Volume 58, Issue 2

In this review, we examine the state-of-the-art technologies (gas and liquid chromatography, mass spectroscopy and nuclear magnetic resonance, etc.) in the well-established area of metabolomics especially as they relate to protozoan parasites.

Chlorazol Black E (CBE) stain has been used for the detection and identification of intestinal parasitic protozoa. In recent years, genotyping of protozoa has been performed to examine pathogenicity and for epidemiologic analysis. In this study, protozoan DNA was amplified from preserved human fecal specimens stained with CBE that were positive for Giardia intestinalis (syn. G. lamblia and G. duodenalis), Chilomastix mesnili, Pentatrichomonas hominis, and Entamoeba histolytica. DNA was amplified from 11 of the 12 (91.6%) samples examined. DNA from CBE-stained smears of G. intestinalis, E. histolytica, and P. hominis was amplified, whereas any amplification product could not be obtained from one of three smears of C. mesnili. Storage term and protozoan number had no association with results of PCR amplification. In genotyping of G. intestinalis, four out of six (66.7%) samples were of genotype AI, while the remaining two (33.3%) samples were of genotype B. The amplified DNA sequences showed high similarity (>99%) with that of G. intestinalis in the GenBank database. These results suggest that DNA remains stable in CBE-stained smears for long term. The present study demonstrates that nuclear extracts from specimens stained with CBE can be amplified by PCR and suggests that specimens stored for extended periods could be applied to genetic and prospective epidemiologic analyses.

The objective of this study was to investigate if there is specific host-parasite association in Chilean populations of Trypanosoma cruzi. For this purpose, two groups of parasites were analyzed, one from chronic chagasic patients, and the other from Triatoma infestans triatomines in three regions of the country. The first group consisted of four types of samples: parasites from peripheral blood of non-cardiopathic T. cruzi infected patients (NB); parasites from their corresponding xenodiagnosis (NX); parasites from peripheral blood of T. cruzi infected cardiopathic patients (CB) and parasites from their xenodiagnostics (CX). The T. infestans sample in turn was from three regions: III, V and M (Metropolitan). The genetic differentiation by the Fisher exact method, the lineage distribution of the samples, the molecular phylogeny and the frequency of multiclonality were analysed. The results show that not only are the groups of T. cruzi clones from Chagas disease patients and vectors genetically differentiated, but also all the sub-groups (NB, NX, CB and CX) from the III, V and M regions. The analysis of lineage distribution was concordant with the above results, because significant differences among the percentages of TcI, TcIII and hybrids (TcV or TcVI) were observed. The phylogenetic reconstruction with these Chilean T. cruzi samples was coherent with the above results because the four chagasic samples clustered together in a node with high bootstrap support, whereas the three triatomine samples (III, V and M) were located apart from that node. The topology of the tree including published T. cruzi clones and isolates was concordant with the known topology, which confirmed that the results presented here are correct and are not biased by experimental error. Taken together the results presented here are concordant with a specific host-parasite association between some Chilean T. cruzi populations.

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585–1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96–95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.

Perumytilus purpuratus is an abundant bivalve located in the intertidal rocky zone of South America that has been considered as a key species of the ecosystem. There are few studies of the host-parasite relationship of this bivalve; thus, this research aims to analyse the spatial and temporal variation in the prevalence of trematodes in P. purpuratus. Bivalves were collected from three localities (El Tabo, Las Cruces and Montemar) of central Chile (33°S, 71°W) during different seasons of 2010. The bivalves were also collected every metre, from the lowest to the highest level of the intertidal rocky zone, to determine the parasite distribution within the localities. Three species of trematodes as sporocyst stages were found: Prosorhynchoides carvajali, Proctoeces sp. and an undetermined fellodistomid species. Of the 37,692 bivalve specimens collected, 2.68% were parasitised. The undetermined fellodistomid species was the most prevalent parasite observed (1.69%). There were little detected differences in the prevalence of some trematode species between seasons. The prevalence of P. carvajali varied between localities, being most prevalent at Montemar. The distribution of trematodes along the rocky zone within the localities was variable, with P. carvajali being more prevalent in the mid-lowest level of the intertidal zone and the undetermined fellodistomid species being more prevalent in the mid-highest level. Both the abundance of definitive hosts and the environmental conditions likely result in different levels of infection by trematodes in P. purpuratus between and within the localities.

Seven species of Cylicocyclus Ihle, 1922 (Nematoda: Strongylidae) were collected from donkeys from Henan Province, China. Five samples of each species were selected for sequencing. Sixteen different internal transcribed spacer (ITS) sequences representing the seven species of Cylicocyclus were obtained. Sequence differences in the first internal transcribed spacer (ITS-1) among species was lower than that of the second internal transcribed spacer (ITS-2). Phylogenetic analyses were conducted using the combined ITS-1 and ITS-2 data sets from the present study and using reference sequences from the GenBank database. The MP and ML trees were similar in topology. The phylogenetic trees were divided into two clades. Clade I included 8 species of Cylicocyclus; within this group, Cylicocyclus leptostomus (Kotlan, 1920) is nested between different samples of Cylicocyclus ashworthi (LeRoux, 1924), suggesting C. ashworthi may represent a species complex. Clade II included Cylicocyclus elongatus (Looss, 1900) and Cylicocyclus ultrajectinus (Ihle, 1920); however, these two species always clustered with the comparative species (Petrovinema poculatum (Looss, 1900) and Poteriostomum imparidentatum Quiel, 1919), suggesting that C. elongatus and C. ultrajectinus represent members of other genera.

