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1.
Pathogens ; 11(5)2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35631041

RESUMO

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.

2.
Parasit Vectors ; 12(1): 115, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890166

RESUMO

BACKGROUND: Cytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics. RESULTS: A 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans. CONCLUSIONS: In surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.


Assuntos
Doenças do Gato/parasitologia , Variação Genética , Lynx/parasitologia , Piroplasmida/genética , Infecções Protozoárias em Animais/parasitologia , Animais , Gatos , Marcadores Genéticos , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética
3.
ACS Infect Dis ; 5(4): 506-514, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30746930

RESUMO

To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.


Assuntos
Malária Falciparum/parasitologia , Peptídeos/química , Peptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Eritrócitos/parasitologia , Humanos , Cinética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
4.
Vet Parasitol ; 263: 49-53, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30389023

RESUMO

Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99-100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.


Assuntos
Antígenos de Protozoários/genética , Doenças do Gato/parasitologia , Piroplasmida/genética , RNA Mensageiro/genética , Esquizontes/genética , Animais , Gatos , DNA de Protozoário/genética , Infecções Protozoárias em Animais/mortalidade , Infecções Protozoárias em Animais/parasitologia , Infecções Protozoárias em Animais/prevenção & controle , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia
5.
Nanomedicine ; 12(6): 1445-51, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27015769

RESUMO

Surface enhanced Raman spectra (SERS) of normal red blood cells (RBCs) and Plasmodium falciparum infected RBCs (iRBCs) at different post invasion time were obtained based on silver nanorod array substrates. Distinct spectral differences were observed due to the cell membrane modification of RBCs during malaria infection. The SERS spectra of ring stage iRBCs had a characteristic Raman peak at Δv=1599cm(-1) as compared to those of normal RBCs, while the trophozoite and schizoid stages had identical SERS spectra with a characteristic peak at Δv=723cm(-1), which is significantly different from ring stage iRBCs, consistent with ongoing modification of the iRBC membrane. Since ring stage iRBCs of P. falciparum are found circulating in blood, such a difference provides a new strategy for rapid malaria detection. The limit of detection as well as the ability to detect a mixed iRBC and RBC solution was also investigated.


Assuntos
Malária/diagnóstico , Análise Espectral Raman , Contagem de Eritrócitos , Eritrócitos , Humanos , Plasmodium falciparum
6.
PLoS One ; 10(5): e0129239, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26010880

RESUMO

Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 µM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.


Assuntos
Antimaláricos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Antimaláricos/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/química , Proteínas de Protozoários/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Malar J ; 13: 31, 2014 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-24467985

RESUMO

BACKGROUND: Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country's diagnostic laboratory; and, (ii) determine the assay's sensitivity and specificity compared to a nested 18S rRNA PCR. METHODS: Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR. RESULTS: Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100% concordance with the results obtained at the reference laboratory in the USA. CONCLUSION: The PET-PCR is a new molecular diagnostic tool with similar performance characteristics as commonly used PCR methods that is less expensive, easy to use, and amiable to large scale-surveillance studies in developing country settings.


Assuntos
Primers do DNA , Malária Falciparum/diagnóstico , Microscopia/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA de Protozoário/genética , Corantes Fluorescentes , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Tanzânia
8.
Am J Vet Res ; 74(6): 901-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23718659

RESUMO

OBJECTIVE: To characterize systemic immune responses in Cytauxzoon felis-infected cats. SAMPLE: Blood and lung samples obtained from 27 cats. PROCEDURES: Cats were allocated into 4 groups: cats that died of cytauxzoonosis, acutely ill C felis-infected cats, healthy survivors of C felis infection, and healthy uninfected cats. Serum concentrations of tumor necrosis factor-α and interleukin-1 ß were measured and serum proteins characterized. Blood smears were stained immunocytochemically and used to assess immunoglobulin deposition. Immunohistochemical expression of CD18 and tumor necrosis factor-α were compared in lung tissues obtained from cats that died and healthy uninfected cats. A real-time reverse-transcription PCR assay for CD18 expression was performed on selected blood samples from all groups. RESULTS: Concentrations of both cytokines were greater and serum albumin concentrations were significantly lower in cats that died of cytauxzoonosis, compared with results for all other groups. Erythrocytes from acutely ill cats and survivors of C felis infection had staining for plasmalemmal IgM, whereas erythrocytes from the other groups did not. Increased staining of C felis-infected monocytes and interstitial neutrophils for CD18 was detected. The real-time reverse-transcription PCR assay confirmed a relative increase in CD18 expression in cats that died of cytauxzoonosis and acutely ill cats, compared with expression in other groups. Immunostaining for TNF-α in lung samples confirmed a local proinflammatory response. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated immunopathologic responses were greater in cats that died of C felis infection than in cats that survived C felis infection.


