Approaches for the successful isolation and cell culture of American Rickettsia species.

Rickettsia are intracellular vector-borne bacteria, which are the etiologic agent of severe infections that could inflict death to their host. The intracellular behaviour of Rickettsia makes the study of its genetics, proteomics and cellular processes very difficult. Hence, isolation remains an important experimental technique that permits the obtention of important yields of bacteria, useful for a broad range of experiments. Isolation of Rickettsia using passages in animals or embryonated eggs has been described for long time; however, it was until the 1990s that faster and more feasible approaches for cell culture were developed. Current isolation approaches are mainly based on shell vial culture, that varies according to the media, atmosphere or temperature conditions. These variations have allowed the establishment of isolates from different pathogenic and non-pathogenic Rickettsia species, using arthropod, animal or human samples. Purification method of bacteria has also witnessed changes alongside the quantification of its load in the resulting isolates, from the laborious and time consuming plaque assays, to the routinary use of real-time polymerase chain reaction (qPCR), which is faster and more accurate. This review discusses various approaches that have been used for the isolation and purification of different Rickettsia species along with the mention of some successful examples. It indicated that a successful strategy for the isolation of Rickettsia requires a careful selection of media, cell lines and culture conditions which now are not as time consuming as used to be.

First molecular evidence of Toxoplasma gondii in opossums (Didelphis virginiana) from Yucatan, Mexico.

Toxoplasma gondii is an obligate intracellular parasite recognized as a causal agent of toxoplasmosis; zoonotic disease endemic in many countries worldwide, including Mexico. Different species of animals participate in the wild cycle infection, including opossums of the species Didelphis virginiana. Thirteen D. virginiana were captured in Yucatan, Mexico. Detection of T. gondii was achieved by Polymerase Chain Reaction, which determined an infection of 76.9% (10/13) in brains. Positive amplicons were sequenced for analysis, this produced results similar to T. gondii with identity and coverage values of 98% and 96-100%, respectively. This study presents the first molecular evidence of the circulation of T. gondii in D. virginiana from Mexico.

Rickettsia rickettsii isolation from naturally infected Amblyomma parvum ticks by centrifugation in a 24-well culture plate technique.

Rocky Mountain spotted fever is an acute illness caused by Rickettsia rickettsii (R. rickettsii) and is transmitted by the bite of ticks of the genera Dermacentor, Amblyomma and Rhipicephalus. The illness results in a high mortality rate and may be easily confused with other febrile syndromes. In Yucatan State, Mexico, childhood cases with a high mortality have been reported. In this work we report the isolation of a Mexican R. rickettsii strain from a tick egg mass using an alternative method for Rickettsia isolation with 24-well plates. We also identified a potential vector of R. rickettsii in the southeast of Mexico, which is Amblyomma parvum.

Rickettsia felis in the Americas.

The authors describe their work in the Americas in Rickettsia felis cases in humans and the presence of Rickettsia felis in vectors.

Identification of Ctenocephalides felis fleas as a host of Rickettsia felis, the agent of a spotted fever rickettsiosis in Yucatan, Mexico.

In search for the vector of the recently recognized spotted fever rickettsiosis of the Yucatán, ticks, fleas, and lice were collected from vegetation and dogs in localities where seropositive persons had been found. The arthropods were examined by polymerase chain reaction (PCR) using primers for the genus-specific 17-kDa protein gene followed by restriction fragment length polymorphism (RFLP) and DNA sequencing. Eleven (20%) of 54 pools of Ctenocephalides felis fleas contained DNA of Rickettsia felis. None of 219 Amblyomma cajennense, 474 Rhiphicephalus sanguineus, 258 Boophilus sp. ticks, and 33 Poliplax species lice contained DNA of Rickettsia. The identity of the rickettsial DNA was confirmed as R. felis by PCR/RFLP for the citrate synthase and outer membrane protein A genes and by DNA sequencing. The results indicate that the host of R. felis in Yucatán is C. felis and suggest that the spotted fever rickettsiosis that has infected >5% of the population of the Yucatán and can present as a dengue-like illness is likely to be caused by R. felis.

Stage specific kinetoplast DNA-binding proteins in Trypanosoma cruzi.

Knowledge regarding kinetoplast DNA organization in all members of the Trypanosomatid family is incomplete. Recently, the presence of kinetoplast-associated proteins in condensing kDNA networks in Crithidia fasciculata has been described and a role for these proteins in the maintenance of these complex structures was suggested. To investigate the presence of protein components in Trypanosoma cruzi kinetoplast, we previously described seven epimastigote kinetoplast-associated proteins. We report here the existence of kinetoplast binding proteins in amastigote and trypomastigote stages of T. cruzi, which could bind both mini and maxicircles components with a stage specific elements for every infective form of the parasite. We propose three major classes of kinetoplast-associated proteins related to the basic processes of this intricate disc structure and suggest a possible function of these binding proteins in the T. cruzi mitochondrial DNA organization.

[Chronic Chagas cardiomyopathy detected in patients at the Regional General Hospital O'Horan, Merida, Yucatan, Mexico]. / Cardiopatiá Chagásica crónica detectada en pacientes del hospital general regional O'Horan, Mérida, Yucatán, México.

The purpose of this study was to determine the frequency of cardiopathy due to Chagas' disease in 36 patients of the cardiology department at the Regional General Hospital O'Horan in Merida, Yucatan. All patients included in the study had cardiac involvement compatible with acute or chronic stages of Chagas' disease. Medical records prepared for each one of the patients included a Chagas' disease targeted clinical history, chest X-ray, electrocardiogram, blood culture and serology using indirect immunofluorescence test. Out of the 36 patients studied, 7 were diagnosed as having Chagas' disease cardiopathy. Grade II cardiomegaly was established in 2 patients while the remaining 5 had grade III cardiomegaly. Conduction abnormalities were established in 6 patients while 2 of these had evidence of necrosis and/or ischemia. Chagas' disease cardiopathy, as our results suggest, is not a rare event in the cardiology ward at the O'Horan Hospital.

Adrenergic ligands trigger intracellular differentiation of trypanosoma cruzi.

Intracellular forms of trypanosoma cruzi, amastigote, could remain in the inner space of mammalian cells for long periods of time and be in contact with various cellular metabolism products. Some of these metabolites could act as signals that trigger parasite differentiation process to trypomastigote form. The present results suggest that increase of intracellular cAMP by adrenergic ligands or cholera toxin in parasite infected cells is able to induce such differentiation process.

Molecular characterization of Mexican stocks of Trypanosoma cruzi using total DNA.

Seventeen Mexican Trypanosoma cruzi stocks and five South American reference strains were analyzed by Hind III restriction fragment length polymorphisms associated with ribosomal RNA gene spacers and by HinfI digestion patterns of total DNA. Our findings demonstrate the occurrence of genetic heterogeneity within these stocks. Hierarchic and non-hierarchic clustering of these molecular characters allowed the formation of groups that correlate with the geographic origin of the stocks. The HinfI digestion pattern permitted the identification of DNA fragments from the kinetoplast maxi-circles, and therefore represents a simple and convenient method for conducting epidemiologic surveys in laboratories in developing countries.