RESUMEN

Cutaneous leishmaniasis (CL) stands out as a significant vector-borne endemic in Pakistan. Despite the rising incidence of CL, the genetic diversity of Leishmania species in the country's endemic regions remains insufficiently explored. This study aims to uncover the genetic diversity and molecular characteristics of Leishmania species in CL-endemic areas of Baluchistan, Khyber Pakhtunkhwa (KPK), and Punjab in Pakistan. Clinical samples from 300 CL patients were put to microscopic examination, real-time ITS-1 PCR, and sequencing. Predominantly affecting males between 16 to 30 years of age, with lesions primarily on hands and faces, the majority presented with nodular and plaque types. Microscopic analysis revealed a positivity rate of 67.8%, while real-time PCR identified 60.98% positive cases, mainly L. tropica, followed by L. infantum and L. major. Leishmania major (p = 0.009) showed substantially greater variation in nucleotide sequences than L. tropica (p = 0.07) and L. infantum (p = 0.03). Nucleotide diversity analysis indicated higher diversity in L. major and L. infantum compared to L. tropica. This study enhances our understanding of CL epidemiology in Pakistan, stressing the crucial role of molecular techniques in accurate species identification. The foundational data provided here emphasizes the necessity for future research to investigate deeper into genetic diversity and its implications for CL control at both individual and community levels.

Asunto(s)

Variación Genética , Leishmaniasis Cutánea , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/parasitología , Pakistán/epidemiología , Humanos , Masculino , Adolescente , Adulto , Femenino , Adulto Joven , Niño , Persona de Mediana Edad , Leishmania/genética , Leishmania/clasificación , Leishmania/aislamiento & purificación , Preescolar , Análisis de Secuencia de ADN , Leishmania tropica/genética , Leishmania tropica/aislamiento & purificación , Leishmania tropica/clasificación , Leishmania major/genética , Leishmania major/clasificación , Leishmania major/aislamiento & purificación , ADN Protozoario/genética , Filogenia , Epidemiología Molecular , Anciano , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Cryptosporidiosis has previously been reported in animals, humans, and water sources in the United Arab Emirates (UAE). However, most reports were only to the genus level, or generically identified as cryptosporidiosis. We aimed to investigate the genetic diversity of Cryptosporidium species occurring in diarrhetic ungulates which were brought to the Central Veterinary Research Laboratory (CVRL) in Dubai. Using a combination of microscopic and molecular methods, we identified five species of Cryptosporidium occurring among ungulates in the UAE, namely C. parvum, C. hominis, C. xiaoi, C. meleagridis, and C. equi. Cryptosporidium parvum was the most prevalent species in our samples. Furthermore, we identified subtypes of C. parvum and C. hominis, which are involved in both human and animal cryptosporidiosis. This is also the first reported occurrence of Cryptosporidium spp. in the Arabian Tahr, to our knowledge. Since the animals examined were all in contact with humans, the possibility of zoonotic spread is possible. Our study correlates with previous reports in the region, building upon the identification of Cryptosporidium sp. However, there is a need to further investigate the endemic populations of Cryptosporidium, including more hosts, sampling asymptomatic animals, and location data.

Asunto(s)

Criptosporidiosis , Cryptosporidium , Diarrea , Variación Genética , Emiratos Árabes Unidos/epidemiología , Animales , Criptosporidiosis/parasitología , Criptosporidiosis/epidemiología , Cryptosporidium/genética , Cryptosporidium/clasificación , Cryptosporidium/aislamiento & purificación , Diarrea/veterinaria , Diarrea/parasitología , Diarrea/epidemiología , Heces/parasitología , Bovinos , Filogenia , Cabras/parasitología , ADN Protozoario/genética

RESUMEN

Toxoplasma gondii, a common protozoan parasite, poses significant public health risks due to its potential to cause toxoplasmosis in humans and can be contracted from pigs, which are considered its critical intermediate host. The aim of this study is to evaluate the prevalence of T. gondii in slaughtered pigs for human consumption, emphasizing the zoonotic implications and the need for improved biosecurity and monitoring practices in pig farming. A total of 1,526 pig samples (1,051 whole blood samples and 384 lung tissue samples from the local slaughterhouse and 91 aborted fetus samples from local farms) were collected throughout the whole country of Korea in 2020. Among them, 6 (0.4%) were found to be infected with T. gondii by nested PCR. When compared by sample type, the prevalence of T. gondii was significantly higher in the aborted fetus samples (2.2%, 2/91) than in the blood (0.3%, 3/1,051) and lung tissue samples (0.3%, 1/384). The B1 gene sequence of T. gondii was similar (97.9-99.8%) to that of the other T. gondii isolates. This study represents the first molecular genotyping survey of T. gondii in the lung tissue of fattening pigs and aborted fetuses in Korea. Our findings indicated the importance of adopting preventive measures including the implementation of rigorous farm hygiene protocols and the promotion of public awareness about the risks of consuming undercooked pork. By addressing the gaps in current control strategies and encouraging the One Health approach, this study contributes to the development of more effective strategies to mitigate the transmission of T. gondii from pigs to humans, ultimately safeguarding public health.

Asunto(s)

Genotipo , Enfermedades de los Porcinos , Toxoplasma , Toxoplasmosis Animal , Animales , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , República de Corea/epidemiología , Porcinos , Toxoplasmosis Animal/epidemiología , Toxoplasmosis Animal/parasitología , Enfermedades de los Porcinos/parasitología , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/transmisión , Prevalencia , Mataderos , Pulmón/parasitología , Reacción en Cadena de la Polimerasa , Humanos , ADN Protozoario/genética , Feto Abortado/parasitología

RESUMEN

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Asunto(s)

Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Sarcocystis , Sarcocistosis , Sensibilidad y Especificidad , Enfermedades de las Ovejas , Animales , Sarcocystis/genética , Sarcocystis/aislamiento & purificación , Sarcocystis/clasificación , Ovinos , Sarcocistosis/veterinaria , Sarcocistosis/diagnóstico , Sarcocistosis/parasitología , Enfermedades de las Ovejas/parasitología , Enfermedades de las Ovejas/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Técnicas de Diagnóstico Molecular/métodos , ARN Ribosómico 28S/genética , ADN Protozoario/genética

RESUMEN

BACKGROUND: Blastocystis, a widely distributed zoonotic protozoan infecting both humans and numerous animals, remains poorly understood with its potential medical and veterinary significance. This study examined the molecular occurrence and genetic variation of Blastocystis in children and calves in Bangladesh to explore cross-species transmission and disease burden. METHODS: In total, 998 DNA samples were investigated, comprising 299 stool DNA from children and 699 fecal DNA from calves, using polymerase chain reaction and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. RESULTS: This study detected Blastocystis in 5.35% of the children and 14.74% of the calves. While slight variations in occurrence rates were observed across different study variables, none were statistically significant. The occurrence was highest among children under 5 years and calves aged 1-3 months. Regarding breed, the Holstein Friesian cross and the Jersey cross exhibited higher rates of infection. Conversely, occurrences were lower among children and calves in Gazipur district. The remaining parameters displayed nearly equivalent percentages of Blastocystis. The subtypes identified in children included ST1, ST3, and ST4, with ST1 comprising 50% of them. ST3 and ST4 were also found in calves, alongside ST10 (55.34%) being the most prevalent. Other subtypes found in calves were ST14, ST21, and ST24-ST26. CONCLUSIONS: This study found that Blastocystis is more common in calves than in children in Bangladesh, with genetic diversity of nine subtypes. The common occurrence of identical variants of two subtypes in both populations suggests potential zoonotic transmission, highlighting the necessity for further molecular investigations and comprehensive measures within the One Health framework to mitigate public health risks.

Asunto(s)

Infecciones por Blastocystis , Blastocystis , Enfermedades de los Bovinos , ADN Protozoario , Heces , Variación Genética , Bovinos , Animales , Bangladesh/epidemiología , Infecciones por Blastocystis/epidemiología , Infecciones por Blastocystis/veterinaria , Infecciones por Blastocystis/parasitología , Blastocystis/genética , Blastocystis/clasificación , Blastocystis/aislamiento & purificación , Humanos , Lactante , Heces/parasitología , Preescolar , Enfermedades de los Bovinos/parasitología , Enfermedades de los Bovinos/epidemiología , Masculino , Femenino , Niño , ADN Protozoario/genética , Filogenia , Análisis de Secuencia de ADN , Reacción en Cadena de la Polimerasa

RESUMEN

Leishmaniasis, a neglected tropical disease caused by parasitic protozoa of the Leishmania genus, remains a global health concern with significant morbidity and mortality. In Thailand, the rising incidence of autochthonous leishmaniasis cases involving Leishmania (Mundinia) martiniquensis and novel Leishmania (Mundinia) orientalis underscores the critical need for accurate diagnosis and effective control strategies. This study presents a sensitive and specific nucleic acid lateral flow immunoassay (NALFIA) that integrates a duplex PCR assay with a lateral flow device (LFD) strip format. Targeting the internal transcribed spacer 1 (ITS1) region, known for its unique combination of conserved and variable sequences, this assay employs primers labeled with biotin, digoxigenin, and fluorescein isothiocyanate (FITC) markers, enabling precise species identification and differentiation of these two Leishmania species. Remarkably, the assay achieves a sensitivity that surpasses agarose gel electrophoresis, detecting as few as 10-2 parasite/µL for L. martiniquensis and 10-4 parasite/µL for L. orientalis. Notably, the assay exhibited reliable specificity, revealing no cross-amplification with other major viscerotropic Leishmania species or reference organisms. Evaluation using 62 clinical samples further confirms the effectiveness of the PCR-LFD assay, with a sensitivity of 100% for L. martiniquensis and 83.3% for L. orientalis, and an excellent agreement (κ value = 0.948) with nested PCR. This integrated assay represents a promising advancement in diagnostic tools, offering rapid and accurate results that can significantly contribute to effective disease management and control. Given the increasing relevance of these Leishmania species in current public health scenarios, this assay serves as a valuable tool for both diagnostic and research applications.

Asunto(s)

Leishmania , Leishmaniasis , Leishmania/genética , Leishmania/aislamiento & purificación , Humanos , Leishmaniasis/diagnóstico , Leishmaniasis/parasitología , Inmunoensayo/métodos , Infecciones por VIH , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa/métodos , ADN Protozoario/análisis , ADN Protozoario/genética , Tailandia/epidemiología

RESUMEN

The skin is an anatomical reservoir for African trypanosomes, yet the prevalence of extravascular parasite carriage in the population at risk of gambiense Human African Trypanosomiasis (gHAT) remains unclear. Here, we conducted a prospective observational cohort study in the HAT foci of Forecariah and Boffa, Republic of Guinea. Of the 18,916 subjects serologically screened for gHAT, 96 were enrolled into our study. At enrolment and follow-up visits, participants underwent a dermatological examination and had blood samples and superficial skin snip biopsies taken for examination by molecular and immuno-histological methods. In seropositive individuals, dermatological symptoms were significantly more frequent as compared to seronegative controls. Trypanosoma brucei DNA was detected in the blood of 67% of confirmed cases (22/33) and 9% of unconfirmed seropositive individuals (3/32). However, parasites were detected in the extravascular dermis of up to 71% of confirmed cases (25/35) and 41% of unconfirmed seropositive individuals (13/32) by PCR and/or immuno-histochemistry. Six to twelve months after treatment, trypanosome detection in the skin dropped to 17% of confirmed cases (5/30), whereas up to 25% of unconfirmed, hence untreated, seropositive individuals (4/16) were still found positive. Dermal trypanosomes were observed in subjects from both transmission foci, however, the occurrence of pruritus and the PCR positivity rates were significantly higher in unconfirmed seropositive individuals in Forecariah. The lower sensitivity of superficial skin snip biopsies appeared critical for detecting trypanosomes in the basal dermis. These results are discussed in the context of the planned elimination of gHAT.

Asunto(s)

Piel , Trypanosoma brucei gambiense , Tripanosomiasis Africana , Humanos , Guinea/epidemiología , Tripanosomiasis Africana/epidemiología , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/diagnóstico , Masculino , Adulto , Femenino , Trypanosoma brucei gambiense/aislamiento & purificación , Estudios Prospectivos , Prevalencia , Piel/parasitología , Piel/patología , Adulto Joven , Persona de Mediana Edad , Adolescente , ADN Protozoario/genética , Niño

RESUMEN

PURPOSE: The present study was conducted to determine the presence of Toxoplasma gondii in donkeys by molecular tests and genetic diversity analysis of the obtained DNA samples from central Kenya. METHOD: A total of 363 blood samples were collected from donkeys in Meru and Kirinyaga Counties, and 96 samples that were previously seropositive for T. gondii using indirect ELISA were subjected to nested PCR based on the amplification of the internal transcribed spacer 1 (ITS-1) gene followed by DNA sequencing and phylogenetic analysis. Genotyping was performed on 15 selected positive samples using multilocus nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP) with eight genetic markers ('SAG 2, 5'SAG 2, Alt. SAG 2, SAG 3, GRA 6, C29-2, BTUB and L358). RESULTS: Toxoplasma gondii DNA was detected in 36.5% (35/96) of the blood samples. The sequences obtained exhibited 98.2-99.5% homology with those deposited in GenBank. Phylogenetic analysis demonstrated that the obtained sequences are conserved and clustered with those of infecting animals from other regions of the world. Eighteen distinct T. gondii haplotypes were identified to be circulating in donkeys from central Kenya. The T. gondii DNA samples exhibited high haplotype diversity (Hd: 0.915) and limited genetic diversity (π = 0.01027). PCR-RFLP of T. gondii DNA-positive samples revealed three different genetic combinations that consisted of alleles I, II and III, indicating the dissemination of atypical genotypes. CONCLUSION: This study demonstrated that T. gondii is widespread in donkeys from Kenya and could be a possible source of infection in humans. These findings are important for designing control strategies for this parasite to improve the livestock sector, which is one of the main sources of livelihood for farmers in Kenya.

Asunto(s)

ADN Protozoario , Equidae , Variación Genética , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Toxoplasma , Toxoplasmosis Animal , Animales , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Toxoplasma/clasificación , Kenia/epidemiología , Equidae/parasitología , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/epidemiología , ADN Protozoario/genética , Reacción en Cadena de la Polimerasa/veterinaria , Genotipo , Análisis de Secuencia de ADN , Haplotipos

RESUMEN

PURPOSE: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. METHODS: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020-2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. RESULTS: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L-) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. CONCLUSION: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone.

Asunto(s)

Enfermedades de los Gatos , Leishmania infantum , Animales , Gatos , Leishmania infantum/genética , Leishmania infantum/aislamiento & purificación , Enfermedades de los Gatos/parasitología , Enfermedades de los Gatos/epidemiología , Factores de Riesgo , Mar Negro , Femenino , Masculino , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Prevalencia , ADN de Cinetoplasto/genética , ADN Protozoario/genética

