Epidemiological Survey of Trypanosoma cruzi Infection in Domestic Owned Cats from the Tropical Southeast of Mexico

Authors

  • M. Jiménez-Coello,

    1.  Laboratorio de Biologia Celular, CA Biomedicina de Enfermedades Infecciosas y Parasitarias. C.I.R. ‘Dr. Hideyo Noguchi’, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
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  • K.Y. Acosta-Viana,

    1.  Laboratorio de Biologia Celular, CA Biomedicina de Enfermedades Infecciosas y Parasitarias. C.I.R. ‘Dr. Hideyo Noguchi’, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
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  • E. Guzman-Marin,

    1.  Laboratorio de Biologia Celular, CA Biomedicina de Enfermedades Infecciosas y Parasitarias. C.I.R. ‘Dr. Hideyo Noguchi’, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
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  • A. Gomez-Rios,

    1.  Laboratorio de Biologia Celular, CA Biomedicina de Enfermedades Infecciosas y Parasitarias. C.I.R. ‘Dr. Hideyo Noguchi’, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
    2.  Departamento de Salud Animal y Medicina Preventiva, CA Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
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  • A. Ortega-Pacheco

    1.  Departamento de Salud Animal y Medicina Preventiva, CA Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico
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A. Ortega-Pacheco. Departamento de Salud Animal y Medicina Preventiva, CA Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autonoma de Yucatan, Km. 15.5 Carr. Merida-Xmatkuil AP. 4-116 Merida, Yucatan, Mexico. Tel.: +52 999 942 32 00; Fax: +52 999 942 32 05; E-mail: opacheco@uady.mx

Summary

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.

Impacts

  •  Cats are important reservoirs of Trypanosoma cruzi in endemic regions and may play an important role in the peridomestic cycle of the disease.
  •  Regular–bad body condition score in cats is a risk factor associated with PCR-positive cases.
  •  Vector control should be implemented in peridomestic households to prevent infection of cats

Introduction

American trypanosomiasis or Chagas disease is an infectious disease considered an important public health problem in Latin American countries where the disease is endemic. The protozoan parasite Trypanosoma cruzi is the aetiological agent transmitted to humans and other animals by triatomine bugs (bloodsucking bugs) (Salazar-Schettino et al., 2005) where a large number of reservoirs are involved. The seroprevalence in humans, other reservoir mammals and vectors varies depending on the geographical area and laboratory tests used. In Mexico by 1997, a total of 18 reservoir mammals were reported (Salazar-Schettino et al., 2005). Very few reports about trypanosomiasis in domestic animals are reported in Mexico, particularly in domestic cats. In Argentina, domestic cats and dogs have been used of sentinels in urban and rural areas to assess the risk of human trypanosomiasis. In those preliminary studies, cats have been shown to be important reservoirs with high probability of infecting peridomiciliary vectors (Cardinal et al., 2007, 2008; Gürtler et al., 2007). However, the precise role of cats in the domestic transmission of T. cruzi and mechanism remain uncertain. The objective of this study was to determine the prevalence of American trypanosomiasis in domestic cats in an urban area of Mexico by serological (ELISA IgG and WB) and molecular (PCR) methods and to evaluate associated risk factors with the presence of disease.

Materials and Methods

Study population

A total of 220 domestic owned cats from the city of Merida Yucatan, Mexico (19°30′ and 21°35′N latitude, and 87°30′ and 90°24′W longitude), were studied. The climate is characterized as tropical subhumid with a mean annual temperature of 25–28°C (range of 15–40°C during the winter and summer, respectively) and relative humidity of approximately 80%. Annual rainfall is 400–1500 mm (INEGI, 2005). Selected cats included in the study were programmed for elective sterilization performed at the Veterinary Medicine Faculty from the Autonomous University of Yucatan. After a written consent of the owner, cats were randomly selected providing they were evenly distributed from the four quadrants of the city of Merida Yucatan, Mexico.

