The Dynamics of Ticks and Capybaras in a Residential Park Area in Southeastern Brazil: Implications for the Risk of Rickettsia rickettsii Infection

Abstract

The bacterium Rickettsia rickettsii causes Brazilian spotted fever (BSF), a highly lethal disease that is transmitted by Amblyomma sculptum ticks in areas where capybaras (Hydrochoerus hydrochaeris) are the tick's major hosts. In this study, we evaluated the expansion of a capybara population in a residential park in São Paulo state, and the implications of such expansion to the occurrence of ticks and BSF. The capybara population was quantified during 2004–2013. In 2012, there was a BSF human case in the area, culminating in the complete fencing of the residential park and the official culling of all capybaras. Quantification of ticks in the environment was performed by dry ice traps from 2005 to 2018. Domestic dogs in 2006–2011 and capybaras in 2012 were serologically tested for the presence of anti-R. rickettsii antibodies. Our results show that capybara numbers increased ≈5 times from 2004 (41 capybaras) to 2012 (230 capybaras). Dry ice traps collected A. sculptum and Amblyomma dubitatum. The number of A. dubitatum adult ticks was generally higher than A. sculptum adults during 2005–2006; however, during 2012–2013, A. sculptum outnumbered A. dubitatum by a large difference. During 2016–2018 (after capybara culling), the number of both species fell close to zero. The low numbers of A. sculptum adult ticks during 2005–2006 coincided with relatively low capybara numbers (<80). Thereafter, in 2012, we counted the highest numbers of both A. sculptum ticks and capybaras (230 animals). All 40 canine blood samples were seronegative to R. rickettsii, in contrast to the 48.3% seropositivity (83/172) among capybaras. Our results support that the emergence of BSF in the residential park was a consequence of the increase of the local capybara population, which in turn, provided the increment of the A. sculptum population. Culling the entire capybara population eliminated the risks of new BSF cases.

Introduction

The bacterium Rickettsia rickettsii is the etiological agent of Brazilian spotted fever (BSF), also known as Rocky Mountain spotted fever in North America, one of the deadliest tick-borne diseases affecting humans in the world (Paddock et al. 2016). In southeastern Brazil, which includes the states of São Paulo, Rio de Janeiro, Minas Gerais, and Espírito Santo, human cases of BSF have occurred since the first half of the 20th century (Del Fiol et al. 2010, Oliveira et al. 2016). The highest occurrence of BSF has been reported in the state of São Paulo, where during the last 5 years (2014–2018) 384 laboratory-confirmed cases were recorded, with a 60% fatality rate (official data from the São Paulo State Health Secretary; www.saude.sp.gov.br).

Epidemiologically, most of the BSF cases in southeastern Brazil have been associated with the transmission by Amblyomma sculptum (formerly Amblyomma cajennense) in areas where this tick species has been maintained chiefly by capybaras (Hydrochoerus hydrochaeris), the largest living rodent of the world (Labruna 2013). In fact, it has been demonstrated that capybaras can act as an efficient amplifying host of R. rickettsii for A. sculptum ticks, a condition necessary for creation of new cohorts of infected ticks (Souza et al. 2009, Polo et al. 2017).

Since the R. rickettsii infection rates of A. sculptum ticks under natural conditions are usually very low, <1% (Guedes et al. 2011, Krawczak et al. 2014, Labruna et al. 2017), and the transovarial transmission of R. rickettsii in A. sculptum ticks is not highly efficient (Soares et al. 2012), the role of amplifying hosts such as capybaras is crucial for the successful maintenance of R. rickettsii in A. sculptum populations (Labruna 2013, Polo et al. 2017). Besides A. sculptum, capybaras from BSF-endemic areas are also frequently infested by Amblyomma dubitatum (formerly Amblyomma cooperi); however, this tick species is usually found infected only by nonpathogenic Rickettsia species, such as Rickettsia bellii or Rickettsia sp. strain Pampulha (Labruna et al. 2004, Perez et al. 2008, Pacheco et al. 2009, Guedes et al. 2011).

The capybara is a herbivorous semiaquatic rodent that lives in social groups and grazes preferably near water bodies; adult capybaras typically weigh around 50–60 Kg (Moreira et al. 2013). During the last two decades, there has been a great expansion of the number of cases of BSF in southeastern Brazil, especially in the state of São Paulo. One of the major factors related to BSF expansion is considered to be the expansion of capybara populations, which has become established in many urban and suburban areas, in direct contact with human dwellings (Labruna 2013, Polo et al. 2015, Souza et al. 2015). In this study, we evaluated the expansion of a capybara population in a residential park in São Paulo State, and the implications of such expansion to the environmental burdens of ticks, and the occurrence of BSF.