Assuntos
Doenças do Gato/parasitologia , Infecções Protozoárias em Animais/imunologia , Animais , Apicomplexa/classificação , Antígenos CD18/metabolismo , Doenças do Gato/imunologia , Doenças do Gato/mortalidade , Gatos , Imunoglobulina M , Interleucina-1beta/sangue , Interleucina-1beta/metabolismo , Pulmão/metabolismo , Infecções Protozoárias em Animais/mortalidade , Infecções Protozoárias em Animais/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
9.
PLoS One ; 8(1): e54986, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23349994

RESUMO

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.


Assuntos
Malária Vivax/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Plasmodium vivax/patogenicidade , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Especificidade da Espécie
10.
Vet Parasitol ; 190(1-2): 29-35, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22776107

RESUMO

Cytauxzoon felis, a tick-borne protozoan parasite, is the causative agent of cytauxzoonosis in domestic cats in the United States. The natural reservoir for this parasite is the bobcat (Lynx rufus), which typically does not develop clinical signs. Although not likely important reservoirs, C. felis has also been detected in pumas (Puma concolor) in Florida and Louisiana. Recent studies suggest that specific genotypes of C. felis that circulate in domestic cats may be associated with variable clinical outcomes and specific spatial locations. In the current study, we investigated the intraspecific variation of the C. felis internal transcribed spacer (ITS)-1 and ITS-2 rRNA regions from 145 wild felids (139 bobcats and six pumas) from 11 states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, South Carolina, Oklahoma, and Pennsylvania). Unambiguous ITS-1 and ITS-2 data were obtained for 144 and 112 samples, respectively, and both ITS-1 and ITS-2 sequences were obtained for 111 (77%) samples. For the ITS-1 region, sequences from 65 samples collected from wild felids were identical to those previously reported in domestic cats, while the other 79 sequences were unique. C. felis from 45 bobcats and one puma had ITS-1 sequences identical to the most common sequence reported from domestic cats. Within the ITS-2 region, sequences from 49 bobcats were identical to those previously reported in domestic cats and 63 sequences were unique (with some occurring in more than one bobcat). The most common ITS-2 sequence from domestic cats was also common in wild felids (31 bobcats and a puma). Samples from three pumas from Florida and two bobcats from Missouri had a 40- or 41-bp insert in the ITS-2 similar to one described previously in a domestic cat from Arkansas. Additionally, a previously undescribed 198- or 199-bp insert was detected in the ITS-2 sequence from four bobcats. Collectively, based on combined ITS-1 and ITS-2 sequences, five different genotypes were detected in the wild felids. Genotype ITSa was the most common genotype (11 bobcats and one puma) and fewer numbers of ITSb, ITSe, ITSg, and ITSi were detected in bobcats. These data indicate that, based on ITS-1 and ITS-2 sequences, numerous C. felis strains may circulate in wild felids.


Assuntos
Doenças do Gato/parasitologia , Variação Genética/genética , Lynx/parasitologia , Piroplasmida/isolamento & purificação , Infecções Protozoárias em Animais/parasitologia , Puma/parasitologia , Animais , Região dos Apalaches/epidemiologia , Sequência de Bases , Doenças do Gato/epidemiologia , Gatos , DNA de Protozoário/análise , DNA de Protozoário/genética , DNA Espaçador Ribossômico/análise , DNA Espaçador Ribossômico/genética , Reservatórios de Doenças , Genótipo , Meio-Oeste dos Estados Unidos/epidemiologia , Dados de Sequência Molecular , Piroplasmida/genética , Prevalência , Infecções Protozoárias em Animais/epidemiologia , Análise de Sequência de DNA , Sudeste dos Estados Unidos/epidemiologia , Especificidade da Espécie
11.
PLoS One ; 7(2): e31848, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22363751

RESUMO

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.