Sample collection

Blood samples (0.5–2 ml) were taken by puncturing the jugular vein; immediately, they were deposited in two vacutainer® (Becton, Dickinson and Company, NJ, USA) tubes, with and without anticoagulant, the first for the DNA extraction and the second for serological evaluation. DNA extraction was performed with the commercial kit DNeasy Blood and Tissue (QIAGEN, Cat no. 69506, Qiagen, MD, USA). Before extraction, a pre-lysis of the blood was conducted as suggested by Jalal et al. (2004). Samples were stored at −20°C until further evaluation. Serum samples were obtained by centrifugation at 400 g for 15 min. Through a questionnaire survey applied to owners and by clinical evaluation, there were obtained data from each cat including age, sex, body condition score (BCS) and whether or not had access to the streets (indoor cat or indoor & outdoor cat). For determination of BCS, a modified method from Bjornvad et al. (2011) was employed considering fat covering and evidence at palpation of ribs, lumbar vertebrae, pelvic bones and all body prominences. Cats were considered with bad, regular or good BCS.

Indirect enzyme-linked immunosorbent assay and western blot

For the specific detection of IgG antibodies against T. cruzi, a commercial indirect enzyme-linked immunosorbent assay (ELISA) was used (WienerLab Chagatest). The technique used was adapted to that described by Jiménez-Coello et al. (2010), using anti-IgG cat antibody labelled with horseradish peroxidase (HRP) (sc-2423; Santa Cruz Inc., Santa Cruz, CA, USA) on 96-well plate coated with recombinant proteins of T. cruzi. Serum samples were diluted to a ratio of 1 : 100 in phosphate-buffered saline (PBS; pH 7.2), and the secondary goat anti-cat IgG-HRP labelled was used at a dilution of 1 : 5000.

Sera from previously evaluated cats and BALB/c artificially infected mice with high anti-IgG antibody titres by ELISA (1 : 1024) and positive results to PCR against T. cruzi were used as positive controls. A sera pool from 10 healthy cats previously tested by triplicate with ELISA IgM, IgG and PCR were used as negative controls. On the basis of the ELISA, subjects were diagnosed as either positive/negative for specific IgG antibodies to T. cruzi. The optical density (OD) was measured in a spectrophotometer at 450 nm (Multiskan Multisoft Primary EIA®) and was used to compute the per cent positivity (PP) using the formula mean OD (sample or negative control) divided by the mean OD value positive control multiplied by 100. Per cent positivity of 15% or above was considered as positive.

For confirmation of the diagnosis only in seropositive cases previously detected by the indirect ELISA, the Western blot (WB) was performed (Jiménez-Coello et al., 2008), in which Tulahuen T. cruzi strain epimastigotes were used as antigen. Samples were considered positives to WB based on an established criterion. A serum sample was considered positive when it recognized at least five antigenic bands from a group of 10 with the highest frequency of recognition; the result was considered indeterminate when the sample recognized of 1–4 antigenic bands and was negative when the serum sample showed no reactivity (Teixeira et al., 1994, Jiménez-Coello et al., 2008).

Molecular detection of Trypanosoma cruzi

The PCR was performed as described (Dorn et al., 1997) using primers for the conserved region of the kinetoplast minicircles of T. cruzi (TC1 and TC2). Assays were performed on purified DNA using the primers TC1 (5′-TTGAACGGCCCTCCCAAAAC-3′) and TC2 (5′-GATTGGGGTTGGTGAAATATA-3′). Amplification conditions for PCR assay were as follows: 3 μl of DNA template, 20 pm of each primer, 0.1 mm of each dNTP, 1 U of Go-Taq enzyme ® Hot Start Polymerase (Promega, WI, USA) and contain 1× PCR Buffer Colourless Go-taq ® Flexi Buffer and MgCl2 at a concentration of 0.2 mm, in a total volume of 25 μl per reaction. An initial denaturation step of 94°C for 3 min was followed by 35 cycles of 94, 55 and 72°C, each for 1 min, and a final 10-min extension at 72°C. The PCR products were electrophoresed on a 1.8% agarose gel, stained with ethidium bromide and visualized by UV transilluminator. As a positive control, genomic DNA was obtained from epimastigotes maintained in an axenic culture as well as genomic DNA purified of whole blood from experimental infected mice with T. cruzi, strain H4, which showed high parasitaemia levels (24 × 106/ml). Both positive controls were used in every PCR set. As a negative control, a heterologous genomic DNA obtained from Salvia hispanica was used.

Statistical analysis

A case was considered positive when two serological tests were positive in owned cats. Also it was considered as a positive PCR case, when the identification of the target of DNA fragment from the parasite was obtained. Prevalence of positive cases to T. cruzi was calculated by ELISA + WB, PCR and both conditions. An univariate analysis (x2) to determine the association between risk factors and PCR positives was used in chi-squared tests. Odds ratio (OR) and 95% confidence intervals were also calculated using the EpiInfo program (Dean et al., 1994). The level of significance was set at < 0.05. To show the dispersion plot points graph of the ELISA absorbance values, the MedCalc software was used (Schoojans et al., 1995).

