Serological occurrence for tick-borne agents in beef cattle in the Brazilian Pantanal.

This study investigated the seropositivity for five different tick-borne agents, namely Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Anaplasma phagocytophilum, and Trypanosoma vivax in beef cattle in the Brazilian Pantanal. The serum samples collected from animals (200 cows; 200 calves) were used in indirect enzyme-linked immunosorbent assays (iELISA) to detect IgG antibodies against A. marginale, B. bovis, B. bigemina, and T. vivax, and Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum. No correlation was observed between seropositivity for C. burnetii and A. phagocytophilum with other agents whereas moderate correlation was observed for A. marginalexB. bigemina x B. bovis. Cows were more seropositive for T. vivax whereas calves were more seropositive for B. bovis and B. bigemina. The highest number of seropositive animals by a single agent was observed for T. vivax (15.2%). Co-seropositivity for T. vivax + A. marginale was higher in cows (25.5%) and for T. vivax + B. bovis + B. bigemina + A. marginale was higher in calves (57.5%). The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, Rhipicephalus microplus, in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, may explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.


Introduction
The Brazilian Pantanal plays an important role in the national economy due to beef cattle production and is also described as one of the most biologically diverse regions in the world (Myers et al., 2000;Alho, 2005). The human occupation of the Pantanal has introduced exotic animals and plants that changed the ecosystem, thus favoring the dissemination of pathogens among the native fauna (Alho, 2005;Fantin-Cruz, 2008). The relationship between hosts, parasites, and the environment when disharmonious can cause an onset of diseases (Herrera et al., 2007). According to Guimarães et al. (2001), ticks are one of the most important ectoparasites and pathogen vectors for farmed animals in the Pantanal region, keeping a close relationship with the changing environmental conditions. Moreover, beef cattle production in the Pantanal has been changing, resulting in increasing cattle density and replacement of native vegetation cover with exotic grasses. Consequently, the growing contact between domestic animals and native fauna also increases the exchange of pathogens (Cançado et al., 2008). Anaplasma marginale, Babesia bovis, Babesia bigemina, Coxiella burnetii, Trypanosoma vivax and Anaplasma phagocytophilum infections can cause serious diseases in cattle and are mainly transmitted by arthropod vectors (Maurin & Raoult, 1999;Fereig et al., 2017). Tick-borne diseases are an important factor affecting livestock production in tropical and subtropical regions worldwide, with considerable economic losses (Marcelino et al., 2012).
Anaplasma marginale is an intra-erythrocyte bacterium that has an economic impact on endemic countries, especially due to the high morbidity and mortality in susceptible cattle herds. Losses result from low weight gain, reduced milk yield, abortion, treatment costs and mortality (Kocan et al., 2003). Brazil is endemic for A. marginale, with a molecular prevalence in domestic ruminants varying from 7.5% to close to or equal to 100% (Silva et al., 2016). Bovine babesiosis is a hemoparasitosis caused by the protozoa Babesia bovis and Babesia bigemina, which cause fever, anemia, anorexia, lethargy, hemoglobinuria, and muscle tremors (Singh et al., 2009). This disease is also economically important due to the high morbidity and mortality of animals, especially among calves, and its effects on weight gain and milk yield, requiring expensive control and prevention measures. Trypanosoma vivax can cause hematological and nervous changes in cattle, as well as abortions and other reproductive disorders (Silva et al., 1996;Batista et al., 2007).
This work investigated the serological occurrence of A. marginale, B. bovis. B. bigemina, C. burnetii, A. phagocytophilum and T. vivax in beef cattle in the Brazilian Pantanal.

Animals and study area
The sampled beef cattle (Bos taurus indicus) herds were reared in an extensive farming system in five different farms located in the Central Region of the Pantanal Sul Matogrossense, Sub-Region of Nhecolândia. The blood samples were collected from 400 animals (200 cows and 200 calves) for a cross-sectional study approved by the National Council for Animal Experimentation Control (CONCEA) and the Ethics Committee on Animal Use (CEUA, FCAV, UNESP, Protocol No. 12375/15). All four sampled farms had animals infested with ticks, Rhipicephalus microplus and Amblyomma sculptum, which were previously identified using already described taxonomic keys (Martins et al., 2010). Some animals presented co-infestation by two or more tick species. Hematophagous dipterans, such as horn flies (Haematobia irritans) and tabanids (Tabanus spp.) were also observed (Souza Ramos et al., 2019a, b;Mendes et al., 2019;De Mello et al., 2019).

Bovine serum sampling
Whole blood samples collected directly from the caudal vein and stored in 10 mL tubes were centrifuged at 2000 x g for 10 minutes to separate the sera. Serum aliquots were distributed in 2 mL microtubes and stored at -20 ºC until further serological tests.

Indirect enzyme-linked immunosorbent assay (iELISA) for detecting IgG antibodies against A. marginale, B. bovis, B. bigemina and T. vivax
The antibodies against B. bovis and B. bigemina were detected by the indirect ELISA technique (iELISA) following the protocol described by Machado et al. (1997), whereas the detection of antibodies to A. marginale followed the technique adapted by Andrade et al. (2004). The A. marginale, B. bovis and B. bigemina total antigens were produced by the Immunodot diagnoses Ltda. (Imunodot Desenvolvimento, Indústria e Comércio de Imunógenos e Produtos de Diagnóstico Veterinários, Jaboticabal, SP), at the optimum concentration of 10 μg/mL, diluted in 0.5 M bicarbonate carbonate buffer and pH 9.6. The presence of IgG antibodies against T. vivax was evaluated following the modified methodology established by Aquino et al. (1999). The T. vivax total antigen was produced in the Laboratory of Immunoparasitology of the Universidade Estadual Paulista (UNESP/FCAV, Jaboticabal, SP) (González et al., 2005) at an optimum concentration of 0.1 μg/mL, also diluted in 0.5M bicarbonate carbonate buffer and pH 9.6. After incubation at 4 ºC for 12 hours, a blockade was performed using PBS Tween 20 (pH 7.2) with added 6% milk powder (Molico ® , Nestlé, Brazil). The plates (Maxisorp ® ; Nunc, Thermo Scientific, Brazil) were incubated in a moist chamber at 37 ºC for 90 minutes. After three washings with PBS-Tween 20 buffer, the positive and negative reference serum and tested serum samples, previously diluted with PBS-Tween 20 solution plus 5% normal Rabbit serum at the following dilutions 1:400 (A. marginale, B. bovis and B. bigemina) and 1:100 (T. vivax) according to Sampaio et al. (2015), were added to the ELISA plates. The plates were incubated again at 37 ºC for 90 minutes. After three washes with PBS-tween 20 buffer, the bovine conjugated IgG (Sigma ® , St. Louis, USA) was added to the ELISA plate, in the 1:30000 dilution in PBS-tween 20 plus 5% of normal rabbit serum, with subsequent incubation and washing. Finally, the substrate of the alkaline phosphatase enzyme, P-nitrophenyl phosphate (Sigma ® , St. Louis, MO, USA) diluted at 1 mg/mL in diethanolamine buffer pH 9.8 (Sigma ® , St. Louis, USA) was added. The iELISA plates were sealed with aluminum foil and incubated at room temperature for 30 minutes for A. marginale, B. bovis and B. bigemina and during 45 minutes for T. vivax. The reading was performed in an ELISA reader (B.T.-100; Embrabio, São Paulo, Brazil), with a 405 nm filter. The cutoff values, calculated as 2.5 times the mean absorbance of the negative control sera (Machado et al., 1997), were as follows 0.248 for B. bovis; 0.256 for B. bigemina; 0.190 for A. marginale; and 0.298 for T. vivax.

Indirect Fluorescent Antibody Test (IFAT) for detecting IgG antibodies against C. burnetii and A. phagocytophilum
Detection of IgG antibodies against C. burnetii was performed using the crude antigen of the C. burnetii At12 strain (Pacheco et al., 2013;Reeves et al., 2006). Serum samples from cattle previously known to be seropositive and/or seronegative for such antigen were used as control (Zanatto et al., 2019a). Detection of IgG antibodies against A. phagocytophilum used the crude antigen obtained from HL-60 cells infected with the Webster strain of A. phagocytophilum, courtesy of Dr. John Stephen Dumler (Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA) (Scorpio et al., 2004). The positive control sample for A. phagocytophilum (1:2560 titer) was obtained from an equine experimentally infected with the Webster strain of A. phagocytophilum in the Department of Veterinary Pathology, UNESP, Jaboticabal, SP, Brazil. Serum samples from healthy cattle were used as negative controls (Sousa et al., 2013). Serum samples were first diluted to 1:64 (C. burnetii) and 1:80 (A. phagocytophilum) in phosphate-buffered saline solution (PBS) (pH 7.2; NaCl 1.3 M, KCl 27 M, Na2HPO4 56 mM, KH2PO4 10 mM, and NaH2PO4 9.2 mM). After dilution, 20 μL and 10 μL serum aliquots were placed in each well of the slides containing antigens against C. burnetii and A. phagocytophilum, respectively, with posterior incubation in a moist chamber at 37 ºC for 30 minutes. After incubation, the slides were washed three times with a wash buffer (phosphate-buffered saline, PBS pH 7.4 + 1% Triton + 1.5 mL of Evans Blue) for five minutes, consecutively, and then dried at room temperature. Then, 20 mL conjugated anti-bovine IgG diluted to 1:200 and marked by Fluorescein isothiocyanate (Sigma-Aldrich ® , St. Louis, Missouri, United States) was added to the slides with C. burnetti antigens and 10 mL conjugated goat anti-horse IgG diluted to 1:64, also marked with fluorescein isothiocyanate (SIGMA ® , St. Louis, Missouri, United States), was added to the slides with A. phagocytophilum antigen. The last incubation in a moist chamber at 37 ºC for 30 minutes was followed by subsequent washing, as previously described. After the slides were left to dry at room temperature, buffered glycerin was added (glycerin and carbonate-bicarbonate buffer 0.5 M, pH 9.6), the slides were covered with laminules and observed under a microscope equipped with fluorescent light and at 400X magnification (Olympus BX-FLA ® , Tokyo, Japan).

Statistical analysis
The correlation between seropositivity for the several investigated agents was estimated by the Pearson Correlation Index (c), using the software The R Project for Statistical Computing (R version 3.4.4). The frequency of co-seropositivity for more than one agent in cows and calves and between male and female calves was analyzed by Chi-Square (x 2 ) and Fisher exact test. P values < 0.05 were considered statistically significant for one of the categories. In order to check the dependence between seropositive animals and farms, the Chi-square test (x 2 ) and Fisher's exact test were used, with P <0.05.

Results
The occurrence of antibodies to the selected tick-borne agents and T. vivax is shown in Table 1. The Chi-Square (x 2 ) test showed that T. vivax seropositivity was higher in cows compared to calves (P < 0.05) while B. bovis and B. bigemina seropositivity was higher for calves (P < 0.05). There was no statistical difference (P > 0.05) between the categories for A. marginale in the Chi-square test. Furthermore, Fisher's exact test indicated no statistical difference for C. burnetii and A. phagocytophilum between the two categories, cows and calves. All results are shown in Table 1. The Pearson Index (c) showed no correlation between the seropositivity for C. burnetii and A. phagocytophilum and the presence of antibodies to the other investigated agents, whereas a moderate correlation was observed for the seropositivity for A. marginale x B. bigemina x B. bovis (Table 2).  (Table 3). Additionally, co-seropositivity for B. bigemina was higher for female calves compared to males (P < 0.05).

Agents Cows Calves
Anaplasma marginale (    The seropositivity of cows for A. marginale, B. bovis and B. bigemina showed significant statistical dependence both in the general comparison of farms and in the comparison of farms to each other (P <0.05). Regarding the seropositivity of cows for B. bovis, a higher dependence relationship was observed when the comparisons were performed taking into account two farms at each time. On the other hand, seropositivity for B. bigemina only showed significant dependence between FC vs FPA farms. There was no statistically significant difference in T. vivax seropositivity among the analyzed farms, since the seropositivity for this agent was high in the five sampled farms. A single cow was seropositive for A. phagocytophilum on Alegria Farm, whereas two others were seropositive for C. burnetii on Porto Alegre and Novo Horizonte farms, respectively. Due to the low number of seropositive animals to A. phagocytophilum and C. burnetii, there was no significant difference between the analyzed farms (Tables 6 and 7; Figure 2).

Discussion
In Brazil, the expanding geographic distribution of T. vivax has resulted from the livestock moving between endemic regions and those where the conditions are favorable (climate favorable to hematophagous dipterans, animals lacking prior immunity to the parasite, and the permanent practice of sharing blood-contaminated needles [fomites] during vaccination and application of oxytocin) (Cadioli et al., 2012;Bastos et al., 2017;Andrade Neto et al., 2019). The results showed that cows are significantly more exposed to T. vivax compared to calves, which may be related to the longer period in contact with hematophagous mechanical vectors and fomites (especially due to shared needles). Hematophagous dipterans (horn and horse flies) were also observed in significant numbers in the sampled properties.
The iELISA results indicated a high occurrence for A. marginale in the studied region. In the Brazilian territory, this pathogen occurrence in domestic ruminants estimated by iElisa can range from 16.3% in the semiarid Sergipe to close to 100% in Minas Gerais, Paraíba, Bahia, Paraná, Pará, Rio de Janeiro, São Paulo, and Goiás, being considered an endemic agent in Brazil (Oliveira et al., 2013;Silva et al., 2016). Calves and cows were also exposed to A. marginale, due to the constant presence of the tick Rhipicephalus microplus, considered a biological vector, and hematophagous dipterans parasitizing both. cows and calves. Additionally, the transplacental transmission may also have contributed to the herd seropositivity, but the occurrence of this transmission mode has not been confirmed among the studied animals. The co-seropositivity for T. vivax x A. marginale was highest among cows, which remained for a longer period in this breeding system compared to calves and were, therefore, more exposed to the common transmission routes, such as the mechanical vectors and contaminated fomites.
Also, the iELISA results indicated moderate exposure to B. bovis (61.25%; 245/400) and B. bigemina (51.25%; 205/400) in the sampled beef cattle. Likewise, Barros et al. (2005) reported similar percentages of seropositivity to B. bovis (63.7%) in among beef cattle in the municipality of Uauá, Bahia, northeastern Brazil. However, the iELISA seropositivity for B. bigemina in the Pantanal region was lower than that found (90.5%; 459/506) among beef cattle in Araguaína, Tocantins (Trindade et al., 2010). According to Mahoney & Ross (1972), endemic stability areas for B. bovis and B. bigemina comprise those regions where the clinical illness incidence is low, and 75% of calves between 6 and 9 months of age are seropositive for Babesia spp. Although clinical cases of bovine babesiosis have not been diagnosed in the animals sampled in the present study, Pupin et al. (2019) reported 41 clinical cases and anatomopathological lesions of 33 babesiosis outbreaks by B. bovis in Zebu cattle of all age groups in the state of Mato Grosso do Sul in the 1995-2017 period.
The Chi-square test showed that calves were significantly (P < 0.05) more seropositive for the studied agents than cows, i.e., the infection frequency was higher for calves. It can be suggested that because the immune system of calves is not yet fully developed, they become, therefore, more susceptible to the infestation by the B. bovis vector tick. It is known that Nellore cattle have innate resistance against the fixation of the R. microplus tick to the skin, which is avoided by licking (Veríssimo et al., 2015). Thus, it is also hypothesized that calves have lower licking reflex compared to cows, thus allowing a higher number of fixed larvae and nymphs, which would have the sporogenic cycle of B bovis and B. bigemina, respectively, activated in their salivary glands (Jalovecka et al., 2018). The moderate correlation found among co-seropositive animals for A. marginale x B. bovis x B. bigemina, is probably due to the constant presence of the R. microplus tick in the herds, the biological vector common to all three agents. Furthermore, calves had a higher co-seropositivity for T. vivax x A. marginale x B. bovis x B. bigemina, which may be related especially to the developing immune system, in addition to contact with common transmission pathways of these agents. The seropositivity of both cows and calves was related to the farm studied, which can be explained by the sanitary conditions of each property, presence of ticks, hematophagous dipterans and reuse of needles.
The beef cattle sampled in the present study showed low exposure to C. burnetti. In fact, there are few available data on the serological occurrence of this agent in cattle in Brazil. Recently, Zanatto et al. (2019a) conducted the first study showing C. burnetii as a pathogen associated with infectious bovine rhinotracheitis and bovine viral diarrhea, N. caninum, Leptospira spp., T. gondii and T. vivax in cattle presenting reproductive disorders in Brazil and reported seropositivity of 13.7% (14/102) among the sampled animals. Coxiella burnetii spreads in the herds via aerosol and is commonly related to the extensive farming system (Parker et al., 2006), although ingesting contaminated food and bites of infected ticks are also defined as alternative transmission routes (Eldin et al., 2017). In this sense, the extensive farming system would favor the parasite transmission, mainly via aerosols from urine, feces, and secretions of the infected animals. Considering that the extensive production of livestock predominates in the study region, it is suggested that the concentration of the agent per m 2 is low, decreasing the risk of transmission among the animals.
However, it is important to keep in mind that the Small Cell Variant (SCV), the resistant form of C. burnetii can survive for long periods in the environment (Coleman et al., 2004). Zanatto et al. (2019b) demonstrated the first serological evidence of exposure to C. burnetii (5.32%, 7/169) of free-living cervids (Mazama gouazoubira and Blastocerus dichotomous) in Latin America, warning about its importance for an eventual Q fever epidemiology. Interestingly, some of the cervids seropositive for C. burnetii were sampled in the Pantanal Brazilian wetland, allowing to raise the possibility that the parasite may be disseminated in the region, despite the low seropositivity found. Furthermore, IFAT detects IgG antibodies against C. burnetii in phase I, commonly found during the chronic phase of the disease (Peacock et al., 1983), indicating the possibility that such animals may be infected. Nevertheless, it is not possible to discard the possibility of cross-reaction with other pathogens .
Also, the IFAT results indicated low seropositivity for A. phagocytophilum in the studied cattle. Sacchi et al. (2012) also found seropositivity (16.78%) for A. phagocytophilum by IFAT in Pantanal deer (B. dichotomous) sampled in the flooded areas of the Paraná River due to the construction of the Porto Primavera hydroelectric plant. However, phylogenetic analysis positioned the found Anaplasma sp. genotype close to Anaplasma platys. Ebani & Bertelloni (2014) reported that cross-serological reaction with other species of Anaplasma and Ehrlichia could generate false-positive results for A. phagocytophilum in IFAT. It is known that the A. phagocytophilum transmission and distribution are related to the presence of vector ticks of the genus Ixodes spp., present only in the USA, Europe, and Asia (Woldehiwet, 2010;Sacchi et al., 2012). The absence of vector ticks in Brazil suggests that seropositivity for A. phagocytophilum of a single sample may result from a cross-reaction with another species of Anaplasma spp. circulating in cattle in the Brazilian territory. Since cervids and beef cattle under extensive farming system can divide the same grazing area in the Pantanal, it is probable that cattle herds are being exposed to the Anaplasma spp. genotype detected in cervids.

Conclusion
The present study revealed that the beef cattle in the Brazilian Pantanal are exposed to tick-borne agents (A. marginale, B. bovis, B. bigemina, C. burnetii, and A. phagocytophilum) and T. vivax. The high seropositivity correlation for A. marginale x B. bovis x B. bigemina is probably due to the presence of the tick biological vector, R. microplus in the studied farms. Common transmission pathways, mediated by hematophagous dipterans and fomites, explain the high co-seropositivity of cows for A. marginale and T. vivax. Low seropositivity to C. burnetii is probably due to the type of breeding system employed (extensive). Seropositivity for A. phagocytophilum in only one animal suggests the occurrence of a cross-serological reaction with another agent of the genus Anaplasma.