Assuntos
Bioensaio/métodos , Malária/parasitologia , Parasitos/genética , Plasmodium knowlesi/genética , Plasmodium knowlesi/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Zoonoses/parasitologia , Animais , Sequência de Bases , Reações Cruzadas/imunologia , Primers do DNA/metabolismo , Genoma de Protozoário/genética , Haplorrinos/parasitologia , Humanos , Limite de Detecção , Parasitos/isolamento & purificação , Plasmodium vivax/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Especificidade da Espécie
12.
PLoS One ; 7(2): e31565, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347496

RESUMO

Plasmodium falciparum malaria remains one of the world's foremost health problems, primarily in highly endemic regions such as Sub-Saharan Africa, where it is responsible for substantial morbidity, mortality and economic losses. Malaria is a significant cause of severe disease and death in pregnant women and newborns, with pathogenesis being associated with expression of a unique variant of the multidomain Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) called VAR2CSA. Here, we characterize the polymorphism of the DBL3X domain of VAR2CSA and identify regions under selective pressure among placental parasites from women living in endemic western Kenya. In addition to significant levels of polymorphism, our analysis reveals evidence for diversification through intra-segmental recombination and novel mutations that likely contributed to the high number of unique VAR2CSA sequence types identified in this study. Interestingly, we also identified a number of critical residues that may be implicated in immune evasion through switching (or toggling) to alternative amino acids, including an arginine residue within the predicted binding pocket in subdomain III, which was previously implicated in binding to placental CSA. Overall, these findings are important for understanding parasite diversity in pregnant women and will be useful for identifying epitopes and variants of DBL3X to be included in a vaccine against placental malaria.


Assuntos
Antígenos de Protozoários/genética , Placenta/parasitologia , Polimorfismo Genético/imunologia , Complicações Parasitárias na Gravidez/imunologia , Recombinação Genética/imunologia , Aminoácidos , Antígenos de Protozoários/imunologia , Feminino , Humanos , Evasão da Resposta Imune , Quênia/epidemiologia , Gravidez , Estrutura Terciária de Proteína
13.
J Clin Microbiol ; 49(7): 2411-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21525225

RESUMO

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/µl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.


Assuntos
Biologia Computacional/métodos , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Parasitologia/métodos , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase/métodos , Animais , Primers do DNA/genética , DNA de Protozoário/genética , Mineração de Dados/métodos , Genoma de Protozoário , Humanos , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Tanzânia , Venezuela
14.
Vet Parasitol ; 175(3-4): 325-30, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21071149

RESUMO

Cytauxzoon felis, a protozoan parasite of wild and domestic felids, is the causative agent of cytauxzoonosis in domestic and some exotic felids in the United States. The bobcat (Lynx rufus) is the natural reservoir for this parasite, but other felids such as Florida panthers (Puma concolor coryii) and domestic cats may maintain long-term parasitemias and serve as reservoirs. Experimentally, two tick species, Dermacentor variabilis and Amblyomma americanum, have demonstrated the ability to transmit C. felis. These two tick species have overlapping distributions throughout much of the southeastern United States. The objective of the current study was to determine the distribution and prevalence of C. felis in free-ranging bobcat populations from 13 states including California, Colorado, Florida, Georgia, Kansas, Kentucky, Missouri, North Carolina, North Dakota, Ohio, Oklahoma, South Carolina, and West Virginia. These states were selected because of differential vector presence; D. variabilis is present in each of these states except for the region of Colorado sampled and A. americanum is currently known to be present only in a subset of these states. Blood or spleen samples from 696 bobcats were tested for C. felis infection by a polymerase chain reaction (PCR) assay which targeted the first ribosomal internal transcribed spacer region (ITS-1). Significantly higher prevalences of C. felis were detected from Missouri (79%, n=39), North Carolina (63%, n=8), Oklahoma (60%, n=20), South Carolina (57%, n=7), Kentucky (55%, n=74), Florida (44%, n=45), and Kansas (27%, n=41) compared with Georgia (9%, n=159), North Dakota (2.4%, n=124), Ohio (0%, n=19), West Virginia (0%, n=37), California (0%, n=26), and Colorado (0%, n=67). In addition to bobcats, seven cougars (Puma concolor) from Georgia, Louisiana, and North Dakota and one serval (Leptailurus serval) from Louisiana were tested for C. felis. Only one cougar from Louisiana was PCR positive, which represents the first report of an infected cougar outside of the Florida panther population. These data also indicate that C. felis is present in North Dakota where infection has not been reported in domestic cats. Based on a nonparametric analysis, prevalence rates were significantly higher in states where there are established populations of A. americanum, which supports recent data on the experimental transmission of C. felis by A. americanum and the fact that domestic cat clinical cases are temporally associated with A. americanum activity. Collectively, these data confirm that bobcats are a common reservoir for C. felis and that A. americanum is likely an epidemiologically important vector.


