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Histopathology of tick-bite lesions in naturally

infested capybaras (Hydrochoerus hydrochaeris)in Brazil

KARIN MARIE VAN DER HEIJDEN1, MATIAS PABLO JUANSZABO2,3,*, MIZUE IMOTO EGAMI4, MARCELO CAMPOSPEREIRA1 and ELIANA REIKO MATUSHIMA1

1Universidade de Sao Paulo, Sao Paulo, SP, Brasil; 2Faculdade de Medicina Veterinaria da Uni-

versidade Federal de Uberlandia, Av. Para, 1720, Campus Umuarama-Bloco 2T, Uberlandia/MG,

CEP 38400-902, Brasil; 3Universidade Estadual Paulista, Jaboticabal, SP, Brasil; 4Universidade

Federal de Sao Paulo, Sao Paulo, SP, Brasil; *Author for correspondence (e-mail: szabo@

famev.ufu.br or szabo@fcav.unesp.br; phone: +55-34-3218-2228; fax: +55-34-3218-2521)

Received 16 March 2005; accepted in revised form 18 October 2005

Key words: Capybara, Heterophils, Histopathology, Ixodidae, Ticks

Abstract. In the present work features of tick-bite lesions were evaluated in capybaras naturally

infested with Amblyomma cajennense and Amblyomma dubitatum ticks. Gross appearance of tick

bite site was characterized by a mild swelling and erythema. Microscopic examination revealed the

cement cone, a tube-like homogenous eosinophilic mass penetrating deep into the dermis. This

structure was surrounded in the dermis by a cellular infiltrate and free eosinophilic granules and

was associated to edema of variable intensity. Necrosis was a common feature deep in the dermis

particularly at the far end of the eosinophilic tube. Hyperplasia, cellular edema and occasionally

necrosis of keratinocytes could be seen at both sides of the ruptured epidermis. Cellular infiltrate

was constituted overwhelmingly by polymorphonuclear leukocytes with eosinophilic granules. In

capybaras cells with such features can be either eosinophils or heterophils (pseudoeosinophils), the

latter being the equivalent of neutrophils of other mammals. Ultrastructural analysis of the cellular

infiltrate revealed the predominance of heterophils over eosinophils. Mononuclear cells and mast

cells and, in lesser numbers, basophils were also seen at skin attachment sites. The presence of

heterophils in the reaction of capybaras against Amblyomma ticks is an outstanding feature but its

role in the reaction to the tick is not known. It is however speculated that capybara heterophils

might be associated with a more permissive environment for tick feeding and pathogen transmis-

sion as already shown for the equivalent cell type, the neutrophil, in the reaction of the dog against

the Rhipicephalus sanguineus tick.

Introduction

The capybara (Hydrochoerus hydrochaeris L.), is the largest living rodent(Pachaly et al. 2001). This animal is under domestication in several States ofBrazil, with the aim of producing a high quality feeding stuff, of sound proteinvalue and low caloric content (Nascimento et al. 2000). Attempts are beingmade to establish rules for the rational breeding of capybaras, under extensive,semi-intensive, and intensive systems. A national law forbid hunting in Brazil

Experimental and Applied Acarology (2005) 37: 245–255

DOI 10.1007/s10493-005-4155-5 � Springer 2005

but allows slaughter of farmed capybaras in registered abattoirs with regularsanitary inspection (Pachaly et al. 2001). On the other hand free-livingcapybaras are increasing in numbers due to lack of natural predators (John2002) and a possible connection between capybaras, ticks and spotted fever, anoften fatal rickettsiosis, is supposed (Lemos et al. 1996b; Dias 2002).

Capybaras can develop massive tick infestations in captivity (Pereira andLabruna 1998) but free-living capybaras have been reported to carry manyticks also (Lemos et al. 1996a). The high infestation levels of capybaras suggestthat this host does not develop an efficient immunity against ticks but there isno experimental data to prove it. This is an important issue to address asresistance to ticks may reduce tick borne-disease transmission (Nazario et al.1998).

