Msp2 Gene Research Articles

Detection and Phylogenetic Characterization of Anaplasma capra: An Emerging Pathogen in Sheep and Goats in China.

Anaplasma capra is an emerging pathogen, which can infect ruminants and humans. This study was conducted to determine the occurrence of A. capra in the blood samples of sheep and goats in China. Using nested polymerase chain reaction (nested-PCR) targeting the gltA gene and conventional PCR targeting the heat shock protein (groEL) gene and the major surface protein4 gene (msp4), A. capra was detected in 129 (8.9%) of 1453 sheep and goat blood samples. The positive rate was higher in goats (9.4%, 89/943) than in sheep (7.8%, 40/510) (χ2 = 1.04, p > 0.05, df = 1). For sheep, A. capra was found in 17 sites from 2 provinces. The prevalence was 28.6% in sheep from Liaoning province, which was higher than in Henan Province (7.3%). For goats, A. capra was detected in 35 sites from 7 provinces. The prevalence varied from 0 to 19.4% in the goat sites examined. The prevalence rates were 19.4, 19.3, 10, 8.8, 6.8, 1.8, and 0% in goats from Guizhou province, Henan Province, Inner Mongolia Autonomous Region, Shanxi Province, Xinjiang Uygur Autonomous Region, Yunnan province, and Gansu province, respectively. Based on the analysis of the A. capra citrate synthase gene (gltA), two variants were identified. Variant I showed a high sequence similarity to the A. capra, which were previously reported in sheep, goats, Ixodes persulcatus, Haemaphysalis longicornis, Haemaphysalis qinghaiensis, and humans. Variant II was only found in Luoyang, Anyang, and Sanmengxia, of Henan province. To our knowledge, this is the first detection of this variant of A. capra in sheep and goat blood in China. Phylogenetic analysis based on groEL and msp4 genes showed that the Anaplasma sp. sequences clustered independently from A. capra and other Anaplasma species with high bootstrap values. We found A. capra DNA in sheep and goats in China, providing evidence that sheep and goats can be infected by A. capra. We also found that this zoonotic pathogen is widely distributed in China. This study provides information for assessing the public health risks for human anaplasmosis.

Bartonella and Rickettsia Infections in Haematophagous Spinturnix myoti Mites (Acari: Mesostigmata) and their Bat Host, Myotis myotis (Yangochiroptera: Vespertilionidae), from Poland

Hematophagous Spinturnix myoti mites and their host, the greater mouse-eared bat (Myotis myotis), were tested for the presence of Bartonella spp., Rickettsia spp., and Anaplasma phagocytophilum. In total, Bartonella spp. DNA was amplified in 28% of 134 mite pools and in 25% of 59 bats tested by PCR targeting a fragment of citrate synthase gltA gen. Adult mites were at least threefold more frequently infected compared to immature stages. The overall infection prevalence among mite pools from cave-dwelling bats was higher than for those collected from attic shelters. Three distinct genotypes were detected. The most prevalent genotype in mites and bats matched closely with Candidatus Bartonella hemsundetiensis identified in bats from Finland and was relatively distant from bat-borne Bartonella strains described in the UK and France. Importantly, most sequences were close to those reported in forest workers from Poland. The presence of identical genotype among S. myoti samples and M. myotis bats suggests that bartonellae can be shared between mites and their bat hosts. In this case, wing mites could serve as vectors, whereas their hosts as reservoirs. One blood sample was positive by PCR for the msp2 gene of A. phagocytophilum. Two mite pools yielded Rickettsia spp. DNA. Both sequences were distinct from any known species but can be classified as spotted fever group Rickettsia spp. Our findings expanded our knowledge on the role of spinturnicid mites in the ecology and epidemiology of bacterial infections associated with vespertilionid bats, especially regarding the genus Bartonella.

First report of Anaplasma marginale infection in goats, Brazil.

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries and is increasingly being detected in other animal species. Rhipicephalus microplus is the main vector of this bacterium and may be found parasitizing small ruminants. In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. Considering that A. marginale infection has been previously molecularly described in sheep, this study has aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from northeastern Brazil. A total of 403 goat blood samples were included in the study. An epidemiological questionnaire was applied to each farm owner addressing age, gender, presence of ticks and multispecies grazing. All samples were screened for A. marginale- and A. ovis-infection using primers targeting the Anaplasma spp. msp4 gene. The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing. Anaplasma spp. were differentiated by sequencing of the repeat region of the msp1α gene. For the statistical analysis the Chi-square or the Fisher’s exact test was used to verify association of the individual factors (age, gender, presence of ticks, and multispecies grazing) with Anaplasma spp. infection. We report the first molecular detection of A. marginale in goats from northeastern Brazil, based on msp1α, msp4 and msp5 gene sequencing analysis. Sequencing of the detected A. marginale msp1α gene revealed the F repeat. Amblyomma parvum and R. microplus were found feeding on animals.

Role of genetic factors and ethnicity on the multiplicity of Plasmodium falciparum infection in children with asymptomatic malaria in Yaoundé, Cameroon

In this cross-sectional study, we investigated host genetic factors and ethnic variation in circulating Plasmodium falciparum merozoite surface protein 2 (msp-2) clones among children with asymptomatic malaria.Isolates from seventy two asymptomatic malaria children were used for genotyping block 3 of msp-2 gene by nested polymerase chain reaction (PCR). Sickle cell trait and glucose-6-phosphate dehydrogenase (G6PD) deficiency were analysed by restriction fragment length polymorphism of DNA products from PCR targeting codons 6 and 68 of the beta-globin (HBB) and G6PD genes respectively. ABO blood group was typed by agglutination method.A total of forty two msp-2 genotypes (20 for 3D7 and 22 for FC27) were detected for an average (standard error of mean) multiplicity of infection (MOI) of 2.45 (0.16). The MOI was statistically the same among the five identified ethnic groups (P = 0.83). The overall prevalence of sickle cell trait and G6PD deficiency were 12.50 % and 22.22 % respectively. MOI was similar between children with Hb AA and Hb AS genotypes (P = 0.42). MOI was significantly high among children with a mutant G6PD genotype (P = 0.017). MOI was significantly higher in blood group O than group A (P = 0.03).Our findings show that although ethnicity and sickle cell trait have no association with MOI, the association was observed with G6PD genotype and ABO group. The results suggest the need for extension and expansion of the current study in order to investigate the mechanisms involved.

Expression of full-length Plasmodium falciparum P48/45 in P. berghei blood stages: A method to express and evaluate vaccine antigens

The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity

Recurrent parasitaemia during follow up of clinical trials of antimalarial drug efficacy results from either recrudescence of parasites surviving treatment or from parasites newly emerging from the hepatic stage of infection. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. There is risk of both false positive and false negative results, leading to misclassification errors. The high-resolution melt (HRM) assay was developed with pairs of conserved primers targeting blocks of merozoite surface protein 1 and 2 (msp1 and msp2) genes, and polymorphisms were compared using sequence-confirmed Plasmodium falciparum DNA samples from laboratory isolates. In this study, the HRM dissociation profiles of msp1 and msp2 amplicons were determined and validated against parasite isolates from malaria patients. The msp1 and msp2 profiles of both laboratory and clinical isolates were reproducibly differentiated by HRM. These rapid assays are performed in a closed-tube system, and so avoid cross-contamination while increasing throughput, which are two major advantages. The HRM assays offer significant gains in simplicity, speed and interpretation of results, and reduced analysis cost, for studies that require discrimination of parasite clones. Assay performance in large-scale studies utilizing DNA samples derived from filter-paper bloodspots should now be evaluated.

Molecular detection and characterization of tick-borne protozoan and rickettsial pathogens isolated from cattle on Pemba Island, Tanzania

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. In Tanzania, detailed studies on tick-borne pathogens in cattle using sensitive molecular detection methods are scarce. In this study, we investigated the occurrence of Theileria spp., Babesia spp., Anaplasma spp. and Ehrlichia spp. in 245 blood samples collected from cattle on Pemba Island, Tanzania. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening revealed overall infection rates of 62.4% for Theileria spp., 17.6% for Babesia bigemina, 15.9% for Anaplasma marginale, 7.4% for Ehrlichia ruminantium and 4.5% for Babesia bovis. Further analysis using sequences of Theileria spp. 18S rRNA revealed infection of cattle with Theileria mutans (68.6%), Theileria taurotragi (48.4%), Theileria parva (41.2%), and Theileria ovis (1.9%). Co-infections of cattle, with up to six tick-borne pathogens, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 genes are conserved among cattle blood samples in Pemba, with 99.3%–100%, 99.6%–100% and 100% sequence identity values, respectively. In contrast, the B. bigemina RAP-1a and B. bovis SBP-2 gene sequences were relatively diverse with 99.5%–99.9% and 66.4%–98.7% sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina RAP-1 and B. bovis SBP-2 gene sequences showed significant differences in the genotypes, as they appeared in separate clades. This study provides important data for understanding the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.

Detection of Babesia spp., Theileria spp. and Anaplasma ovis in Border Regions, northwestern China.

Babesia spp., Theileria spp. and Anaplasma ovis are important intracellular agents that are transmitted by tick bites. However, Babesia spp., Theileria spp. and A.ovis in ticks have not been systematically reported along the border of northwestern China. In this study, a total of 1,084 adult ticks, including 134 Haemaphysalis punctata, 337 Hyalomma asiaticum, 233 Dermacentor nuttalli, 69 Rhipicephalus turanicus and 265 Dermacentor marginatus were collected from 11 counties or cities of Xinjiang Uygur Autonomous Region. The ticks were identified from morphological and molecular characteristics. Two fragments of 18S rRNA gene were used to determine the species level of Babesia and Theileria. Msp4 gene encoding major surface protein 4 was used to determine A.ovis. Of the 1,084 samples, five species of Babesia (B.occultans, B.caballi, B.motasi, B.major and Babesia sp. detected in this study), two kinds of Theileria (Theileria ovis and Theileria sp. detected in this study) and A.ovis with six phylogenic branches were detected in the border of northwestern China. Babesia occultans, first found in China, was first molecularly detected in D.nuttalli. Babesia caballi and Babesia sp. detected in this study were first molecularly detected in Hy.asiaticum. Genotype III of A.ovis was predominant in the border regions of northwestern China.

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes

BackgroundThe Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.MethodsUsing mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.ResultsMetacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.ConclusionsThe data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

MOLECULAR ANALYSIS OF PBMSP-1 GENE IN ERYTHROCYTE OF MICE INFECTED WITH Plasmodium berghei

This study aimed to analyze MSP-1 gene in mice erythrocyte infected with Plasmodium berghei using polymerase chain reaction (PCR). The result showed the existence of 462 bp DNA band which was assumed to encode MSP-1 protein with molecular weight of 19 kDa that can only be found in erythrocyte infected with Plasmodium berghei. BLASTn analysis showed that PbMSP-1 obtained in this study have 100% similar identity with mRNA of PbMSP-1 partial sequence (462 bp), 93% similarity with PbMSP-1 complete sequence (5750 bp and 5376 bp), and 87% similarity with PbMSP-1 incomplete sequence (333 bp).

Molecular investigation of Anaplasma spp. in domestic and wildlife animals in Peninsular Malaysia.