Assuntos
DNA de Protozoário/genética , Reservatórios de Doenças/parasitologia , Felidae/parasitologia , Lynx/parasitologia , Piroplasmida/patogenicidade , Infecções Protozoárias em Animais/epidemiologia , Animais , Piroplasmida/genética , Reação em Cadeia da Polimerase , Prevalência , Infecções Protozoárias em Animais/parasitologia , Estados Unidos/epidemiologia
15.
Vet Parasitol ; 172(3-4): 311-6, 2010 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-20605334

RESUMO

The objectives of the current study were to assess the prevalence of Cytauxzoon felis infection among a population of domestic cats that were clinically healthy but at higher risk for parasite exposure and to determine if the strains present in these asymptomatically infected cats were genetically unique as compared to those present both in domestic cats that were fatally infected and in the natural reservoir host, the bobcat. Using real-time PCR analysis targeting a portion of the parasite 18S rRNA gene specific for C. felis, 27/89 (30.3%) high-risk asymptomatic domestic cats from Arkansas and Georgia, and 34/133 (25.6%) bobcats from Arkansas, Georgia and Florida, were identified as positive for C. felis infection. Conventional PCR analysis was performed on all positive samples, targeting the C. felis ribosomal internal transcribed spacer regions 1 and 2 (ITS1, ITS2) in order to utilize the ITS sequences as markers to assess the genotype variability of the parasite population. Within the asymptomatically infected domestic cat samples, 3 genetically distinct parasite populations were identified. The C. felis ITS sequences from asymptomatic cats were identical to those previously reported from clinically ill infected cats, and 2 of the 3 sequence types were also present in infected bobcat samples. While sequence diversity exists, evaluation of the ITS region does not appear to be useful to verify pathogenicity of C. felis strains within host species. However, the presence of asymptomatic C. felis infections in clinical healthy domestic cats warrants further investigation to determine if these cats can serve as a new reservoir for C. felis transmission.


Assuntos
Doenças do Gato/epidemiologia , Doenças do Gato/parasitologia , Lynx/parasitologia , Piroplasmida/genética , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/parasitologia , Animais , Gatos , DNA Espaçador Ribossômico/genética , Variação Genética , Genótipo , Dados de Sequência Molecular , Piroplasmida/isolamento & purificação , Prevalência , RNA Ribossômico 18S/genética
16.
Infect Genet Evol ; 10(6): 834-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19879379

RESUMO

Plasmodium merozoites attach to and invade red blood cells (RBCs) during the erythrocytic cycle. The invasion process requires recognition of RBC surface receptors by proteins of the Plasmodium Duffy binding like erythrocyte binding like (DBL-EBP) family. Clones and isolates of Plasmodium falciparum have varying abilities to utilize different RBC receptors, and multiple distinct pathways so far identified depend on glycophorins A, B, C, and as yet unidentified receptors. At present, five members of the DBL-EBP family have been identified in the P. falciparum genome, based on gene structure and amino acid sequence homology. The cardinal features of this family consist of conserved 5' and 3' cysteine-rich regions (regions II and VI, respectively) whose cysteine residues are highly conserved along with the majority of aromatic amino acids. In contrast to the single DBL-EBP family member in Plasmodium vivax, in P. falciparum all DBL-EBP family members have a duplication of the conserved 5' cysteine-rich region denoted as the F1 and F2 domains. These cysteine-rich regions are considered crucial in recognition of erythrocyte receptors and it has been shown that several bind to glycophorins on the erythrocyte surface. Several studies, on both field isolates and laboratory strains have uncovered a relatively high degree of sequence polymorphism in the DBP-EBL genes. This study is now extended to include field isolates collected from sites within Kenya. DNA isolated from blood samples of infected patients was utilized to amplify the region I sequence of ebl-1 gene in order to investigate polymorphism in the region immediately adjacent to the 5' cysteine-rich domains, and to determine the prevalence of an insertion mutant that effectively knocks out the gene.