Characterization of the relationship between the tick and host immunesystem is fundamental to an understanding of tick biology and pathogentransmission. Ticks and pathogens inoculated at feeding sites on the host haveto deal with an inflammatory reaction modulated by the immune response andsuch reaction can be an important barrier against parasites. On the other handticks are known to alter host reactions by means of saliva secretion (Ribeiro1995). Thus, description of reactions at tick feeding sites, are important for theunderstanding of this host-parasite relationships. However most studiesdescribing lesions at tick attachment sites addresses experimental and artificialinfestations of laboratory rodents and that of domestic animals. At the sametime and not much is known about reaction of other animals. In the presentwork microscopic features of tick-bite lesions of capybaras were evaluated, ahost associated with Brazilian Spotted Fever (Labruna et al. 2004).

Material and methods

Period and locality of sample collection

Ticks and skin samples were collected from May 1994 to March 1998. Localityof tick and skin sampling, number of hosts and tick species found are shown inTable 1. Sampling localities included capybaras bred in a University (Piraci-caba-SP), from a Zoo (Sao Bernardo do Campo-SP), free living animals in afarm (Ibitinga-SP), animals bred for commercial purposes (Santo Antonio daPatrulha-RS, Guararapes- SP and Salmourao-SP) and from a conservationistbreeder (Carapicuiba-SP). All ticks on capybaras were from naturally occur-ring infestations.

Sample collection

Authorization for sample collection was obtained separately for each locality.Tick and skin samples were obtained either immediately after slaughter of

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commercially bred capybaras in an abattoir or from anesthetized animals.Xylazine (Rompun�, Bayer do Brasil S.A.) and Ketamine (Ketalar�, AcheLab. Farmaceuticos S/A) was used for anesthesia of animals in an initial doseof 1 and 5 mg/kg, respectively.

Tick sampling

All animals were examined for the presence of ticks and representative samplesof approximately 10 adult ticks were randomly collected and stored in 70�ethanol until identification in laboratory. Intensity of tick infestation ofcapybaras was visually scored at semi-quantitative levels as follows: 0 (no tickfound); + (1 to 10 ticks); ++ (11 to 100); +++ (more than 100).

Skin sampling

Parasitized skin samples (n = 17) were obtained as described previously(Szabo and Bechara 1999). Briefly, biopsies were taken from partially engorgedfemale tick feeding sites with the aid of a 5 mm diameter punch. Control skinsamples (n = 6) were obtained at non-parasitized sites. Skin samples wereimmediately immersed in fixative (buffered formalin, pH 7.0).

Histological processing

Skin samples were kept for 24 h in the fixative, embedded in paraffin andprocessed according to routine histological techniques. Each biopsy was seri-ally sectioned at a thickness of 4 lm, and stained with Hematoxylin–eosin andMay Grunwald-Giemsa.

Table 1. Localities of tick sampling, number of hosts examined, tick species found and infestation

levels.

Town and state of sampling Number of

capybaras

Tick species Infestation level

of hosts

Carapicuiba-SP (23�33¢¢ S, 46�53¢ W) 4 � 0

Sao Bernardo do Campo-SP (23�45¢¢ S, 46�30¢ W) 5 � 0

Santo Antonio da Patrulha-RS (29�47¢¢ S, 50¢32¢ W) 14 � 0

Piracicaba-S P (22�44¢¢ S, 47�38¢ W) 5 A. cajennense +++

Guararapes-SP (21�14¢¢ S, 50�39¢W) 12 A. cajennense +++

Ibitinga-SP (21�46¢¢ S, 48�48¢W) 12 A. cajennense +++

A. dubitatum

Salmourao-SP (21�33¢¢ S, 50�54¢ W) 9 A. cajennense +++

0 = non-infested; +++ = more then 100 ticks per animal.