Acari ectoparasites were collected from bats during 12 months in the Rio Negro farm (19°34′22″S and 56°14′36″W), Aquidauana, Mato Grosso do Sul. A total of 654 bats belonging to the families Phyllostomidae, Noctilionidae, Molossidae, Vespertilionidae and Emballonuridae were captured. Only 136 bats of nine genera and 11 species were parasitised. Periglischrus iheringi Oudemans was the most abundant mite species, and this prevalence may be related to the low degree of host specificity of this species and due to the broad geographical distribution of its hosts. The greatest mean intensity was found to Periglischrus torrealbai Machado-Allison on Phyllostomus discolor Wagner (Phyllostomidae) and Periglischrus tonatii Herrin and Tipton associated with Lophostoma silviculum d’Orbigny (Phyllostomidae), which also had the highest prevalence of infestation.

We studied variation of ectoparasite load in a free ranging populations of Mehely’s horseshoe bat (Rhinolophus mehelyi) on five successive occasions in a nursery roost in western Iran. In total, 87 Rhinolophus mehelyi were captured. The patterns of abundance differed greatly among parasite species but total parasite load was markedly higher in pregnant females in spring and early summer and lower in solitary males. On average, 90% of bats were infested by Eyndhovenia sp. with a mean intensity of 13.79 individuals per bat. Penicillidia sp. and one species from Streblidae were found in 66.7% and 11.49% of bats with parasite load of 2.31 and 1.8 parasite per bat, respectively. Using ratio of forearm length to body mass as an indication of bat health the correlation coefficient between parasite load and the health indicator was 0.002 for males and 0.06 for females indicating that parasite load has no apparent impact on bat’s health.

Amoebiasis is one of the most important infectious diseases afflicting mainly tropical and subtropical countries. This study was carried out in the Sharjah Emirate, UAE in order to accurately detect and differentiate Entamoeba histolytica, Entamoeba dispar and E. moshkovskii in fecal samples collected from the Sharjah municipality public health clinic by ELISA and nested polymerase chain reaction (PCR). One hundred and twenty specimens were examined and the PCR was positive for E. histolytica, E. dispar and E. moshkovskii (collectively referred to as Entamoeba complex) in 19.2% (23 out of 120). Of those, 10% (12/120) were mono — infection with E. histolytica; 2.5% (3/120) with E. dispar; and 2.5% (3/120) E. moshkovskii. The nested PCR also detected mixed infections by both E. histolytica and E. dispar in 3.3% (4/120) and E. dispar and E. moshkovskii in 0.8% (1/120). The TechLab ELISA kit failed to detect E. histolytica in any of the E. histolytica PCR positive samples. Overall, the percentage of E. histolytica including those found in mixed infections was 13.3% (16/120). Compared to nested PCR, microscopy was found to have an overall sensitivity of 52.2% and a specificity of 75.2% for detection of Entamoeba complex. The present study indicates that E. histolytica is present in the UAE with an average incidence rate of 13.3%. However, larger studies need to be conducted in order to confirm these findings. We propose the use of PCR in both the routine diagnosis of amoebiasis and epidemiological survey in the UAE.

The parasitic infections of market derived Osteolaemus tetraspis from the rainforest and Varanus ornatus from locations in the savanna-mosaic and the rainforest of southern Nigeria were investigated. Parasites recovered from O. tetraspis included members of the Pentastomida, Trematoda and Nematoda. An undescribed pentastomid belonging to the family Sebekidae was recovered from O. tetraspis. The same parasite was also found to parasitize V. ornatus from the rainforest. Other parasites found in O. tetraspis were Pseudoneodiplostomum thomasi, Dujardinascaris sp. and larva of a Camallanus sp. Varanus ornatus from the rainforest and the derived savanna had some parasites including Duthiersia fimbriata, an unidentified pseudophyllidean cestode and Tanqua tiara in common. Cosmocerca ornata and Oswaldocruzia hoepplii were restricted to hosts from the derived savanna while the unidentified trematode occurred only in lizards from the rainforest. The unidentified pseudophyllidean cestode bears a close resemblance to Probothriocephalus, a cestode previously reported only from deep water teleosts. Pseudoneodiplostomum thomasi and Duthiersia fimbriata are new locality records for Nigeria.

Not only do males and females of many species vary in their responses to certain parasitic infections, but also to treatments such as vaccines. However, there are very few studies investigating differences among sexes following vaccination and infection. Here we demonstrate that female Sprague-Dawley rats vaccinated with cDNA encoding a recently discovered cysteine proteinase of Fasciola hepatica (FhPcW1) develop considerably lower liver fluke burdens after F. hepatica infection than their male counterparts. This is accompanied by differences in the course of their immune responses which involve different eosinophil and monocyte responses throughout the study as well as humoral responses. It is evident that host gender influences the outcome of parasitic infections after vaccination and research on both sexes should be considered when developing new treatments against parasites.

In this work we record the highest number of bat flies species among those already performed in the Brazilian cerrado and discuss the associations and patterns of parasitism of these species and their hosts. A total of 1,390 ectoparasitic flies were collected, belonging to 24 species of Streblidae and one of Nycteribiidae, parasitizing 227 bats of 15 species. Among the species found, the presence of Trichobius sp. on Lonchophylla mordax and the first occurrence of Hershkovitzia sp. on Thyroptera devivoi are highlighted. Lophostoma species presented the highest proportion of individuals with infracommunities and the highest values of parasitological indexes. The high number of bat fly species and hosts, as well as the high values for rates of parasitism and infracommunities, suggests that this area of cerrado has good shelter conditions for these species. The abundance of species and high rates of parasitism detracts from the hypothesis that a higher mean intensity of ectoparasites results from lower competition among flies for hosts in areas with lower ectoparasite species richness. Biogeographical and historical factors of host populations, besides the number of host species and individuals sampled, may contribute to species number and intensity of parasitism.

The liver fluke Fasciola hepatica infects a wide range of hosts, and has a considerable impact on the agriculture industry, mainly through infections of sheep and cattle. Further, human infection is now considered of public health importance and is hyperendemic in some regions. The fluke infection causes considerable damage to the hosts’ liver. However, the mechanisms of liver destruction have not yet been completely elucidated. In the present report we incubated a human liver cell line in the presence of either F. hepatica excretory/secretory material (FhES) or recombinant phosphoglycerate kinase (FhPGK). Dosedependent cytotoxicity in the presence of FhES was observed, indicating that FhES is capable of killing human hepatocytes, supporting a role for FhES in damaging host liver cells during infection; while treatment with a recombinant intracellular protein — FhPGK, had no impact on cell survival.

This study aimed to determine whether asymptomatic horses naturally infected with Theileria equi retain infected erythrocytes in the spleen and whether the presence of the hemoparasite in this organ is associated with parasitemia. We collected samples from 25 adult horses without clinical signs of any disease. From each animal, we collected whole blood samples from the jugular vein and a splenic puncture blood sample. All samples were submited to blood cell counts and detection of Theileria or Babesia. DNA extraction and PCR were performed in all samples for identification of piroplasm infection (T. equi and B. caballi). From the 25 horses evaluated for piroplasm detection by PCR, seven horses (28%) were positive in jugular vein blood but negative in splenic blood samples, five horses (20%) were positive in splenic blood samples but negative in jugular vein blood samples, and 13 horses (52%) were positive in both jugular vein and splenic blood samples. The hematological evaluation revealed anemia in 13 of 25 (52%) infected horses, lymphopenia in five (20%), neutrophilia in two (8%), neutropenia in one (4%), and thrombocytopenia in one (4%) infected horse. The present study demonstrated that several (20%) of the asymptomatic piroplasm carrier horses did not show parasitemia, but show infected erythrocytes in the spleen.

A high infestation of the monogenean Gussevia tucunarense in a cultivation of bujurqui-tucunare was reported. The prevalence was 100%. The mean intensity and abundance of the parasite was 164.4 of parasites per individual. This is the first report of a high infestation by G. tucunarense in C. semifasciatus cultured from the Peruvian Amazon.

The public health importance of Kudoa infection in fish remains unclear. Recently in Japan a Kudoa species, K. septempunctata, was newly implicated as a causative agent of unidentified food poisoning related to the consumption of raw olive flounder. Other marine fishery products are also suspected as causative raw foods of unidentified food poisoning. For this study, we detected kudoid parasites from sliced raw muscle tissues of a young Pacific bluefin and an adult yellowfin tuna. No cyst or pseudocyst was evident in muscles macroscopically, but pseudocysts were detected in both samples histologically. One substitution (within 1100 bp overlap) and ten substitutions (within 753 bp overlap) were found respectively between the partial sequences of 18S and 28S rDNAs from both isolates. Nucleotide sequence similarity searching of 18S and 28S rDNAs from both isolates showed the highest identity with those of K. neothunni from tuna. Based on the spore morphology, the mode of parasitism, and the nucleotide sequence similarity, these isolates from a Pacific bluefin and a yellowfin tuna were identified as K. neothunni. Phylogenetic analysis of the 28S rDNA sequence revealed that K. neothunni is classifiable into two genotypes: one from Pacific bluefin and the other from yellowfin tuna. Recently, an unidentified kudoid parasite morphologically and genetically similar K. neothunni were detected from stocked tuna samples in unidentified food poisoning cases in Japan. The possibility exists that K. neothunni, especially from the Pacific bluefin tuna, causes food poisoning, as does K. septempunctata.