The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Ancient parasitic DNA reveals Toxascaris leonina presence in Final Pleistocene of South America.

Parasitological analysis of coprolites has allowed exploring ecological relationships in ancient times. Ancient DNA analysis contributes to the identification of coprolites and their parasites. Pleistocene mammalian carnivore coprolites were recovered from paleontological and archaeological site Peñas de las Trampas 1.1 in the southern Puna of Argentina. With the aim of exploring ancient ecological relationships, parasitological analysis was performed to one of them, dated to 16 573-17 002 calibrated years BP, with 95.4% probability. Parasite eggs attributed to Toxascaris sp. by morphological characters were isolated. DNA of coprolite and eggs was extracted to molecular identification. Ancient mitochondrial DNA analysis confirmed the zoological origin of the coprolite as Puma concolor and that of parasite eggs as Toxascaris leonina. This is the oldest molecular parasite record worldwide, and it supports the presence of this parasite since the Pleistocene in America. These findings have implications for the biogeographic history of parasites and for the natural history of the region.

First mitochondrial and nuclear DNA sequences of Lamanema chavezi (Nematoda: Molineidae): Novel findings to improve its identification in feces from South American camelids.

Lamanema chavezi (Family Molineidae) is a parasitic nematode of South American camelids (SACs). A few studies have reported this parasite in SACs, mainly in domestic camelid species (llama and alpaca). Parasite identification by means of copro-parasitological methods is non-invasive and allows performing epidemiological studies. However, egg misidentification and difficulty to culture third-stage larvae do not allow identifying nematodes to species level. In contrast, molecular tools allow identifying eggs of gastrointestinal nematodes more accurately. However, the little genomic information available in databases for some species prevents an accurate diagnosis. In the present work, L. chavezi females present in feces of llamas from northwestern Argentina were molecularly characterized to obtain genomic information and improve parasitological diagnosis of L. chavezi-like eggs present in guanaco feces from southeastern Argentina. An 833-bp fragment of nuclear ribosomal DNA (rDNA) and a 434-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from both L. chavezi females and eggs were amplified and sequenced. Comparison between sequences from females and eggs showed 99-99.6% identity to rDNA and 99.5-96.1% to the cox1 gene fragments, confirming egg morphological assignment. A higher divergence between sequences was observed in the cox1 fragment, with a maximum variation of 3.9%. The examination of eggs found in guanaco feces from southeastern Argentina and their specific molecular identification represent the first record for this host in Argentine Patagonia and contribute to improving the diagnosis of gastrointestinal nematodes in SACs, mainly in wild camelids.

First morphological and molecular analysis of Eucoleus boehmi like eggs in dogs from Argentina.

The canid parasites Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi) parasitize the lower and the upper respiratory tract, respectively. Reports and descriptions of these nematodes are scarce in Argentina, possibly due to misdiagnosis of morphologically similar trichuroids eggs, and the lack of knowledge about the species of Eucoleus in this geographical area. Scanning electron microscopy is a useful tool for identification of E. boehmi eggs based on the characteristics of the shell structure which differentiate between species. Molecular analysis complements morphological identification. Until now, there are no studies based on the analysis of E. boehmi eggs in Argentina. The aim of the present work was to study by morphological, morphometric, and molecular analysis, eggs attributable to E. boehmi isolated from dogs naturally infected in Mar del Plata city, Argentina. Eggs isolated from two dog fecal samples were analyzed by light and scanning electron microscopy. A fragment of mitochondrial DNA (mtDNA) of the cytochrome c oxidase subunit I gene (cox1) from eggs was sequenced, and phylogenetic analysis was performed in this study. According to morphological results based on the wall surface ultrastructure, the eggs studied were assigned to E. boehmi. Molecular analysis supported the morphological identification. The divergence of 9-12% with the European isolated could suggest a new geographical genetic variation of E. boehmi, but also question the possible existence of cryptic species. This is the first characterization of E. boehmi eggs in dogs from Argentina.

Correction: Metformin exhibits preventive and therapeutic efficacy against experimental cystic echinococcosis.

[This corrects the article DOI: 10.1371/journal.pntd.0005370.].

Metformin exhibits preventive and therapeutic efficacy against experimental cystic echinococcosis.