Assuntos
Região 5'-Flanqueadora/genética , Sistema do Grupo Sanguíneo Duffy/genética , Mutagênese Insercional , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Antígenos de Protozoários/genética , Sequência de Bases , Estudos Transversais , Frequência do Gene , Humanos , Quênia , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Mutagênese Insercional/estatística & dados numéricos , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/genética , Homologia de Sequência do Ácido Nucleico
17.
J Vet Diagn Invest ; 21(4): 493-8, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19564497

RESUMO

Cytauxzoon felis infection in domestic cats has historically been nearly 100% fatal. However, increasing reports of domestic cats that survive cytauxzoonosis and reports of asymptomatic cats with C. felis infections suggest the existence of different parasite strains that vary in pathogenicity. The objective of the current study was to obtain epidemiologic information about cytauxzoonosis through genotypic characterization of archived histologic specimens from domestic cats with C. felis infections that were diagnosed in Georgia between 1995 and 2007. Such retrospective data on genetic variability will provide an historic context for current studies of C. felis genotype frequencies. Cytauxzoon felis DNA was obtained from formalin-fixed, paraffin-embedded tissues from infected cats diagnosed with cytauxzoonosis at necropsy. Genetic characterization of C. felis was performed using sequence analysis of the polymerase chain reaction-amplified ribosomal internal transcribed spacer regions 1 and 2 (ITS1, ITS2). Eleven different combined ITS1 and ITS2 sequences were identified, the majority of which were identical to those previously reported in fatally infected cats from Georgia. The findings of the current study document the existence of genetically distinct C. felis populations in historical samples and, together with data from contemporary samples, demonstrate a diverse population structure for C. felis.


Assuntos
Doenças do Gato/parasitologia , Variação Genética , Piroplasmida/genética , Infecções Protozoárias em Animais/parasitologia , Animais , Doenças do Gato/epidemiologia , Gatos , DNA Intergênico/genética , DNA de Protozoário/genética , Georgia/epidemiologia , Infecções Protozoárias em Animais/epidemiologia , Estudos Retrospectivos
18.
J Vet Diagn Invest ; 21(1): 59-63, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19139502

RESUMO

Although cytauxzoonosis has historically been nearly 100% fatal in domestic cats, increasing number of reports of infected cats that demonstrate less-severe disease suggest the existence of different strains of Cytauxzoon felis. To test this hypothesis, the genetic variability of C. felis was examined in blood samples from naturally infected domestic cats from Arkansas and Georgia by using the first and second ribosomal internal transcribed spacer regions (ITS1, ITS2) as markers to assess genotypic variability. In addition, the clinical outcome of infection (survival vs. fatal disease) was analyzed. Within the C. felis ITS1 region, there were a total of 8 single nucleotide polymorphisms (SNP) and a single nucleotide insertion. Within the ITS2 region, there were a total of 4 SNPs and a single 40 base pair insertion. When taken together, the ITS1 and ITS2 sequence data defined a total of 11 different sequences and 3 unique genotypes. One unique ITS1-ITS2 genotype was detected in samples submitted exclusively from Arkansas, and a second unique genotype was submitted exclusively from Georgia. There was a significant association between infection with C. felis that contained particular ITS genotypes and survival of the infected domestic cat. The identification of unique C. felis genotypes obtained from different geographic areas and the association of particular ITS genotypes with the outcome of infection suggest the existence of parasite strains that may vary in pathogenicity to the domestic cat and offer an explanation for the survival of some infected cats in more recent case studies.


