RESUMO
Despite reports that food-borne parasitic infections have been increasing worldwide, the methodologies employed to detect food contamination by helminths are still largely based on methodologies used to detect these pathogens in feces and water. This study sought to improve the diagnosis of parasitic contaminants in lettuce by standardizing a method for detecting helminth eggs and larvae and estimating their percentage of recovery. Sanitized lettuces were artificially contaminated with different amounts of Ascaris suum and hookworm eggs and larvae. To standardize the method, we tested liquid extractors, vegetable washing steps, and spontaneous sedimentation times. Higher percentages of egg and larvae recovery were obtained using 1 M glycine as the liquid extractor, manual shaking for 3 min and 2 h of sedimentation. Five different levels of artificial contamination (ten replicates each; n = 50) were tested using these standardized conditions, yielding an average recovery of 62.6 % (±20.2), 51.9 % (±20.0), and 50.0 % (±27.3) for A. suum eggs, hookworm eggs, and larvae, respectively. Tests were performed with a different matrix to evaluate the performance of the method. Furthermore, collaborative analytical studies performed by different laboratories produced satisfactory results. The method for the identification of helminth eggs and larvae proposed in this study proved to be simpler and more efficient than previously published procedures, thereby demonstrating its potential contribution to health surveillance and epidemiological studies.
Assuntos
Ancylostomatoidea/isolamento & purificação , Ascaris suum/isolamento & purificação , Contaminação de Alimentos/análise , Lactuca/parasitologia , Contagem de Ovos de Parasitas/métodos , Animais , Fezes/parasitologia , Humanos , Larva , OócitosRESUMO
Single nucleotide polymorphisms at codons 167, 198, and 200 in the ß-tubulin isotype 1 gene have been associated with benz-imidazole resistance. Until now, the only mutation observed in Ancy-lostoma caninum was at codon 200 of this gene. However, the standard-ized methodologies used to detect mutations in this species are faulty. The objective of this study was to standardize a molecular technique based on amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) for detecting the mutation at codon 200 in the A. caninum ß-tubulin isotype 1 gene. Controls were synthesized both for the absence of the mutation, using conventional PCR, and for the presence of the mutation, using the Megaprimer-PCR technique. After standardization of the ARMS-PCR using the controls, the technique was validated through an analysis of 75 A. caninum DNA samples, fol-lowed by sequencing. The results revealed that the developed technique has high sensitivity, specificity, and reproducibility, which allow its ap-plication in the field.
Assuntos
Ancylostoma/genética , Resistência a Medicamentos/genética , Mutação , Reação em Cadeia da Polimerase/normas , Polimorfismo de Nucleotídeo Único , Tubulina (Proteína)/genética , Ancylostoma/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Códon , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
BACKGROUND: Presence of tumor markers in serum might be connected to the number of secreting cells and with the stage of the neoplasm. However, there are few studies regarding these markers in veterinary clinical oncology. OBJECTIVES: To determine the serum concentrations of cancer antigen 15.3 (CA 15.3), carcinoembryonic antigen (CEA), and lactate dehydrogenase (LDH) in female dogs with different stages of mammary cancer. ANIMALS: Ninety female dogs, including 30 that were healthy, 40 that had nonmetastatic cancer, 12 with regional metastasis, and 8 with distant lymph node metastasis. METHODS: Prospective case-controlled observational study. Serum samples were collected to measure CA15.3, CEA, and LDH from 60 female dogs with mammary cancer during mastectomy and 30 healthy female dogs during routine check-up. CA15.3 and CEA were determined by chemiluminescent immunoassay and LDH by ultraviolet kinetic method. Western blotting analysis was performed to confirm the specificity and possible cross-reactivity of human CA15.3 and CEA antibodies with canine serum. Group data were compared by ANOVA followed by Student-Newman-Keuls and Tukey's tests. Correlations were investigated by Pearson and Spearman tests. RESULTS: CEA, CA15.3, and LDH were measurable in all groups. Higher serum concentration of CA15.3 and LDH was associated with regional and distant metastases. There was a significantly higher serum CA15.3 concentration in animals with lymph node metastasis when compared with animals without metastasis. There were no significant differences in CEA among groups. Expression of CA15.3 and CEA in canine serum was confirmed by Western blotting. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum CA15.3 can be used to distinguish nonmetastatic from metastatic carcinomas.
Assuntos
Antígeno Carcinoembrionário/sangue , Doenças do Cão/sangue , Regulação Neoplásica da Expressão Gênica/fisiologia , L-Lactato Desidrogenase/sangue , Neoplasias Mamárias Animais/sangue , Adenocarcinoma/sangue , Adenocarcinoma/metabolismo , Adenocarcinoma/veterinária , Animais , Biomarcadores Tumorais , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Carcinoma/sangue , Carcinoma/metabolismo , Carcinoma/veterinária , Estudos de Casos e Controles , Doenças do Cão/metabolismo , Cães , Feminino , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Neoplasias Mamárias Animais/classificação , Neoplasias Mamárias Animais/metabolismoRESUMO
This paper reports the development and use of a Real Time PCR for detection of Babesia canis canis, B. canis rossi, and B. canis vogeli in endemic areas of Brazil. The sequences of the internal transcribed spacer (ITS) of several organisms were aligned and five primers and four probes were designed for amplification of a fragment (around 125 bp) which differentiates subspecies of B. canis. Blood samples collected from dogs living in farms in three distinct rural regions within the State of Minas Gerais (Lavras, Belo Horizonte and Nanuque) were tested. Blood samples had been collected during a dry season (Lavras, n=100; Belo Horizonte, n=50; Nanuque, n=102); the dogs were re-sampled in the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=29; Nanuque, n=66). From each sample, DNA was extracted and Giemsa stained smears were microscopically examined for direct detection of Babesia parasites. B. canis vogeli was the only subspecies found, with an overall prevalence of 9.9% during the dry season and 10.8% during the rainy season. Dogs living in Nanuque and Belo Horizonte showed significantly higher prevalence rates than those living in Lavras (13.7%, 12.0% and 5.0%, respectively). The Real Time PCR developed proved to be appropriate to detect B. canis subspecies in endemic areas.