RESUMEN

PURPOSE: Dientamoeba fragilis (D. fragilis) is a common intestinal protozoan with a global distribution. In the present study, we aimed to determine genetic diversity of D. fragilis isolates with multilocus sequence typing (MLST) in the southwest of Turkey and analyse the clinical findings. MATERIALS AND METHODS: The study included faecal samples from 200 individuals in Aydin, Turkey. The positivity of D. fragilis was determined with 18 S rRNA gene-based PCR assay. Six nested-PCR reactions were set to amplify partial D. fragilis housekeeping genes in the positive samples. The sequences were aligned with the references from GenBank to detect nucleotide polymorphisms and haplotypes. Additionally, the clinical findings and demographic characteristics of patients were statistically analysed between D. fragilis-infected and non-infected cases. RESULTS: The positivity of D. fragilis was 16% (32 out of 200 cases) with 18 S rRNA based-PCR, and all were classified as "genotype 1". The analysis of six MLST loci revealed different haplotypes only at one locus; the remaining five loci exhibited no polymorphisms. The haplotypes in the present study were identical to at least one previously reported reference, except the locus "large subunit of RNA polymerase II" locus. There were no significant differences in any of the clinical findings or demographic characteristics between the infected and non-infected groups. CONCLUSIONS: Our study revealed a low genetic diversity of D. fragilis isolates from Turkey, like other countries including Italy, Denmark, the UK, Australia, and Brazil. The high degree of sequence similarity in housekeeping genes indicated the clonal distribution of D. fragilis.

Asunto(s)

Dientamoeba , Dientamebiasis , Variación Genética , Tipificación de Secuencias Multilocus , Dientamoeba/genética , Dientamoeba/aislamiento & purificación , Dientamoeba/clasificación , Turquía/epidemiología , Humanos , Dientamebiasis/parasitología , Dientamebiasis/epidemiología , Masculino , Femenino , Adolescente , Niño , Adulto , Adulto Joven , ARN Ribosómico 18S/genética , Heces/parasitología , Persona de Mediana Edad , Preescolar , Haplotipos , Genotipo , ADN Protozoario/genética , Reacción en Cadena de la Polimerasa , Anciano , Filogenia , Lactante

RESUMEN

The aim of this study was to estimate the molecular infection prevalence of Toxoplasma gondii in sheep liver tissues destined for human consumption. A total number of 224 liver tissues were collected from slaughtered sheep in Sejnane slaughterhouse (Northwest Tunisia). PCR was used to detect T. gondii DNA in liver tissues followed by phylogenetic analysis of amplicons. The phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences.The overall molecular prevalence of T. gondii in sheep livers was 25% (56/224). The highest molecular prevalence of T. gondii was recorded in sheep aged of less than one year old (27.3%; 52/190). Infection prevalence was significantly higher in Noire de Thibar breed (33%; 17/51) compared to other breeds (p = 0.023). There were no differences depicted according to sheep's gender. The T. gondii sequences obtained in the present study (GenBank accession numbers: OR509829 and OR509830) were 98.40-100% homologous to T. gondii sequences published in the GenBank. These results highlight a high level of T. gondii contamination of tissues destined for human consumption. Further studies are needed to improve our knowledge on different genotypes of T. gondii that infect Tunisian sheep population.

Asunto(s)

Hígado , Filogenia , Enfermedades de las Ovejas , Toxoplasma , Toxoplasmosis Animal , Animales , Túnez/epidemiología , Toxoplasma/genética , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , Ovinos , Toxoplasmosis Animal/parasitología , Toxoplasmosis Animal/epidemiología , Enfermedades de las Ovejas/parasitología , Enfermedades de las Ovejas/epidemiología , Hígado/parasitología , ADN Protozoario/genética , Femenino , Masculino , Humanos , Prevalencia , Reacción en Cadena de la Polimerasa/veterinaria

RESUMEN

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL.

Asunto(s)

Proteínas HSP70 de Choque Térmico , Leishmaniasis Cutánea , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Humanos , Proteínas HSP70 de Choque Térmico/genética , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y Especificidad , ADN Protozoario/genética , Leishmania/genética , Leishmania/aislamiento & purificación

RESUMEN

Iodamoeba is a single-celled intestinal parasite, which is common in humans in certain parts of the world, and also in pigs. For the first time, we provide DNA-based evidence of goat, dromedary, fallow deer, and donkey as hosts of Iodamoeba and show that Iodamoeba-specific nucleotide sequences from these four hosts do not appear to overlap with those of humans, unlike those from pigs. We moreover show that similar strains of Iodamoeba can be found in Madagascar, Western Sahara, and Ecuador and that intra-sample diversity is typically extensive across even small fragments of DNA in both human and non-human hosts.

Asunto(s)

Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Especificidad del Huésped , Animales , Humanos , Filogenia , Ecuador , Madagascar , ADN Protozoario/genética , Equidae/parasitología , Amebozoos/genética , Amebozoos/clasificación , Datos de Secuencia Molecular , Ciervos/parasitología , Camelus/parasitología , Cabras/parasitología , Análisis de Secuencia de ADN , Porcinos

RESUMEN

Prostomateans, as common inhabitants in diverse aquatic environments, are among the simplest ciliate lineages, and serve as trophic links in food webs. However, only a few members are well-known and thoroughly studied, and the diversity of this group remains elusive. The unique genus Plagiocampa has a long history of research, but few studies have been performed using up-to-date methods. In the present work, Plagiocampa longis Kahl, 1927 and Plagiocampa minima Kahl, 1927, collected from Chinese coastal habitats, were investigated based on microscopical observation, protargol staining, and SSU rRNA gene sequencing. Their ciliature and morphometric data as well as gene sequences are documented. Phylogenetic analyses revealed that the family Plagiocampidae is likely monophyletic and has a closer relationship with parasitic Cryptocaryon.

Asunto(s)

Cilióforos , Filogenia , Cilióforos/clasificación , Cilióforos/genética , Cilióforos/citología , Cilióforos/aislamiento & purificación , China , ARN Ribosómico 18S/genética , ADN Ribosómico/genética , ADN Protozoario/genética , Análisis de Secuencia de ADN

RESUMEN

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 µmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993-0.996, p < 0.001), indicating the LAMP method is in the "better" range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection.

Asunto(s)

Enfermedades de los Bovinos , Coccidiosis , Colorimetría , Neospora , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Enfermedades de las Ovejas , Neospora/genética , Neospora/aislamiento & purificación , Animales , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , Coccidiosis/veterinaria , Coccidiosis/diagnóstico , Coccidiosis/parasitología , Colorimetría/veterinaria , Colorimetría/métodos , Ovinos , Bovinos , Enfermedades de las Ovejas/parasitología , Enfermedades de las Ovejas/diagnóstico , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/parasitología , Técnicas de Diagnóstico Molecular/veterinaria , Técnicas de Diagnóstico Molecular/métodos , ADN Protozoario/genética , Reacción en Cadena de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa/métodos

RESUMEN

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

Asunto(s)

Búfalos , Reacción en Cadena de la Polimerasa , Animales , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Bovinos , Caballos , Perros , Babesia/aislamiento & purificación , Babesia/genética , Sensibilidad y Especificidad , Trypanosoma/aislamiento & purificación , Trypanosoma/genética , ADN Protozoario/genética , Theileria/aislamiento & purificación , Theileria/genética , ADN/sangre , ADN/aislamiento & purificación , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/parasitología , Enfermedades de los Bovinos/sangre , Enfermedades de los Perros/sangre

