A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

Background: Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Results: Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. Conclusion: The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended. © 2017 The Author(s)

Phylogenetic relationships of the Amblyomma cajennense complex (Acari: Ixodidae) at mitogenomic resolution

The genus Amblyomma is the third most diverse in the number of species within the Ixodidae, with practically half of its species distributed in the Americas, though there are also species occurring in Africa, Asia, and Australia. Within the genus, there are several species complexes with veterinary and public health importance. The Amblyomma cajennense complex, in the Americas, is represented by six species with a wide distribution, from Texas to northern Argentina. We combined two sequencing techniques to generate complete mitogenomes of species belonging to the Amblyomma cajennense complex: genome skimming and long-range PCRs sequencing methods. Thus, we generated seven new mitochondrial genomes for all species of the Amblyomma cajennense complex, except for Amblyomma interandinum. Genetic distances between the mitogenomes corroborate the clear differentiation between the five species of the Amblyomma cajennense complex. The phylogenetic relationships of these species had previously been evaluated by combining partial nuclear and mitochondrial genes and here these relationships are corroborated with a more robust framework of data, which demonstrates that the conjunction of mitochondrial and nuclear partial genes can resolve close relationships when entire genes or genomes are unavailable. The gene order, structure, composition, and length are stable across these mitogenomes, and they share the general characteristics of Metastriata. Future studies should increase the number of available mitogenomes for this genus, especially for those species from the Indo-Pacific region and Africa, by means of a better understanding of their relationships and evolutionary process.EEA RafaelaFil: Cotes-Perdomo, Andrea P. Museo Nacional de Ciencias Naturales (MNCN-CSIC), Departamento de Biodiversidad y Biología Evolutiva; EspañaFil: Cotes-Perdomo, Andrea P. University of South-Eastern. Faculty of Technology. Natural Sciences and Environmental Health Department. Natural Sciences and Maritime Sciences; NoruegaFil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Nava, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria. Instituto de Investigación de la Cadena Láctea (IDICAL); ArgentinaFil: Castro, Lyda R. Universidad del Magdalena. Facultad de Ciencias Básicas. Grupo de investigación Evolución, Sistemática y Ecología Molecular (GIESEMOL); ColombiaFil: Rivera-Paéz, Fredy A. Universidad de Caldas. Facultad de Ciencias Exactas y Naturales. Departamento de Ciencias Biológicas. Grupo de Investigación en Genética, Biodiversidad y Manejo de Ecosistemas (GEBIOME); ColombiaFil: Cortés-Vecino, Jesús A. Universidad Nacional de Colombia. Facultad de Medicina Veterinaria y de Zootecnia. Grupo de Investigación Parasitología Veterinaria; ColombiaFil: Uribe, Juan E. Museo Nacional de Ciencias Naturales (MNCN-CSIC). Departamento de Biodiversidad y Biología Evolutiv; EspañaFil: Uribe, Juan E. Smithsonian Institution. National Museum of Natural History. Invertebrate Zoology Department; Estados Unido

Caso probable de fiebre manchada (Rickettsia felis) transmitida por pulgas

Rickettsia felis is the etiologic agent of flea-borne spotted fever, with Ctenocephalides felis as its main vector and reservoir. Typically, the disease presents as acute fever associated with headache, asthenia, generalized maculo-papular rash, and in some cases, an inoculation eschar. In recent years, R. felis has acquired an important role in the etiology of the acute febrile syndrome; it is indeed an emerging infectious disease, albeit underdiagnosed. Indirect immunofluorescence assay (IFA) is currently the reference diagnostic method. However, this technique has limitations related to the cross reactivity among different species of rickettsiae. Herein, we describe a case of a 16 year-old patient with an acute febrile syndrome secondary to probable infection with R. felis. doi: http://dx.doi.org/10.7705/biomedica.v33i0.723Rickettsia felis es el agente etiológico de la fiebre manchada transmitida por pulgas, cuyo principal vector y reservorio es Ctenocephalides felis. Típicamente, la enfermedad se presenta como fiebre aguda asociada a cefalea, astenia, exantema máculo-papular generalizado y, en algunos casos, conescara de inoculación. En los últimos años, R. felis ha venido adquiriendo un papel importante en la etiología del síndrome febril agudo, calificándola como una enfermedad emergente y subdiagnosticada. La inmunofluorescencia indirecta es actualmente el método diagnóstico de referencia. Sin embargo, esta técnica presenta limitaciones relacionadas con la reacción cruzada que existe entre las diferentesespecies del género Rickettsia. En el presente reporte se describe el caso de un paciente de 16 años con síndrome febril agudo secundario a infección probable por R. felis. doi: http://dx.doi.org/10.7705/biomedica.v33i0.723

