High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil / Alta diversidade genética de Anaplasma marginale em bovinos de leite no nordeste do Brasil

Abstract Anaplasma marginale is an obligate intracellular Gram-negative bacterium found in ruminants' erythrocytes and is the etiological agent of bovine anaplasmosis. The bacterium's genetic diversity has been characterized based on sequences of major surface proteins (MSPs), such as MSP1α. The aim of the present study was to investigate the genetic diversity of A. marginale in cattle in the state of Maranhão, northeastern Brazil. To this end, 343 blood samples were harvested and subjected to iELISA assays using the recombinant surface protein MSP5. Out of 343 blood samples, 235 (68.5%) were randomly chosen and submitted to DNA extraction, qPCR and conventional PCR targeting the msp1α gene to determine amino acid sequences and classify the genotypes. The iELISA results showed 81.34% seropositivity (279/343), whereas qPCR revealed 224 positive samples (95.32%). Among these qPCR-positive samples, 67.4% (151/224) were also positive in the cPCR. Among the 50 obtained sequences, 21 strains had not been previously reported. Regarding the genotypes, H (26/50) and E (18/50) were identified most often, while genotypes F and C were only identified twice each and B and G once each. In conclusion, high prevalence and genetic diversity for A. marginale were observed in dairy cattle herds in the state of Maranhão.

Resumo Anaplasma marginale é uma bactéria Gram-negativa intracelular obrigatória de eritrócitos de ruminantes e responsável pela anaplasmose bovina. A diversidade genética de A. marginale tem sido caracterizada com base nas sequências das principais proteínas de superfície (MSPs), como a MSP1α. O objetivo deste estudo foi investigar a diversidade genética de A. marginale em bovinos no estado do Maranhão, Nordeste do Brasil. Dessa forma, 343 amostras de sangue foram submetidas ao ensaio iELISA, utilizando-se a proteína recombinante MSP5. Das 343 amostras de sangue, 235 (68,5%) foram escolhidas aleatoriamente e submetidas à extração de DNA, qPCR e PCR convencional para gene msp1α, para determinação das sequências de aminoácidos e classificação dos genótipos. Os resultados do iELISA mostraram 81,34% de soropositividade (279/343), enquanto qPCR revelou 224 amostras positivas (95,32%). Dentre estas na qPCR, 67,4% (151/224) mostraram-se positivas no PCR convencional. Das 50 sequências obtidas, 21 cepas não haviam sido relatadas anteriormente. Em relação aos genótipos, H (26/50) e E (18/50) foram os mais frequentes, enquanto os genótipos F e C foram identificados apenas duas vezes cada, e B e G uma vez cada. Em conclusão, alta prevalência e marcante diversidade genética de A. marginale foram observadas em rebanhos leiteiros no estado do Maranhão.

Role of toll-like receptor 4 in eliciting adaptive immune responses against recombinant BCG expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum

Objective: To determine the role of toll-like receptor 4 (TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) were injected with phosphate buffered saline T80, BCG or rBCG intraperitoneally, in the presence or absence of a TLR-4 inhibitor; TAK-242. Enzyme-linked immunosorbent assay was carried out for serum total IgG, IgG1, IgG2a and IgG2b determination. Spleens were also harvested and splenocytes cultured for determination of intracellular cytokines; IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG, and the subclasses IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than BCG and phosphate buffered saline immunised mice in the absence of TAK-242. A significant rise in total IgG occurred with more booster immunisations. The level of IgG2a was highest, followed by IgG2b, then IgG1. The production of both IL-4 and IFN-γ was also highest in the rBCG immunised groups. These significant rises were inhibited in the presence of TAK-242. Conclusions: We present evidence of the role of TLR-4 in the increased production of total IgG, IgG1, IgG2a and IgG2b, as well as IL-4 and IFN-γ in response to our rBCG construct.

Pam3CSK4 enhances adaptive immune responses to recombinant Mycobacterium bovis bacille Calmette-Guérin expressing Plasmodium falciparum C-terminus merozoite surface protein-1

Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme-linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.

