Ability of real-time PCR for differential diagnosis of various forms of cutaneous leishmaniasis: a comparative study with histopathology.

OBJECTIVE: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify Leishmania tropica parasites in paraffin-embedded tissue samples. RESULTS: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The quantitative real-time PCR (qPCR) quantified parasite loads were highly correlated with microscopic results (r = 0.598; P < 0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite load than chronic CL lesions), but there was no difference in the parasite load according to the patients' age and sex as well as location of the lesions. In contrast to Ridley scoring system (P < 0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P = 0.549), which indicates the superiority of histopathological evaluation for chronic forms differentiation.

A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria.

BACKGROUND: The decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains. METHODS: We observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1-86 and mdr1-184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments. RESULTS: By varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (HE) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1-86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed. CONCLUSIONS: Persistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria.

Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation.

BACKGROUND: Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity. METHODS: A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen. RESULTS: The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100% agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing. CONCLUSION: Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.

Establishment of Canine-Derived Giardia duodenalis Isolates in Culture.

Researchers continue to rely on axenic cultivation of Giardia duodenalis trophozoites in vitro to study the life cycle and host-parasite interactions of G. duodenalis and to develop vaccines and drugs to prevent and treat giardiasis. The majority of in vitro studies of G. duodenalis have used a small subset of isolates, mostly of assemblage A, and these isolates are usually originally isolated from humans. The most commonly used isolate for lab studies is known as WB. Canine giardiasis is a disease of veterinary importance, but it may also be of relevance in zoonotic transmission. Few G. duodenalis isolates from dogs have been adapted to in vitro culture, probably because the methods used are not suitable for the canine-specific genotypes that tend to dominate in most dog populations. In the current study, an experimental approach to cultivating canine-derived isolates of G. duodenalis was attempted by modification of the standard protocol based on physiological differences between the human and canine digestive system. An adapted method is described for improving the rate of in vitro excystation of cysts isolated from dogs by chemically weakening the cyst wall. A new canine-derived assemblage A G. duodenalis isolate was successfully adapted to axenic culture by using this method; the dog apparently had a mixed infection of assemblages A and D, but the assemblage A successfully outcompeted the assemblage D under conditions of in vitro culture. Based on the results, reasons regarding why humans do not seem to be suitable hosts for G. duodenalis in assemblages C and D are discussed.

Detection of Mixed Infections with Plasmodium spp. by PCR, India, 2014.

In 8 malaria-endemic states in India, mixed Plasmodium spp. infections were detected by PCR in 17.4% (265/1,521) of blood samples that microscopy had shown to contain only P. falciparum. The quality of microscopy must be improved because use of PCR for detection of malaria parasites is limited in rural areas.

Survey of Wild and Domestic Mammals for Infection with Leishmania infantum following an Outbreak of Desert Zoonotic Visceral Leishmaniasis in Jiashi, People's Republic of China.

In 2008 and 2009, an outbreak of desert-subtype zoonotic visceral leishmaniasis occurred in Jiashi county, Xinjiang, China. So far, no animal reservoir has been identified for this type of visceral leishmaniasis. Therefore, we surveyed the most common mammals (wild and domestic) for Leishmania infections during the outbreak in 2008 and 2009 in order to identify the source of the visceral leishmaniasis in this region. Spleen, liver, bone marrow and blood samples collected from 86 wood mice (Apodemus sylvaticus), 61midday jirds (Meriones meridianus) and 27 Yarkand hares (Lepus yarkandensis) were tested for the presence of Leishmania by microscopy, culture and PCR. All of the animals were found to be negative for Leishmania infections; On the other hand, Leishmania DNA was detected in blood samples collected from livestock reared in the outbreak area: 30.36% (17/56) of sheep, 21.57% (11/51) of goats, 17.78% (8/45) of cattle, and 21.62 (8/37) of donkeys were positive for Leishmania DNA by PCR. The amplified kDNA sequences from the livestock samples matched Leishmania DNA sequences isolated from patients with visceral leishmaniasis in the study area. We suggest that these domestic mammals are a possible reservoir host for Leishmania infantum in the outbreak area.

Subtyping Cryptosporidium ubiquitum,a zoonotic pathogen emerging in humans.

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.

RPS8--a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. We compared the intron-exon structure and DNA sequences of the RPS8 gene from Babesia and Theileria spp. isolates in China. Similar to 18S rDNA, the 40S ribosomal protein S8 gene, RPS8, including both coding and non-coding regions is a useful and novel genetic marker for defining species boundaries and for inferring phylogenies because it tends to have little intra-specific variation but considerable inter-specific difference. However, more samples are needed to verify the usefulness of the RPS8 (coding and non-coding regions) gene as a marker for the phylogenetic position and detection of most Babesia and Theileria species, particularly for some closely related species.

Application of a qPCR assay with melting curve analysis for detection and differentiation of protozoan oocysts in human fecal samples from Dominican Republic.

A quantitative polymerase chain reaction assay with melt curve analysis (qPCR-MCA) was applied for the detection of protozoan oocysts in 501 human fecal samples collected in Dominican Republic. Samples were subjected to qPCR using universal coccidia primers targeting 18S rDNA to detect oocysts followed by MCA to identify oocyst species based on amplicon melting temperature. Putative positive samples were also tested by conventional PCR and microscopy. Cystoisospora belli (×3), Cryptosporidium parvum (×3), Cryptosporidium hominis (×5), Cryptosporidium meleagridis (×1), Cryptosporidium canis (×1), and Cyclospora cayetanensis (×9) were detected by qPCR-MCA and confirmed by sequencing. This assay consistently detected 10 copies of the cloned target fragment and can be considered more efficient and sensitive than microscopy flotation methods for detecting multiple species of oocysts in human feces. The qPCR-MCA is a reliable protozoan oocyst screening assay for use on clinical and environmental samples in public health, food safety and veterinary programs.

Identification of novel and zoonotic Cryptosporidium species in fish from Papua New Guinea.

There is still limited information on the distribution of Cryptosporidium species and genotypes in fish. The present study investigated the prevalence of Cryptosporidium species in cultured freshwater (n=132), wild freshwater (n=206) and wild marine (n=276) fish in Papua New Guinea (PNG) by PCR screening at the 18S rRNA locus. A total of seven fish (2 cultured freshwater, 1 wild freshwater and 4 wild marine fish) were identified as positive for Cryptosporidium. Specifically, Cryptosporidium was found in four different host species (Nile tilapia, Oreochromis niloticus; silver barb, Puntius gonionotus; mackerel scad, Decapterus maracellus and oblong silver biddy, Gerres oblongus), giving an overall prevalence of 1.14% (95% CI: 0.3-2%, n=7/614). Of the seven positive isolates, five were identified as C. parvum and two were a novel piscine genotype, which we have named piscine genotype 8. Piscine genotype 8 was identified in two marine oblong silver biddies and exhibited 4.3% genetic distance from piscine genotype 3 at the 18S locus. Further subtyping of C. parvum isolates at the 60 kDa glycoprotein (gp60) locus identified 3 C. parvum subtypes (IIaA14G2R1, IIaA15G2R1 and IIaA19G4R1) all of which are zoonotic and a C. hominis subtype (IdA15G1). The zoonotic Cryptosporidium were identified in fish samples from all three groups; cultured and wild freshwater and wild marine fish. Detection of Cryptosporidium among aquaculture fingerlings warrants further research to gain a better understanding of the epidemiology of Cryptosporidium infection in cultured fish. The identification of zoonotic Cryptosporidium genotypes in fish from PNG has important public health implications and should be investigated further.

Toxoplasma gondii in free-ranging wild small felids from Brazil: molecular detection and genotypic characterization.

Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals.

Molecular characterization of Leishmania spp. in reservoir hosts in endemic foci of zoonotic cutaneous leishmaniasis in Iran.

Zoonotic cutaneous leishmaniasis (ZCL) is an expanding disease and a public health issue in Iran. In the present study, rate of natural infection of rodent populations with Leishmania was investigated in six endemic foci including 28 villages in Golestan, Esfahan, Yazd, Fars, Khuzestan and Ilam provinces. A total of 593 rodents were captured and identified as Rhombomys opimus (n = 325), Meriones libycus (n = 171), Meriones persicus (n = 27), Tatera indica (n = 37), Nesokia indica (n = 12), Rattus rattus (n = 13) and Mus musculus (n = 8). Microscopic examinations of Giemsa-stained smears showed that 108 out of 593 (18.2%) rodents were infected with Leishmania spp., whereas infection of 186 out of 593 (31.4%) rodents with Leishmania was then confirmed by ITS1-PCR. The highest rate of infection was found in R. opimus (prevalence of 35%) and M. libycus (31%). Based on Restriction Fragment Length Polymorphism (RFLP), 145 (78%) of 186 samples detected as Leishmania DNA were identified as L. major, 8 (4%) samples as L. turanica and 33 (18%) as mixed infection (L. major and L. turanica). Samples from infected rodents were inoculated subcutaneously at tail base of BALB/c mice. In 35 of them, nodules and ulcers containing amastigotes appeared at the inoculation site. The samples prepared from infected rodents were cultured in NNN medium and only two samples werepositive. Rhombomys opimus, M. libycus, M. persicus, T. indica and N. indica were confirmed as reservoir hosts of ZCL in the studied regions. Leishmania major infection was usually accompanied L. turanica in naturally infected gerbils (R. opimus and M. libycus) in Golestan, Esfahan and Fars provinces.

Host-parasite incongruences in rodent Eimeria suggest significant role of adaptation rather than cophylogeny in maintenance of host specificity.

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes.

Growing diversity of trypanosomatid parasites of flies (Diptera: Brachycera): frequent cosmopolitism and moderate host specificity.

Widely distributed, highly prevalent and speciose, trypanosomatid flagellates represent a convenient model to address topics such as host specificity, diversity and distribution of parasitic protists. Recent studies dealing with insect parasites of the class Kinetoplastea have been focused mainly on trypanosomatids from true bugs (Heteroptera), even though flies (Diptera, Brachycera) are also known as their frequent hosts. Phylogenetic position, host specificity and geographic distribution of trypanosomatids parasitizing dipteran hosts collected in nine countries on four continents (Bulgaria, Czech Republic, Ecuador, Ghana, Kenya, Madagascar, Mongolia, Papua New Guinea and Turkey) are presented. Spliced leader (SL) RNA gene repeats and small subunit (SSU) rRNA genes were PCR amplified from trypanosomatids infecting the gut of a total of forty fly specimens belonging to nine families. While SL RNA was mainly used for barcoding, SSU rRNA was utilized in phylogenetic analyses. Thirty-six different typing units (TUs) were revealed, of which 24 are described for the first time and represent potential new species. Multiple infections with several TUs are more common among brachyceran hosts than in true bugs, reaching one third of cases. When compared to trypanosomatids from heteropteran bugs, brachyceran flagellates are more host specific on the genus level. From seven previously recognized branches of monoxenous trypanosomatids, the Blastocrithidia and "jaculum" clades accommodate almost solely parasites of Heteroptera; two other clades (Herpetomonas and Angomonas) are formed primarily by flagellates found in dipteran hosts, with the most species-rich Leishmaniinae and the small Strigomonas and "collosoma" clades remaining promiscuous. Furthermore, two new clades of trypanosomatids from brachyceran flies emerged in this study. While flagellates from brachyceran hosts have moderate to higher host specificity, geographic distribution of at least some of them seems to be cosmopolitan. Moreover, the genus Angomonas, so far known only from South America, is present on other continents as well.

Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+)-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N)/d(S) ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.

Anopheles moucheti and Anopheles vinckei are candidate vectors of ape Plasmodium parasites, including Plasmodium praefalciparum in Gabon.

During the last four years, knowledge about the diversity of Plasmodium species in African great apes has considerably increased. Several new species were described in chimpanzees and gorillas, and some species that were previously considered as strictly of human interest were found to be infecting African apes. The description in gorillas of P. praefalciparum, the closest relative of P. falciparum which is the main malignant agent of human malaria, definitively changed the way we understand the evolution and origin of P. falciparum. This parasite is now considered to have appeared recently, following a cross-species transfer from gorillas to humans. However, the Plasmodium vector mosquito species that have served as bridge between these two host species remain unknown. In order to identify the vectors that ensure ape Plasmodium transmission and evaluate the risk of transfer of these parasites to humans, we carried out a field study in Gabon to capture Anopheles in areas where wild and semi-wild ape populations live. We collected 1070 Anopheles females belonging to 15 species, among which An. carnevalei, An. moucheti and An. marshallii were the most common species. Using mtDNA-based PCR tools, we discovered that An. moucheti, a major human malaria vector in Central Africa, could also ensure the natural transmission of P. praefalciparum among great apes. We also showed that, together with An. vinckei, An. moucheti was infected with P. vivax-like parasites. An. moucheti constitutes, therefore, a major candidate for the transfer of Plasmodium parasites from apes to humans.

Genotypes and mouse virulence of Toxoplasma gondii isolates from animals and humans in China.

BACKGROUND: Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. METHODOLOGY/PRINCIPAL FINDINGS: Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). CONCLUSION/SIGNIFICANCE: A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.

Giardia and Cryptosporidium on dairy farms and the role these farms may play in contaminating water sources in Prince Edward Island, Canada.

BACKGROUND: Cattle represent a reservoir for Giardia and Cryptosporidium and may contaminate water sources. OBJECTIVES: To determine the distribution of Cryptosporidium and Giardia on dairy farms and in water bodies near the farms. FARMS AND WATER SOURCES: Twenty dairy farms and 20 wells and 13 surface water samples associated with dairy farms. METHODS: Proportions of samples positive for Cryptosporidium or Giardia were determined by a direct immunofluorescence assay. Fecal and water samples were taken at different times. RESULTS: Thirty-two (95% CI: 29-35%) and 14% (95% CI: 12-17%) of fecal samples, and 100 (95% CI: 96-100) and 55% (95% CI: 32-77%) of herds, were positive for Giardia and Cryptosporidium, respectively. Giardia duodenalis assemblage E was detected in high proportions (90%) of fecal samples. Cryptosporidium bovis predominated (51%) in all cattle. C. andersoni predominated in adult cattle (53%), whereas the predominant species in animals < 2 months and 2-6 months was C. bovis, respectively. Only calves < 2 months of age were positive for C. parvum. In 46% (95% CI: 19-75%) and 85% (95% CI: 55-98%) of surface water, concentrations of Giardia cysts and Cryptosporidium oocysts were higher in downstream, than in upstream, locations of farms, whereas only 1 groundwater sample was positive for Cryptosporidium. CONCLUSIONS: This sample of dairy cattle was predominantly infected with nonzoonotic species and genotypes of Cryptosporidium, Giardia, or both. More studies are needed to determine if the presence of Giardia or Cryptosporidium in surface water was associated with shedding in animals from nearby farms.

Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey.

Assessing vector-parasite relationship is important in understanding the emergence of vector-borne diseases and the evolution of parasite diversity. This study investigates avian Plasmodium parasites in mosquitoes collected from Kayseri province in Central Anatolian, Turkey and determines the haemosporidian parasite lineages from these mosquito species. A total of 6153 female mosquitos from 6 species were collected from 46 sites during June-August of 2008 and 2009. Each mosquito's head-thorax and abdomen were separated, categorized with respect to species and collection area and pooled for DNA extraction. A total of 1198 genomic DNA pools (599 thorax-head, 599 abdomen) were constituted of which 128 pools (59 thorax-head, 69 abdomen) were positive for avian haemosporidian parasites (Plasmodium and Haemoproteus) by Nested-PCR analysis. Culex pipens, Aedes vexans, Culex theileri and Culiseta annulata were positive with minimum infection rates (MIRs) of 16.22 and 18.15, 4.72 and 5.98, 5.18 and 10.36, 10.64 and 10.64 in their thorax-head and abdomen parts, respectively. No avian haemosporidian DNA was detected from Culex hortensis and Anopheles maculipennis. Phylogenetic analyses of the partial cytb gene of avian haemosporidian mt-DNA from 13 positive pools revealed that 11 lineages in four phylogenic groups were Plasmodium and the other two were Haemoproteus. Our results suggest that Cx. pipiens could probably be the major vector of avian Plasmodium in Central Turkey. This is the first report of molecular detection and characterization of avian Plasmodium lineages from mosquitoes in Turkey.

Molecular identification of tick-borne pathogens in Nigerian ticks.

A molecular epidemiology investigation was undertaken in two Nigerian states (Plateau and Nassarawa) to determine the prevalence of pathogens of veterinary and public health importance associated with ticks collected from cattle and dogs using PCR, cloning and sequencing or reverse line blot techniques. A total of 218 tick samples, Amblyomma variegatum (N=153), Rhipicephalus (Boophilus) decoloratus (N=45), and Rhipicephalus sanguineus (N=20) were sampled. Pathogens identified in ticks included piroplasmids (Babesia spp., Babesia bigemina and Babesia divergens), Anaplasma marginale and Rickettsia africae. Piroplasmids were identified in A. variegatum, A. marginale was found in R. decoloratus, while R. africae was detected in all tick species examined. Ehrlichia spp. and Theileria spp. were not identified in any of the ticks examined. Of the 218 ticks examined, 33 (15.1%) contained pathogen DNA, with the presence of B. divergens and R. africae that are zoonotic pathogens of public health and veterinary importance. The variety of tick-borne pathogens identified in this study suggests a risk for the emergence of tick-borne diseases in domestic animals and humans, especially amongst the Fulani pastoralists in Plateau and Nassarawa states of Nigeria.