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

"Background: Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Results: Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. Conclusion: The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended. © 2017 The Author(s).

Zoonotic nematode contamination in recreational areas of Suba, Bogotá

Objective: Establishing the presence of zoonotic gastrointestinal nematodosin soil samples in recreational areas in Suba (one of the largest localities of Bogotá, Colombia) between July 2005 and June 2006. Methodology: The presence of several gastrointestinal parasites\u27 eggs, larvae and oocysts were determined by initial soil sedimentation and later flotation with Sheather solution; some of them were responsible for important zoonotic diseases affecting the human population. Results: Parasites were identified in 376 samples: Ancylostoma larvae (10,7 %), Ancylostoma eggs (0,6 %), Toxocara eggs (5,8 %), Strongyloides eggs (3,3 %), Dipylidium eggs (0,06 %) and Sarcocystis oocysts (0,06 %). Other parasites identified were Toxascaris eggs (0,9 %), Spirocerca eggs (0,25 %) and Isospora oocysts (0,25 %). Some larvae and eggs present in 522 samples could not be classified and there was no evidence of parasites in 702 samples (45 %). 94,2 %, (n=49) of the recreational area was thus polluted. Conclusions: The high percentage of recreational areas so polluted indicated that these places would constitute a risk factor for the transmission of parasitic diseases to animals and human beings. Due to this sanitary problem\u27s relevance in public health programmes, government policy must be determined involving communities so that people are educated to improve their sanitary practices in recreational places regarding caring for their pets

Diversity of Blastocystis Subtypes in Horses in Colombia and Identification of Two New Subtypes

Blastocystis is a common intestinal protist in humans and animals worldwide. Wild and domestic animals are thought to be reservoirs of Blastocystis subtypes that also infect humans. There are limited studies on the prevalence and subtype distribution of Blastocystis in horses. In this study, 185 fecal samples were collected from horses (1 month to 17 years of age) in four regions of Colombia (Sabana de Bogotá, Costa Atlántica, Llanos Orientales, and Bogotá D.C.). Blastocystis presence and subtypes were determined by PCR and next generation amplicon sequencing. Eighty-one (43.8%) horses were positive for Blastocystis, with positive horses in all four regions. Molecular characterization identified 12 Blastocystis subtypes, 10 known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26), and 2 novel subtypes (ST33 and ST34). The validity of the novel subtypes was confirmed via phylogenetic and pairwise distance analyses of the full-length SSU rRNA gene sequences. Mixed subtype infections were common (55.6% of Blastocystis-positive horses). ST10 was the most prevalent subtype, present in 82.8% of Blastocystis-positive horses. Potentially zoonotic subtypes were identified in 88.9% of the Blastocystis-positive horses. This constitutes the most comprehensive study of Blastocystis in horses. Our findings indicate that horses harbor potentially zoonotic subtypes and could contribute to the transmission of Blastocystis to humans

Evaluating the Effect of Sarconesiopsis magellanica (Diptera: Calliphoridae) Larvae-Derived Haemolymph and Fat Body Extracts on Chronic Wounds in Diabetic Rabbits