RESUMEN

Giardia duodenalis is an intestinal pathogen that is found globally. Children are more susceptible and often suffer severe consequences after infection. Despite this, the health effects of this pathogen continue to be poorly understood and neglected. In Wenzhou, Zhejiang province, China, stool samples were obtained from 1032 children who were admitted to Yuying Children's Hospital. Out of these, 684 presented with diarrhea, while 348 were asymptomatic. The stool samples were screened for G. duodenali by targeting the small subunit of the ribosomal RNA (SSU rRNA) gene. Subtypes of G. duodenalis were identified via amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triosephosphate isomerase (tpi) genes in samples positive for the G. duodenalis. The findings indicated the presence of G. duodenalis in 0.9 % (9/1032) of the samples, with 9/684 (1.3 %) of the samples originating from children with diarrhea and none from the asymptomatic samples. All 9 samples that tested positive for G. duodenalis were determined to be of assemblage A. Of these, 6 samples were effectively genotyped at all 3 loci, resulting in the identification of 3 distinct MLGs: MLG-AII1 (n = 1), MLG-AII2 (n = 4), and MLG-AII2 (n = 1), all belonging to G. duodenalis assemblage AII. This was the first study that confirmed G. duodenalis infections in children residing in southern Zhejiang, China, with comparatively low rates of infection. The detection of G. duodenalis assemblage AII indicates a possibility of transfer from one human to another. The parasite's effect on the health of young children requires special attention and consideration.

Asunto(s)

Diarrea , Heces , Genotipo , Giardia lamblia , Giardiasis , Tipificación de Secuencias Multilocus , Humanos , Giardiasis/parasitología , Giardiasis/epidemiología , Giardia lamblia/genética , Giardia lamblia/clasificación , Giardia lamblia/aislamiento & purificación , China/epidemiología , Preescolar , Diarrea/parasitología , Heces/parasitología , Femenino , Masculino , Lactante , Niño , Proteínas Protozoarias/genética , Triosa-Fosfato Isomerasa/genética , Filogenia , Glutamato Deshidrogenasa/genética , ADN Protozoario/genética , Prevalencia

RESUMEN

Phlebotomine sand flies are recognized as a primary vector of Leishmania and are also suspected vectors of Trypanosoma. The transmission cycle of these parasites relies on the distribution of sand fly vectors, parasites, and reservoir animals. This study aimed to detect Leishmania and Trypanosoma DNA and identify the sources of bloodmeals in post-feeding sand flies captured across Thailand. A total of 42,911 field female sand flies were collected from 11 provinces across Thailand using CDC light traps. Among these, 253 post-feeding sand flies were selected for analysis. The predominant species in this study was Sergentomyia khawi (33.60 %). The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR), specific to the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal RNA (SSU rRNA) gene regions were used to detect the presence of Leishmania and Trypanosoma DNA, respectively. Additionally, cytochrome c oxidase subunit I (COI) gene region was utilized to identify the sources of host bloodmeals. Leishmania DNA was not detected in any specimens. The analysis of SSU rRNA sequences revealed the presence of Trypanosoma DNA (11.46 %, 29/253) in sand fly samples. Among these samples, T. noyesi (1.58 %, 4/253) was identified in Idiophlebotomus longiforceps and Phlebotomus asperulus, Trypanosoma Anura01+02/Frog2 (1.18 %, 3/253) in Se. khawi, and Trypanosoma Anura04/Frog1 (8.70 %, 22/253) in Se. khawi, Se. hivernus and Grossomyia indica. Bloodmeal analysis utilizing the COI gene revealed a diverse range of vertebrate hosts' blood, including bird, bat, frog and sun skink. Our findings confirm the presence of Trypanosoma DNA and identify the sources of bloodmeals from vertebrate hosts in various sand fly species, suggesting their potential as possible vectors for Trypanosoma in Thailand. Furthermore, our study is the first to provide molecular evidence using the COI gene to identify frogs as a host blood source for sand flies in Thailand. Further studies focusing on the isolation of live parasites in sand flies to confirm vector potential and examining the role of animal reservoirs will enhance our understanding of the host-parasite relationship and enable more efficient control for disease transmission.

