Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. Chagas disease, caused by the protozoan Trypanosoma cruzi, represents a health and social threat to an estimated number of eight million people, affecting mainly neglected populations in endemic areas and emerging in non endemic countries by migratory movements. Parasite genetic diversity is related to geographical distribution and transmission cycles and might play a role in clinical manifestations as well as in anti-parasitic chemotherapy response. T. cruzi has been classified into six Discrete Typing Units (DTUs), after consensus reached among experts in the field. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is needed. Available typing schemes are usually applied to cultured parasite stocks, because they are not sensitive enough to be used in biological specimens. Only nested PCR procedures could directly type biological samples, but are prompt to contamination and require a high number of reactions. Thus, we developed a multiplex Real-Time PCR using TaqMan probes (MTq-PCR) for DTU typing in a single or a second round of amplification. It proved useful to determine DTUs in cultured stocks, vector and reservoir specimens, as well as in patients´samples, especially in those from individuals with acute, congenital infection or Chagas reactivation. It is amenable to quantification and automation for kit production.

A survey in a major Mexican city (Merida) and three rural communities was conducted to generate information regarding the size and structure of the owned-dog populations and people's opinions about the dogs and how they took care of them. Household characteristics and dog population size, health and reproductive issues were compared between the two kinds of communities: urban and rural. A telephone survey was conducted in Merida city whereas personal interviews were used in the rural communities. Local veterinarians were also interviewed to evaluate their influence on the dog populations in Merida city. The ratio of people to dogs was 3.4:1 in the city, and 1.7:1 to 4.6:1 in the different rural communities. In general it was more common to find a dog-owning household in the city of Merida (72.8%) than in the rural areas (63.6%, 65.5% and 71.1%), and in the city more households had adequate fences to restrain dogs. Larger families were more likely to own a dog than small families. Households of med...

To assess the risk of zoonotic pathogen transmission as function of stray dog presence and health status, a cross-sectional study was carried out in a large city of southern Mexico that lacks comprehensive strategies for the control of stray canine populations. The photographic capture-recapture method was used to estimate the density of dogs/km². In the same way, dog feces from 14 public parks of the city were collected to determine the prevalence and intensity of infection with gastrointestinal parasites. The canine population was estimated between 65 and 80 thousand dogs, with a population density of 1,081 dogs/km², mostly males (71.4%). A high proportion of dogs (72.3%) were found to be in good body condition score (BCS 3). The person:dog ratio was 2.3. The likelihood of being in the BCS 2 category was lower in areas with a higher density of dogs. All feces collected from the parks contained eggs of intestinal parasites, most of them with a medium (42.9%) to high (35.7%) infection intensity, notably Ancylostoma caninum. It was recorded that cases with a low-intensity of GI infection showed polyparasitism (35.7%) associated with A. caninum. There is a large population of stray dogs that roam freely in the streets of Campeche city with access to sources of food, which is reflected by their good BCS, and dogs do not have access to preventive medicine programs (de-worming) and thus contaminate public parks with feces with significant parasitic egg loads of zoonotic importance.

This investigation determined the presence and prevalence of the zoonotic agents Leptospira interrogans , Trypanosoma cruzi , and Aspergillus spp. in the stray dog population (a total of 224 stray dogs) in an urban area of Southern Mexico. Blood serum samples were taken from all dogs, and root hair samples were taken from dogs with skin lesions and partial alopecia. IgG antibodies for L. interrogans from 10 serovars were detected using the microscopic agglutination test. Immunofluorescence antibody test and Western blot assay were used for serologic diagnosis of T. cruzi. The Sabouraud medium was used to isolate Aspergillus spp. Prevalence of L. interrogans was 4.9%, which was determined by identifying only serovars Pyrogenes, which accounted for 3.6%, and Tarassovi, which constituted 1.3%, with titers from 1:100 to 1:800. Additionally, T. cruzi antibodies were detected in 4.5% of the dogs. Skin lesions were found in 43% of the dogs (98/224), and 35 cultures were positive for Aspergillus spp. (35.7%, p < 0.05, 95% confidence interval 2.45–3.67), identified as A. niger (82.8%), A. flavus (14.3%), and A. terreus (2.9%). This study demonstrates the presence of certain zoonotic agents (bacteria, protozoa, and fungi) in stray dogs living within the studied area. Dogs play an important role in the transmission of diseases that are potentially harmful to humans. Although the prevalence of canine leptospirosis and trypanosomiasis is not high in Southern Mexico compared with other tropical regions of Mexico, the presence of these zoonotic agents in the stray dog population demonstrates that the stray dog population in this region is a significant reservoir and potential source of infection in humans. Special care should be taken when handling stray dogs that exhibit skin lesions with partial alopecia, since a pathological Aspergillus sp. fungus may be present.

American trypanosomiasis is an important zoonotic disease which affects more than 15 million persons in America. In Mexico, Chagas' disease is widely distributed in the country mostly in states with tropical weather conditions, including Yucatan. A cross-sectional study was performed on serum samples from 35 dogs and their owners (n = 75) from the south area of Merida city. Specific IgG antibodies against Trypanosoma cruzi (T. cruzi) using enzyme-linked immunosorbent assay, Immunofluorescence antibody test (IFI) and Western blot (WB) were detected. The overall percentage of seropositivity was 34% in dogs and 8% for sampled owners. Some owners brought samples of insect vectors found in their households, which were kept with BALB/c mice as a source of food and which were evaluated by polymerase chain reaction. All tested insects were positive to T. cruzi, and BALB/C mice were IFI and WB positive after 45 days in contact with these vectors. Further investigations showed that there is a high risk of infection with Chagas disease in dogs which spend the night outdoors (P < 0.05), with low body conditions score and older dogs. Risk factors identified to be associated to the infection in owners were gender and occupation (bricklayers, P < 0.05). The presence of seropositive dogs in houses where vectors are well-adapted represents a high risk for humans to become infected when bitten by a vector infected by a positive dog.