Detection of anti-Trypanosoma spp. antibodies in cattle from southern Brazil

Abstract Bovine trypanosomosis, caused by Trypanosoma vivax, is a disease that originated in Africa and currently affects cattle in several South American countries, including almost all Brazilian states. Despite the reports on T. vivax infection in southern Brazil, data on its circulation status is currently unavailable. In this study, we aimed to detect anti-Trypanosoma spp. IgG antibodies in cattle from Rio Grande do Sul and suggest areas with T. vivax transmission risk. A total of 691 serum samples from cattle in the intermediate regions of Rio Grande do Sul were analyzed using indirect immunofluorescence assay (IFA). The overall seroprevalence of anti-Trypanosoma antibodies in cattle was 24.6% (170/691). The detection rate ranged from 0-37.3%, with a high prevalence in the intermediate regions of Ijuí (37.3%), Uruguaiana (30.7%), and Passo Fundo (28.9%). Thus, these regions were suggested as possible bovine trypanosomosis risk areas due to the high seroprevalence. This is the first serological study to determine Trypanosoma spp. infection status in cattle from Rio Grande do Sul, providing data on the epidemiology of trypanosomosis in the state.


Introduction
Trypanosomes are flagellate protozoans belonging to the family Trypanosomatidae and genus Trypanosoma.Various parasitical species belonging to Trypanosoma, such as Trypanosoma brucei, Trypanosoma congolense, Trypanosoma evansi, and Trypanosoma vivax, cause trypanosomosis, a livestock disease prevalent in Africa, Latin America, and Asia (Stevens & Brisse, 2004).In South America, T. vivax is the most important causative agent of trypanosomosis in cattle, causing losses in the cattle industry.Other species, such as non-pathogenic T. theileri and T. evansi, also infect cattle but rarely cause diseases (Radostitis et al., 2007).Economic losses in Brazilian cattle-rearing are associated with animal mortality following outbreaks, and indirect interferences of subclinical and undiagnosed infections that reduce weight gain and milk production as well as cause abortion, infertility, and other reproductive disorders, thus implicating livestock productivity (Otte et al., 1994;Seidl et al., 1999;Jones & Dávila, 2001).
African dissemination of T. vivax includes cyclical transmission by Glossina spp., with T. vivax developing in their digestive tract.Since Glossina spp. is absent in South America, the major transmission methods include non-cyclical or mechanical transmission by blood-sucking flies (Otte & Abuabara, 1991), such as tabanids (horseflies) and Stomoxys calcitrans (stable flies).Although not confirmed, cyclical transmission may involve one or more vector species (Otte & Abuabara, 1991).Furthermore, iatrogenic transmission through fomites and transplacental infections have also been described (Cadioli et al., 2012).T. vivax infects various domestic and wild species, including sheep, goats, horses, and cervids, which can act as important reservoirs (Dávila et al., 2003).
In South America, T. vivax infection manifests variable virulence and pathogenicity levels (Gardiner & Mahmoud, 1992), causing non-specific clinical signs, such as severe anemia, weight loss, edema, immunosuppression, and reproductive failure.Some acute cases can develop various neurological disorders that eventually cause the death of the affected animals (Gonzatti et al., 2014).Asymptomatic and chronic infections are common in cattle from endemic regions and can be reactivated by nutritional and physical stress, concomitant disease, pregnancy, and lactation (Garcia et al., 2016).No single clinical sign is pathognomonic and the disease may simulate many other infections (Büscher, 2014); therefore, it is easily overlooked or confused with other diseases.
In southern Brazil, despite reports of T. vivax infection in cattle (Silva et al., 2009) and naturally infected horses (Silva et al., 2011), no data on pathogen circulation status in Rio Grande do Sul (RS) have been recorded.RS is geographically located between territories that have characteristics that may favor the spread of the disease.The region of Argentina, which borders Brazil, has numerous cattle herds and the culture of practicing rodeos, in addition, there are several reports of animal trafficking to Brazil.This can provide a risk factor for the introduction of diseases to brazilian heards.Thus, understanding the actual T. vivax circulation in RS is important.This study aimed to detect anti-Trypanosoma spp.antibodies in cattle in the regions of RS and elucidate the trypanosomosis transmission risk areas in the region under study.