Role of toll-like receptor 4 in eliciting adaptive immune responses against recombinant BCG expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum

Objective:To determine the role of toll-like receptor 4 (TLR-4) in eliciting cellular and humoral immune responses against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum.Methods:Six groups of mice (n=6 per group) were injected with phosphate buffered saline T80,BCG or rBCG intraperitoneally,in the presence or absence of a TLR-4 inhibitor;TAK-242.Enzyme-linked immunosorbent assay was carried out for serum total IgG,IgG1,IgG2a and IgG2b determination.Spleens were also harvested and splenocytes cultured for determination of intracellular cytokines;IL-4 and IFN-γ via enzyme-linked immunosorbent assay.Results:The production of total IgG,and the subclasses IgG 1,IgG2a and IgG2b was significantly higher in rBCG-immunised mice than BCG and phosphate buffered saline immunised mice in the absence of TAK-242.A significant rise in total IgG occurred with more booster immunisations.The level of IgG2a was highest,followed by IgG2b,then IgG1.The production of both IL-4 and IFN-γ was also highest in the rBCG immunised groups.These significant rises were inhibited in the presence of TAK-242.Conclusions:We present evidence of the role of TLR-4 in the increased production of total IgG,IgG1,IgG2a and IgG2b,as well as IL-4 and IFN-γ in response to our rBCG construct.

Pam3CSK4 enhances adaptive immune responses to recombinant Mycobacterium bovis bacille Calmette-Guérin expressing Plasmodium falciparum C-terminus merozoite surface protein-1

Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme-linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

Immune response and biochemistry of calves immunized with rMSP1a ( Anaplasma marginale) using carbon nanotubes as carrier molecules / Resposta imunológica e bioquímica de bezerros imunizados com rMSP1 a (Anaplasma marginale) utilizando nanotubos de carbono como moléculas carreadoras

Abstract Vaccination against Anaplasma marginale has been considered an important control strategy for bovine anaplasmosis. Recently, mice immunized with rMSP1 a linked to carbon nanotubes (MWNT) showed significant immune responses, generating a new possibility for use of an inactivated vaccine. The objective of this study was to investigate the cellular and humoral responses in calves immunized with MWNT+rMSP1a , associated with inactivated vaccine of A. marginale produced in vitro, and evaluate the toxic effects of the MWNT on renal and hepatic function. rMSP1a was covalently linked to MWNT. Inactivated vaccine (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. Twenty-four Holstein calves were divided (four groups) and immunized subcutaneously with PBS and non-carboxylated MWNT (control, G1), AmUFMG2 (G2), MWNT+rMSP1a (G3), and AmUFMG2 with MWNT+rMSP1a (G4). Blood samples were collected for total leukocyte counts, biochemical profiling and evaluation of the cellular and humoral response. Immunization with MWNT+rMSP1a induced increase in the total number of leukocytes, NK cells, in the lymphocyte populations and higher levels of antibodies compared to calves immunized only with AmUFMG2. Furthermore, MWNT did not induce changes in the biochemical profile. These data indicate that MWNT+rMSP1a were able to induce the immune responses more efficiently than AmUFMG2 alone, without generating toxicity.

Resumo Vacinação contra Anaplasma marginale tem sido considerada uma importante estratégia de controle da anaplasmose bovina. Recentemente, camundongos imunizados com rMSP1a funcionalizada à nanotubos de carbono (MWNT) apresentaram resposta imune significante, gerando nova possibilidade para o uso da vacina inativada. O objetivo desse estudo foi investigar a resposta celular e humoral em bezerros imunizados com MWNT+rMSP1a, associado com a vacina inativada de A. marginale produzida in vitro, e avaliar os efeitos tóxicos dos MWNT nas funções hepática e renal. rMSP1 a foi ligada covalentemente aos MWNT. Vacina inativada (AmUFMG2) foi produzida através do cultivo de A. marginale em células IDE8. Vinte e quatro bezerros Holandeses foram divididos (quatro grupos) e imunizados subcutaneamente com: PBS e MWNT não-carboxilados (controle, G1), AmUFMG2 (G2), MWNT+rMSP1 a (G3), e AmUFMG2 com MWNT+rMSP1a (G4). Amostras de sangue foram coletadas para contagem de leucócitos, perfil bioquímico e avaliação da resposta celular e humoral. Imunização com MWNT+rMSP1a induziu aumento dos leucócitos totais, células NK, na população de linfócitos e altos níveis de anticorpos comparado com animais imunizados apenas com AmUFMG2. Além disso, MWNT não induziu alterações no perfil bioquímico. Esses dados indicam que MWNT+rMSP1a foram capazes de induzir eficientemente a resposta imune comparado com AmUFMG2 sozinho, sem gerar toxicidade.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

