Use of the Indirect Immunofluorescent Antibody Test (IFAT) and Blood Culture to Detect Trypanosoma cruzi Infections in Raccoons from Georgia

Michael J. Yabsley, Gayle Pittman Noblet, and Oscar J. Pung

Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634-0326 USA (Yabsley and Noblet) and Department of Biology and Institute for Arthropodology and Parasitology, Georgia Southern University, Statesboro, Georgia 30460-8042 USA (Pung)

Abstract: Eighty-six raccoons (Procyon lotor) trapped in four southeastern counties in Georgia were tested for Trypanosoma cruzi by blood culture and by the indirect immunofluorescent antibody test (IFAT). Cultures from 25 raccoons (30.1%) were positive for epimastigotes, whereas 49 of the serum samples (56.9%) were positive for anti-T. cruzi antibodies. Of the 25 culture-positive raccoons, 24 tested positive with the IFAT (96% sensitivity), while an additional 23 culture-negative raccoons were seropositive.

Key words: Trypanosoma cruzi, Raccoons, Wildlife, Serological test, IFAT, Survey, Culture

Introduction

Trypanosoma cruzi, the etiological agent of American trypanosomiasis or Chagas' disease, is important from both a medical and veterinary perspective. In both dogs and humans, T. cruzi is a known cause of fatal myocarditis during the chronic phase of disease. Although apparently underdiagnosed, T. cruzi infections in dogs have been reported from the southern states of Texas, Louisiana, Oklahoma, Georgia, South Carolina and Virginia. ¹ For humans in the United States, autochthonous vector-transmitted Chagas' disease has been reported only from California and Texas. However, a recent acute infection was diagnosed in an 18-month-old infant from rural Tennessee, using a polymerase chain reaction assay. Interestingly, the family dog had an anti-T. cruzi IFAT titer of 1:1024. ² Consequently, knowledge of T. cruzi occurrence in wildlife as reservoir hosts in the United States is important.

Since the first isolation of T. cruzi from wild animals in Georgia³ in 1958, additional culture surveys have reported prevalence rates for raccoons of 22-43%. ^4,5 In the Southeast and adjacent states, reports of prevalences range from as low as 2% in northern Florida and southern Georgia to as high as 62% in Oklahoma. ^3,6

Previous prevalence studies for T. cruzi in raccoons have included only the culture of whole blood samples and direct parasitological examination of blood smears. But direct isolation of T. cruzi from animals, either by xenodiagnosis or blood culture, has limitations due to low sensitivity and unsuitability for testing of large numbers of animals in the field. For humans, and to a lesser extent mammalian reservoir hosts, serological tests have been used widely in South America to study the epidemiology of T. cruzi. In North America, however, studies with various wildlife (including raccoons, opossums, woodrats, armadillos, and small rodents) have involved primarily the culture of blood. ⁶

The most common serological assays used for human infections with T. cruzi are the indirect hemagglutination (IHA), complement-fixation (CF), direct agglutination (DA), indirect immunofluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA). Common problems associated with the first three tests include cross-reactivity with other pathogens and low sensitivity. Since both the IFAT and ELISA are reported to be very sensitive and specific for detecting infections with T. cruzi,^7,8 the IFAT was chosen as the serological test of choice for the present study of raccoons as wildlife reservoir hosts in southeast Georgia.

Materials and Methods

From September 1992 through September 1994, 86 raccoons were trapped at the following locations in southeast Georgia: Fort Stewart Military Base in Bryan County (n = 17), the city of Statesboro and surrounding areas in Bulloch County (n = 11), the Harrold Nature Preserve in Candler County (n = 5) and St. Catherine's Island in Liberty County (n = 53; St. Catherine's Island is a 5,600 ha barrier island accessible only by boat). Live traps^a baited with canned cat food in the afternoon were checked the next morning. Captured animals were anesthetized by intramuscular injection of xylazine^b (0.25 mg/kg body weight) mixed with ketamine hydrochloride^c (25 mg/kg). Blood obtained by cardiac puncture or from the femoral vein was placed in heparinized Vacutainer^® tubes. ^d For purposes of culture, 1 ml of blood was added to 9 ml of Liver Infusion Tryptose (LIT) medium⁹ to allow for growth of any trypanosomes present. A thin blood smear prepared from each animal was stained with Giemsa and examined by light microscopy. Plasma separated from blood was stored at -80° C for serological testing.

The IFAT was used to detect anti-T. cruzi antibodies by the method of Camargo. ¹⁰ Antigen was prepared from 11-day old Brazil strain epimastigotes cultured in LIT medium. Epimastigotes were washed three times with phosphate buffered saline (PBS) and then resuspended in 1% formalin in PBS. Antigen was placed in both circles on commercial serological test slides^e and dried in an oven at 50° C for 30 minutes. Slides were stored in a glass jar with CaCl₂ at -4° C for no longer than 1 month prior to serological testing.

A commercial fluorescein conjugated goat anti-raccoon IgG^f was used for the IFAT. Aliquots of 150 m l were kept frozen at -80° C and diluted in 0.1% solution of Evans blue^g before use. Evans blue counterstains the epimastigotes red which facilitates visualization by fluorescent microscopy (Figure 1). Slides were examined under a Ziess microscope equipped with a 50W Hg illuminator (488 nm excitation and a 520 nm band-pass filter).

Optimal serum (1:40) and conjugate (1:10) concentrations were determined by checkerboard titration, using known positive- and negative-control raccoon serum. Positive controls consisted of pooled serum samples collected from seven culture-positive raccoons, and negative controls were pooled sera of 39 raccoons trapped near the Toronto Zoo in Ontario, Canada, which is outside the range of any known vector for T. cruzi. Endpoint titers of IFAT positive samples were determined by testing doubling dilutions from 1:40 to 1:1280. Amount of fluorescence was classified on a scale of 1-4, with 3 and 4 being counted as positive (Figure 2).

Figure 1. A negative control reaction. Note the lack of fluorescence and the red hue due to counterstaining with 0.1% Evans blue (40X).	Figure 2. Positive control sample. Note the green fluorescence due to fluorescein conjugated anti-raccoon antibodies binding to anti-T. cruzi antibodies present in the serum (40X).

Results

Forty-nine of 86 raccoons (56.9%) tested by IFAT were positive for antibodies to T. cruzi. Raccoons trapped on St. Catherine's Island (Liberty County) had an infection rate of 66%, the highest for all counties examined. One animal on the island had a titer of 1: 640, the highest observed in this study. In contrast, 42.4% of raccoons from the mainland counties (Bryan, Bulloch, and Candler) were seropositive for T. cruzi, with a range in prevalence of 27.3-52.9% (Table 1). Infected animals were found in each locale surveyed.

When serological data were compared with previously reported culture data for these same animals,^4,5 results for 59 of 86 samples (68.6%) tested with IFAT were consistent with those obtained from culture of blood, with 24 samples being positive and 35 being negative by both methods (Table 2). For three cultures contaminated with fungi, serum from one raccoon was IFAT negative, while serum from the two other raccoons was IFAT positive. Differences between IFAT and culture results occurred for 24 samples, with 23 raccoons testing positive with the IFAT while being negative by culture. Only one of 37 raccoons (2.7%) that tested negative with the IFAT was confirmed positive for T. cruzi by culture.

Discussion

Limited serological testing of wildlife in North America has been conducted with varying success. The direct agglutination test (DAT) was used to survey a group of armadillos for antibodies to T. cruzi. ¹¹ Of 80 armadillos tested, 23 were positive both by culture and DAT; an additional seven culture-negative animals tested positive with DAT. In contrast, DAT was reported as unsatisfactory for detecting infections in experimentally infected opossums. ¹² Serological testing of opossums in 1966 with seven different techniques, including the complement fixation test which is used widely with humans, also was reported as unsatisfactory. ¹³ Possible explanations for limited testing of wildlife populations could be the need for host species-specific conjugates and time involved in trapping hosts.

No apparent serological testing for anti-T. cruzi antibodies in raccoons has been conducted except for one ongoing study in South Carolina where 50% of trapped animals tested positive by IFAT (Yabsley and Noblet, unpublished data); prevalences in that study ranged from 42% in the foothills/mountains to 56% in coastal regions. In the present study, 49 of the 86 Georgia raccoons (56.9%) were positive for anti-T. cruzi antibodies when tested by IFAT, indicating either a current or past infection with the parasite. In contrast, only 30.1% of these animals were culture-positive. Since infection with T. cruzi is life long, with parasites eventually entering a chronic stage characterized by low parasitemia, such results are not surprising, as isolation of the parasite by culture becomes very difficult under these circumstances.

Aberrant results obtained from one raccoon that tested negative with the IFAT, but was culture positive, included an estimate of 6,000 trypomastigotes/ml of blood based on examination of stained smears. This animal possibly was recently infected with T. cruzi, and insufficient time had elapsed for development of a detectable antibody response to the parasite by IFAT. Similarly, dogs experimentally infected with T. cruzi did not exhibit a detectable antibody response during the first 26 days of infection. ⁷ For experimental T. cruzi infection of opossums, antibodies were not detected until 4-5 weeks post infection (PI), while peak parasitemia was observed 2-3 weeks PI. ¹⁴

Results of the present study demonstrate that the IFAT serological test is very sensitive and can be used to detect T. cruzi infections in raccoon reservoir hosts. Ongoing studies will include testing raccoon serum samples with the ELISA and examination of results for sensitivity and specificity relative to IFAT and blood culture techniques.

Sources and Manufacturers

a. Tomahawk Live Trap Co. , Tomahawk, Wisconsin (USA)
b. Mobay Corp. , Animal Health Division, Shawnee, Kansas (USA)
c. Aveco Co. Inc. , Fort Dodge, Iowa (USA)
d. Beckton Dickinson, Rutherford, New Jersey (USA)
e. Fisher Scientific, Rome, Georgia (USA)
f. Kirkegaard & Perry Laboratories, Gaithersburg, Maryland (USA)
g. Sigma Chemical Co. , St. Louis, Missouri (USA)

Acknowledgments

The authors would like to acknowledge Dr. Ian Barker at the University of Guelph, Ontario (Canada) for the raccoon sera samples used as negative controls and Dr. Rick Tarleton at the University of Georgia, Athens, Georgia (USA) for the Brazil strain of T. cruzi used as antigen. This research was funded in part by a Clemson University Research Grant.

References

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