RESUMEN
Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples-after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar's test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.
RESUMEN
Ixodid ticks transmit many obligate intracellular Rickettsial species. Several previous studies have identified Rickettsia species in the northeastern and southern part of China, but few reports on the prevalence of infection of spotted fever group Rickettsiae (SFGR) in ticks in southwest China are available. Here, we investigated SFGR in 394 adult ticks of five species including Dermacentor nuttalli, Dermacentor silvarum, Haemaphysalis longicornis, Ixodes sinensis and Ixodes persulcatus, collected in the border region between China and Burma in Yunnan Province. PCR was used to detect the presence of the citrate synthase (gltA) gene of Rickettsia species. SFGR was found in 12.1% (15/124) of I. persulcatus ticks, which was significantly higher than the 7.2% (7/97) positive D. nuttalli, 5.4% (3/56) D. silvarum, 5.6% (4/72) H. longicornis and 4.4 (2/45) I. sinensis. A portion of the gltA and ompA gene data subjected to phylogenetic analysis revealed that the detected SFGR clustered into two species, Rickettsia raoultii and the new Rickettsia species Candidatus Rickettsia jingxinensis. Detection of both Rickettsia spp. in this region indicates a potential public health threat posed by SFGR infection in Yunnan Province.
Asunto(s)
Ixodidae/microbiología , Filogenia , Rickettsia/aislamiento & purificación , Animales , China , Genes Bacterianos , Rickettsia/clasificación , Rickettsiosis ExantemáticasRESUMEN
BACKGROUND & OBJECTIVES: : Bartonella henselae causes infections which closely resemble febrile illness and chronic diseases such as tuberculosis and haematological malignancies. There are not many studies on Bartonella infections from India. The present study was undertaken to diagnose B. henselae infection in diverse clinical conditions in a tertiary care hospital in north India. METHODS: A total of 145 patients including those with fever and lymphadenopathy, infective endocarditis and neuroretinitis were enrolled in the study. Whole blood, serum and lymph node aspirate and valvular vegetations if available, were obtained. Samples were plated on chocolate agar and brain-heart infusion agar containing five per cent fresh rabbit blood and were incubated at 35°C for at least four weeks in five per cent CO2with high humidity. Immunofluorescent antibody assay (IFA) was done for the detection of IgM antibodies in the serum using a commercial kit. Whole blood was used to perform polymerase chain reaction (PCR) for the citrate synthase gene (gltA). RESULTS: IFA was positive in 11 of 140 (7.85%) patients and PCR was positive in 3 of 140 (2.14%) patients. Culture was negative in all the cases. A higher incidence of Bartonella infection was seen in patients with fever and lymphadenopathy (n=30), seven of whom were children. In ophthalmological conditions, four cases were IFA positive. INTERPRETATION & CONCLUSIONS: The present study shows that the threat of Bartonella infection is a reality in India. It is also an important treatable cause of fever and lymphadenopathy in children. Serology and PCR are useful tests for its diagnosis. Clinicians should consider. BARTONELLA: infection in the differential diagnosis of febrile illnesses and chronic diseases.
Asunto(s)
Infecciones por Bartonella/sangre , Bartonella henselae/aislamiento & purificación , Citrato (si)-Sintasa/sangre , Linfadenopatía/sangre , Zoonosis/sangre , Adolescente , Adulto , Animales , Anticuerpos Antibacterianos/sangre , Infecciones por Bartonella/microbiología , Infecciones por Bartonella/transmisión , Bartonella henselae/patogenicidad , Enfermedad por Rasguño de Gato/epidemiología , Enfermedad por Rasguño de Gato/transmisión , Gatos , Niño , Reservorios de Enfermedades , Femenino , Humanos , India/epidemiología , Linfadenopatía/microbiología , Linfadenopatía/patología , Masculino , Persona de Mediana Edad , Conejos , Ratas , Centros de Atención Terciaria , Adulto Joven , Zoonosis/epidemiología , Zoonosis/microbiología , Zoonosis/patologíaRESUMEN
Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere.