Lilian de Oliveira Guimarães

Plasmodium (Novyella) nucleophilum from an Egyptian Goose in São Paulo Zoo, Brazil: microscopic confirmation and molecular characterization.

Graphical abstract

The genetic diversity of Plasmodium malariae and Plasmodium brasilianum from human, simian and mosquito hosts in Brazil.

Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from Plasmodium brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from simian Plasmodium species. This study presents the first molecular epidemiological data for P. malariae and P. brasilianum from Brazil obtained from different hosts and uses them to explore the genetic diversity in relation to geographical origin and hosts. By using microsatellite genotyping, we discovered that of the 14 human samples obtained from areas of the Atlantic forest, 5 different multilocus genotypes were recorded, while in a sample from an infected mosquito from the same region a different haplotype was found. We also analyzed the longitudinal change of circulating plasmodial genetic profile in two untreated non-symptomatic patients during a 12-months interval. The circulating genotypes in the two samples from the same patient presented nearly identical multilocus haplotypes (differing by a single locus). The more frequent haplotype persisted for almost 3 years in the human population. The allele Pm09-299 described previously as a genetic marker for South American P. malariae was not found in our samples. Of the 3 non-human primate samples from the Amazon Region, 3 different multilocus genotypes were recorded indicating a greater diversity among isolates of P. brasilianum compared to P. malariae and thus, P. malariae might in fact derive from P. brasilianum as has been proposed in recent studies. Taken together, our data show that based on the microsatellite data there is a relatively restricted polymorphism of P. malariae parasites as opposed to other geographic locations.

Diversity and distribution of avian malaria and related haemosporidian parasites in captive birds from a Brazilian megalopolis

BackgroundThe role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited.MethodsIn 2011–2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections.ResultsThe overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals). In all, infections with parasites of the genus Plasmodium (overall prevalence 97.6%) predominated when compared to those of the genus Haemoproteus (2.4%). In total, 14 cytochrome b (cytb) lineages of Plasmodium spp. and 2 cytb lineages of Haemoproteus spp. were recorded. Eight lineages were new. One of the reported lineages was broad generalist while others were reported in single or a few species of birds. Molecular characterization of Haemoproteus ortalidum was developed.ConclusionThis study shows that many species of birds are at risk in captivity. It is difficult to stop haemosporidian parasite transmission in zoos, but is possible to reduce the infection rate by treating the infected animals or/and while keeping them in facilities free from mosquitoes. Protocols of quarantine should be implemented whenever an animal is transferred between bird maintaining institutions. This is the first survey of haemosporidians in captive birds from different orders maintained in zoos. It is worth emphasizing the necessity of applying practices to control these parasites in management and husbandry of animals in captivity.

Survey of Plasmodium spp. in free-ranging neotropical primates from the Brazilian Amazon region impacted by anthropogenic actions.

This study investigated Plasmodium spp. infection in free-ranging neotropical primates from Brazilian Amazon regions under the impact of major anthropogenic actions. Blood samples from 19 new world primates were collected and analyzed with microscopic and molecular procedures. The prevalence of Plasmodium infection was 21.0% (4/19) and PCR positive samples were identified as P. brasilianum. Considering the social-economic changes that the Amazon is facing, the prevalence of P. brasilianum infection highlights the necessity to closely monitor the movement of both human and non-human primate populations, in order to mitigate pathogen exposure and the introduction of new agents into previously naïve areas.

Anopheles (Kerteszia) cruzii (DIPTERA: CULICIDAE) IN PERIDOMICILIARY AREA DURING ASYMPTOMATIC MALARIA TRANSMISSION IN THE ATLANTIC FOREST: MOLECULAR IDENTIFICATION OF BLOOD-MEAL SOURCES INDICATES HUMANS AS PRIMARY INTERMEDIATE HOSTS

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

First molecular screening of Plasmodium species in ungulates from Southern Brazil

ObjectiveDespite malaria epidemiology has been extensively studied in primates, few studies were conducted in ungulates. After half a century without descriptions of Plasmodium spp. in deer since its first identification, recent research has rediscovered Plasmodium on ungulates in Africa, Asia, North America and South America, including Central Brazil. Here, a captive herd was evaluated in southern Brazil using light microscopy and PCR. DNA samples were tested for fragment amplification of two Plasmodium spp. genes: mitochondrial cytochrome b and small subunit ribosomal RNA.ResultsAll analyses were negative. However, the tests were performed on samples that were collected at a single time point, and parasitemia may fluctuate over the parasite’s life cycle. Thus, the possibility of occult infection cannot be ruled out. Despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the first molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from different ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region.

Influence of polymorphisms in toll-like receptors (TLRs) on malaria susceptibility in low-endemic area of the Atlantic Forest, São Paulo, Brazil

In low-endemic areas for malaria transmission, asymptomatic individuals play an important role as reservoirs for malarial infection. Understanding the dynamics of asymptomatic malaria is crucial for its efficient control in these regions. Genetic host factors such as Toll-like receptor (TLR) polymorphisms may play a role in the maintenance or elimination of infection. In this study, the effect of TLR polymorphisms on the susceptibility to malaria was investigated among individuals living in the Atlantic Forest of São Paulo, Southern Brazil. A hundred and ninety-five Brazilian individuals were enrolled and actively followed up for malaria for three years. Twenty-four polymorphisms in five toll-like receptor (TLR) genes were genotyped by RFLP, direct sequencing or fragment analysis. The genotypes were analyzed for the risk of malaria. Ongoing Plasmodium vivax or P. malariae infection, was identified by the positive results in PCR tests and previous P. vivax malaria, was assumed when antiplasmodial antibodies against PvMSP119 were detected by ELISA. An evaluation of genomic ancestry was conducted using biallelic ancestry informative markers and the results were used as correction in the statistical analysis. Nine SNPs and one microsatellite were found polymorphic and three variant alleles in TLR genes were associated to malaria susceptibility. The regression coefficient estimated for SNP TLR9.-1237.T/C indicated that the presence of at least one allele C increased, on average, 2.3 times the malaria odds, compared to individuals with no allele C in this SNP. However, for individuals with the same sex, age and household, the presence of at least one allele C in SNP TLR9.-1486.T/C reduced, on average, 1.9 times the malaria odds, compared to individuals with no allele C. Moreover, this allele C plus an S allele in TLR6.P249S in individuals with same sex, age and ancestry, reduced, on average, 4.4 times the malaria odds. Our findings indicate a significant association of TLR9.-1237.T/C gene polymorphism with malarial infection and contribute to a better knowledge of the role of TLRs in malaria susceptibility in an epidemiological setting different from other settings.

Infectious Diseases in Free-Ranging Blonde Capuchins, Sapajus flavius, in Brazil

The main threats to primates worldwide are the degradation, fragmentation, and loss of their habitats; hunting (especially for bushmeat); and illegal trade. For many species, the most important threat is forest fragmentation, resulting in small populations that are restricted to isolated forest patches. In this situation, primates are particularly vulnerable to disease. The Endangered blonde capuchin (Sapajus flavius) is now restricted to a few forest patches in Northeast Brazil. We investigated the occurrence of parasites and bacterial diseases in one of three free-ranging groups of S. flavius in a small forest patch in Paraíba state, Northeast Brazil. We tested for antibodies against Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Leptospira spp. (24 strains), and Brucella spp.. We used molecular analysis to detect Plasmodium spp., and evaluated blood smears for the presence of hemoparasites. All individuals tested negative for Leptospira spp. and B. abortus, but 8 of 48 (16%) presented antibodies for both Leishmania spp. and T. cruzi. We identified antibodies to T. gondii in 12% of the individuals tested. Plasmodium brasilianum infection was present in 4% of the individuals tested, and blood smears showed microfilariae parasites in 46% of the individuals tested. The occurrence of these infectious diseases in S. flavius may pose a significant threat in terms of reduced recruitment and poor survival rates, and an understanding of the influence of pathogens is crucial for the management of small populations of primates.