Metformin (Met) is an anti-hyperglycemic and potential anti-cancer agent which may exert its anti-proliferative effects via the induction of energetic stress. In this study we investigated the in vitro and in vivo efficacy of Met against the larval stage of Echinococcus granulosus. Metformin showed significant dose- and time-dependent killing effects on in vitro cultured protoscoleces and metacestodes. Notably, the combination of Met together with the minimum effective concentration of ABZSO had a synergistic effect after days 3 and 12 on metacestodes and protoscoleces, respectively. Oral administration of Met (50 mg/kg/day) in E. granulosus-infected mice was highly effective in reducing the weight and number of parasite cysts, yet its combination with the lowest recommended dose of ABZ (5 mg/kg/day) was even more effective. Coincidentally, intracystic Met accumulation was higher in animals treated with both drugs compared to those administered Met alone. Furthermore, the safe plant-derived drug Met exhibited remarkable chemopreventive properties against secondary hydatidosis in mice. In conclusion, based on our experimental data, Met emerges as a promising anti-echinococcal drug as it has proven to efficiently inhibit the development and growth of the E. granulosus larval stage and its combination with ABZ may improve the current anti-parasitic therapy.

Reprint of "Survey and first molecular characterization of Echinococcus granulosus sensu stricto (G1) in Pampas fox (Lycalopex gymnocercus) in Buenos Aires province, Argentina".

Echinococcosis is a zoonosis caused by tapeworms of the genus Echinococcus. Echinococcus granulosus sensu lato (s. l.) has a world-wide distribution and its transmission is primarily maintained in a synanthropic cycle with dogs as definitive hosts and livestock species as intermediate hosts. However, many wild canids also function as definitive hosts for E. granulosus s. l. Echinococcosis in humans is mainly caused by E. granulosus sensu stricto (s. s.) G1 genotype. In the present work, we expanded the epidemiological study on echinococcosis reported cases in Pampas fox (Lycalopex gymnocercus) to provide a prevalence estimate for rural areas of southern Buenos Aires province, Argentina. Ninety-five whole intestines were analyzed using the sedimentation and counting technique with a result of 83 foxes (87.37%) harboring at least one helminth species. E. granulosus s. l. adults were found in one Pampas fox (1.05%). These adult helminthes were E. granulosus s. s. (G1) according to the genotyping analysis of a 450-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.

Survey and first molecular characterization of Echinococcus granulosus sensu stricto (G1) in Pampas fox (Lycalopex gymnocercus) in Buenos Aires province, Argentina.

Echinococcosis is a zoonosis caused by tapeworms of the genus Echinococcus. Echinococcus granulosus sensu lato (s. l.) has a world-wide distribution and its transmission is primarily maintained in a synanthropic cycle with dogs as definitive hosts and livestock species as intermediate hosts. However, many wild canids also function as definitive hosts for E. granulosus s. l. Echinococcosis in humans is mainly caused by E. granulosus sensu stricto (s. s.) G1 genotype. In the present work, we expanded the epidemiological study on echinococcosis reported cases in Pampas fox (Lycalopex gymnocercus) to provide a prevalence estimate for rural areas of southern Buenos Aires province, Argentina. Ninety-five whole intestines were analyzed using the sedimentation and counting technique with a result of 83 foxes (87.37%) harboring at least one helminth species. E. granulosus s. l. adults were found in one Pampas fox (1.05%). These adult helminthes were E. granulosus s. s. (G1) according to the genotyping analysis of a 450-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.

The Pampas fox (Lycalopex gymnocercus) as new definitive host for Spirometra erinacei (Cestoda: Diphyllobothriidae).

Spirometra erinacei, Faust, Campbell and Kellogg, 1929, is a pseudophyllidean cestode of the family Diphyllobothriidae. The genus Spirometra is cosmopolitan and these parasites infect carnivores, specially felids and canids. In Argentina, S. erinacei and S. mansonoides have been reported sporadically only in domestic definitive hosts. The Pampas fox, Lycalopex gymnocercus, is the most abundant native carnivore in southern South America, where it inhabits grasslands and open woodlands and areas highly modified by extensive ranching and agricultural activities. This report describes the first finding of S. erinacei infecting Pampas fox, and provides an estimate prevalence of this cestode in rural areas of southern Buenos Aires province, Argentina based on 78 complete Pampas fox intestine samples analysis. This study found a 15.4% of prevalence of S. erinacei in small intestine (adult stage) and a 21.8% in fecal samples (egg stage). In the present work, the first case of S. erinacei in a wild definitive host from Argentina was reported expanding the list of definitive hosts of S. erinacei in South America.

Epidemiological studies on Echinococcus in Pampas fox (Lycalopex gymnocercus) and European hare (Lepus europaeus) in Buenos Aires province, Argentina.

In Argentina, hydatid disease caused by Echinococcus granulosus is widespread. The south of Buenos Aires province, Argentina, is one of the three regions where hydatidosis is endemic. Although domestic dogs and sheep are considered to be the main hosts for E. granulosus, the potential role of wildlife in the local transmission of E. granulosus has not been investigated. The aim of this study was to estimate the hydatidosis/echinococcosis prevalence in European hare (Lepus europaeus) and Pampas fox (Lycalopex gymnocercus), two abundant species with a strong predator-prey relationship in rural areas of Buenos Aires province using different diagnostic tests. A total of 61 fox intestines were examined, finding that 52 (85.2%) harbored at least one helminth species. However, no adult or immature form of Echinococcus sp. was found in the intestinal contents. Coproparasitological analysis and Copro-ELISA followed by Copro-PCR were used as supplementary diagnostic tests. Only one (1.7%) of 59 fecal samples was positive to Taeniidae eggs by coproparasitological analysis, but this same sample was negative by the Copro-ELISA test. The analysis by Copro-ELISA showed 6 of 57 (10.6%) positive samples, but the Copro-PCR tests carried out on these samples were negative to E. granulosus. A total of 6,808 lungs, 3,576 livers, and 3,542 hearts of hunted hares were examined and palpated, but no structure resembling hydatid cysts were detected. Our results suggest that hares and Pampas foxes are not currently important wild reservoirs of E. granulosus in the studied area.

First palaeoparasitological record of a dioctophymatid egg in an archaeological sample from Patagonia.

The collection of parasitological information from ancient material requires an exhaustive study of samples. In 2005, cestode and nematode eggs were found in a coprolite sample tentatively assigned to a canid. The sample was obtained from the layer of the archaeological site located in Cerro Casa de Piedra, Santa Cruz Province, Argentina, and dated from 6540±110 years before present. The aim of the present work was to reexamine this fixed sample in order to confirm the presence of these parasites. The palaeoparasitological results support our previous findings. Interestingly, another parasite was also confirmed: a dioctophymatid nematode. Dioctophyma renale has been reported in several modern carnivores in the Southern Hemisphere but in ancient materials, it has only been reported in human coprolites from Switzerland. This report constitutes the first evidence of the presence of a dioctophymatid nematode parasite dioctophymatid nematode in American pre-Columbian times. The results obtained in this work show the importance of revising earlier palaeoparasitological results.

Genetic diversity of Anaplasma marginale in Argentina.

Bovine anaplasmosis caused by Anaplasma marginale is a worldwide major constraint to cattle production. The A. marginale major surface protein 1 alpha (msp1alpha) gene contains a variable number of tandem repeats in the amino terminal region and has been used for the characterization of pathogen genetic diversity. This study reports the first characterization of A. marginale genetic diversity in Argentina based on msp1alpha genotypes and its putative relationship with Rhipicephalus (Boophilus) microplus infestations. Herein, we analyzed whole blood bovine samples from anaplasmosis outbreaks in R. microplus infested (9 samples) and eradicated/free (14 samples) regions. Sequence analysis revealed the existence of 15 different msp1alpha genotypes with 31 different repeat units. Six new repeat sequences were discovered in this study and 13/31 (42%) repeats were unique to Argentinean strains. The analysis of msp1alpha repeat sequences according to R. microplus infestations resulted in three repeat groups: (i) found in tick-infested regions (20 repeats), (ii) found in tick free regions (6 repeats) and (iii) randomly distributed (5 repeats). Moreover, A. marginale msp1alpha genetic diversity was higher in tick-infested regions than in tick free areas. These results, together with previous evidence suggesting that A. marginale msp1alpha repeat units co-evolved with the tick vector, might represent an evidence of the role of tick-mediated transmission for the generation of pathogen genetic diversity.

Flow cytometry to evaluate Anaplasma marginale parasitemia using a fluorescent nucleic acid stain.

In this work we describe a flow cytometry-based method using SYTO16 (a DNA intercalating agent) to quantify Anaplasma marginale-infected erythrocytes in blood from bovine animals. The linearity and reproducibility of the results obtained with SYTO16 labeling followed by flow cytometry analysis make it a suitable approach for measurement of parasitemia in A. marginale infections.

Improved molecular tools for detection of Babesia bigemina.

Molecular detection of Babesia bigemina involves a nested PCR protocol and reverse line blot hybridization (RLBH) assay based on the 18S gene. In this study, we report the development of molecular tools for improving B. bigemina detection in bovine blood-a one-step PCR assay based on the amplification of rap-1a paralogous and a new RLBH Babesia spp. 18S probe. The one-step PCR assay is highly specific, with an estimated analytical sensitivity corresponding to 0.00002% parasitemia. The RLBH assay, with a new B. bigemina probe, allows the detection of all tested B. bigemina isolates showing no cross-hybridization with B. bovis 18S gene. By developing this highly specific and sensitive one-step PCR and upgrading the RLBH assay for B. bigemina, we have improved molecular assays which, together with serologic methods, provide valuable tools for epidemiologic studies of bovine babesiosis.