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American trypanosomiasis is an infectious disease of importance for public health and caused by the protozoa Trypanosoma cruzi mainly transmitted by triatomine bugs. The precise role of cats in the peridomestic transmission of T. cruzi and the mechanism by which cats become infected remain uncertain. The objective of this work was to determine the prevalence of T. cruzi infection in domestic cats from an urban area of tropical Mexico by serological and molecular methods and evaluate associated risk factors. A total of 220 domestic cats from Merida Yucatan, Mexico, were studied. Animals older than 3 months were blood sampled. Serum and DNA were obtained. Specific T. cruzi IgG antibodies were detected using a commercial indirect ELISA with an anti-cat antibody HRP labelled. Positive cases were confirmed by Western blot (WB). Polymerase chain reaction (PCR) was also performed using the primers TC1 and TC2. From the 220 cats, 8.6% had antibodies against T. cruzi using ELISA test and later confirmed by WB. In 75 cats (34%), the sequence of ADNk of T. cruzi was amplified. The bad–regular body condition was the only risk factor associated with PCR positive to T.cruzi (P < 0.001). In Mexico, there are no previous epidemiological reports that demonstrate the importance of the cat as a reservoir of T. cruzi. Few individuals were identified with a serological response because they were probably at an early stage of infection or antibodies were not detected because they could be immunocompromised (FIV, FeLV or others). It is necessary to monitor PCR-positive patients and conduct further studies for better understanding of the epidemiology and pathogenesis of Chagas disease in domestic cats.
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- Materials and Methods
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In this study, the seroprevalence of Chagas disease in cats (8.63%) was determined using two WHO-approved tests (ELISA and WB) for humans (Otani et al., 2009). ELISA is considered the first choice for screening purposes and one of the pair of techniques recommended for diagnostic studies in dog populations. In the present study, as no commercial kits designed for cats are available, the Chagatest (WienerLab, Argentina) was used and results latter confirmed by WB considering it has been proved before in dogs with good results (Jiménez-Coello et al., 2008, 2010). There are no epidemiological studies in Mexico demonstrating the role of cats as a reservoir of T. cruzi, so this is the first report on its type. Comparing our results with the 78.6% obtained in Argentina (Wisnivesky-Colli et al., 1985), seroprevalence is well below. These difference may be in part because in that study, the low-specificity indirect hemagglutination inhibition and complement fixation tests were used, and secondly, because the number of cats sampled was too small (n = 14). However, the role of cats in the transmission of the disease is very different than dogs because cats are not permanent residents of the households.
More recently, in the same region, Cardinal et al. (2006, 2007, 2008) reported a prevalence of IgG antibodies against T. cruzi in cats by ELISA, immunofluorescence and indirect hemagglutination inhibition of 4.5% (n = 109), ranged from 2.1% to 6.6% depending on the control measures against the vector used in every studied area. In areas with high control of the vector, the lower prevalence in cats was found. There are very few studies reporting the infection of cats with T. cruzi using PCR. In most cases, serological detection (ELISA and IFA in particular) and xenodiagnosis are used.
An isolated study in cats using PCR was performed by Cardinal et al. (2008) to determine the type of lineage of T. cruzi from the faeces of triatomines. The xenodiagnosis from four serologically positive cats showed an infection with T. cruzi II lineage, which is associated with the domestic cycle of the disease (Guhl and Lazdins-Helds, 2007; Kirchhoff, 2009).
In this survey, we found a low prevalence of specific antibodies than circulating genome of T. cruzi in the evaluated cats, probably due to the phase of the disease when samples were collected. Cats that were PCR positive and serologically negative could have been at an early stage of the disease, and therefore, no IgG antibodies were produced at the moment of the sampling.
Some animals may successfully control the parasite by mounting a more effective immune response to the numerous variable surface antigens, and this may be the mechanism for developing immune response. Domestic cats are apparently infected with T. cruzi, but no evidence of clinical cases are seen, or a higher proportion of positive serological cases is recorded as is observed in other mammal species from this region (dogs, opossums, rats and many others), and this may indicate a different immune response in this specie, especially when immunocompromised. Grant (1998) mentioned that cats with Felv infection have been regularly observed for Complement (C’) level fluctuations, but C’ levels and C’ consumption are factors in the pathogenesis of other viral infections such as feline infectious peritonitis (FIP), feline herpesvirus type-1 (FHV), and probably FIV and parasitic infections. Complement-dependent antibodies lyse viruses and other pathogenic organisms, but they are less effective if C’ levels are severely reduced or not available. This may explain our serological results. More immunological and immunopathological studies in cats are required to clarify the role of T. cruzi in cats.