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Section analysis

Analysis of tissue samples was performed under light microscopy. Only thosesections displaying the central tick attachment lesion, characterized by thepresence of the cement cone of the tick in the dermis, were analyzed. Generaltissue features were evaluated on Hematoxylin–eosin stained sections. Totalcell counts were made on sections stained by May-Grunwald & Giemsa. Forthis purpose, cells from three areas of 0.0052 mm2 surrounding the tick cementcone in the dermis were counted. Means of each area were used for furtheranalysis. The counting area was delimited by a Reichert integrating graticule(Austria/PK 6, 3· mn) on oil immersion fields (objective 100 · ). Differentialcell counts were performed on the same sections and areas used for total cellcounts.

Ultrastructural identification cells at tick attachment sites

Eosinophils cannot be distinguished from heterophils under light microscopysince both are polymorphonuclear and both exhibit eosinophilic granules in thecytoplasm. Ultrastructural analysis was used to distinguish these cell types attick attachment sites in capybaras. For this purpose skin fragments from tickattachment sites were fixed in 2.0% phosphate buffered glutaraldehyde. Rou-tine double fixation and embedding in Araldite 502 were used (Luft 1961). Thinsections were obtained with the aid of a Sorvall 5000 ultramicrotome, stainedwith uranyl acetate and lead citrate and examined in a Carl Zeiss EM-900electron microscope.

Statistical analysis

Cell numbers in the dermis at tick-attachment sites and non-parasitized dermiswere compared and significance assessed by analysis of variance (p<0.05).

Results

Tick species and infestation levels

Capybaras were infested in four out of seven localities studied (Table 1). Inthese four localities all animals were infested and infestation levels were alwayshigh. Although the threshold for high infestation levels was set at 100 ticks perinspected animal, many capybaras were infested with several hundred ticks.Tick identification solely was reported previously in a previous report (van derHeijden et al. 2003). From the two tick species identified, Amblyommacajennense was found in four and Amblyomma dubitatum in one locality.

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Gross and microscopic lesions

The gross appearance of the tick bite site was characterized by a mild swellingand erythema which increased when several female ticks were attached closetogether. Microscopic examination of the centre of the lesion revealed thecement, a tube-like homogenous eosinophilic mass starting from the surface ofthe skin, at a ruptured epidermis site, and penetrating deep into the dermis.This structure many times contained fragments of tick mouthparts, was sur-rounded in the dermis by a cellular infiltrate and free eosinophilic granules andwas associated to edema of variable intensity. Necrosis was a common featuredeep in the dermis particularly at the far end of the eosinophilic tube.Hyperplasia, cellular edema and occasionally necrosis of keratinocytes couldbe seen at both sides of the ruptured epidermis. Results of total and differentialcell counts of infested sites were compared to control, non-infested, skin cellcounts and are presented in Figure 1. Cellular infiltrate was constituted over-whelmingly by polymorphonuclear leukocytes, and from these heterophils/eosinophils were the main component (Figure 2). Mononuclear cells and mastcells and, in lesser numbers, basophils were also seen at skin attachment sites.

Ultrastructural analysis

Ultrastructural analysis of the cellular infiltrate of four different parasitizedskin fragments revealed the predominance of heterophils characterized by theiroval shape, lobulated nucleus, heterochromatin bound to internal membraneof the nucleus and central euchromatin and an abundant cytoplasm rich inround, oval or elongated homogenous electron-opaque granules (Figure 3).

