Dynamics of Leishmania spp. infection in dogs from an unaffected region in transition to a visceral leishmaniasis transmission area, Rio Grande do Sul, Brazil.

In Brazil, visceral leishmaniasis (VL) has been expanding and urbanizing, mainly in non-endemic areas such as the State of Rio Grande do Sul. Considering that infected dogs are the main reservoirs of VL in urban areas, the present study aimed to evaluate the propagation of canine visceral leishmaniasis (CVL) infection from an unaffected region in transition to a VL transmission area. For this, 1159 and 1087 samples of canine serum from 2015 and 2021, respectively, were analyzed, using the indirect immunofluorescence test. In addition, necropsy reports between 2007 and 2021 were evaluated. The results showed a prevalence of anti-Leishmania spp. antibodies of 7.5% in the samples from 2015, while in 2021 samples, it was 23.5%, with an incidence of 0.4 cases per 100 dogs. It is noteworthy that in 2007, there was no record of CVL as the cause of death in the pathological reports, and in 2021, 41 diagnoses were made with the protozoan being a determinant of the death of the animal. These values indicate an increasing trend in the prevalence and incidence coefficients of CVL. The results of this study allowed us to verify the spread of the disease from an unaffected region to a transmission area of the agent, as well as provide subsidies for health authorities to implement improvements in the CVL control program in the municipality, to mitigate the emergence of human cases of the disease.

PCR-RLFP characterization of Leishmania spp. in domestic animals from the south-western border of Brazil.

The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.

Risk and protective factors of Leishmaniasis in the rural area of the western border region of Rio Grande do Sul, Brazil.

BACKGROUND: The Leishmaniases are on the top of the global list of tropical neglected diseases. The number of infected dogs in South America is estimated in millions and correlated to disease cases in humans, especially in Brazil. Equines may get infected too and can play a role in the epidemiological chain. Thus, the aim of the present study was to evaluate risk and protective factors of leishmaniasis in rural areas of the western border region of Rio Grande do Sul state, Brazil by Leishmania spp. protozoa molecular detection and serological evaluation (ELISA) in equine and canine blood samples. This work included nine farms around the city of Uruguaiana. Epidemiologic information regarding farm characteristics and biologic material collection of canine (22) and equine (91), totalizing 113 samples was collected. The polymerase chain reaction (PCR) technique was used to detect Leishmania spp. in biological samples. Variables related to the farm were collected and evaluated through descriptive analysis followed by chi-square test and a logistic regression analysis. RESULTS: Nineteen positive samples (19/113 - 16,81%) were detected, being 18 equines and 1 canine, in six of the nine farms included in the study. No animal showed clinical signs of the disease. According to the variables analyzed, when compared each characteristic separately, the presence of abundant vegetation and poor hygiene demonstrated to be risk factors to Leishmania infection in rural areas. The logistic regression showed that excellent general hygiene, proximity to the weir and trimmed grass were protective factors (p=0.038, p=0.001 and p=0.014, respectively). Having excellent hygiene represents a 70% lower chance of getting infected, keeping the grass cut protects the animal by more than 90% and the proximity of the weir represents a protective factor of 96%. CONCLUSIONS: The presence of Leishmania infection in the western border region of Rio Grande do Sul was 16,81% and it was influenced by farm characteristics. The role of the excellent general hygiene as a protective factor is extremely relevant in the leishmaniases prevention.

Molecular detection of Leishmania spp. in Brazilian cross-border south region mammalian hosts.

Visceral leishmaniasis is an endemic zoonotic disease identified especially in developing territories. Brazil's northeast, southeast and midwest have been endemic for several years; currently, the infection is spreading to the south. Dogs are the main reservoirs; however, other mammal species have also been infected. Herein, we have identified the infecting Leishmania species in dogs and horses from the south of Brazil, a new outbreak of the infection. Blood samples were collected in the urban area of Uruguaiana city. DNA was extracted from peripheral blood, kinetoplast DNA (kDNA) and ribosomal DNA (rDNA) fragments were obtained by polymerase chain reaction (PCR) and sequenced. Out of 123 samples, 25 of them (14 dogs and 11 horses) were positive for Leishmania spp. Sequence alignment and phylogenetic analysis revealed that the kDNA in positive samples was similar to four species previously reported: L. infantum/L. chagasi, L. donovani, L. major. Despite kDNA minicircles regions are very useful due to high sensitivity to Leishmania spp. DNA detection, the sequence polymorphism among minicircles can be an obstacle to interspecific differentiation. Our results suggest that these strains are circulating in Brazil south region cross-border and indicate the susceptibility of new outbreak for visceral leishmaniasis infection in horses domiciled in endemic region for canine and human visceral leishmaniasis.

Assessment of Leishmania infantum infection in equine populations in a canine visceral leishmaniosis transmission area.

BACKGROUND: Leishmaniosis, zoonosis that produces significant public health impacts, is caused by Leishmania infantum. Canines are the main domestic reservoir and, besides humans, other species of mammals could be infected when living in endemic areas. In this study, we detected equine Leishmania infantum infections in a canine visceral leishmaniosis transmission area and evaluated the clinical, haematological, biochemical and oxidative stress disorders. This study was conducted in Uruguaiana, Rio Grande do Sul, south of Brazil. Peripheral blood samples were collected from 124 animals (98 horses and 26 dogs) of both genders and several breeds after they underwent general and dermatologic examinations. RESULTS: Twenty five Leishmania infantum infected animals (20.16%), 14 horses and 11 dogs were detected by PCR (Polymerase Chain Reaction) amplification of kinetoplast DNA regions with 96% homology to Leishmania infantum (GenBank Accession No. L 19877.1). The clinical and haematological alterations of infected equines were skin lesions, nodules, lymphadenopathy, decreased levels in red blood cells and haematocrit (p < 0.05) and increase in urea serum concentration (p < 0.05), while CVL presented a decrease in red blood cells counts (p < 0.05), increase in lymphocytes (p < 0.05), and decrease in neutrophil-lymphocyte ratio (p < 0.05). Oxidative stress markers of plasma protein carbonyl and plasma lipid peroxidation were not statistically significant (p > 0.05) in both species. CONCLUSIONS: To our knowledge, this has been the first leishmaniosis equine survey performed in south of Brazil, caused by Leishmania infantum that were able to initially identify haematological and biochemical changes in the species, even in asymptomatic animals. We present evidence supporting those findings of haematological and biochemical changes could be related to infection. Surprisingly, the clinical manifestations of equine infection were similar to those found in canine visceral leishmaniosis. The equine population could be play an important role in the cycle of leishmaniosis in south Brazil and consequently indicates a great risk of public health. This evaluation of infected animals is important to establish the clinical and laboratory parameters involved in the disease progression.

Evaluation of the tickcide, genotoxic, and mutagenic effects of the Ruta graveolens L (Rutaceae) / Avaliação dos efeitos carrapaticida, genotóxico e mutagênico da Ruta graveolens L (Rutaceae)

Current analysis investigated the tickcide effects of the aqueous extract and chloroform fractions of Ruta graveolens L. (rue) on engorged females of Rhipicephalus microplus, as well as their genotoxic and mutagenic effects on human leukocytes. The best tickcide activity (non-dependent dose) and genotoxic / mutagenic effects (dependent-dose) were observed on exposure to chloroform fractions. Results suggest that extract fractions of R. graveolens L are efficient against R. microplus, although the fraction and the tested concentrations show genotoxic and mutagenic potential for human leukocytes.

O efeito carrapaticida do extrato aquoso e frações da Ruta graveolens L. (arruda) sobre teleóginas de Rhipicephalus microplus, bem como seu potencial genotóxico sobre leucócitos humanos foram investigados neste trabalho. A melhor atividade carrapaticida (dose não dependente) e efeito genotóxico / mutagênico (dose dependente) foram observados nas frações clorofórmicas. Os resultados encontrados sugerem que frações clorofórmicas do extrato de Ruta graveolens L. são eficazes contra R. microplus, embora a fração e as concentrações testadas apresentem potencial genotóxico e mutagênico para células leucocitárias humanas.

Metarhizium anisopliae chitinase CHIT30 is involved in heat-shock stress and contributes to virulence against Dysdercus peruvianus.

Entomopathogenic fungi are able to produce several chitinases, which serve a variety of biological functions, such as fungal cell wall organization and the degradation of exogenous chitin for nutrition or insect infection processes. In this study, we analyzed the contribution of the CHIT30 chitinase from Metarhizium anisopliae in morphogenetic development and virulence as a model of chitinase function. The analysis of chi3 gene expression revealed transcript accumulation in response to heat-shock stress conditions as well as cultivation in medium supplemented with chitin. Null chi3 mutants were constructed to determine the biological role of CHIT30. No substantial differences in the secreted chitinase activity could be detected between the wild type and the Δchi3 mutant. However, both endochitinase and exochitinase activities were diminished in the mutant strain following heat-shock treatment, suggesting that CHIT30 is involved in heat-shock adaptation. Mutants lacking CHIT30 chitinase showed reduced virulence against the cotton stainer bug Dysdercus peruvianus, indicating that the CHIT30 chitinase plays a role in the infection process of M. anisopliae.

Teste de ELISA indireto para o diagnóstico sorológico de pitiose / Indirect ELISA for the serodiagnostic of pythiosis

A pitiose, doença granulomatosa de eqüinos causada pelo oomiceto Pythium insidiosum, tem como característica a evolução rápida seguida de morte dos animais. Estas mortes muitas vezes são causadas por diagnósticos errôneos ou demorados quando os doentes já não respondem ao tratamento. Este trabalho teve por objetivo a padronização do ensaio imunoenzimático indireto (ELISA) para diagnóstico sorológico de pitiose em eqüinos e coelhos, visando a diminuição de erros e de tempo necessário para o diagnóstico. Para o desenvolvimento e validação do teste foram utilizadas 72 amostras de soro de eqüinos saudáveis e 44 soros de eqüinos com pitiose confirmada. Os resultados da validação do ELISA para eqüinos foram: sensibilidade 97,72 por cento, especificidade 90,27 por cento, valor preditivo positivo 86 por cento, valor preditivo negativo 98,4 por cento e eficiência de 93,1 por cento. Para coelhos, o teste foi padronizado com 48 amostras de soro de animais saudáveis e 24 amostras de coelhos imunizados com antígenos de P. insidiosum. Os resultados foram: sensibilidade 91,66 por cento, especificidade 95,83 por cento, valor preditivo positivo 91,66 por cento, valor preditivo negativo 95,83 por cento e eficiência de 94,44 por cento. Os resultados deste trabalho demonstram que o ensaio imunoenzimático indireto é um método seguro e eficaz para o diagnóstico sorológico da pitiose.

Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks.

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.

In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed