Sylvatic Isolates Research Articles

Multilocus sequence typing: genetic diversity in Trypanosoma cruzi I (TcI) isolates from Brazilian didelphids

BackgroundTrypanosoma cruzi is a protozoan parasite characterized by extensive genetic heterogeneity. There are currently six recognised, genetically distinct, monophyletic clades designated discrete typing units (DTUs). TcI has the broadest geographical range and most genetic diversity evidenced by a wide range of genetic markers applied to isolates spanning a vast geographical range across Latin America. However, little is known of the diversity of TcI that exists within sylvatic mammals across the geographical expanse of Brazil.ResultsTwenty-nine sylvatic TcI isolates spanning multiple ecologically diverse biomes across Brazil were analyzed by the application of multilocus sequence typing (MLST) using four nuclear housekeeping genes. Results revealed extensive genetic diversity and also incongruence among individual gene trees. There was no association of intralineage genotype with geography or with any particular biome, with the exception of isolates from Caatinga that formed a single cluster. However, haplotypic analyses of METIII and LYT1 constitutive markers provided evidence of recombination events in two isolates derived from Didelphis marsupialis and D. albiventris, respectively. For diversity studies all possible combinations of markers were assessed with the objective of selecting the combination of gene targets that are most resolutive using the minimum number of genes. A panel of just three gene fragments (DHFR-TS, LYT1 and METIII) discriminated 26 out of 35 genotypes.ConclusionsThese findings showed geographical association of genotypes clustering in Caatinga but more characteristically TcI genotypes widely distributed without specific association to geographical areas or biomes. Importantly, we detected the signature of recombination events at the nuclear level evidenced by haplotypic analysis and incongruence.

Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses.

The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian/American ZIKV lineage.

Evaluación de la variabilidad genética de aislamientos colombianos de Trypanosoma cruzi mediante marcadores microsatélites

IntroductionChagas disease which is caused by the protozoan Trypanosoma cruzi, is a major public health problem in Latin American countries with a different distribution of the parasite across the continent in which nearly 15 million people are infected and 28 million are at risk. Genetic profiling of T. cruzi has shown great diversity and variability of the parasite allowing its classification into six discrete typing units (DTUs I-VI) in which TcII to TcVI are well characterized by different molecular markers. However, the presence of four subgroups according to the transmission cycle has only been reported in DTU T. cruzi I. ObjectiveTo evaluate the genetic variability evidenced within TCI by the use of microsatellite markers. Materials and methodsSeven microsatellite loci were tested in fifteen Colombian isolates from vectors, reservoirs, and humans by means of PCR and automatic laser fluorescent sequencer (ALF). Data were analyzed using a population genetic data analysis software (Arlequin® 3.1 and Microsat®). ResultsVariability among the isolates was demonstrated with 24 alleles, from which twelve had never been reported before. However, none of the microsatellite loci were able to support the idea of genotypes within TcI strains. The parameters of expected and observed heterozygocity allowed us to determine presence of two populations (domestic and sylvatic isolates); likewise, the linkage disequilibrium helped with the construction of two physical maps for the loci analyzed. ConclusionsWe corroborated the high genetic variability displayed by TcI populations suggesting a pattern of intraspecific variation in Colombia.

Morphological and molecular characterization of sylvatic isolates of Trichinella T9 obtained from feral raccoons (Procyon lotor)

Morphological and molecular characterization of sylvatic isolates of Trichinella T9 obtained from feral raccoons (Procyon lotor)

Major cysteine protease (cruzipain) in Z3 sylvatic isolates of Trypanosoma cruzi from Rio de Janeiro, Brazil

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is represented by a set of parasites which circulate between man, vectors, domestic and wild animals. Recently, our group isolated from Triatoma vitticeps strains of T. cruzi that were characterized as belonging to the Z3 phylogenetic lineage. Since very little is known about the biological and/or biochemical markers of sylvatic Z3 isolates, we have studied the protein and protease profiles of distinct Z3 isolates designated as SMM10, SMM53, SMM88, and SMM98. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both quantitative and qualitative differences were observed in the protein profiles of these strains. All strains produced an acidic cysteine protease of 45 kDa, resembling cruzipain activity. The strain SMM10 synthesized an additional 55 kDa metalloprotease. Using Western blotting and anti-cruzipain antibody to detect cruzipain-like molecules, a 40-kDa reactive molecule was identified in all strains; in the strain SMM10, an 80-kDa protein was also reacted. Studies about cruzipain isoforms from sylvatic parasites could be valuable tools in the comprehension of the genetic variability in the pathogenesis of Chagas' disease.

Predominance of Trypanosoma cruzi I among Panamanian sylvatic isolates

Trypanosoma cruzi is throughout Panama, which is in agreement with the widespread of the sylvatic vectors implicated in the transmission. Eco-epidemiological changes in some regions of the country have led to a successful dissemination of the palm-tree Attalea butyracea and a possible adaptation of the primary vector of Chagas’ disease to human settlements. These facts might increase both vector-human contact and human infection with different potentials T. cruzi genotypes and make therefore necessary a study to disclose Panamanian T. cruzi make-up. In this study, 71 T. cruzi isolates from Rhodnius pallescens were analyzed using mini-exon gene and sequence-characterized amplified region markers. The analyzed strains were T. cruzi lineage I. This finding along with prior results indicates that T. cruzi I is the principal genotype circulating in both sylvatic and domestic/peridomestic cycles and consequently responsible for the disease in the country.

Dengue Emergence and Adaptation to Peridomestic Mosquitoes

Phylogenetic evidence suggests that endemic and epidemic dengue viruses (DENV), transmitted among humans by the anthropophilic mosquitoes Aedes aegypti and Ae. albopictus, emerged when ancestral, sylvatic DENV transmitted among nonhuman primates by sylvatic Aedes mosquitoes adapted to these peridomestic vectors. We tested this hypothesis by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DENV-2 to Ae. albopictus and Ae. aegypti. First and second-generation offspring of mosquitoes from different geographic regions in the Americas and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DENV-2 isolates from West Africa, Southeast Asia, and Oceania. Both Aedes species were highly susceptible (up to 100% infected) to endemic/epidemic DENV-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DENV-2 strains. Our findings support the hypothesis that adaptation to peridomestic mosquito vectors mediated dengue emergence from sylvatic progenitor viruses.

Trypanosoma cruzi infection in Leontopithecus rosalia at the Reserva Biológica de Poco das Antas, Rio de Janeiro, Brazil.

Wild golden lion tamarins (Leontopithecus rosalia) - endangered primates that are native to the Brazilian Atlantic coastal forest - were surveyed for the presence of Trypanosoma cruzi with the use of Giemsa-stained blood smears, hemocultures and an indirect immunofluorescence assay (IFAT). Positive IFAT with titers ranging from 1:20 to 1:1280 were observed in 52% of the 118 wild tamarins examined and the parasite was isolated from 38 tamarins. No patent parasitemia was observed among the tamarins from which T. cruzi was isolated. Serum conversion and positive hemoculture was observed for three animals that had yielded negative results some months earlier, which indicates that T. cruzi is actively transmitted among tamarins. In contrast to observations with other sylvatic isolates, those from the tamarins were significantly more virulent and most of them produced mortality in experimentally infected Swiss mice. Some variation in the kDNA restriction profiles among the isolates was observed. Electrophoresis with GPI, G6PDH, IDH, MDH and ME enzymes showed a Z2 profile.

Genetic, Phenotypic, and Behavioral Variation in North American Sylvatic Isolates of Trichinella

Two restriction endonucleases (Cla I and Hpa II) produced polymorphic repetitive DNA profiles which were used in a clustering analysis to quantify the level of genetic variation among 14 North American sylvatic isolates of T5 Trichinella. Differences in genetic profiles reflected phenotypic differences in parasite reproductive success as measured by an isolate's reproductive capacity index in natural hosts. Two genetically distinct isolates of the T5 genotype and T. spiralis were used to infect white-footed mice Peromyscus leucopus for a behavioral analysis. Three behavioral characteristics were tabulated from videotapes: time to emerge from cage, time spent inside cage, and number of rears (index of exploratory activity). Mice infected with 400 T. spiralis larvae showed a significant decrease in exploratory activity and significant increases in time to emerge and time spent inside cage, whereas mice infected with 200 larvae displayed no differences in behavior. Mice infected with a T5 isolate from a raccoon (R9) also displayed decreased exploratory activity. In contrast, mice infected with the other T5 isolate from a coyote (C26) showed relatively constant rearing levels but spent less time inside their cages during the late period of the infection. Results suggest that different genetic strains of Trichinella induce varied types of behavioral modifications upon their hosts.