Materials and Methods

Ethical statements

This work was authorized by the Environment State Secretary of the state of São Paulo (authorization no.96/2012), the “Instituto Chico Mendes de Conservação da Biodiversidade” (authorization SISBIO no. 59260-1), and was approved by the Ethical Committee in Animal Research of the Faculty of Veterinary Medicine of the University of São Paulo (protocol Nos. 3104/2013, and 3686180917).

Study area and capybara counting

This study was performed in a residential park area at Itu Municipality (13°15′S, 47°22′W), São Paulo State, southeastern Brazil. The residential park has an area of 484 ha, which contains four lakes, and ≈400 homes interposed by open grass lands and conserved forest areas (Krawczak et al. 2014). The construction of the residential park and its artificial lakes started during the late 1970s, when the first individual capybaras were observed in the area, possibly after immigration through water courses from outside the residential park. This type of immigration was possible until 2011, when the residential park was completely fenced, including the water courses, which were fenced with an iron screen fence that has prevented the passage of medium- to large-sized animals in both directions until present time.

Because of the large size of the capybara body, estimation of capybara population can be achieved by direct counting of the individuals. In the current residential park, such approach was facilitated because capybaras tended to graze daily at the open grass lands, where they usually accepted human presence. Therefore, to estimate the capybara population in the residential park, we performed direct countings during the years of 2004–2008 and 2011–2013. In each year, the total number of capybaras was estimated by performing several countings at weekly intervals during 2–3 months; the highest number of capybaras in a single counting was considered the estimated number of the capybara population for that year.

In September 2012, a severe case of BSF occurred in a child few days after being infested by ticks while playing near one of the lakes of the residential park, inhabited by free-ranging capybaras (Krawczak et al. 2014). This human case triggered an official sanitary program in the residential park, which consisted in the capture and culling of the entire capybara population of the area, as previously reported (Krawczak et al. 2014). Capybara culling started in December 2012 and lasted till October 2015, when the last individual capybara was captured. After this period, field surveys aiming to visualize and to count capybaras were performed until 2018, to certify the absence of capybaras in the residential park.

Environmental tick burdens

Quantification of ticks in the environment was performed by dry ice (CO₂) traps, as previously described (Oliveira et al. 2000). For this purpose, dry ice traps were armed in the forest areas surrounding the three major lakes of the residential park, which coincided with the area inhabited by most of the capybara population of the study site. Tick quantifications were performed at 10 occasions, being 1 in each of the years 2005, 2006, 2012, 2013, and 2016, 3 occasions at 2017, and 2 at 2018. The number of dry ice traps that were armed in each occasion varied from 3 to 60. All ticks from each trap were brought to the laboratory, where they were identified to species following Onofrio et al. (2006), Martins et al. (2010), and Martins et al. (2016).

Nutria capture

After 2015, when no capybara was seen in the residential park anymore, field works revealed the presence of several nutrias (Myocastor coypus) in the same area previously occupied by capybaras. To verify if nutrias (rodents weighing up to 5–7 Kg) were serving as suitable hosts for ticks or if they had been exposed to R. rickettsii infection, we performed field work with the purpose of capturing these animals. For this purpose, three tomahawk traps (60 × 30 × 35 cm) were armed at the margins of the three major lakes of the residential park. These traps were weekly baited with fresh sugar cane and corn and were left armed for 6 months in each of the years 2017 and 2018.

Trapped nutrias were anesthetized with an intramuscular injection of ketamine (10 mg/Kg) and xylazine (0.2 mg/Kg) to collect ticks and blood. The whole nutria body was checked for the presence of ticks, which were collected and sent to the laboratory for taxonomic identification following Martins et al. (2010). A blood sample was collected through the femoral vein, centrifuged, and the separated serum was kept frozen until analysis. After returning from anesthesia, nutrias were released at the same capture site. Each nutria received a subcutaneous microchip to monitor future recaptures.

Serological analyses

In 2006, blood samples were collected from 30 dogs that were reared unrestrained in the residential park; these animals had free access to the capybara-inhabited areas. In 2011, blood samples were collected from additional 10 dogs under the same condition. From December 2012 to May 2013, blood samples were collected from 172 capybaras of the residential park (during the culling program), as described in a previous report (Krawczak et al. 2014).

Canine and capybara blood samples were tested by the indirect immunofluorescence assay (IFA) using crude antigen of R. rickettsii strain Taiaçu, as previously described (Labruna et al. 2007, Krawczak et al. 2014). Animal sera were diluted in 2-fold increments with phosphate-buffered saline, starting from the 1:64 dilution. Slides were incubated with fluorescein isothiocyanate-labeled sheep anti-capybara IgG (CCZ, São Paulo, Brazil) or rabbit anti-dog IgG (Sigma, St Louis, MO). Positive and negative control sera were derived from R. rickettsii experimentally infected capybaras and dogs from the studies of Souza et al. (2009) and Piranda et al. (2008), respectively.

The results of the capybara sera have been previously reported (Krawczak et al. 2014). Because of the phylogenetic relatedness of capybaras and nutrias (they are both Caviomorpha rodents), sera obtained from nutrias were also tested by IFA using the same protocol described above for capybaras; however, in this case, the positive control serum was from a guinea pig (Cavia porcellus) previously infected with R. rickettsii (Piranda et al. 2008), to certify that the anti-capybara conjugate would work for caviomorpha rodents different from capybaras.

Statistical analyses

Due to the variation among the number of dry ice traps that were used for collecting ticks in each field work, values are presented as the mean number of individuals of each tick species collected per trap in each field work. These numbers were compared between collections for each tick species, or between tick species in each field work, by the Student's t-test (when variables showed normal distribution by the Kolmogorov–Smirnov test) or by the nonparametric Mann–Whitney test. Analyses were run in the Minitab Release 18 program.

Results

The estimated number of capybaras in the residential park, in each of the sampled years, is presented in Table 1. Capybara number increased ≈5 times during an 8-year period, from 2004 (when 41 capybaras were counted) to 2012, when 230 capybaras were counted and the culling program was initiated because of a BSF case in that same year. With the culling program, the last individual capybara was captured in December 2015, and thereafter, no capybara was seen in the residential park during the countings of 2016, 2017, and 2018. It must be emphasized that the whole residential park has been completely fenced since 2011, what has prevented that new capybaras immigrate from outside the park.

Table 1.

Estimated Number of Capybaras in a Residential Park in Itu Municipality, State of São Paulo, Brazil, from 2004 to 2018

Year	No. of counted capybaras
2004	41
2005	56
2006	78
2007	84
2008	114
2011	167
2012^a	230
2013^a	44
2015^a	1
2016	0
2017	0
2018	0

Culling of the entire capybara population of the residential park started in December 2012 and lasted till October 2015, when the last individual was captured.

Dry ice traps collected two tick species in the residential park: Amblyomma sculptum and Amblyomma dubitatum. The number of ticks collected in each of the 10 occasions is presented in Table 2. All larvae were retained as Amblyomma spp. because we could not identify them to species level at the time the study was done. Similarly, nymphs collected in 2005, 2012, 2013, and 2016 were not saved for morphological identification to species level; hence, they were retained as Amblyomma spp.

Table 2.

Number of Ticks Collected by Dry Ice Traps in a Residential Park in Itu Municipality, State of São Paulo, Brazil, from 2005 to 2018

Date of collection		No. of dry ice traps			No. of collected ticks
Date of collection			Amblyomma sculptum		Amblyomma dubitatum		Amblyomma spp.^a
Year	Month		Adults	Nymphs	Adults	Nymphs	Nymphs	Larvae
2005	May	3	2		10		19	68
2006	January	60	46	123	227	916		0
2012	October	20	660		42		3000	0
2013	December	11	143		8		41	0
2016	March	10	17		1		6	949
2017	March	12	7	1	3	11		0
2017	June	7	0	0	0	0		0
2017	September	6	0	0	0	27		0
2018	May	6	0	0	0	5		0
2018	September	6	0	0	0	0		0

Refers to those ticks that could not be identified to species level, which include all collected larvae and nymphs from 2005, 2012, 2013, and 2016.

Since different numbers of dry ice traps were armed along the sampled years, our comparisons relied on the number of ticks per trap. In addition, since immature ticks could not be identified to species in some years, our statistical analyses included only adult ticks. In this regard, it can be seen in Table 3 that the number of A. dubitatum adult ticks was generally higher than A. sculptum adults during 2005–2006; however, during 2012–2013, A. sculptum outnumbered A. dubitatum by a large difference.

Table 3.

