Molecular Characterization of Trypanosoma cruzi in Infected Meccus pallidipennis in the Southern Region of the State of Mexico, Mexico.

PCR amplification and sequencing of Trypanosoma cruzi (T. cruzi) spliced-leader intergenic region of the mini-exon gene intergenic region (SL-IR) fragment was performed on intestinal tissue and fecal content DNA extracted from 19 Meccus pallidipennis (M. pallidipennis) specimens collected in the southern region of the State of Mexico. DNA sequence analysis from 49 bp T. cruzi SL-IR showed that all 19 samples corresponded to haplotype TcIa, and all of them were identical to GenBank sequence JQ028863. When extending the analysis to the whole 256 bp amplified sequence of the SL-IR, we found six sequences with a C insertion at position 10, one of which also presented a mutation (T/C) at position 54. One more sequence had an insertion (T) at position 223. Our findings suggest that two dominating TcIa clones are present in M. pallidipennis in the southern region of the State of Mexico. Interestingly, the SL-IR region of the dominating genotype was 100% identical to a circulating clone from Costa Rica present in humans, dogs, Triatoma dimidiata, and Panstrongylus rufotuberculatus. Future regional studies should explore the presence of this haplotype in humans and domestic animals.

Isolation and molecular characterization of Corynebacterium pseudotuberculosis from sheep and goats in Mexico.

The pathogenic bacteria of Corynebacterium pseudotuberculosis caused a chronic contagious infectious disease of the caseous lymphadenitis or pseudotuberculosis. Globally, isolates obtained from different injuries that affect sheep and goats, have been identified by fully or partially gene sequencing. However, in Mexico there is no complete study to identify by molecular and phylogenetic techniques the circulating isolates as well as its virulence factors. Therefore, in the present study we reported the identification of 57 isolates of C. pseudotuberculosis by bacteriological tests and the amplification of 16S rRNA, rpoB and pld genes, as well as, genes involved in virulence and pathogenicity: Fag A, Fag B, Fag C, Fag D and hsp60. Phylogenetic analysis was performed based on the partial sequence of the rpoB gene. Genes involved in virulence and pathogenicity were identified in the 98.2% of the isolates. Regarding the phylogenetic analysis, were identified the species and subspecies to which they belong of all the tested isolates. The phenotypic and genotypic characterization will allow to establish preventive and prophylactic measures aimed to the creation of effective immunogens against Corynebacterium pseudotuberculosis.

Antibiotics susceptibility of quinolones against Salmonella spp. strains isolated and molecularly sequenced for gyrA gene.

Drug-resistant Salmonella is frequently detected in most parts of the world, and its rate of resistance has increased significantly in recent years. However, this study aimed to evaluate the minimum inhibitory concentration (MIC, determined with the Kirby-Bauer method) of quinolones in 86 Salmonella spp. strains isolated from pigs. Both the inside and outside of the QRDR region of strains were sequenced. The DNA sequence of the QRDR region of Salmonella spp. revealed the mutations S83F, D87N and S83Y. The region outside the QRDR showed a mutation in L582G. Forty-five isolates of Salmonella ssp. were categorized as quinolone-resistant; out of these, 16 corresponded to Salmonella enterica and isolates showed intermediate resistance (6.25%) to nalidixic acid. Three isolats (18.6%) were resistant to ampicillin; two (12.5%) were resistant to carbenicillin. Moreover, three (18.7%) isolates were resistant to gentamicin, nitrofurantoin and pefloxacin, and 8 (50%) were resistant to trimethoprim/sulfamethoxazole. Six percent of Salmonella spp. strains showed less resistance to antimicrobial agents compared to S. Thyphimurium (18%). The resistance to individual quinolones varied by serotypes. For S. anatum and S. Reading, it was 12.25%, and for S. choreaeaesuis, S. typhimurium monofasica, 6.25%. In contrast, S. agona, S. bredeney and S. london were sensitive to these antibiotics. In conclusion, quinolones have become the drugs of choice for the treatment of severe Salmonella infections. The study of mutations outside the QRDR region opens up new insights about the resistance of Salmonella to fluoroquinolones.

Detection of Quinolone Resistance in Salmonella typhimurium Pig Isolates Determined by gyrA Gene Mutation Using PCR- and Sequence-Based Techniques within the gyrA Gene.

BACKGROUND: The emergence of reduced susceptibility to fluoroquinolones among Salmonella enterica serotype Typhimurium isolates leading to clinical failure of treatment poses a great therapeutic challenge. METHODS: The current study is focused on the evaluation of the minimum inhibitory concentration (MIC) of quinolones in 29 Salmonella typhimurium of 86 Salmonella spp. strains, obtained from pigs from the State of Mexico. The MIC was performed with the Kirby-Bauer method. On the other hand, the GyrA gene was sequenced. The present study was undertaken to describe the resistance profiles and fluoroquinolone resistance mechanism of Salmonella Typhimurium. RESULTS: The DNA sequence of the gyrA genes from Salmonella enterica serovar typhimurium revealed strong similarity between gyrA and its counterpart in Escherichia coli. The sequencing of quinolone resistance-determining region (QRDR) of the gyrA gene showed the presence of mutation at either S83 or at D87 in almost all the Salmonella typhimurium isolates. CONCLUSION: This mutation, although phenotypically expressed as decreased susceptibility to fluoroquinolones goes undetected by the disk diffusion method using the present method of Kirby-Bauer. Hence, it can increase morbidity and mortality due to delay in appropriate antibiotic treatment.

Experimental-Theoretic Approach to Drug-Lymphocyte Interactome Networks with Flow Cytometry and Spectral Moments Perturbation Theory.