Assuntos
Doenças do Gato/parasitologia , Eucariotos/genética , Infecções Protozoárias em Animais/parasitologia , Animais , Arkansas/epidemiologia , Doenças do Gato/epidemiologia , Gatos , DNA de Protozoário/genética , Eucariotos/patogenicidade , Variação Genética , Georgia/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Infecções Protozoárias em Animais/epidemiologia , Virulência
19.
J Vet Diagn Invest ; 20(4): 485-8, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18599854

RESUMO

Repeated polymerase chain reaction (PCR) testing of 3 asymptomatic domestic cats were positive for Cytauxzoon felis DNA, suggesting persistent infection. Two cats initially presented with clinical signs consistent with acute cytauxzoonosis and, in both cases, signs of illness resolved after treatment. Parasitemia was detected in peripheral blood smears from these cats upon presentation with illness and, at subsequent follow-up appointments, in the absence of clinical illness. Polymerase chain reaction analysis was positive for C. felis from blood sampled at each time point. A third cat, a housemate of a cat fatally infected with C. felis, was preventatively treated for infection at the time of the housemate cat's death. This contact cat, having never shown signs of clinical illness consistent with cytauxzoonosis infection, had no detectable parasitemia but was positive for C. felis on repeated PCR testing. Detection of asymptomatically infected cats allows for the possibility of a yet unrecognized population of infected domestic cats that may have the capacity to serve as an additional reservoir host for C. felis, altering the currently accepted paradigm of C. felis transmission to domestic cats through bobcats as the reservoir host. In cases of very low parasitemia, more sensitive means of parasite detection, such as PCR testing, may be necessary to detect infected cats. Increased detection of asymptomatically infected cats will aid in understanding the epidemiology of C. felis infection and enhance the ability to prevent this highly fatal infectious disease of domestic cats.


Assuntos
Doenças do Gato/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Infecções Protozoárias em Animais/diagnóstico , Animais , Antiprotozoários/uso terapêutico , Doenças do Gato/tratamento farmacológico , Gatos , Feminino , Imidocarbo/uso terapêutico , Masculino , Parasitemia , Infecções Protozoárias em Animais/sangue , Infecções Protozoárias em Animais/tratamento farmacológico
20.
Malar J ; 7: 42, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18312657

RESUMO

BACKGROUND: Malaria during pregnancy is characterized by the sequestration of malaria-infected red blood cells (iRBC) in the intervillous spaces of the placenta, often accompanied by the infiltration of maternal mononuclear cells, causing substantial maternal and foetal/infant morbidity. The iRBC bind to receptors expressed by the syncytiotrophoblast (ST). How ST responds to this interaction remains poorly understood. Because it is known that ST is immunoactive and can respond to infectious agents, the consequences of this ST-iRBC interaction should be investigated. METHODS: An in vitro system was used to assess the biochemical and immunological changes induced in ST by ST-adherent iRBCs. Changes in ST mitogen-activated protein kinase (MAPK) activation were assessed by immunoblotting and mRNA expression levels of selected cytokine and chemokines in primary ST bound by iRBC were determined using real-time, reverse transcription PCR. In addition, secreted cytokine and chemokine proteins were assayed by standard ELISA, and chemotaxis of PBMC was assessed using a two-chamber assay system. RESULTS: Following iRBC/ST interaction, ST C-Jun N-terminal kinase 1 (JNK1) was activated and modest increases in the mRNA expression of TGF-beta and IL-8/CXCL8 were observed. In addition, this interaction increased secretion of MIF and MIP-1alpha/CCL3 by ST and induced migration of PBMC towards iRBC-stimulated ST. CONCLUSION: Results from this study provide the first evidence that ST participates in shaping the local immunological milieu and in the recruitment of maternal immune cells to the maternal blood space during placental malaria infection.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/imunologia , Trofoblastos/imunologia , Trofoblastos/parasitologia , Animais , Western Blotting , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Ensaios de Migração Celular , Quimiotaxia de Leucócito , Técnicas de Cocultura , Citocinas/sangue , Citocinas/genética , Citocinas/imunologia , Feminino , Expressão Gênica , Humanos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Placenta/citologia , Placenta/imunologia , Placenta/parasitologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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