Seroprevalence of Antibodies against Trypanosoma cruzi in Brown Rats (Rattus norvegicus) from Grenada, West Indies

Chagas disease is an arthropod borne parasitic disease of humans and animals caused by infection with Trypanosoma cruzi. Chagas disease is prevalent in Latin America and the Caribbean nations. Rats (Rattus species) are considered a reservoir host in transmission of the disease. The aim of this study was to estimate the prevalence of antibodies against T. cruzi in brown rats (Rattus norvegicus) from Grenada. A total of 145 rat sera were examined for T. cruzi antibodies using a qualitative immunochromatographic screening test: Chagas Stat PakTM (Chembio Diagnostic System, Inc. Medford NY, USA). A seroprevalence of 10.3% (15/145) for T. cruzi antibodies was found. Results from this study indicate a moderate exposure level of R. norvegicus to T. cruzi in Grenada. Further research to find out the presence of the insect vector near the rat colony and the relationship of reservoir host in disease transmission is indicated.

Chagas disease, a vector-borne disease, is an important public health problem in Central and South America and in nearly all the countries facing the Caribbean basin (Petana, 1978). Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi and is transmitted by blood sucking triatomine bugs. In the mammalian host, T. cruzi amastigotes multiply in muscle cells and other nucleated cells of the body. Amastigotes released by the rupture of the cells change into trypomastigotes which circulate in the blood. Trypomastogotes may invade other cells of the host or are ingested by triatomine bugs as they blood feed. Trypomastigotes multiply and undergo metamorphosis in the hind gut of the bug and are passed in the feces (Bowman, 1999). Infection to humans and other animal hosts through infected feces of triatomine bugs is by way of the oral, nasal and conjuctival mucosa or abrasion of the skin. Other routes of transmission to humans are by transfusion of infected blood, transplant of infected organs, or transmission from an infected mother to her child at birth (Alejandro et al., 2013). T. cruzi infects many mammalian species as reservoir hosts. Dogs, opossums and rats (Rattus species) are considered the most important reservoir hosts (Alejandro et al., 2013). Chagas disease in humans in South America and Caribbean nations is correlated with infection in dogs (Rosypal et al., 2007, Crisante et al., 2006Pineda et al., 2011). Chickweto et al. (2014 reported 10.5% sero-prevalence of T. cruzi in stray and pet dogs in Grenada. An earlier study found 4.3% of pet and stray dogs from Grenada had antibodies to T. cruzi (Rosypal et al., 2010).
There is paucity of information on infection in rats from Caribbean nations. Rats were found infected with T. cruzi in Mexico (Gurmersindo et al., 2018), Medagascar (Rahelirina et al., 2010), and Venezuela (Herrera et al., 1997). The presence of T. cruzi in a wild caught rat was reported from Trinidad (Downs, 1963).
As far as authors are aware, there is no published literature on T. cruzi infection in rats from Grenada. The aim of this research was to determine the seroprevalence of T. cruzi in brown rats (R. norvegicus) in Grenada.

Ethical approval
The project (Detection of zoonotic pathogens in brown rats (Rattus norvegicus) in Grenada) was approved by the Institutional Animal Care and Use Committee (IACUC # 16009-R) of the St. George's University, Grenada.

Study area
Grenada is the southernmost country in the Caribbean Sea with an area of 348.5 Km 2 . The country with low hills, small trees, shrubs and tropical climate is most suitable for rat population.

Collection of rats
One hundred forty five rats were collected live from 1 st May to 14 th July 2017, using traps (45cm l × 15cm w × 15 cm h) with cheese and various local fruits as bait. Attempts were made to trap the rats from and near the residential buildings. Traps were placed two days per week in the evening and visited in the morning the next day. Traps with rats were covered with black cloth, transported to the necropsy laboratory of the School of Veterinary Medicine and were anesthetized using 1-2% isoflurane in oxygen via (portable vet anesthesia machine isoflurane vaporizer VET CE) manufacturer DRE (Avante Health Solution Company, USA).

Collection of samples
The anesthetized rats were examined for their physical health and weighed. Gender was also recorded. Rats below 100g were grouped as young and over 100g as adult following the methodology used by Panti-May et al. (2012). Blood was collected from the heart through the thoracic wall and rats were exsanguinated this way. Sera were separated from the blood by centrifugation at 1500g for 15 minutes at room temperature and stored at -80 °C till tested.

Test method
A rapid immune-chromatographic screening test (Chagas Stat Pak™, Chembio Diagnostic System, Inc. Medford NY, USA)was used for antibody detection in sera of rats. This assay has been used previously for detection of anti-T. cruzi antibodies in wildlife, including several rodent species (Charles et al., 2013, Yabsley et al., 2009. Screening was performed on sera of 145 rats according to the manufacturer's directions. Briefly, 5 µl of rat serum was aliquoted onto the sample well of the test device and 6 drops (~240µl) of provided sample diluent was slowly pipetted into the sample well. Results were read after 15 minutes. According to the test procedure, within 15 minutes, a purple control line and a second purple line in the test area indicated that result was positive. The presence of only a single purple line in the control region signified a negative result.

RESULTS AND DISCUSSION
Antibodies to T. cruzi were found in 15 rats (15/145) 10.3% (CI 95% from 5.91 to 16.49). The Chagas Stat Pak™ is a qualitative assay, so antibody titers could not be determined. A higher prevalence of antibodies to T. cruzi was found in St. David (13.3%) than in St. Georges (7.1%). Male and female rats demonstrated equal prevalence of antibodies (10.0% male and 10.8% female). Among the rats tested, 12.0% of adult rats had antibodies to T. cruzi, but none of the young rats tested positive by the Chagas Stat Pak™. The serological results according to parish, sex and age are presented in table 1.
In the present study we found 10.30% seropositive R. norvegicus in 2 parishes of Grenada. There was no significant difference in the positive rats between St. George and St. David parish. The seroprevalence of antibodies to T. cruzi in rats in Grenada found in the present study was similar to pet and stray dogs (Chikweto et al., 2014), however, in a previous study of dogs Rosypal et al. (2010) found 4.3% seroprevalence of T. cruzi antibodies in Grenada.  Carolyn et al. (2017) quoted an infection rate ranging from 5% to 57% in R. rattus in Latin America. Fifty seven percent of R. rattus were demonstrated positive for T. cruzi infection in the Republic of Panama (John and Johnson, 1970). We are not comparing our seroprevalence of antibodies to T. cruzi in rats with results of previous researchers in different countries. The variation in prevalence in different countries could be due to the various diagnostic tests used. Different techniques were used for the diagnosis of T. cruzi by previous researchers, including: direct blood smear microscopy, culture, IHA analysis, complement fixation test, ELISA, and molecular techniques. Although good correlation between the infected vector triatomines and infected rats was shown previously in Mexico (Gurmercindi et al., 2018), it was not possible to determine this correlation in the present study as vector triatomines were not collected and examined for T. cruzi infection.
During the present study, we did not find significant differences between young and adults, and between male and female rats positive for T. cruzi antibodies. No mention in the literature was found regarding differences in sex among T. cruzi infected rats. Previous researchers (Elizabeth et al., 2000;Pascutti et al., 2003) reported young rats (approximately 30 days old) more sensitive than adults. Pascutti et al. (2003) showed that increased resistance in adult rats seems to be the result of a more appropriate antibody production.

CONCLUSION AND RECOMMENDATION
Although, no human cases of Chagas disease have been reported in Grenada, the presence of antibodies in rats and dogs found in separate studies in Grenada may suggest a risk factor for humans. The prevention and control of T. cruzi in endemic countries is partially through the control of vector triatomines and reservoir hosts. Further research in Grenada is suggested to collect and study the vector triatomines near the population of rats to find out the relationship of infection in rats with vector. The results of this study will help in formulation of prevention and control of Chagas disease policy in Grenada and the region.