Msp2 Gene Research Articles

Molecular detection and characterization of Anaplasma spp. in sheep and cattle from Xinjiang, northwest China.

BackgroundAnaplasmosis is caused by obligate intracellular bacteria in the genus Anaplasma. These bacterial pathogens are transmitted by ticks and impact both human and animal health. This study was conducted to determine the prevalence and molecular characterization of Anaplasma spp. in ruminants sampled in Xinjiang, northwest China.MethodsA survey was performed in August 2012 in rural areas of six counties in Xinjiang province. A total of 250 blood samples from ruminants were collected and tested for the presence of Anaplasma spp. by PCR. Positive samples were genetically characterized based on the 16S rRNA and msp4 genes.ResultsThe results showed a high prevalence of Anaplasma spp. in ruminants, with at least three different Anaplasma species detected (A. phagocytophilum, A. bovis and A. ovis). The mean prevalence of single infection with each species was 17.6% (A. phagocytophilum), 4.8% (A. bovis) and 40.5% (A. ovis). Coinfection occurred in 20 (8.0%) animals. Phylogenetic analysis of the 16S rRNA gene of A. bovis and A. phagocytophilum revealed a higher degree of genetic diversity for the latter. The results for A. ovis showed genotypic variation among geographic regions in China. In addition, a closely related isolate to the canine pathogen A. platys was identified in ruminants.ConclusionsThis survey revealed a high prevalence of Anaplasma sp. infections in sheep and cattle in the northwestern border regions of China, indicating the potential risk of transboundary disease.

Genetic diversity of Plasmodium falciparum isolates based on MSP-1 and MSP-2 genes from Kolla-Shele area, Arbaminch Zuria District, southwest Ethiopia.

BackgroundThe genetic diversity of Plasmodium falciparum has been extensively studied in various countries. However, limited data are available from Ethiopia. This study was conducted to evaluate the extent of genetic diversity of P. falciparum in Kolla-Shele, in the southwest of Ethiopia.MethodsA total of 88 isolates from patients with uncomplicated P. falciparum attending Kolla-Shele Health Centre was collected from September to December, 2008. After extraction of DNA by Chelex® method, the samples were genotyped by using nested-PCR of msp1 (block 2) and msp2 (block 3) including their allelic families: K1, MAD20, RO33 and FC27, 3D7/IC1, respectively.ResultsAllelic variation in both msp1 and msp2 were identified in the 88 blood samples. For msp1 67% (59/88) and msp2 44% (39/88) were observed. K1 was the predominant msp1 allelic family observed in 33.9% (20/59) of the samples followed by RO33 and MAD20. Of the msp2 allelic family 3D7/IC1 showed higher frequency (21.5%) compared to FC27 (10.3%). A total of twenty-three alleles were detected; of which, eleven were from msp2 and twelve from msp2 genes. Fifty-nine percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 1.8 (95% CI: 1.48-2.04). The heterozygosity index was 0.79 and 0.54for msp1 and msp2, respectively. There was no statically significant difference in the multiplicity of infection by either age or parasite density (P > 0.05).ConclusionThis genetic diversity study showed the presence of five allelic types in the study area, with dominance K1 in the msp1 family and 3D7/IC1 in the msp2 family. Multiple infections were observed in nearly 60% of the samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0604-8) contains supplementary material, which is available to authorized users.

Genetic diversity in MSP-1 gene of Plasmodium falciparum and its association with malaria severity, parasite density, and host factors of asymptomatic and symptomatic patients in Papua, Indonesia

Background: Genetic diversity of Plasmodium falciparum represents a major issue in understanding the several aspects of malaria infection and diseases. The merozoite surface protein (MSP)-1 gene of P. falciparum codes for a major asexual blood-stage antigen, presently proposed as a major malarial vaccine candidate. Objective: To determine the length of the DNA bands and genetic diversity of the MSP-1 gene of P. falciparum, which is associated with malaria severity and host factors in population of the asymptomatic and symptomatic patients in Papua, Indonesia. Materials and Methods: A total of 152 isolates from the blood samples of the patients infected with P. falciparum were analyzed by microscopic examination. DNA was extracted from MSP-1 gene and nested PCR amplification carried out. The active case detection was conducted in Koya and Skow villages, whereas passive detection was obtained from general hospitals. Result: The main allele of the gene MSP-1, which related to the severity of malaria, the parasite density, and host factors of the population with a single infection, was found to MAD20 that was higher in mild malarial cases. The family alleles K1, MAD20, and RO33 of the MSP-1 gene of P. falciparum were found to be in large numbers in severe malaria and light malaria of the symptomatic patients. In the asymptomatic patients, it was found that the alleles of MSP-1 (K1, MAD20, and RO33) were more numerous when the parasite density was less than 10,000/μL of blood. Conclusion: There was no significant correlation between the genetic diversity of the MSP-1 gene of P. falciparum with malaria severity, parasite density, age group, and gender of asymptomatic patients, but there was a significant correlation with the age groups and gender in the symptomatic patients. The K1, MAD20, and RO33 alleles showed a higher density in asymptomatic patients, but K1 and RO33 alleles were higher at severe and mild malaria and MAD20 was higher at mild malaria cases. Therefore, MSP-1 gene allele could be used as a genetic marker to determine the genetic diversity of P. falciparum and as a candidate molecular vaccine.

Diversity of ankA and msp4 genes of Anaplasma phagocytophilum in Slovenia

Granulocytic anaplasmosis is a tick transmitted emerging disease in Europe and worldwide. The agent, Anaplasma phagocytophilum is transmitted by ticks of the genus Ixodes and causes infections in humans and domestic animals. The analysis of different target genes showed that in nature several genetic variants of A. phagocytophilum were present. The purpose of our study was to genetically characterize A. phagocytophilum strains from eight humans, 16 dogs, 12 wild boars, one bear and 18 tick pools from Slovenia. Therefore, the ankA and msp4 genes of A. phagocytophilum were chosen. The same genetic ankA and msp4 variant of A. phagocytophilum was detected in humans, wild boar and a part of the pooled ticks indicating that it circulates in a zoonotic cycle between wild boar and ticks. In dogs, three ankA variants of A. phagocytophilum were detected. One of them was identical to the one that was found in humans. In contrast, all dogs harboured the same msp4 variant as humans and wild boar. In ticks, numerous ankA and msp4 variants were present.

Plasmodium falciparum genotype diversity in artemisinin derivatives treatment failure patients along the Thai-Myanmar border.

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.

Longitudinal study of risk factors for anaplasmosis and transplacental transmission in herd cattle

Anaplasma marginale , bacteria that occurs in tropical and subtropical areas throughout the world, is responsible for causing a disease that is one of the most constraint to cattle production in many countries. The present work aimed to investigate potential risk factors for anaplasmosis and the occurrence of transplacental transmission by molecular and serological assays in cattle. A total of 22 calves were randomly sampled and monitored during four years; the presence of infection/exposure to A. marginale was assessed by a semi-nested PCR assay targeting msp-5 gene of A. marginale , indirect ELISA for detection of IgG antibodies to A. marginale and blood smears. The A. marginale infection prevalence data were analyzed in relation to risk factors, using chi-squared, odds ratio and multiple logistic regressions. DNA amplification results revealed a frequency of infection with A. marginale of 41% (9/22) in the newborn calves before colostrum suckling. However, only 14% (3/22) of newborn calves were seropositive for A. marginale . All calves were positive to A. marginale by PCR and microscopic examination of blood smears before the two and fourth month of age, respectively. The main risk factors associated with seroprevalence were breed (OR=36.2), tick infestation (OR=3.44) and stocking density (OR=3.28). The results indicated that exposure of cattle to A. marginale was common in dairy herds and endemic instability situation probably is due to inadequate antibody production in cows or genetic variability of pathogen. This study still demonstrated that in addition to transmission of A. marginale by ticks and flies, the transplacental transmission is very important and should be the target of disease control programs in Brazil.

Genetic variability of Anaplasma phagocytophilum in ticks and voles from Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia

Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilum was assayed in Ixodes persulcatus, Ixodes trianguliceps ticks and Myodes spp. voles from two I. persulcatus/I. trianguliceps sympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilum was found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. trianguliceps ticks removed from voles, 1/12 (8.3%) of I. persulcatus removed from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESL operon sequence analysis of positive samples revealed three distinct A. phagocytophilum genetic groups previously identified in ticks and mammals in Russia. Genetic group 1 was found in 6/36 (16.7%) of sequenced positive blood samples; this group was previously revealed in I. persulcatus and Myodes spp. voles in different regions of Russia. Genetic group 2 was found in 30/36 (83.3%) of sequenced positive blood samples and all positive I. trianguliceps; this group was previously revealed only in Myodes spp. voles and common shrews (Sorex araneus) in I. persulcatus/I. trianguliceps sympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatus and one blood sample; this group was previously revealed in I. persulcatus and Siberian chipmunks (Tamias sibiricus). We suppose that I. trianguliceps is the most probable vector for A. phagocytophilum of group 2. Analysis of the msp4 gene, intergenic region DOV1, and some other genetic loci has shown that isolates from different genetic groups significantly differ in all studied loci and that A. phagocytophilum of group 2 is closely related to A. phagocytophilum isolates revealed in voles and I. trianguliceps in Europe. A. phagocytophilum of groups 1 and 2 are the most similar to each other, while A. phagocytophilum of group 3 clusters with European A. phagocytophilum isolates from I. ricinus and various mammalian species.

Genetic diversity of the msp-1, msp-2, and glurp genes of P lasmodium falciparum isolates along the Thai-Myanmar borders

To study the genetic diversity at the msp-1, msp-2, and glurp genes of Plasmodium falciparum (P. falciparum) isolates from 3 endemic areas in Thailand: Tak, Kanchanaburi and Ranong provinces. A total of 144 P. falciparum isolates collected prior to treatment during January, 2012 to June, 2013 were genotyped. DNA was extracted; allele frequency and diversity of msp-1, msp-2, and glurp genes were investigated by nested polymerase chain reaction. P. falciparum isolates in this study had high rate of multiple genotypes infection (96.5%) with an overall mean multiplicity of infection of 3.21. The distribution of allelic families of msp-1 was significantly different among isolates from Tak, Kanchanaburi, and Ranong but not for the msp-2. K1 and MAD20 were the predominant allelic families at the msp-1 gene, whereas alleles belonging to 3D7 were more frequent at the msp-2 gene. The glurp gene had the least diverse alleles. Population structure of P. falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci, 3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI. Isolates from Kanchanaburi had different structures; the most prevalent alleles were K1 and RO33. The present study shows that P. falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp-1 and msp-2 and diversity but different from Kanchanaburi isolates. These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.

First molecular identification and genetic characterization of Anaplasma ovis in sheep from Tunisia

The aims of this study consist of first estimating the molecular prevalence of Anaplasma ovis with specific species loop-mediated isothermal amplification (LAMP) method in sheep (n=204) from Northern and Central Tunisia. This study also characterizes the genotypes of this Anaplasma species by partial msp4 gene sequence. The molecular prevalence of A. ovis used to record 70.1%. Prevalence rate was significantly higher in El Alia locality (governorate of Bizerte, Northern Tunisia), where it indicated 91.1%, than in Sbikha locality (governorate of Kairouan, Central Tunisia), where it indicated 49.5%. In Sbikha, A. ovis prevalence varied significantly according to two factors, i.e., gender and age. In fact, ewes were significantly more infected than ram lambs. Ticks collected on animals allowed recovering 271 ixodid ticks from El Alia. Ticks belonged to 3 species of Rhipicephalus genus: first, R. turanicus (59.78%) which was the dominant species and which was followed by R. sanguineus (36.16%) and R. annulatus (4.06%). In Sbikha, an absence of ticks was noted in all of the examined sheep. The analysis of A. ovis msp4 sequences revealed four different genotypes; two of them were previously described in Italy and two new other genotypes were classified in an independent cluster. The present work is the first published report of A. ovis infection in sheep in Tunisia. The results indicate the presence of A. ovis at a high rate; hence, the need for a survey into the medical and economic impact of the disease, and the identification of vectors of this bacterium in Tunisia must be made.

First report of Anaplasma phagocytophilum and Babesia microti in rodents in Finland.

Tick-borne diseases pose an increasingly important public health problem in Europe. Rodents are the reservoir host for many tick-transmitted pathogens, including Anaplasma phagocytophilum and Babesia microti, which can cause human granulocytic anaplasmosis and babesiosis, respectively. To estimate the presence of these pathogens in rodents in Finland, we examined blood samples from 151 bank voles (Myodes glareolus) and demonstrate, for the first time, that A. phagocytophilum and B. microti commonly infect bank voles (in 22% and 40% of animals, respectively) in Finland. Sequence analysis of a fragment of 18S rRNA showed that the B. microti strain isolated was identical to the Munich strain, which is considered to be nonzoonotic. The A. phagocytophilum strain (based on a fragment of the msp4 gene) was identical to one found earlier in rodents in the United Kingdom that is transmitted by the tick Ixodes trianguliceps, all the life stages of which feed on small mammals. The infection probability of B. microti in the bank voles was the greater the older the individual was, and males were more often infected than females. A. phagocytophilum infection probability first increased and then decreased with the age of individual without any difference between sexes. While these pathogens presumably pose a limited zoonotic risk to humans in Finland, they might have important interactions with other rodent pathogens and therefore affect infection dynamics of, for example, zoonotic pathogens.

Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail-anchored proteins.

The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1(-/-) mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.

Diversity and population structure of Plasmodium falciparum in Thailand based on the spatial and temporal haplotype patterns of the C-terminal 19-kDa domain of merozoite surface protein-1

BackgroundThe 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum (PfMSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of PfMSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, PfMSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown.MethodsSixty-three isolates of P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of PfMSP-119 haplotypes in individual populations. The PfMSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite.ResultsFive conserved polymorphic positions, which accounted for five distinct haplotypes, of PfMSP-119 were identified. Differences in the prevalence of PfMSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual PfMSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries.ConclusionThe major MSP-119 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.

Genotyping of Anaplasma phagocytophilum strains from Poland for selected genes.

Anaplasma phagocytophilum is a veterinary and medical tick-borne pathogen of vertebrates which invades granulocytes. The potential reservoirs of Anaplasma include game animals and small mammals. The aim ofthis study was to gain insight into the variability ofnucleotide sequences of ribosomal and selected protein-coding genes (ankA, msp4 and the groESL heat-shock protein operon) present in isolates of A. phagocytophilum collected from wild ruminants (Cervus elaphus and Capreolus capreolus) in Poland. Fragments of the genes were amplified using PCR and sequenced. High DNA variability was found in fragments of the msp4 and groESL genes (15 and 8 alleles, respectively), whereas ankA and 16S rRNA demonstrated very little or no variability (2 and 1 alleles, respectively). Unique allelic profiles were determined for the examined groups and allowed distinguishing 6 A. phagocytophilum genotypes, 2 of which were reported only in the roe deer (C. capreolus), and 1 in the red deer (C. elaphus).

Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico

BackgroundPlasmodium vivax is a protozoan parasite with an extensive worldwide distribution, being highly prevalent in Asia as well as in Mesoamerica and South America. In southern Mexico, P. vivax transmission has been endemic and recent studies suggest that these parasites have unique biological and genetic features. The msp1 gene has shown high rate of nucleotide substitutions, deletions, insertions, and its mosaic structure reveals frequent events of recombination, maybe between highly divergent parasite isolates.MethodsThe nucleotide sequence variation in the polymorphic icb5-6 fragment of the msp1 gene of Mexican and worldwide isolates was analysed. To understand how genotype diversity arises, disperses and persists in Mexico, the genetic structure and genealogical relationships of local isolates were examined. To identify new sequence hybrids and their evolutionary relationships with other P. vivax isolates circulating worldwide two haplotype networks were constructed questioning that two portions of the icb5-6 have different evolutionary history.ResultsTwelve new msp1 icb5-6 haplotypes of P. vivax from Mexico were identified. These nucleotide sequences show mosaic structure comprising three partially conserved and two variable subfragments and resulted into five different sequence types. The variable subfragment sV1 has undergone recombination events and resulted in hybrid sequences and the haplotype network allocated the Mexican haplotypes to three lineages, corresponding to the Sal I and Belem types, and other more divergent group. In contrast, the network from icb5-6 fragment but not sV1 revealed that the Mexican haplotypes belong to two separate lineages, none of which are closely related to Sal I or Belem sequences.ConclusionsThese results suggest that the new hybrid haplotypes from southern Mexico were the result of at least three different recombination events. These rearrangements likely resulted from the recombination between haplotypes of highly divergent lineages that are frequently distributed in South America and Asia and diversified rapidly.

Polymorphism of the merozoite surface protein-1 block 2 region in Plasmodium falciparum isolates from Mauritania.

BackgroundThe genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Mauritania. The present study examined and compared the genetic diversity of P. falciparum isolates in Mauritania.MethodsPlasmodium falciparum isolates blood samples were collected from 113 patients attending health facilities in Nouakchott and Hodh El Gharbi regions. K1, Mad20 and RO33 allelic family of msp-1 gene were determined by nested PCR amplification.ResultsK1 family was the predominant allelic type carried alone or in association with Ro33 and Mad20 types (90%; 102/113). Out of the 113 P. falciparum samples, 93(82.3%) harboured more than one parasite genotype. The overall multiplicity of infection was 3.2 genotypes per infection. There was no significant correlation between multiplicity of infection and age of patients. A significant increase of multiplicity of infection was correlated with parasite densities.ConclusionsThe polymorphism of P. falciparum populations from Mauritania was high. Infection with multiple P. falciparum clones was observed, as well as a high multiplicity of infection reflecting both the high endemicity level and malaria transmission in Mauritania.

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Prevalence of Anaplasma phagocytophilum in ticks collected from migratory birds in Danube Delta, Romania

Wild birds are hosts for several species of ticks, contributing to the maintenance of their local populations in delimited geographic areas. migratory birds play important roles as distributors of ticks within and between distant territories, including continents. Ticks collected from birds are responsible for hosting a significant number of human pathogens. The extensive wetland complex of the Danube Delta provides an internationally important stopover site for millions of birds, belonging to 300 different species, travelling annually. The aim of this study was to detect Anaplasma phagocytophilum in feeding ticks collected from migratory birds along four migratory seasons. Ticks were collected from the birds with forceps and preserved in 96% ethanol for later examination using a separate vial for each bird. A total of 1436 birds in 56 species (15 families) of Passeriformes and 3 non-Passeriformes bird species were captured. A total of 400 ticks were collected and identified as larvae (n = 191; 47.75%), nymphs (n = 201; 50.25%) or adult females (n = 8; 2%). No adult males were found. The ticks belonged to four species (Ixodes ricinus, I. arboricola, I. redikorzevi and Haemaphysalis punctata). Ixodes ricinus was the most common tick (369/400 ticks, 92.25% of the total collected), with a total of 181 larvae, 180 nymphs and 8 females. For detection of Anaplasma phagocytophilum all 400 ticks were examined by PCR, targeting the msp2 gene. Anaplasma phagocytophilum specific DNA was detected in 2 larvae and 2 nymphs of, I. ricinus, from 3 birds prevalence 1.08-%. The infection was absent from the other species of collected ticks The bird species that carried ticks infected with A. phagocytophilum were Turdus merula, Erithacus rubecula and Fringilla coelebs. Several other studies have demonstrated the presence of A. phagocytophilum in ticks collected from birds. Comparing our data with these results, we confirm the low prevalence of A. phagocytophilum, showingthat migratory birds, despite being without reservoir competencemight be important hosts for the dispersal of infected ticks.

Genetic Diversity of Variable Region Block 2 in the Merozoite Surface Protein-1 (MSP1) in Plasmodium falciparum Field Isolates from South-East of Iran

Merozoite surface protein 1 of (PfMSP-1) is a leading malaria vaccine candidate. However, extensive genetic diversity of this gene in field isolates of represents a major obstacle for the development of an effective vaccine against malaria. The present study was aimed at analysing genetic polymorphisms of K1, MAD20 and RO33 allelic types of MSP-1 block 2 among isolates from Sistan and Baluchestan, Iran. In this study a total of 94 infected persons from Sistan and Baluchistan Province of Iran, were included. Blood samples were collected from March 2011 to September 2012. Block 2 of the MSP-1 gene was genotyped by allele-specific nested polymerase chain reaction (PCR) after DNA extraction. Eighty-nine (94.7%) of the 94 samples were successfully amplified; 7 distinct MSP-1 genotypes were identified by size differences on agarose gels. MAD20 was the predominant MSP-1 allelic family identified in 46.1% (41/89) of the samples while RO33 family had the least frequency (7.9%). A total of 9/89 (10.1%) samples exhibited multiple infections with two alleles at PfMSP-1. The present study shows that the level of genetic diversity is relatively low in south-east of Iran and most of infections are composed of one clone, which is consistent with an area of low malaria transmission. These data are useful for malaria prevention and control in Iran.

Molecular epidemiology of the emerging zoonosis agent Anaplasma phagocytophilum (Foggie, 1949) in dogs and ixodid ticks in Brazil.

BackgroundAnaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil.MethodsWe tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum.ResultsAmong the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs.ConclusionsThis is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.

Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia

BackgroundDespite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed.MethodsBlood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections.ResultsConsiderable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases.ConclusionsThe parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.