We can combine experimental techniques like Flow Cytometry Analysis (FCA) with Chemoinformatics methods to predict the complex networks of interactions between organic compounds and targets in the immune system. In this work, we determined experimentally the values of EC50 = 17.82 µg/mL and Cytotoxicity = 20.6 % for the anti-microbial / anti-parasite drug Dermofural over Balb/C CD9 lymphocytes using flow cytometry. After that, we developed a new Perturbation-theory model for Drug-Cell Target Interactome in Lymphocytes based on dispersion-polarization moments of drug structure. The models correctly classifies 34591 out of 42715 (Accuracy = 80.9%) cases of perturbations in assay endpoints of 11492 drugs (including both train and validation series). Each endpoint correspond to one out of 2616 assays, 38 molecular and cellular targets, 77 standard type measures, in four possible (human and rodents).

Identification and molecular characterization of Corynebacterium xerosis isolated from a sheep cutaneous abscess: first case report in Mexico.

BACKGROUND: Corynebacterium xerosis is a commensal organism found in skin and mucous membranes of humans. It is considered an unusual pathogen, and it is rarely found in human and animal clinical samples. Here we describe the isolation of C. xerosis from a 4-months-old Pelifolk lamb located in Tesistán, central western Mexico. This microorganism should be considered for differential diagnosis in cutaneous abscessed lesions in sheep, as it represents a zoonotic risk factor for human infection in sheep farms. CASE PRESENTATION: The animal exhibited a hard-consistency, 5 cm diameter abscess, without drainage, in the neck. The presumptive clinical diagnosis was caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis. Samples were obtained by puncture and cultured in 8 % sheep blood agar under microaerophilic conditions. Colonies were non-haemolytic, brown-yellowish and showed microscopic and biochemical features similar to C. pseudotuberculosis, except for the urea test. A multiplex-PCR for the amplification of partial sequences of the pld, rpoB and intergenic fragment from 16S to 23S genes suggested that isolate could be C. xerosis, which was later confirmed by sequencing analysis of the rpoB gene. CONCLUSIONS: This study shows for the first time isolation and molecular characterization of C. xerosis from a clinical sample of an ovine cutaneous abscess in Mexico. This finding highlights the need for differential diagnosis of this pathogen in ovine skin abscesses, as well as epidemiological and control studies of this pathogen in sheep farms.

Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model.

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines.

Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico.

The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzyme-linked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87% (n = 246). Seropositive animals were found in six out of nine (66.6%) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51%. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico.

Testing the efficacy of a multi-component DNA-prime/DNA-boost vaccine against Trypanosoma cruzi infection in dogs.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. METHODS: We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. RESULTS: Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8(+) T cell proliferation and IFN-γ production, and suppression of phagocytes' activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. CONCLUSIONS: Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease.

Prevalence of Trypanosoma cruzi in dogs (Canis familiaris) and triatomines during 2008 in a sanitary region of the State of Mexico, Mexico.

American trypanosomiasis is a public health problem in Latin America and southern parts of the United States. Infection in triatomines (vector) and domestic dogs (reservoir host) is a good indicator of Trypanosoma cruzi circulation and human risk of infection. The State of Mexico, Mexico, has been considered free of T. cruzi, and no detailed epidemiologic study has been conducted to assess the intricacies of the transmission cycle of the parasite in the region. Such studies would enhance our understanding of the epidemiology of T. cruzi infection in this geographic region and provide regional sanitary authorities with stronger fundamental knowledge for making decisions and allocating funds for Chagas disease control programs in the State of Mexico. The objective of this study was to determine the prevalence of T. cruzi infection in dogs (seroprevalence) and triatomines (fecal parasites) in a previously identified, discrete endemic region of parasite circulation and to widen our studies in the Tejupilco Sanitary Region located in the southern part of the State of Mexico. Dog blood samples (n=102) were analyzed for the presence of anti-T. cruzi antibodies by two assays, namely indirect hemagglutination assay and enzyme-linked immunosorbent assay. Triatomines (n=88) were collected and fecal aliquots were analyzed for the presence of parasites by light microscopy. Average seroprevalence in dogs in the Tejupilco Sanitary region was 24.5%, and the overall triatomine infection rate was 34.01%. Triatoma pallidipennis was the only triatomine species found in this region. Our data demonstrate that T. cruzi is actively circulating in the Tejupilco Sanitary Region and emphasize the requirement for epidemiologic surveillance programs throughout the putative endemic areas of the State of Mexico.

Risk factors associated with triatomines and its infection with Trypanosoma cruzi in rural communities from the southern region of the State of Mexico, Mexico.

Trypanosoma cruzi prevalence in triatomines and risk factors associated to the presence of the insect were studied in 990 rural houses in the southern region of the State of Mexico, Mexico. In each house, triatomines were collected, and information related to house construction material was obtained. T. cruzi infection was diagnosed in all triatomines. A primary screening was performed using 2 x 2 contingency tables of exposure variables. All variables with P

Trypanosoma cruzi circulating in the southern region of the State of Mexico (Zumpahuacan) are pathogenic: a dog model.

Here we describe clinical and pathologic evidence of Chagas disease caused in dogs by circulating Trypanosoma cruzi from a newly recognized endemic area in Mexico. We show that the Zumpahuacan isolate, although less virulent than the Sylvio-X10 reference strain that caused acute myocarditis and death, was pathogenic in dogs. Dogs infected with the Zumpahuacan isolate exhibited electrocardiographic alterations, left- and right-ventricle dilation, and hydropericardium. Histologically, diffused perimysial and endomysial lymphoplasmacytic cell infiltration, cardiomyocyte necrosis, and amastigote nests were noted in Zumpahuacan-infected dogs. These findings suggest that the risk of T. cruzi infection and Chagas disease is present in the State of Mexico, and further research is needed to identify the T. cruzi bio-types circulating in southern State of Mexico.