Anaplasma spp. are Gram-negative obligate intracellular, tick-borne bacteria which are of medical and veterinary importance. Little information is available on Anaplasma infection affecting domestic and wildlife animals in Malaysia. This study investigated the presence of Anaplasma spp. in the blood samples of domestic and wildlife animals in Peninsular Malaysia, using polymerase chain reaction (EHR-PCR) assays targeting the 16S rRNA gene of Anaplasmataceae. High detection rates (60.7% and 59.0%, respectively) of Anaplasma DNA were noted in 224 cattle (Bos taurus) and 78 deer (77 Rusa timorensis and one Rusa unicolor) investigated in this study. Of the 60 amplified fragments obtained for sequence analysis, Anaplasma marginale was exclusively detected in cattle while Anaplasma platys/Anaplasma phagocytophilum was predominantly detected in the deer. Based on sequence analyses of the longer fragment of the 16S rRNA gene (approximately 1000 bp), the occurrence of A. marginale, Anaplasma capra and Candidatus Anaplasma camelii in cattle, Candidatus A. camelii in deer and Anaplasma bovis in a goat was identified in this study. To assess whether animals were infected with more than one species of Anaplasma, nested amplification of A. phagocytophilum, A. bovis and Ehrlichia chaffeensis DNA was performed for 33 animal samples initially screened positive for Anaplasmataceae. No amplification of E. chaffeensis DNA was obtained from animals investigated. BLAST analyses of the 16S rDNA sequences from three deer (R. timorensis), a buffalo (Bubalus bubalis) and a cow (B. taurus) reveal similarity with that of Candidatus Anaplasma boleense strain (GenBank accession no.: KX987335). Sequence analyses of the partial gene fragments of major surface protein (msp4) gene from two deer (R. timorensis) and a monitor lizard (Varanus salvator) show the detection of a strain highly similar (99%) to that of A. phagocytophilum strain ZJ-China (EU008082). The findings in this study show the occurrence of various Anaplasma species including those newly reported species in Malaysian domestic and wildlife animals. The role of these animals as reservoirs/maintenance hosts for Anaplasma infection are yet to be determined.

Plasmodium falciparum msp1 and msp2 genetic diversity and allele frequencies in parasites isolated from symptomatic malaria patients in Bobo-Dioulasso, Burkina Faso

BackgroundIn Burkina Faso, malaria remains the overall leading cause of morbidity and mortality accounting for 35.12% of consultations, 40.83% of hospitalizations and 37.5% of deaths. Genotyping of malaria parasite populations remains an important tool to determine the types and number of parasite clones in an infection. The present study aimed to evaluate the merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) genetic diversity and allele frequencies in Bobo-Dioulasso, Burkina Faso.MethodDried blood spots (DBS) were collected at baseline from patients with uncomplicated malaria in urban health centers in Bobo-Dioulasso. Parasite DNA was extracted using chelex-100 and species were identified using nested PCR. Plamodium falciparum msp1 and msp2 genes were amplified by nested polymerase chain reaction (PCR) and PCR products were analyzed by electrophoresis on a 2.5% agarose gel. Alleles were categorized according to their molecular weight.ResultsA total of 228 blood samples were analyzed out of which 227 (99.9%) were confirmed as P. falciparum-positive and one sample classified as mixed infection for P. malaria and P. falciparum. In msp1, the K1 allelic family was predominant with 77.4% (162/209) followed respectively by the MAD20 allelic family with 41.3% and R033 allelic family with 36%. In msp2, the 3D7 allelic family was the most frequently detected with 93.1 % compared to FC27 with 41.3%. Twenty-one different alleles were observed in msp1 with 9 alleles for K1, 8 alleles for MAD20 and 4 alleles for R033. In msp2, 25 individual alleles were detected with 10 alleles for FC27 and 15 alleles for 3D7. The mean multiplicity of falciparum infection was 1.95 with respectively 1.8 (1.76–1.83) and 2.1 (2.03–2.16) for msp1 and msp2 (P = 0.01).ConclusionsOur study showed high genetic diversity and allelic frequencies of msp1 and msp2 in Plasmodium falciparum isolates from symptomatic malaria patients in Bobo-Dioulasso.

Genetic, host and environmental factors associated with a high prevalence of Anaplasma marginale