Samples positive to both serological and PCR tests suggest that cats were probably at an indeterminate or chronic stage with a remission of the disease in which parasites can be found in peripheral blood, or probably cats experienced a new re-infection. As in humans, PCR-positive and serologically negative cats could be detected in an acute infection or a reactivation of parasitemia because of immunosuppression (caused by a viral disease or old age) (Ferreira and Borges, 2002; Braz, 2008). However, these results just reflect the stage of the disease when individuals were sampled. It is important to consider that the sensitivity of PCR may vary or fail, because of the extremely low parasite load in chronically infected individuals, meaning that a small sample of blood taken for DNA extraction may not contain an enough parasite genome to be detected (Kirchhoff, 2009).
As for the association of serological test with PCR, the results observed in this study were similar to those previously described in humans (Antas et al.,1999; Jiménez-Cardoso et al., 2000; Salomone et al., 2003); results obtained from these studies were from different serological tests like ELISA and IFA, where PCR did not show a good agreement in the indeterminate and chronic stages of disease. Serological tests recognize specific T. cruzi antibodies because of an immune response, whereas PCR recognized the presence of the parasite’s genome (Antas et al., 1999).
It is known that the PCR may have a sensibility between 50% and 90%, while its specificity is close to 100%. The heterogeneity of PCR has not been fully explained, and this may be because of the time elapsed between sample collection and processing, the extraction method used (if used guanidine or not, or if the samples were heated during the extraction) or the primers used (Brasil et al., 2010). For these reasons, it is not recommended to use molecular or serological testing alone. When using their combination, more information on the immunological and parasitological status of the patient may be obtained.
In this study, DNA samples were extracted using a commercial kit recommended by a group of experts in the diagnosis of T. cruzi (Schijman et al., 2011). From the DNA samples purified from whole blood, a pre-lysis was performed as recommended (Jalal et al., 2004), so the sample volume was 10 times higher than the traditional extraction method; this modification may have allowed to detect a greater number of positive cases of T. cruzi using the PCR technique.
Because PCR has a greater ability to detect the presence of the parasite, it should not be used as a diagnostic method for indeterminate or chronic cases. However, it can be an excellent tool for monitoring possible failures during treatment or determine a possible referral of the infection during a patient’s clinical immunosuppression.
Regular to bad body condition of cats was the only factor associated with the presence of T.cruzi genome. In a study in dogs, it is suggested that an animal in good body condition may control the parasitemia of T. cruzi more efficiently than one in poor body condition. Body condition can affect xenodiagnosis studies (on the absence of high parasitaemia), but not the serological diagnosis as moderate malnutrition does not affect the immune response (Petersen et al., 2001), and this may explain partially cats that were PCR positive but serologically negative. In other studies, it has been reported that individuals with poor physical condition may have further periods of reactivation of protozoas (i. e. T. cruzi or T. gondii), because of a deficient immune system response (Gomes et al., 1994; Petersen et al., 2001).
In eastern Mexico, the food preferences of the vectors of T. cruzi do not include cats (Brenière et al., 2004). However, in the southeast of Mexico, which is the region where this study was conducted, the cat was considered in thirteenth place of food preference of Triatoma dimidiata (Quintal and Polanco, 1977). Consequently, when increasing the density of cats, an increase in potential reservoirs and maintenance of the parasite is expected (Gürtler et al., 2009).
The domestic cat population studied here represents an important reservoir of the parasite, as cats are constantly interacting with humans and are a source of maintenance of the parasite in both populations (Gürtler et al., 1993, 1997, 1998, 2007). Cats have more hunting habits to survive (Robertson, 1998) and may be infected by hunting and ingesting the vector. The oral route of infection has been documented in other mammals (Yaeger, 1971; Yoshida, 2008). It has been described that an animal can become infected by eating meat infected with T. cruzi amastigote pseudocysts (Miles, 2009). Souza et al. (1997) mentioned that an animal with wild and periurban habits cannot be overlooked as an element favouring the possibility of oral transmission. It has also been reported the experimental infection in opossums by oral administration of T. cruzi in cell cultures containing a controlled amounts of amastigotes. Levels of parasitaemia observed in animals orally infected were low (mild) compared with controls intravenously infected with metacyclic trypomastigotes (animals developed parasitaemia levels classified as severe) (Roellig et al., 2009).
A high birth rate of cats allowed a steady flow of babies that may contract the disease as fast as in the dogs (Gürtler et al., 2007). Besides, the vertical transmission of the disease in rats and mice is well known (Andrade, 1982; Moreno et al., 2003; Alarcón et al., 2009) and may have a similar behaviour in dogs and cats. From these observations, it is clear that more research is needed on the epidemiology of the disease especially on the role of other important reservoir such as cats where the precise mechanism of infection and immunological response remain unclear.