Biochemical, Biological, and Genetic Characterization of a Sylvatic Isolate of Trichinella

Biological, biochemical, and genetic relationships of Trichinella isolates were assessed and compared from 3 species of Illinois fur-bearing mammals. Tongue muscle collected from 1987 through 1989 from 323 raccoons (Procyon lotor), 9 red fox (Vulpes fulva), and 1 coyote (Canis latrans) were digested and Trichinella muscle larval prevalences and mean intensities (larvae/g) determined. The prevalence and mean intensity of tongue muscle-stage larvae were 2.8% and 44.4% and 326 and 2 larvae/g for raccoon and red fox, respectively. The single coyote examined for muscle larvae was negative. Seven of 13 Trichinella isolates (5 raccoon, 2 red fox) were maintained and amplified in mice. Comparative analyses of DNA from larvae of these isolates and from other Trichinella isolates were performed by dot-blot hybridization using specific repetitive DNA probes. DNA from the 7 Illinois sylvatic isolates reacted with the Trichinella T5 isolate DNA probe pUPB-3.7 and did not cross-react with the Trichinella spiralis probe pBP-2. The sylvatic isolates of Trichinella were also analyzed using 27 allozymes. Their allozymic patterns were similar to Trichinella reference strain T5 from Pennsylvania, except for mannose phosphate isomerase. These results suggest that the Illinois wildlife isolates belong to the Trichinella T5 genotype according to the classification system established by the International Trichinella Reference Centre and as defined here by positive hybridization to the pUPB-3.7 probe. Results from Southern blot analyses using rRNA as a probe and allozyme patterns revealed some heterogeneity among geographical isolates of the T5 genotype.

Biological Characterization of Trichinella Isolates from Various Host Species and Geographical Regions

Forty isolates of Trichinella collected from 5 continents were compared for 7 biological characters: newborn larvae produced per female worm cultured in vitro at the seventh, eighth, and ninth day postinfection, host muscle nurse cell development time, reproductive capacity index in rats and chickens, and resistance of muscle larvae to freezing. The isolates also were compared by analyses of an environmental character of the location from which they were isolated: the isotherms for January and July. By factorial analysis of correspondence of the biological and environmental data, the 40 isolates were grouped into 8 gene pools (T1-T8). The environmental temperature-related distribution was more evident for the sylvatic isolates (T2, T3, T5, T6, T7, T8), than for T1, which was isolated from domestic pigs, and for T4, a bird-adapted, nonencapsulating genetic type. The 8 biological groups correlated closely with the 8 gene pools previously identified on the basis of allozyme analysis. These results support the concept that the genus Trichinella is composed of at least 5 distinct gene pools or sibling species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella sp. (T3), Trichinella pseudospiralis (T4), and Trichinella nelsoni (T7), and 3 other groups of uncertain taxonomic status (i.e., T5, T6, and T8).

The Use of the Polymerase Chain Reaction to Identify Porcine Isolates of Trichinella

A method was developed to identify domestic isolates of Trichinella using the polymerase chain reaction. Oligonucleotide primers, based on the repetitive DNA sequence (pPRA) from the P1 isolate of Trichinella, were used to amplify genomic DNA from 13 domestic isolates and tested against sylvatic isolates of Trichinella. Pattern differences were observed among domestic isolates, indicating divergence of this repetitive sequence. The primers were specific for domestic Trichinella as no amplification was detected for sylvatic isolates or Trichinella pseudospiralis. It was possible to identify an isolate from a single larva following digestion or in situ in muscle tissue.

A repetitive DNA probe specific for a North American sylvatic genotype of Trichinella

A partial genomic DNA library constructed in pUC 13 using DNA from a sylvatic isolate of Trichinella spiralis (T. spiralis T5) was differentially screened with radiolabeled homologous genomic DNA and with DNA from T. spiralis T1. One clone was identified and designated pUPB-3.7 which, by slot blot and Southern blot analyses, reacted specifically with T. spiralis T5 DNA and did not cross-react with DNA from any other T. spiralis genotype. The 482-bp repetitive sequence which is 70% rich in A and T residues, comprises at least 2.7% of the parasite genome and can detect as little as 0.4 ng of DNA. When used to assess the prevalence of T. spiralis T5 in Indiana wildlife, DNA from 19 of 20 independently obtained sylvatic isolates reacted positively with the pUPB-3.7 probe indicating that within this geographical locality, T. spiralis T5 is the predominating genotype in wild mammals

Molecular Characterization of Sylvatic Isolates of Trichinella spiralis

Genetic relationships of 20 Trichinella isolates from Indiana wildlife were assessed and compared to Trichinella isolated from an infected swine herd. Trichinella larvae were isolated from coyotes, mink, raccoons, and red foxes. The larvae were maintained and amplified in white mice (ICR) and wild mice (Peromyscus leucopus). Differences in phenotypic characters of sylvatic isolates in the 2 laboratory hosts included an approximately 10-30-fold increase in parasite fecundity in wild mice. DNA for each isolate was extracted from Trichinella larvae and analyzed by dot-blot hybridization using a repetitive DNA probe pBP2 that recognizes DNA sequences specific for swine Trichinella. The probe hybridized only to Trichinella from swine and a single coyote isolate. Restriction endonucleases were used to digest DNA and the resulting fragments were separated by gel electrophoresis. Based on the presence of repetitive DNA sequences in the Trichinella genome, distinctive banding patterns were seen among the isolates. Trichinella isolated from swine had a pattern distinct from all sylvatic isolates except 1 from a coyote. Because this coyote was from the same general locality as the swine Trichinella outbreak, it was concluded that the isolate represents transmission of swine trichinellosis to the wildlife population. Further analysis using the enzyme Cla I identified unique banding patterns for wild isolates, suggesting that the sylvatic group is a genetically heterogeneous complex.

Trichinella spiralis: Genetic evidence for synanthropic subspecies in sylvatic hosts

Isolates of the nematode genus Trichinella from sylvatic hosts differ in their potential to reproduce in domestic swine. The structure of the genomic DNA from 13 sylvatic isolates from North America and 5 pig isolates, 4 from North America and 1 from Asia, was examined and correlated with the infectivity of the isolate for domestic pigs. DNA restriction fragment length differences, identified by ethidium bromide staining and by hybridization with 32P-labeled ribosomal RNA, served as molecular markers to classify each isolate. All 5 pig isolates and 8 of 13 sylvatic isolates had a high infectivity and reproductive capacity in pigs. All isolates that were highly infectious for pigs regardless of host origin had similar DNA characteristics and were classified operationally as T. spiralis spiralis (pig) and those of the second group as T. spiralis ssp. A DNA clone of repetitive DNA from T. s. spiralis, pBP2, was selected from a library of genomic DNA in plasmid pUC8. When used as a probe, pBP2 hybridized only to the DNA of T. s. spiralis isolates, thus making it a useful diagnostic reagent to predict whether new isolates are highly infectious for pigs (i.e., T. s. spiralis). These results show that T. s. spiralis occurs in wild mammals and this should be considered a serious obstacle to efforts to eradicate trichinellosis from domestic swine.

A Comparison of Three Geographical Isolates of Trichinella spiralis from the Mid-Atlantic United States

Three isolates of Trichinella spiralis (black bear: 41o50'N, 79o00'W, 1982; grey fox: 41o00'N, 76o00'W, 1982; domestic pig: 39o80'N, 75o30'W, 1983) from the mid-Atlantic United States were characterized in Crl: COBS CFW (SW) mice during 10 successive 40-day generations. Over 10 generations, the isolates differed as follows: worm position of the ursine isolate was significantly more posteriad compared to the porcine isolate; sex ratio of the vulpine isolate was significantly lower compared to the ursine and porcine isolates; females of the porcine isolate produced significantly more newborn larvae in vitro than the sylvatic isolates; both the larvae per gram (LPG) and reproductive capacity index (RCI) were significantly higher for the porcine isolate; and male worms of the vulpine isolate were significantly smaller than those of the porcine or ursine isolates. No differences were observed among isolates for percentage of inoculum recovered, length of female worms, or uterine length. The fecundity of the isolates in hamsters, gerbils, multimammate rats, Sprague-Dawley rats, and Peromyscus sp. was examined also. Based upon RCI and LPG, the porcine isolate was the most fecund in all hosts except gerbils. This higher fecundity was often manifest as increased morbidity and mortality in hosts infected with this isolate. The relationship between the virulent nature of the porcine isolate and the enhanced transmission of T. spiralis are discussed.