Assuntos
Babesia/classificação , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Babesiose/epidemiologia , Babesiose/parasitologia , Babesiose/veterinária , Brasil/epidemiologia , Doenças do Cão/epidemiologia , Cães , Incidência , Prevalência , Especificidade da EspécieRESUMO
Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free-living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co-infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross-protection does not occur in these deer. Immunological cross-reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.
Assuntos
Infecções por Anaplasmataceae/veterinária , Anaplasmataceae/isolamento & purificação , Cervos , Doenças Transmitidas por Carrapatos/veterinária , Infecções por Anaplasmataceae/epidemiologia , Infecções por Anaplasmataceae/microbiologia , Animais , Brasil/epidemiologia , Feminino , Masculino , Doenças Transmitidas por Carrapatos/microbiologiaRESUMO
Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6 percent) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2 percent) cases. Ten years post-cure, 39 (52 percent) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.
Assuntos
Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem , Anticorpos Antiprotozoários/sangue , Imunidade Celular/imunologia , Leishmaniose Visceral/imunologia , Anticorpos Antiprotozoários/imunologia , Estudos Transversais , Técnica Indireta de Fluorescência para Anticorpo , Testes IntradérmicosRESUMO
Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6%) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2%) cases. Ten years post-cure, 39 (52%) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.
Assuntos
Anticorpos Antiprotozoários/sangue , Imunidade Celular/imunologia , Leishmaniose Visceral/imunologia , Adolescente , Adulto , Anticorpos Antiprotozoários/imunologia , Criança , Pré-Escolar , Estudos Transversais , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lactente , Testes Intradérmicos , Masculino , Adulto JovemRESUMO
This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75 days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2 weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.
Assuntos
Anaplasma marginale/patogenicidade , Anaplasmose/microbiologia , Doenças dos Bovinos/microbiologia , Anaplasma marginale/genética , Anaplasma marginale/imunologia , Anaplasmose/prevenção & controle , Animais , Brasil , Bovinos , Doenças dos Bovinos/prevenção & controle , Eritrócitos/imunologia , Eritrócitos/microbiologia , Genótipo , Masculino , Distribuição AleatóriaRESUMO
Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.
Assuntos
Regulação da Expressão Gênica , Proteínas de Helminto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estágios do Ciclo de Vida , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/química , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n=92; Belo Horizonte, n=50; Nanuque, n=102) and the subsequent rainy season (Lavras, n=71; Belo Horizonte, n=28; Nanuque, n=66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.
Assuntos
Babesia/isolamento & purificação , Babesiose/veterinária , Doenças do Cão/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Babesia/genética , Babesiose/sangue , Babesiose/epidemiologia , Babesiose/parasitologia , Sequência de Bases , Brasil/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/genética , Doenças do Cão/sangue , Doenças do Cão/epidemiologia , Cães , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , População Rural , Estações do Ano , Estudos Soroepidemiológicos , Carrapatos/parasitologiaRESUMO
In a previous work Ancylostoma caninum sex-specific genes had being identified, however without confirmation, by using an non specific RT-PCR comparing male and female cDNA. In this work more fragments had been identified and a semi-quantitative RT-PCR carried out in order to confirm the sex-specific character of the transcripts obtained. Four fragments were confirmed as sex-specific or as being expressed more in one sex than the other.
Assuntos
Ancylostoma/genética , Ancilostomíase/veterinária , Doenças do Cão/parasitologia , Ancylostoma/isolamento & purificação , Ancilostomíase/parasitologia , Animais , Cães , Feminino , Masculino , RNA de Helmintos/química , RNA de Helmintos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de Proteína , Caracteres SexuaisRESUMO
Schistosoma are dioecious digenetic trematodes carrying a large (270 Mb) genome. Gaining knowledge about the genome of these parasites is of importance for the understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the host's immune system. This review will provide an update on the Schistosoma Gene Discovery Program, which is part of the Schistosoma Genome Project created in 1992. One of the main objectives of this program is the discovery and characterisation of new genes of Schistosoma mansoni and Schistosoma japonicum in an attempt to search for new targets for drugs and vaccine development. The success of the Schistosoma Gene Discovery Program is demonstrated by the number of catalogued genes, that now reaches 15 to 20% of the full gene complement of its genome.
Assuntos
Mapeamento Cromossômico , Genoma de Protozoário , Schistosoma/genética , Animais , Etiquetas de Sequências Expressas , Feminino , Expressão Gênica , MasculinoAssuntos
Regulação da Expressão Gênica no Desenvolvimento , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Animais , Biblioteca Gênica , Genes de Helmintos , Humanos , Estágios do Ciclo de Vida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/fisiopatologiaRESUMO
ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.
Assuntos
Etiquetas de Sequências Expressas , Genes de Helmintos , Schistosoma mansoni/genética , Animais , DNA Complementar/genética , DNA de Helmintos/genética , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Schistosoma mansoni/crescimento & desenvolvimentoRESUMO
A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.
Assuntos
DNA Complementar/genética , DNA de Helmintos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Frequência do Gene , Schistosoma mansoni/genética , Animais , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Schistosoma mansoni/crescimento & desenvolvimentoRESUMO
Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor.