Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.

Resumo Riquetsioses do Grupo da Febre Maculosa são doenças emergentes. Em algumas destas doenças, os cães domésticos agem como sentinelas. Estudos sorológicos caninos têm demonstrado que a dispersão de patógenos rickettsiais está concentrada em áreas rurais, sendo a soroprevalência maior onde as rickettsioses humanas são endêmicas. Na cidade do Rio de Janeiro, a vegetação de Mata Atlântica vem sendo devastada pela urbanização. Nesse contexto, objetivou-se detectar a presença de Rickettsia spp. em áreas urbanas da Zona Oeste do Rio de Janeiro. Amostras de soro obtidas de 130 cães foram testadas, utilizando-se a Imunofluorescência Indireta. Carrapatos coletados desses cães foram testados, utilizando-se a reação em cadeia da polimerase. Observou-se que as taxas de reações sorológicas contra R. rickettsii e R. parkeri nessa área de estudo excederam a prevalência das áreas rurais e não endêmicas, destacando-se a importância dos cães como sentinelas urbanos das rickettsioses. A possibilidade de contato com capivaras e gambás favoreceu a exposição à Rickettsia spp., reforçando a hipótese de ligação entre a paisagem local e o ciclo silvestre de transmissão riquetsial. O carrapato Rhipicephalus sanguineus sensu lato foi encontrado com maior frequência. Amostras com positividade pela PCR mostraram similaridade com R. rickettsii e R. felis, um patógeno emergente raramente descrito em carrapatos. Observou-se circulação riquetsial em áreas urbanas e em carrapatos obtidos do ambiente doméstico, os quais podem estar envolvidos na epidemiologia dessas bactérias.

Abstract Spotted fever group rickettsioses are emerging diseases. In some of these diseases, domestic dogs act as sentinels. Canine serological studies have demonstrated that rickettsial dispersion is concentrated in rural areas, seroprevalence being higher where human rickettsioses are endemic. In Rio de Janeiro, the Atlantic forest vegetation has been devastated by urbanization. In this context, we aimed to detect Rickettsia spp. in urban areas of the West Zone of Rio de Janeiro. Sera from 130 dogs were tested by Indirect Immunofluorescence Assay, and ticks collected from these dogs were tested by polymerase chain reaction. We found the rate of serological reactions against R. rickettsii and R. parkeri in our study area to exceed those of rural and non-endemic areas, highlighting the importance of dogs as urban sentinels. The possibility of contact with opossums and capybaras increased the chances of exposure to Rickettsia spp., reinforcing the hypothetical link between the landscape and the rickettsial wild cycle. Rhipicephalus sanguineus sensu lato was the tick most frequently observed. PCR-positive samples showed similarity with R. rickettsii and R. felis, an emerging pathogen rarely reported from ticks. We observed that rickettsiae circulate in urban places and ticks from indoor environments, which may be involved in bacterial epidemiology.

Resumo Hoplias malabaricus (Characiformes, Erythrinidae), traíra, é um peixe neotropical de água doce que tem significante impacto na economia e saúde pública. De abril de 2016 a abril de 2018, foram adquiridos 45 espécimes de H. malabaricus comercializados no município de Magé, Estado do Rio de Janeiro, Brasil. Os peixes foram necropsiados e filetados para investigação da presença de larvas de nematoides. Vinte dos peixes coletados estavam parasitados por 347 larvas de nematoides, identificadas taxonomicamente como larvas de quarto estágio de Eustrongylides sp. usando-se dados morfológicos, morfométricos e moleculares, apresentando os seguintes valores: prevalência de 44,44%, intensidade média de 17,35, abundância média de 7,71, e amplitude de variação da infecção de 2-40. Os sítios de infecção foram musculatura, mesentério, cavidade abdominal e serosas do intestino, estômago e fígado. Este é o primeiro registro de larvas de Eustrongylides sp. parasitando H. malabaricus no Estado do Rio de Janeiro.

Abstract Hoplias malabaricus (Characiformes, Erythrinidae), trahira, is a neotropical freshwater fish of economic and public health significance. A total of 45 specimens of H. malabaricus commercialized in the municipality of Magé, state of Rio de Janeiro, Brazil, were acquired between April 2016 and April 2018 to investigate the presence of nematode larvae. Twenty of the fish were found parasitized by 347 fourth-stage nematode larvae identified taxonomically as Eustrongylides sp. using morphological, morphometric and molecular data. The parasitic indices were: prevalence 44.44%, mean intensity 17.35, mean abundance 7.71, and range of infection 2-40. Infection sites were musculature, mesentery, abdominal cavity, and serosa of intestine, stomach and liver. This is the first report of Eustrongylides sp. larvae parasitizing H. malabaricus in the state of Rio de Janeiro.

Resumo Rickettsia rickettsii é o agente causador da Febre Maculosa Brasileira (FMB), doença na qual humanos e cães são susceptíveis. Os cães são sentinelas nos inquéritos sorológicos, contudo, a doença canina é raramente descrita. Assim sendo, objetivou-se avaliar a infecção natural por Rickettsia spp. do Grupo da Febre Maculosa (GFM) em cães e carrapatos obtidos de domicílios próximos a fragmentos de mata, caracterizando áreas de interface doméstico–silvestre. Amostras de 115 cães e 135 ixodídeos foram avaliadas pela reação em cadeia da polimerase (PCR) tendo como alvo o gene gltA de Rickettsia spp. e o gene ompA das espécies do GFM. Um cão (0,87%; 1/115) foi positivo para R. rickettsii. Este animal apresentou alterações clínicas e laboratoriais inespecíficas (trombocitopenia, hiperproteinemia, linfonodos edemaciados, emagrecimento, anorexia e letargia). Rickettsia parkeri foi identificada em 2,96% (4/135) dos carrapatos (Amblyomma sculptum, A. aureolatum e Rhipicephalus sanguineus). Este estudo confirmou a presença de bactérias do GFM em locais preservados e não endêmicos, onde populações domésticas e silvestres interagem. Reforçamos o fato do cão ser susceptível à infecção natural por R. rickettsii. Embora este seja um achado raro, medidas preventivas devem ser tomadas contra a FMB nas áreas estudadas. Em última análise, a infecção por R. parkeri possivelmente está sendo demonstrada pela primeira vez em A. sculptum.

Abstract Rickettsia rickettsii is the causative agent of Brazilian spotted fever (BSF), for which humans and dogs are both susceptible. Dogs are sentinels in serological surveys, however, canine disease is rarely reported. Therefore, we aimed to evaluate natural infection by spotted fever group (SFG) Rickettsia spp. in dogs and ticks collected from domiciles close to forest fragments, featuring domestic–wildlife interface areas. Samples from 115 dogs and 135 ixodids were assessed by polymerase chain reactions (PCR) targeting the gltA gene for Rickettsia spp. and the ompA gene for the SFG rickettsial species. One dog (0.87%; 1/115) was positive for R. rickettsii. This dog presented nonspecific laboratory and clinical abnormalities (thrombocytopenia, hyperproteinemia, lymph node enlargement, emaciation, anorexia, and lethargy). Rickettsia parkeri was identified in 2.96% (4/135) of the ticks (Amblyomma sculptum, A. aureolatum, and Rhipicephalus sanguineus). This study confirmed the presence of SFG bacteria in non-endemic and preserved locations, where domestic and wild populations interact. We reinforce the fact that the dog is susceptible to natural R. rickettsii infection. Although this is a rare finding, preventive measures should be taken against BSF in the studied areas. Finally, R. parkeri infection is possibly being demonstrated in A. sculptum for the first time.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.