Anaplasma marginale is the most prevalent vector-borne pathogen in the livestock industry in Colombia, causing economic losses of approximately USD 4.2 million per year. The present study reports the seasonal transmission patterns, genetic diversity and phylogeographic traits of A. marginale strains in cattle and buffaloes from Colombian livestock areas. A three-point longitudinal survey was designed to evaluate the above characteristics of farms in the Caribbean and Orinoquía regions. The A. marginale prevalence was evaluated in 1432 cattle blood samples, 152 buffalo blood samples and the hemolymph of 439 ticks using semi-nested PCR (sn-PCR) targeting the msp5 gene. The molecular prevalence in cattle and buffaloes was 54.8% and 13.1%, respectively, with higher values during the wet and late wet seasons. Factors such as age and production system were significantly associated with the infection. Rhipicephalus microplus was the only carrier of A. marginale DNA, with an infection rate of 17.2%. On the other hand, the tandem repeat and microsatellite analyses of the msp1α gene showed high genetic diversity and new tandem repeats that suggested strain adaptation to different transmission modes. Phylogeographic analysis using the msp4 gene showed a relationship between Colombian isolates and Mexican, Brazilian, Venezuelan, European and Asian isolates, as well as two worldwide haplogroups that were associated with the geographical origin of each isolate. In conclusion, this study shows that A. marginale occurs under enzootic stability in both hosts, with a high prevalence of infection during wet months and in animals dedicated to beef production. The genetic variability analyses suggest that a high strain diversity is associated with multiple selective pressures in the study area, while phylogeographic traits suggest a high genetic similarity between Mexican and South American strains.

Kloning dan Analisis Bioinformatika Gen MSP1 Plasmodium falciparum Isolat Kota Jayapura

Cloning gene involves the construction of a recombinant plasmid that inserted in a competent cell. On the other hand, genetic engineering requires bioinformatic analysis to be converted into tabulation and data interpretation. The study, titled "cloning block 2 MSP1 gene of Plasmodium falciparum isolate Jayapura city and bioinformatics analysis" is aimed to improve the technique of cloning the MSP1 gene of P. falciparum, initiated the creation of DH5α competent cells, ligations and transformations, plasmid isolation, confirmation the recombinant plasmid and able to perform bioinformatics analysis and construct phylogenetic tree. This study began with the manufacture of E. coli DH5α competent cells, MSP1 gene ligation in pJET1.2/blunt vector and transformation by using the heat shock transformation method, plasmid isolation of alkali lysis method, then plasmid confirmed by PCR and sequencing method, further sequence analysis and phylogenetic tree construction. The results showed that confirmation of MSP1 gene presence in pJET1.2/blunt with PCR was successful. From a total of 4 positive colonies grown in liquid culture, then isolated plasmid and confirmed with PCR obtained electroferogram bands with a size about 1049 bp indicates the presence of MSP1 gene in plasmid. Based on the results, cloning of MSP1 gene using pJET1.2/blunt cloning vector and competent cell E. coli DH5α has been successfully performed. Bioinformatics analysis of sequencing result and phylogenetic tree were constructed successfully with 2 clusters isolate of malaria patients from Jayapura city. Key words: Bioinformatics, cloning gene, heat shock transformation, MSP1, P. falciparum.

Genetic characterization of Anaplasma marginale strains from Tunisia using single and multiple gene typing reveals novel variants with an extensive genetic diversity

Anaplasma marginale, which is responsible for bovine anaplasmosis in tropical and subtropical regions, is a tick-borne obligatory intraerythrocytic bacterium of cattle and wild ruminants. In Tunisia, information about the genetic diversity and the phylogeny of A. marginale strains are limited to the msp4 gene analysis. The purpose of this study is to investigate A. marginale isolates infecting 16 cattle located in different bioclimatic areas of northern Tunisia with single gene analysis and multilocus sequence typing methods on the basis of seven partial genes (dnaA, ftsZ, groEL, lipA, secY, recA and sucB). The single gene analysis confirmed the presence of different and novel heterogenic A. marginale strains infecting cattle from the north of Tunisia. The concatenated sequence analysis showed a phylogeographical resolution at the global level and that most of the Tunisian sequence types (STs) formed a separate cluster from a South African isolate and from all New World isolates and strains. By combining the characteristics of each single locus with those of the multi-loci scheme, these results provide a more detailed understanding on the diversity and the evolution of Tunisian A. marginale strains.

Multiplicity and molecular epidemiology of Plasmodium vivax and Plasmodium falciparum infections in East Africa

BackgroundParasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology.MethodsSymptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing.ResultsA total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44–65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI.ConclusionsThe study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.

A Caenorhabditis elegans protein with a PRDM9-like SET domain localizes to chromatin-associated foci and promotes spermatocyte gene expression, sperm production and fertility.

To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 C. elegans presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence in situ hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed C. elegans protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes. SET-17 drives the transcription of spermatocyte-specific genes from four genomic clusters to promote spermatid development. SET-17 is concentrated in stable chromatin-associated nuclear foci at actively transcribed msp (major sperm protein) gene clusters, which we term msp locus bodies. Our results reveal the function of a PRDM9/7-family SET-domain protein in spermatocyte transcription. We propose that the spatial intranuclear organization of chromatin factors might be a conserved mechanism in tissue-specific control of transcription.

Correction: Sequence variation in Plasmodium falciparum merozoite surface protein-2 is associated with virulence causing severe and cerebral malaria.

[This corrects the article DOI: 10.1371/journal.pone.0190418.].

First report of Anaplasma ovis in pupal and adult Melophagus ovinus (sheep ked) collected in South Xinjiang, China

BackgroundMelophagus ovinus (sheep ked) is a blood-feeding ectoparasite that belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and mainly parasitizes sheep. The life-cycle of M. ovinus consists of three stages: larva, pupa and adult. It has a worldwide distribution and has been found in four provinces of China, especially South Xinjiang. In addition to causing direct damage to animal hosts, M. ovinus serves as a vector for disease transmission. In this study, our aim was to investigate the presence of Anaplasma spp. in pupal and adult M. ovinus.MethodsA total of 93 specimens (including eight pupal specimens) of M. ovinus collected in South Xinjiang were selected for isolation of genomic DNA, followed by PCR amplification and sequencing of the msp4 gene of Anaplasma spp. The sequences were analyzed in MEGA 7.0 software and via online BLAST.ResultsPCR and sequencing results showed that all the specimens collected in 2013 were free of Anaplasma spp., whereas three and 25 specimens (including five pupal specimens) collected in 2016 and 2017, respectively, tested positive for Anaplasma spp. The analysis of 24 msp4 gene sequences (from four pupal specimens) confirmed the presence of A. ovis in M. ovinus specimens collected in South Xinjiang, China. The detected A. ovis isolates belong to Genotypes II and III.ConclusionsTo the best of our knowledge, this is the first report of the detection of A. ovis DNA in pupal M. ovinus, confirming the vertical transmission of A. ovis in M. ovinus and the potential of M. ovinus to serve as a vector for A. ovis.

Molecular detection of Anaplasma phagocytophilum-like Anaplasma spp. and pathogenic A. Phagocytophilum in cattle from South Korea

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis and tick-borne fever in domestic ruminants. Differential diagnosis of zoonotic and pathogenic tick-borne diseases like granulocytic anaplasmosis is important for the efficient implementation of control programs. Thus, the differentiation of pathogenic A. phagocytophilum from non-pathogenic A. phagocytophilum-like (APL) Anaplasma spp. is essential. Recent molecular analyses of APL revealed its distinct phylogenetic position from A. phagocytophilum. This study was conducted to detect A. phagocytophilum and genetically related strains in 764 cattle in South Korea using PCR and restriction fragment length polymorphism assays. APL clade A and A. phagocytophilum were identified in 20 (2.6%) and 16 (2.1%) cattle, respectively, with 16 cattle (2.1%) displaying co-infection. The 16S rRNA sequences of APL clade A were similar (98.3–99.9%) to those clustered in the APL clade A from eastern Asia. The A. phagocytophilum 16S rRNA sequence shared 98.6–100% identity to those of the A. phagocytophilum group. We used PCR to amplify the groEL and msp2 genes from the 20 samples positive for the 16S rRNA gene and found that 16 were positive for the groEL sequences in the APL clade A, which showed identity (82.8–84.4%) to those clustered in the APL clade A from Japan. Amplification of msp2 was unsuccessful. The co-infection results suggested sequence diversity in Anaplasma spp. Till date, both A. phagocytophilum and APL have been reported to be distributed separately in several animals throughout South Korea. This report is the first co-detection of A. phagocytophilum and APL in Korean cattle using molecular methods. Further studies are needed to provide additional molecular background and trace the evolutionary tree of Anaplasma species in animals and ticks.