Comparisons of the Numbers of Adult Ticks Collected by Dry Ice Traps in a Residential Park in Itu Municipality, State of São Paulo, Brazil, from 2005 to 2018

Year	No. of dry ice traps	Mean number ± standard deviation (range in parentheses) of collected adult ticks per trap^†
Year	No. of dry ice traps	A. sculptum	A. dubitatum
2005	3	0.7 ± 0.6 (0–1)^a	3.3 ± 2.1 (1–5)^a,c
2006	60	0.8 ± 1.3 (0–5)^a	3.8 ± 4.2 (0–20)^a ^*
2012	20	33.0 ± 33.7 (4–140)^b	2.1 ± 3.0 (0–11)^a,c ^*
2013	11	13.0 ± 14.4 (0–40)^c	0.7 ± 1.7 (0–5)^c,d ^*
2016	10	1.7 ± 3.1 (0–8)^a	0.1 ± 0.3 (0–1)^b,d
2017^‡	25	0.3 ± 1.2 (0–6)^a	0.1 ± 0.4 (0–2)^b,d
2018^‡	12	0.0 ± 0.0 (0–0)	0.0 ± 0.0 (0–0)

†

Values followed by different letters in the same column indicate that the numbers of ticks per trap was statistically different (p < 0.05) between the years for the same tick species; asterisk (^*) indicates that the numbers of ticks per trap was statistically different (p < 0.05) between A. sculptum and A. dubitatum in the same year.

‡

During these years, there were two or three different dates of tick collection by dry ice traps (Table 2); for the present analysis, data were merged for each of the 2 years.

During 2016–2018 (after capybara culling), the numbers of both tick species were always very low and statistically similar. Considering each species separately, the numbers of A. dubitatum were relatively constant during 2005–2013, followed by a significant decrease during 2017–2018. In contrast, the numbers of A. sculptum showed a marked increase from the 2005 to 2006 to 2012. In 2013, there was a half-decrease of this number, followed by a drop close to zero during 2016–2018.

Comparing the number of adult ticks per trap with the number of capybaras in the residential park, it can be seen in Figure 1 that the low numbers of A. sculptum adult ticks during 2005–2006 coincided with relatively low capybara numbers (<80). On the contrary, in 2012, we counted the highest numbers of both A. sculptum ticks and capybaras (230 animals). In 2013, when the culling program had already started and the number of capybaras had dropped to 44, the number of A. sculptum ticks had decreased by about half. Then, when no capybaras were present in the residential park anymore, during 2016–2018, the numbers of A. sculptum dropped to zero or near zero.

FIG. 1.

Comparisons of the tick environmental burdens (mean number of Amblyomma sculptum and Amblyomma dubitatum that were captured by dry ice traps) with the number of capybaras present in a residential park in Itu municipality, state of São Paulo, Brazil, along the years that both ticks and capybaras were quantified.

Four different nutrias (no recapture) were trapped during 2017–2018. Among them, one had no ticks, and the other three had 1, 2, and 5 nymphs of A. dubitatum. No A. sculptum or adult ticks were found on the nutrias.

All canine blood samples from 2006 (30 dogs) and 2011 (10 dog) were seronegative to R. rickettsii (their sera did not react at the 1:64 dilution). Similarly, all four nutrias from 2017 to 2018 were also seronegative. Guinea pig positive control serum reacted satisfactory with the anti-capybara conjugate. Among the 172 capybaras sampled during 2012–2013, 83 (48.3%) were seroreactive to R. rickettsii, with endpoint titers varying from 64 to 8192; these results with capybara sera have been published elsewhere (Krawczak et al. 2014).

Discussion

In 2012, a human case of BSF was confirmed in the residential park of the present study. That case motivated the study of Krawczak et al. (2014), who showed that 48.3% (83/172) of the capybaras were seroreactive to R. rickettsii. In addition, Krawczak et al. (2014) successfully isolated a viable strain of R. rickettsii from A. sculptum ticks and estimated that the R. rickettsii infection rate in the A. sculptum ticks of the residential park was 0.2%. Very low infection rates (<1%) by R. rickettsii in A. sculptum populations have been a common feature among BSF-endemic areas in southeastern Brazil (Guedes et al. 2011, Labruna et al. 2017).

The direct detection of R. rickettsii in the A. sculptum population of the residential park supports the results of the 2012 capybara serosurvey, in which many seropositive capybaras had high endpoint titers (≥1024) to R. rickettsii (Krawczak et al. 2014). On the contrary, our canine serosurveys on unrestrained dogs of the residential park during 2006 and 2011 failed to detect any seropositive animal, indicating that these dogs had not been exposed to R. rickettsii-infected ticks. Since unrestrained dogs are considered suitable sentinels for R. rickettsii infection in A. sculptum populations (Sangioni et al. 2005, Horta et al. 2007, Vianna et al. 2008), we can infer that at least until 2006, when we tested a sample of 30 dogs, the A. sculptum population of the residential park was not infected by R. rickettsii. While this condition was also possible in 2011, our canine sample of that year was small (only 10 dogs), precluding a more reliable inference.

Considering that the A. sculptum population of the residential park was not infected by R. rickettsii in 2006, but it was in 2012, we can infer on two factors that might have had a major role in this BSF-endemic shift during this 6-year interval: (i) the ≈3 × increase of the capybara population, from 78 animals in 2008 to 230 animals in 2012; and (ii) the ≈40× increase of the A. sculptum population, from 0.8 adult/trap in 2008 up to 33.0 adults/trap in 2012 (Table 3).

It is well known that the increase of tick density is directly related to higher density of tick major hosts and to higher suitable environmental conditions for the tick free-living stages (Norval and Lightfoot 1982, Randolph 2004, Paddock and Yabsley 2007). The landscape of the capybara-inhabited areas in the residential park (forest patches and open grass lands surrounding lakes) did not go through noteworthy alterations during the study period (data not shown). On the contrary, the number of capybaras varied greatly, increased up to three times, as stated above. Based on these statements, we conclude that the ≈40× increase of the A. sculptum population in the study area was a consequence of the ≈3× increase of the capybara population, which was indeed related to a higher birth rate during the 2006–2012 period.

Recent studies based on mathematical models concluded that capybara birth rate variation had the greatest impact on the number of R. rickettsii-infected A. sculptum ticks; that is, higher the capybara birth rate, higher the R. rickettsii infection rate in ticks (Polo et al. 2017, 2018). In this sense, we can infer that the shifted status of the BSF risk in the residential park from 2006 to 2012 was triggered by the increase of the capybara birth rate during this period, which also culminated in the extraordinary increase of the A. sculptum population. Obviously, for this condition to have occurred, the bacterium R. rickettsii had to have been introduced in the residential park when the conditions were favorable for its maintenance, namely, under higher capybara birth rates and A. sculptum population.

Polo et al. (2017) demonstrated by mathematical models that the introduction of a R. rickettsii-infected capybara carrying at least one infected tick is enough to establish R. rickettsii infection in an A. sculptum population that is sustained by at least 50 susceptible (seronegative) capybaras. Considering that the residential park was not completely fenced until 2011, we can infer that until that year, at least one R. rickettsii-infected capybara carrying at least one infected tick entered the residential park, resulting in the establishment of the rickettsial infection of the local A. sculptum population. This statement is corroborated by the fact that the Itu region (where the present residential park is located) has been a BSF-endemic area since the beginning of this century (Pinter et al. 2011).

Due to the imminent risks of new BSF cases after the first one in 2012, culling of the entire capybara population was implemented as a measure to suppress the tick population, and consequently, to eliminate risks of new BSF cases in the residential park. Our results of tick samplings by dry ice traps confirmed that culling was successful because environmental burdens of A. sculptum dropped to zero or near zero 17 months after the removal of the last capybara in December 2015. Interestingly, only a few nymphs of A. dubitatum were captured during the last four occasions, in which we armed dry ice traps, from June 2017 to September 2018. This result was corroborated by our findings of only a few nymphs of A. dubitatum on nutrias during 2017 and 2018. Since we did not collect any larvae or adult ticks during this same period, we conclude that by the end of 2018, neither A. dubitatum nor A. sculptum was established in the residential park anymore.

Conclusions

The emergence of BSF in the residential park was a consequence of the local increase of the capybara population, which in turn, provided an increment of the A. sculptum population. Culling the entire capybara population resulted in the local suppression of the environmental burdens of A. sculptum and A. dubitatum, eliminating the risks of new BSF cases among residents of the residential park.

Footnotes

Acknowledgments

We are grateful to the technician Eduardo Bonini of the “Superintendência de Controle de Endemias” (SUCEN) of the state of São Paulo, Sorocaba region, for having done some of the dry ice trap collections of the present study and to Lina C. Binder for her assistance in some of the IFA procedures. This work has received financial support from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP grant 2013/18046-7).

Author Disclosure Statement

No conflicting financial interests exist.

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