Materials and Methods
A total of 691 serum samples were obtained from the sera bank of Laboratório de Doenças Parasitárias of Universidade Federal de Santa Maria (LADOPAR/UFSM) from 2020-2022.Samples were from taurine (Bos taurus), zebu (Bos indicus), and crossbreed cattle in the intermediate regions of RS: Uruguaiana, Santa Maria, Porto Alegre, Passo Fundo, Ijuí, Caxias do Sul, Santa Cruz do Sul, and Pelotas (IBGE, 2017).The total number of samples was determined based on statistical analysis using the Epi Info (v7.2.5) system according to the cattle population of RS (IBGE, 2022).The confidence interval was 95%, with 3% standard error.
Indirect immunofluorescence assay (IFA) described by Camargo (1966) was used to detect IgG antibodies against Trypanosoma spp. with some modifications.Briefly, IFA was performed using a microscopic strain infected with T. vivax trypomastigotes.The serum samples were diluted in phosphate buffered saline (pH 7.2) to 1:80 (Cuglovici et al., 2010).Parasitol 2024; 33(1): e013723   3/8 Detection of anti-Trypanosoma spp. in cattle Commercial fluorescein-labeled anti-bovine IgG © (Rabbit Anti-Bovine IgG FITC®, F7884, Sigma Bio Sciences, St. Louis, Missouri, USA) secondary antibody was diluted to 1:400.The samples were incubated with both the primary and secondary antibodies for 50 min at 37 °C in a dark and humid chamber.Confirmed positive and negative serum samples diluted to 1:80 were used as controls.The slides were observed at 400× magnification under a fluorescence microscope (Optiphase INV403F).The samples were considered reactive when the trypomastigotes revealed total fluorescence, showing binding between the FITC-labeled secondary antibody and the antigen-antibody complex.Serum that did not show fluorescence was considered non-reactive.
We conducted an exploratory data analysis of the samples.The investigation involved calculating the absolute frequencies and prevalence of cattle trypanosomosis.Additionally, contingency tables were constructed, considering each herd's intermediate region and the cattle breeding.To assess the presence of significant differences in the prevalence of these variables, we performed the Fisher's exact test, considering 5% as the significance level.

Results
Anti-Trypanosoma spp.IgG was detected in 24.6% (170/691) cattle serum samples.The detection rate in the study region was 0-63.6% (Table 1).A total of 24 herds were sampled, of which 9 and 5 were dairy cattle and beef cattle, respectively, and 10 herds did not report this information.At least one positive sample was detected in 21 herds (89.6%).The detection frequency in the positive herds ranged from 4.2-63.6%.The highest detection frequencies were observed in three dairy herds from Passo Fundo (63.6%, 7/11; 46.1%, 6/13; and 45.4%, 10/22), one herd from Porto Alegre (39.1%, 9/23), and one herd from Pelotas (39.3%, 22/56) (Table 1).Regarding the intermediate regions, the higher prevalences were found in Ijuí (37.3%),Uruguaiana (30.7%), and Passo Fundo (28.9%), respectively (Table 2 and Figure 1).The lowest prevalence was observed in Pelotas (24.3%),Santa Cruz do Sul (23.0%),Porto Alegre (17.5%),Santa Maria (5.7%), and Caxias do Sul (0%) (Table 2 and Figure 1).When calculating prevalence ratio using Santa Maria as reference, we verify that the prevalence is 6.4 times higher in Ijuí than in herds from this region.In addition, besides Caxias do Sul, which did not present any prevalent case, all the regions showed a prevalence more than three times higher than Santa Maria.The Fisher's exact test corroborates this result since it returns a p-value smaller than 0.001, leading to the rejection of the hypothesis that the prevalences are equal in all intermediate regions at the significance level of 5%.The Cattle breeding comparison also leads to significant differences (p-value=0.006 in the Fisher's exact test).It means that bovine trypanosomiasis differs within the categories of cattle breeding.The prevalence ratio shows that bovine trypanosomiasis in beef cattle is 1.7 times higher than in cattle with unspecified breeding.For dairy cattle, the prevalence ratio indicates that its prevalence is 1.5 times higher than in cattle with unspecified breeding.