We evaluated extracts taken from S. magellanica third instar larvae fat body and haemolymph using a diabetic rabbit model and compared this to the effect obtained with the same substances taken from Lucilia sericata larvae. Alloxan (a toxic glucose analogue) was used to induce experimental diabetes in twelve rabbits. Dorsal wounds were made in each animal and they were infected with Staphylococcus aureus and Pseudomonas aeruginosa. They were then treated with haemolymph and lyophilized extracts taken from the selected blowflies’ larvae fat bodies. Each wound was then evaluated by using rating scales and histological analysis. More favourable scores were recorded on the PUSH and WBS scales for the wounds treated with fat body derived from the larvae of both species compared to that obtained with haemolymph; however, wounds treated with the substances taken from S. magellanica had better evolution. Histological analysis revealed that treatment led to tissue proliferation and more effective neovascularisation in less time with both species’ fat body extracts compared to treatment with just haemolymph. The results suggest the effectiveness of the substances evaluated and validate them in the animal model being used here as topical agents in treating chronic wounds

Evaluating Sarconesiopsis magellanica blowfly-derived larval therapy and comparing it to Lucilia sericata-derived therapy in an animal model

Larval therapy is used as alternative treatment for hard-to-heal chronic and infected wounds. Lucilia sericata is the most used blowfly species. However, it has been shown recently that Sarconesiopsis magellanica larval excretions and secretions have potent antibacterial activity; this blowfly belongs to the Calliphoridae family.The present work has dealt with evaluating larval therapy using S. magellanica on wounds induced in diabetic rabbits and its action was compared to the effect induced by L. sericata. Twelve New Zealand White rabbits (. Oryctolagus cuniculus) were used; they were divided into 4 groups, the first two being treated with larval therapy derived from both aforementioned necrophagous blowflies, an antibiotic was used in the third and the fourth was used as control. All the animals were wounded on the back and infected with Pseudomonas aeruginosa and Staphylococcus aureus.Samples of the secretion from each animal's infected wound were taken and sown on blood agar. The colony forming units were then counted. The PUSH scale was used for the macroscopic evaluation of the wounds.Bacterial control was encountered 48. h post-treatment in the treatments involving larval therapy and to a lesser extent with the antibiotic. Likewise, wound debridement was quicker and more efficient with larval therapy compared to the antibiotic group; however, wound closing time was 23 days in all treatments. The group treated with S. magellanica larvae had relatively quicker evolution until the proliferation phase and the start of maturation, even though there were no significant differences between both blowfly species evaluated here regarding treatments by the end of the treatment period.The present study has validated the diabetic rabbit model for inducing chronic wounds regarding larval therapy and has likewise confirmed the effectiveness of S. magellanica-derived larval therapy as an alternative for curing and healing wounds. © 2015 Elsevier B.V

Evaluating Sarconesiopsis magellanica blowfly-derived larval therapy and comparing it to Lucilia sericata-derived therapy in an animal model

Larval therapy is used as alternative treatment for hard-to-heal chronic and infected wounds. Lucilia sericata is the most used blowfly species. However, it has been shown recently that Sarconesiopsis magellanica larval excretions and secretions have potent antibacterial activity; this blowfly belongs to the Calliphoridae family.The present work has dealt with evaluating larval therapy using S. magellanica on wounds induced in diabetic rabbits and its action was compared to the effect induced by L. sericata. Twelve New Zealand White rabbits (. Oryctolagus cuniculus) were used; they were divided into 4 groups, the first two being treated with larval therapy derived from both aforementioned necrophagous blowflies, an antibiotic was used in the third and the fourth was used as control. All the animals were wounded on the back and infected with Pseudomonas aeruginosa and Staphylococcus aureus.Samples of the secretion from each animal's infected wound were taken and sown on blood agar. The colony forming units were then counted. The PUSH scale was used for the macroscopic evaluation of the wounds.Bacterial control was encountered 48. h post-treatment in the treatments involving larval therapy and to a lesser extent with the antibiotic. Likewise, wound debridement was quicker and more efficient with larval therapy compared to the antibiotic group; however, wound closing time was 23 days in all treatments. The group treated with S. magellanica larvae had relatively quicker evolution until the proliferation phase and the start of maturation, even though there were no significant differences between both blowfly species evaluated here regarding treatments by the end of the treatment period.The present study has validated the diabetic rabbit model for inducing chronic wounds regarding larval therapy and has likewise confirmed the effectiveness of S. magellanica-derived larval therapy as an alternative for curing and healing wounds. © 2015 Elsevier B.V