PCR-RFLP for Rapid Subtyping of Plasmodium vivax Korean Isolates

Vivax malaria reemerged in Korea in 1993 and the outbreak has been continued with fluctuating numbers of annual indigenous cases. Understanding the nature of the genetic population of Plasmodium vivax circulating in Korea is beneficial for the knowledge of the nationwide parasite heterogeneity and in the implementation of malaria control programs in the country. Previously, we analyzed polymorphic nature of merozoite surface protein-1 (MSP-1) and MSP-3α in Korean P. vivax population and identified the Korean P. vivax population has been diversifying rapidly, with the appearance of parasites with new genetic subtypes, despite the recent reduction of the disease incidence. In the present study, we developed simple PCR-RFLP methods for rapid subtyping of MSP-1 and MSP-3α of Korean P. vivax isolates. These PCR-RFLP methods were able to easily distinguish each subtype of Korean P. vivax MSP-1 and MSP-3α with high accuracy. The PCR-RFLP subtyping methods developed here would be easily applied to massive epidemiological studies for molecular surveillance to understand genetic population of P. vivax and to supervise the genetic variation of the parasite circulating in Korea.

Allele genetypes and homology analysis of MSP-1 and CSP gene of Plasmodi-um vivax in Shandong Province / 中国血吸虫病防治杂志

Objective To analyze the genotypes and homology of MSP?1 and CSP gene of Plasmodium vivax in Shandong Province,so as to provide the evidence for case traceability. Methods A total of 12 blood samples were collected from P. vivax?infected cases in Shandong Province in 2011. Parasite genomic DNA was extracted. Primers were designed according to MSP?1 and CSP gene sequences of P. vivax. Then Nested PCR,enzyme digestion,sequencing and sequence alignment,and homolo?gous analysis were performed. Results The MSP?1 gene of all the 12 samples from P. vivax?infected cases were detected with a 470 bp PCR amplification band,and 350 bp and 120 bp enzyme digestion fragments,which were identified as type Sal?1. An analysis of phylogenetic tree of MSP?1 gene showed that the sequences of 9 indigenous case samples in Shandong Province were located in the same branch,one case sample infected from India was located in the same branch with India strains. All the 12 P. vivax?infected samples covered GDRA(D/A)GQPA sequences in CSP gene,which were identified as type PV?Ⅰ. Of the CSP gene among 12 P. vivax?infected samples,10 samples of indigenous case in Shandong Province and one sample of the case in?fected in Guangdong Province were detected with both 560-840 bp and 150-230 bp PCR amplification bands,which were iden?tified as temperate zone family strain of type PV?Ⅰ. However,one sample from the case infected in India was detected only with a 560-840 bp band,which was identified as tropical zone family strain of PV?Ⅰ. An analysis of phylogenetic tree of CSP gene showed that the sequences of 10 samples from the indigenous cases in Shandong Province and one sample from the case infected in Guangdong Province were located in the same branch,one sample from the case infected in India was located in the same branch with India and Indonesia strains. Conclusion Of all the indigenous isolates in Shandong Province,MSP?1 gene is geno?typed type Sal?1,CSP gene is genotyped temperate zone family strain of type PV?Ⅰ,with a high homology found among the in?digenous isolates.

Outbreak of anaplasmosis associated with the presence of different Anaplasma marginale strains in dairy cattle in the states of São Paulo and Goiás, Brazil / Surto de anaplasmose associado com a presença de diferentes cepas de Anaplasma marginale em gado leiteiro dos estados de São Paulo e Goiás, Brasil