Results

From the 220 cats, 19 (8.6%) had IgG antibodies against T. cruzi, using indirect ELISA (Fig. 1); when tested by WB, all 19 ELISA-positive samples were confirmed. An example of WB is shown in Fig. 2. Ten cats were positive only to ELISA + WB and nine to both serological tests and PCR (Table 1). Samples were considered positive by the criteria established by Teixeira et al. (1994) and Ramos-Ligonio et al. (2006).

Figure 1.

 Serum anti-IgG absorbance estimated by Indirect ELISA assay assay (Chagatest, Wiener Lab) in owned cats from Merida Yucatan, Mexico.

Figure 2.

 Western Blot results from seropositive cats by Indirect-ELISA. Lanes: (1) control (−) serum from rabbits not infected with Trypanosoma cruzi, (2) control (+) serum from rabbit infected with T. cruzi, (3) strip without primary antibody, (4–11) Positive cat samples. Arrows show five different proteins recognized which are not present in negative control.

Table 1.   Serological and Molecular diagnostic results of Trypanosoma cruzi infection in owned cats from Merida Yucatan, Mexico which were included in this survey
 PCR (+) (%)PCR (−) (%)Total of positives by ELISA & WBTotal of positives by PCRTotal of positives ELISA & WB + PCRTotal
Indirect IgG ELISA (−)66 (30%)135 (61.3%)    
Indirect IgG ELISA (+) and Western Blot (+) 9 (4.09%) 10 (4.54%)    
   19 (8.6%)75 (34.09%)(66 + 9 + 10) = 85
38.6%
100% (220)

From the 220 samples, 75 (34.1%) amplified a 235-bp fragment corresponding to the expected size sequence of T. cruzi ADNk. From these 75 positive PRC samples, 66 were only PCR positive and nine samples were PCR + ELISA positives (Table 1). Figure 3 shows an agarose gel stained with ethidium bromide in which there is an example of PCR results; in lanes 7–10, two cases of DNA amplification are observed from positive cats.

Figure 3.

 Gel electrophoresis in 1.8% agarose stained with ethidium bromide, the DNA amplification products of cats with primers specific for TC1 and TC2 Trypanosoma cruzi. Lanes: (1) control (−) no DNA, (2) control (+) DNA from epimastigotes of T. cruzi cultivated in LIT medium, (3–4) Sero negative cats (5) Seropositive and PCR positive case. (6–8) Seronegative and PCR positive cases.

From the risk factors evaluated, a higher frequency of infection in males (OR 1.43) and cats that had free roaming (OR 1.7) was observed. However, only the regular–bad body condition was associated with the presence of infection (< 0.0001, OR 4.2, CI 1.4–8.8) (Table 2).

Table 2.   Risk factor associated with PCR-positivity for Trypanosoma cruzi infection in owned cats from Merida Yucatan, Mexico
Risk factor n PositivePrevalence (%)ORIC
(95%)
P-value
Sex
 Male652538.41.370.7–2.40.37
 Female1555032.31
Age (years)
 <1862832.51
 1–31013736.61.1
 >333824.20.80.41
Body Condition Store (BCS)
 Good671014.91
 Regular-Bad1536542.54.21.4–8.80.0001
Roaming
 In door & out  door1585937.31.70.8–3.20.10
 In door only621625.01

Discussion

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.

Conclusion

Trypanosoma cruzi is present in the studied domestic cat population. PCR is a useful tool for early and specific diagnosis in cats, and it may also be recommended together with serological tests for the parasite detection, but PCR should not be used in clinical practice for chronic Chagas disease diagnosis. Further studies are needed to better understand the role that cats play in the maintenance of T. cruzi and the pathogenesis and immune response in cats, especially those with poor body condition.

Acknowledgments

We gratefully acknowledge to the Consejo Nacional de Ciencia y Tecnología (CONACYT-Licenciatura 102204-2008), SISTPROY FMVZ-09-001 for the financial support. We also acknowledge to PROGRAMA DE IMPULSO Y ORIENTACIÓN A LA INVESTIGACIÓN (PRIORI) 2009-2010 for the financial support to the participant student.

Conflicts of interest

The authors have declared no potential conflicts.

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