Asunto(s)

ADN Protozoario , Leishmania , Psychodidae , Trypanosoma , Animales , Tailandia/epidemiología , Trypanosoma/genética , Trypanosoma/aislamiento & purificación , Trypanosoma/clasificación , Femenino , Leishmania/genética , Leishmania/aislamiento & purificación , Leishmania/clasificación , ADN Protozoario/genética , Psychodidae/parasitología , Insectos Vectores/parasitología , Filogenia , Reacción en Cadena de la Polimerasa , ADN Espaciador Ribosómico/genética , Complejo IV de Transporte de Electrones/genética , Sangre/parasitología

RESUMEN

Migratory birds play an important role in the cross-regional transmission of zoonotic pathogens. Assessing the presence of zoonotic pathogens carried by migratory birds is critical for disease control. However, information about Blastocystis infection in the migratory birds is very limited. Thus, we conducted this study with the aim to explore the occurrence, prevalence and subtyping of Blastocystis in four breeds of migratory birds in northeastern China. From October 2022 to April 2023, a total of 427 fresh fecal samples were obtained from four breeds of migratory birds in five nature reserves in northeastern China, and screened for Blastocystis by PCR amplification. Twenty-one (4.92 %) of the studied samples were confirmed Blastocystis-positive, and two known zoonotic subtypes ST6 and ST7 were founded, with ST7 being the major subtype. Until now, we firstly reported the infection status and subtyping of Blastocystis in the migratory Greater White-Fronted Goose, White Stork, Oriental White Stork and Bean Goose in China. More importantly, these findings present further data on the genetic diversity and transmission routes of Blastocystis and further arouse public health concerns about this organism.

Asunto(s)

Migración Animal , Enfermedades de las Aves , Aves , Infecciones por Blastocystis , Blastocystis , Heces , Animales , Blastocystis/genética , Blastocystis/clasificación , Blastocystis/aislamiento & purificación , China/epidemiología , Infecciones por Blastocystis/veterinaria , Infecciones por Blastocystis/epidemiología , Infecciones por Blastocystis/parasitología , Enfermedades de las Aves/parasitología , Enfermedades de las Aves/epidemiología , Aves/parasitología , Heces/parasitología , Prevalencia , Filogenia , Variación Genética , Reacción en Cadena de la Polimerasa , ADN Protozoario/genética

RESUMEN

Blastocystis is one of the most common zoonotic intestinal protozoa with global distribution and can cause gastrointestinal syndrome mainly characterized by diarrhea. School children are the main susceptible population. No epidemiological data on Blastocystis among school children in Hainan, the only tropical island province in China. Between March 2021 and June 2023, 1973 fecal samples were collected from school children across three regions in Hainan province. Blastocystis was examined by amplifying the small subunit ribosomal RNA (SSU rRNA) gene via polymerase chain reaction (PCR), and subtypes were identified through DNA sequencing and phylogenetic analysis. The overall prevalence of Blastocystis was 7.3 % (144/1973). The differences in infection rates across different regions, nationalities, and educational stages are statistically significant (P < 0.001). Five subtypes were identified, of which ST3 was the dominant subtype (60.4 %; 87/144), followed by ST1 (27.8 %; 40/144), ST7 (10.4 %; 15/144), ST6 (0.7 %; 1/144), and ST2 (0.7 %; 1/144). 42 known sequences and 15 novel sequences were identified including eight new variations of the ST1 (ST1-16∼ST1-23) with similarities ranging from 98.3 % to 99.78 % and seven new variations of the ST7 (ST7-7∼ST7-13) with similarities ranging from 97.7 % to 99.79 % by intra-subtype genetic polymorphisms analysis. The results evaluate the public health risks of Blastocystis among school children in Hainan and the sources of infection were discussed, providing important basic data for the effective prevention and control of intestinal parasitic diseases in Hainan.

Asunto(s)

Infecciones por Blastocystis , Blastocystis , Heces , Filogenia , Blastocystis/genética , Blastocystis/clasificación , Blastocystis/aislamiento & purificación , Humanos , China/epidemiología , Infecciones por Blastocystis/epidemiología , Infecciones por Blastocystis/parasitología , Niño , Prevalencia , Femenino , Masculino , Heces/parasitología , Adolescente , ADN Protozoario/genética , Análisis de Secuencia de ADN , Reacción en Cadena de la Polimerasa , Genotipo , Preescolar