Discussion
Trypanosomosis, caused by T. vivax, is considered a non-endemic disease in cattle in southern Brazil.Currently, this species is only considered endemic in some Pantanal and Amazon rainforest regions.However, it sporadically infects various species throughout southern Brazil regions.T. vivax infects cattle and horses with clinical signs in the central cities of RS (Silva et al., 2009(Silva et al., , 2011)).Nevertheless, since serological studies have not been performed using serum samples collected in RS, no data on cattle trypanosomosis in RS is available.This is the first bovine serological study on anti-Trypanosoma spp.antibody detection and frequency among all regions of RS, highlighting the importance of widespread disease-causing pathogen analysis for the differential diagnosis of agents that cause hemolytic, neurological, or reproductive disorders.
Though RS is not a T. vivax endemic region, the presence of anti-Trypanosoma spp.antibodies over almost all intermediate regions (7/8) indicated that trypanosomes have spread throughout the cattle from RS and may be associated with symptomatic or asymptomatic infections in beef and dairy herds.According to these findings, there is a risk of trypanosomosis outbreaks in the state, mainly in the cities of intermediate regions with high seroprevalence detection.The presence of vectors and favorable conditions for their development (mainly S. calcitrans), food restriction, inadequate transport conditions, and thermal stress in dairy cattle herds is associated with the needle-sharing practice during oxytocin application in lactating cows; these factors, among other, favor disease epidemiology in cattle herds in Brazil (Cadioli et al., 2012).
The detection rate in RS (24.6%) is lower than that in endemic areas such as the Pantanal and Amazon rainforests (93.1-98.5%;Guedes et al., 2008;De Mello et al., 2019), but higher than that in enzootic unstable areas for T. vivax (11.90-15.99%;Guerra et al., 2013).However, the seroprevalence results can vary according to the study region, herd, seasonality, and cattle breeding management type.
The difference in the prevalence of antibodies against anti-Trypanosoma spp. in different regions in RS support defining areas for trypanosomosis transmission risk.The western intermediate regions of Uruguaiana and Ijuí had the highest serological prevalence, with over 30% detection frequency.
Several circumstances contribute to trypanosome dissemination over a region.The western RS is characterized by a high cattle population with proprieties that have animal trafficking characteristics.The national and international live livestock trade represents a serious risk factor; moreover, the introduction of animals infected with subclinical or chronic trypanosomosis is the main risk factor for T. vivax dissemination from endemic to non-endemic regions.Further studies on naturally infected herds have demonstrated that T. vivax induces chronic asymptomatic infection in cattle from endemic areas (Ventura et al., 2001;Dávila et al., 2003).Another concerning observation in animal trade is the occurrence of horses as T. vivax reservoirs, as observed in Paraguayan regions that border southwest Brazil and Argentina (Suganuma et al., 2022).In both animal species, obligatory T. vivaxmediated trypanosomosis diagnosis is not required for international or national trade.
Territorial proximity facilitates pathogen dispersion from different locations, which is an epidemiologically important factor because western RS borders Santa Catarina state and Argentina, where T. vivax was detected in cattle (Paoletta et al., 2018;Silva et al., 2022).Some serological studies using symptomatic cattle from the western region of Santa Catarina detected 39.0% (57/146) seropositive animals, suggesting T. vivax existence in these areas.In the Argentine territory, T. vivax outbreaks have been described in cattle from Formosa and Mesopotamia Argentina.The agro-ecological conditions of these neighboring regions are similar to those of western RS, with potential risk of dispersion among the Pampa biome regions that border Brazil (Abdala et al., 2021;Monzon et al., 2013;Paoletta et al., 2018).Moreover, the main intermediate regions (Ijuí, Uruguaiana, and Passo Fundo) where anti-Trypanosoma spp.antibodies were detected in this study, border these locations in Santa Catarina and Argentina.Thus, it is important to suggest the intermediate regions of Ijuí, Uruguaiana, and Passo Fundo as risk areas for trypanosomosis in cattle from RS.
Immunological cross-reactivity between trypanosome species may occur, mainly between T. vivax and T. evansi, which share many common antigens (Uzcanga et al., 2004).Their coexistence in southern Brazil makes precise interpretation of serological tests difficult, necessitating the use of molecular tools to determine the species involved.Furthermore, the new species discovered in Argentina may also be present in Brazilian herds and must be considered without knowing its influence on immunological tests.Therefore, antibodies should be associated with specific species with caution; the antibodies found in this study are not considered T. vivax-specific.Despite the high detection rate in cattle from the same herd, anti-Trypanosoma spp.IgG antibodies were observed in many dairy herds from Passo Fundo, Porto Alegre, and Pelotas.Rainy periods and/or high humidity contribute to developing mechanical vectors in the environment, as well as improving the possibility of increasing the infection rate of cattle from the same herd.This phenomenon was also observed by Cuglovici et al. (2010), where seroprevalence ratios in dairy cattle herds from Igarapé and Minas Gerais were 7.4-48.0%,with the highest incidence was correlated with an increased S. calcitrans population during rainy seasons.Thereby, S. calcitrans can corroborate trypanosomosis epidemiology in RS, as it is a widely distributed ectoparasite in RS.
In summary, detecting antibodies against Trypanosoma spp. in cattle from RS provides evidence that these herds have been exposed to these pathogens.Further studies on trypanosomosis epidemiology should attempt to conclusively link the pathogens to animal production losses.Owing to the possibility of cross-reactions with other trypanosomes in serological methods, combined parasitological and molecular methods would provide a clear understanding of the specific species infecting the seropositive animals and the epizootiological situation of the trypanosomes present in cattle herds from RS.

Conclusion
This is the first serological study of trypanosomosis in cattle from RS.This study detected antibodies against Trypanosoma spp. in cattle from almost all intermediate regions, thus suggesting a possible risk of trypanosomosis outbreaks, mainly in animals from the western RS.The intermediate regions from Ijuí, Uruguaiana, and Passo Fundo were suggested as risk areas for bovine trypanosomosis due to high seroprevalence.These findings highlight the need for an effective surveillance program to diagnose and prevent trypanosomosis spread, thus reducing disease impact on livestock productivity, which may otherwise be overlooked and underdiagnosed in RS.

Figure 1 .
Figure 1.Map of Rio Grande do Sul (right) showing Trypanosoma spp.seroprevalence in cattle according to the intermediate regions.(Left) Map showing T. vivax occurrence in South America: upper Rio Paraguay basin, including portions of the Brazilian, Bolivian, and Paraguayan Pantanal regions (blue); Alto Paraná, Amambay, Canindeyú, and Concepción regions of Paraguay (pink); western Santa Catarina (purple); Formosa region of Argentina (red); Mesopotamia Argentina (orange); and Rio Grande do Sul (green).

Table 1 .
Absolute frequencies and prevalences by herd for the serological detection of Trypanosoma spp. in cattle serum samples collected from the Rio Grande do Sul intermediate regions.

Table 2 .
Contingency tables and prevalences by intermediate region and cattle breeding.