Abstract The present study reports the genetic diversity of Anaplasma marginale during anaplasmosis outbreaks in rural properties of the states of Goiás and São Paulo, Brazil. Mortality rates of 3.5% (37/1,050) in calves, 4.7% (45/954) in heifers and 1.1% (25/2,200) in lactating cows were observed in a cattle herd of the municipality of Mambaí, state of Goiás, central-western Brazil. In a cattle herd from the municipality of Lins, state of São Paulo, in southeastern Brazil, none of the animals died, despite presenting clinical signs suggestive of bovine anaplasmosis and exhibiting a drastic decrease in milk production. Thus, blood samples were collected from 100 animals with clinical signs suggestive of bovine anaplasmosis in the municipalities of Mambaí and Lins. Based on the microsatellite structure of the MSP1a of A. marginale, the genotypes E and H were observed in Lins, and the C, D and E genotypes were found in Mambaí. The analysis of the tandem repeat structures of the MSP1a showed nine different strains (τ-10 -15, α-β2, α-β3-13, α-β2 192, τ-β-100, α-β2-Γ, 193-β-100, 191-13-Γ and 191-13-18) in Lins and two (α-β3-Γ and E-F-φ2-F2) in Mambaí. Three new tandem repeats of MSP1a (191, 192 and 193) were described. The τ-10-15 and α-β3-Γ strains were predominantly associated with the occurrence of clinical anaplasmosis and mortality in calves, heifers and lactating cows.

Resumo O presente estudo relata a diversidade genética de Anaplasma marginale durante surtos de anaplasmose bovina no Brasil em propriedades localizadas nos Estados de Goiás e São Paulo. No rebanho bovino de Mambaí, Estado de Goiás, Centro-oeste do Brasil, observaram-se taxas de mortalidade de 3,5% (37/1050) nos bezerros; 4,7% (45/954) nas novilhas e 1,1% (25/2200) nas vacas em lactação. No rebanho bovino de Lins, Estado de São Paulo, Sudeste do Brasil, embora os animais tenham apresentado sinais clínicos sugestivos de anaplasmose bovina, culminando em redução drástica da produção leiteira, nenhum animal veio a óbito. Assim, amostras de sangue de 100 bovinos com sinais clínicos sugestivos de anaplasmose foram coletadas em Mambaí-GO e Lins-SP. Baseando-se na estrutura do microssatélite da MSP1a de A. marginale, observou-se a presença dos genótipos E e H em Lins e C, D e E em Mambaí. A análise da estrutura em “tandem repeats” da MSP1a mostrou nove diferentes estirpes (τ-10 -15, α-β2, α-β3-13, α-β2 192, τ-β-100, α-β2-Γ, 193-β-100, 191-13-Γ e 191-13-18) em Lins e duas (α-β3-Γ e E-F-φ2-F2) em Mambaí. Três novos “tandem repeats” da MSP1a (191, 192 e 193) foram descritos. Foi observado predomínio das estirpes τ-10-15 e α-β3-Γ associado à ocorrência de anaplasmose clínica e mortalidade em bezerras, novilhas e vacas em lactação.

Allelic Diversity of MSP1 Gene in Plasmodium falciparum from Rural and Urban Areas of Gabon

The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families' frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (+/-0.95) higher compared to that found at Owendo (1.55+/-0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.

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

Objective: 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. Methods: 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. Results: 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. Conclusions: 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.

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

Objective: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. Methods: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. Results: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. Conclusions: 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.

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.

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

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Status of allele frequency and diversity of Plasmodium falciparum msp1, msp2 and glurp before implementation of an artemisinin-based combined therapy in Northwestern Colombia / Estado de la frecuencia alélica y la diversidad de Plasmodium falciparum msp1, msp2 y glurp antes de la implementación de terapia combinada con artemisina en el Noroccidente de Colombia

Introduction: The status of msp1, msp2 and glurp allele frequency and the diversity of Plasmodium falciparum in Northwestern Colombia before the implementation of an artemisinin-combined therapy have been explored only by a few authors and in a relatively small number of samples from this highly endemic region. Objective: To evaluate the frequency of msp1, msp2, and glurp alleles and the diversity of P. falciparum in two Colombian regions before the use of an artemisinin-combined therapy. Methods: This study was part of a major anti-malarial efficacy trial designed as a random, clinically-controlled study for which 224 subjects were recruited. Region 2 of msp1 and msp2 (central region) were amplified by a nested PCR; glurp (region R2) was amplified by a semi-nested PCR. Results: For msp1, five genotypes were observed, representing the K1, MAD20, and RO33 allelic families. All samples corresponded to a MAD20 150 bp allele. For msp2 (IC family), two alleles were detected and for glurp, eight were observed. A total 33 haplotypes were detected. Conclusions: Analysis of glurp can be used to successfully genotype parasite populations in the new studies in Colombia aimed at exploring Plasmodium spp population dynamics. In addition, analysis of msp1 and msp2 can also be of value for comparisons with past studies, but not when the objective is to study parasites obtained from the same patient in a reduced period of time; for instance, during treatment efficacy studies.

Introducción: La frecuencia alélica y la diversidad de msp1, msp2 y glurp de Plasmodium falciparum en el Noroccidente de Colombia antes de la introducción de la terapia combinada con artemisinina, han sido exploradas por pocos autores y en números relativamente bajos de muestras clínicas de esta región de alta endemicidad. Objetivo: Evaluar la frecuencia alélica y la diversidad de msp1, msp2 y glurp de P. falciparum en dos regiones colombianas antes del uso de la terapia combinada con artemisinina. Métodos: El estudio fue parte de un ensayo mayor sobre la eficacia de la terapia antimalárica con un diseño clínico aleatorio controlado, en el cual 224 sujetos fueron reclutados. La región 2 de msp1 y msp2 (región central) se amplificaron por medio de una PCR anidada; glurp (región RII) fue amplificada con una PCR semi-anidada. Resultados: En msp1, se observaron 5 genotipos que representaron las familias alélicas K1, MAD20, y RO33. Todas las muestras correspondieron al alelo 150 bp de MAD20. En msp2 (familia IC), se detectaron 2 alelos y en glurp, se observaron 8. Un total de 33 haplotipos fueron detectados. Conclusiones: El análisis de glurp puede ser utilizado para genotipificar exitosamente poblaciones parasitarias en nuevos estudios en Colombia con el objeto de explorar la dinámica de las poblaciones de Plasmodium spp. Adicionalmente, el análisis de msp1 y msp2 puede también ser de valor para comparar con estudios pasados, pero no cuando el objetivo es estudiar parasitos obtenidos de un mismo paciente en un periodo reducido de tiempo, por ejemplo, durante estudios de eficacia terapéutica.

Immune response of calves inoculated with proteins of Anaplasma marginale bound to an immunostimulant complex / Resposta imune de bezerros inoculados com proteínas de Anaplasma marginale ligadas a um complexo imunoestimulante

Despite our current knowledge of the immunology, pathology, and genetics of Anaplasma marginale, prevention in cattle is currently based on old standbys, including live attenuated vaccines, antibiotic treatment, and maintaining enzootic stability in cattle herds. In the present study, we evaluated the use of an immunostimulant complex (ISCOMATRIX) adjuvant, associated with a pool of recombinant major surface proteins (rMSP1a, rMSP1b, rMSP4 and rMSP5) to improve the humoral immune response triggered in calves mainly by IgG2. Ten calves were divided in three groups: 4 calves were inoculated with the ISCOMATRIX/rMSPs (G1); 2 calves were inoculated with ISCOMATRIX adjuvant (G2); and 4 calves received saline (G3). Three inoculations were administered at 21-day intervals. In G1, the calves showed significant increases in total IgG, IgG1 and IgG2 levels 21 days after the second inoculation, compared to the control group (p < 0.05), and G1 calves remained above the cut-off value 28 days after the third inoculation (p < 0.05). The post-immunized sera from calves in G1 reacted specifically for each of the rMSPs used. In conclusion, the ISCOMATRIX/rMSPs induced antigen-specific seroconversion in calves. Therefore, additional testing to explore the protection induced by rMSPs, both alone and in conjunction with proteins previously identified as subdominant epitopes, is warranted.

Apesar dos avanços da imunologia, patologia e genética de Anaplasma marginale, a prevenção em bovinos ainda é baseada nas vacinas vivas atenuadas, na terapia com antibiótico e estabilidade enzoótica dos rebanhos bovinos. No presente estudo, avaliou-se o uso de um complexo imunoestimulante (ISCOMATRIX), associado às proteínas recombinantes de superfície (rMSP1a, rMSP1b, rMSP4 e rMSP5) para melhorar a resposta imune humoral desencadeada em bezerros, principalmente por IgG2. Dez animais foram divididos em três grupos: 4 bezerros foram inoculados com o ISCOMATRIX/rMSPs (G1), 2 bezerros foram inoculados com ISCOMATRIX adjuvante (G2) e 4 bezerros receberam salina (G3). Três doses vacinais foram administradas em intervalos de 21 dias. No G1, os bezerros apresentaram aumentos significativos nos níveis de IgG total, IgG1 e IgG2 21 dias após a segunda inoculação, em comparação com o grupo de controle (p <0,05). Nos bezerros do G1 esses níveis de anticorpos permaneceram acima do ponto de corte 28 dias após a terceira inoculação (p < 0,05). Os soros pós-imunização de bezerros do G1 reagiram especificamente com cada uma das rMSPs utilizadas. Em conclusão, o ISCOMATRIX/rMSPs induziu soroconversão antígeno-específica em bezerros. Portanto, se justifica a realização de ensaios adicionais para explorar a proteção induzida pela rMSPs, tanto sozinhas como em conjunto com novas proteínas identificadas com epítopos subdominantes.

Detection of genetic diversity of Anaplasma marginale isolates in Minas Gerais, Brazil / Detecção de diversidade genética de isolados Anaplasma marginale em Minas Gerais, Brasil

Bovine anaplasmosis, caused by the tick-borne rickettsiaAnaplasma marginale, is endemic in tropical and subtropical regions of the world and results in economic losses in the cattle industry. Major surface proteins (MSPs) have been used as markers for the genetic characterization of A. marginale strains and demonstrate that many isolates may occur in a given geographic area. However, in Brazil, little is known about the genetic diversity of A. marginale isolates within individual herds. This study was designed to examine the genetic variation among A. marginale infecting calves in a farm in the south of Minas Gerais State, Brazil. Blood samples collected from 100 calves were used to prepare Giemsastained smears that were microscopically examined for the presence of A. marginale. From each blood sample, DNA was extracted and analyzed by a polymerase chain reaction (PCR), followed by sequencing to determine diversity among the isolates. Examination of blood smears showed that 48% of the calves were infected with A. marginale, while the real-time PCR detected 70.2% positivity. Congenital infections were found in four calves. The microsatellite and tandem repeat analyses showed high genetic diversity among the isolates.

A anaplasmose bovina, causada pela rickettsia Anaplasma marginale e transmitida por carrapatos, é endêmica em regiões tropicais e subtropicais no mundo e causa grandes perdas econômicas na indústria de bovinos. Proteínas principais de superfície (MSPs) foram usados como marcadores para a caracterização genética de amostras de A. marginale, demonstrando que diferentes isolados podem ocorrer numa certa região geográfica. Porém, no Brasil pouco se sabe sobre a variedade genética de isolados de A. marginale em rebanhos individuais. Este estudo teve como objetivo investigar a ocorrência de variação genética entre bezerros infectados com A. marginale numa fazenda do sul de Minas Gerais, Brasil. Amostras de sangue coletadas de 100 bezerros foram utilizadas para o preparo de esfregaços sanguíneos, corados pelo Giemsa, para detecção da infecção por A. marginale. Amostras de DNA extraídas de cada amostra foram analisadas através de PCR seguido de sequenciamento. O exame dos esfregaços demonstrou que 48% dos bezerros estavam infectados com A. marginale, enquanto que o PCR detectou 70,2% de positividade. Infecção congênita foi detectada em quatro bezerros. As análises de microsatélites e 'tandem repeats' comprovaram uma grande diversidade genética entre os isolados.

Characterization of the Population Demographics and the MSP-1 Block 2 Allele Gene Frequencies of <i>P. falciparum</i> Infected Individuals in Davao, Philippines

<i>Plasmodium falciparum</i> is one of the causative agents of malaria in humans. This parasite causes the most severe forms of the disease. In order to combat the disease, it is important to have knowledge about the parasite and its interaction with its host. In this study, we profiled 74 patients admitted to hospital in Tagum, Davao, Philippines who were confirmed to be infected with <i>P. falciparum</i>. We correlated the age, sex and parasite load with malaria severity and show that among these, only sex is correlated with disease severity in this population. In addition, we profiled the MSP-1 block 2 allele distribution in the population and found that the most abundant allele form was K1, followed by MAD20. The RO33 allele form was the rarest allele in this population.