjected to DNA extraction and amplification of 18S gene. A total of 16 sequences were obtained and analyzed using BLAST. Sequences were identified as Colpoda steiini, one as Colpoda aspera, one to Colpoda sp., and one as "ciliated". Four identified as Parabodo caudatus, two as Urostipulosphaera sp., one as Helkesimastix sp., and one as a Euglena-like. The presence of clinical signs was observed in 16 spiders. The intestinal protozoa that affect armed spiders were identified for the first time as an initial step for understanding the parasitic diseases in these organisms.

Molecular Diagnosis and Prevalence of Trypanosoma vivax (Trypanosomatida: Trypanosomatidae) in Buffaloes and Ectoparasites in the Brazilian Amazon Region.

Trypanosoma vivax Ziemann is a parasite that affects both wild and domestic ungulates and is transmitted mechanically via tabanids and other blood-sucking insects in the Americas. A total of 621 blood samples from water buffaloes (Bubalus bubalis (Linnaeus) (Artiodactyla: Bovidae), and 184 ectoparasite samples (Amblyomma cajennense (Fabricius) sensu stricto and Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), and Haematopinus tuberculatus (Burmeister) (Phthiraptera: Haematopinidae)) were obtained from 60 farms in the State of Pará, Brazilian Amazon. Twelve buffalo blood samples (1.89%) and 11 ectoparasites (6%) were positive for T. vivax based on the cathepsin L-like gene. All sequences were 99% similar to T. vivax from northeastern Brazil (EU753788) in amplified PCR assays on each of the hosts tested.