Discussion

Capybaras from the present study were mostly captive and either displayedvery high infestation levels or were not infested at all. This indicates that underartificial breeding conditions tick infestations are prone to attain high levels incapybaras unless they are maintained totally tick-free. Two tick species wereidentified on the animals. The most prevalent, A. cajennense, is a commonNeotropical tick which feeds mainly on horses, capybaras and tapirs eventhough it may be found on many other hosts (Pereira and Labruna 1998;Labruna et al. 2001). This tick species is also the most aggressive to humans inBrazil and is considered the main vector of Rickettsia rickettsii, the causativeagent of spotted fever in the Neotropical region, a severe human disease(Pereira and Labruna 1998). A. dubitatum a synonym to A. cooperi (Camicas1998) is the well-known Neotropical tick of capybaras throughout SouthAmerica (Evans et al. 2000). It can occasionally attach to humans (Guimaraeset al. 2001) and the isolation of a spotted fever group rickettsiae from

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A. dubitatum of a capybara in Brazil has already been described (Lemos et al.1996b).

Lesions induced by the ticks were centred on the cement, a structure secretedby acini types II and III of the salivary gland of the parasite to firmly secure thetick to the host via the mouthparts during the long feeding process (Wikel1996). Ticks recovered from capybara were all from genus Amblyomma,characterized by long mouthparts (hypostome) and which is fully inserted inthe dermis of the host during feeding (Kemp et al. 1982). Epithelial hyper-plasia, edema and necrosis were confined to the immediate surroundings of tickattachment and may have been caused, either by parasite induced tissuedamage, the immune inflammatory reaction to the tick, or both.

Figure 1. Total and differential cell counts at tick attachment sites of naturally infested capybaras.

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Inflammatory cell infiltrate at the tick attachment site was composed over-whelmingly by polymorphonuclear cells containing acidophilic granules in thecytoplasm but also by mast cells and a few basophils. Mast cells and basophilsare cells rich in cytoplasmatic granules containing histamine which increasesvascular permeability (Kumar 2002). Thus these cells must have contributed tothe observed edema in the dermis.

Heterophils are found in birds, reptiles, and amphibians but also in rabbitsand are the equivalent leukocyte to neutrophils of other mammals (Canfield1998). Thus animals have either neutrophils or heterophils. Capybaras haveheterophils and not neutrophils as demonstrated by cytochemical techniques ofblood smears (van der Heijden et al. 2003). At the same time it is not possibleto distinguish heterophils from eosinophils under light microscopy in Hema-toxylin-eosin or May-Grunwald Giemsa stained sections. Both cell types areseen in these sections as polymorphonuclear leukocytes with large reddishgranules. In this work ultrastuctural analysis was used to differentiate them attick attachment sites. The distinction between neutrophils or heterophils andeosinophils is important because these cell types differ functionally. Neu-trophils predominate for the first 6–24 h in most forms of acute inflammationmoreover bacterial infections induce a relatively selective neutrophilia. Eosin-ophils, on the other hand, are characteristically found in inflammatory sitesaround parasitic infections or as part of immune reactions mediated by IgE,typically associated with allergies (Kumar et al. 2002).

Van der Heijden et al. (2003) analyzed the blood parameters of the sameanimals from the present work and observed that tick infested capybaras hadincreased levels of circulating eosinophils but decreased levels of circulating

Figure 2. Photomicrographs of tick attachment site on a capybara. Notice the presence of many

polymorphonuclear cells containing acidophilic granules in the cytoplasm (heterophils or eosin-

ophils) (objective 40· ).

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heterophils when compared to non-infested hosts. At the same time resultsfrom this work showed that at tick attachment site heterophils predominate.Although there is no straightforward explanation for that, it can be supposedthat blood levels of heterophils were lowered by large amounts of cellsmigrating to the intensely infested skin. Eosinophils on the other hand, mighthave had their migration specifically blocked by tick saliva or infestedcapybaras might also have had higher levels of endoparasites which increasedeosinophilia, a matter that was not investigated.

Mechanisms of resistance against ticks are poorly understood (Willadsenand Jongejan 1999), however it was observed in various experimental

Figure 3. Electron micrographs of skin dermal region of tick infested capybara. Notice the

presence of heterophil granulocytes showing sections of lobulated nucleus and cytoplasm rich in

round, oval or elongated homogenous electron-opaque granules (a,b). Collagen fibers (b – arrows)

are seen around the heterophil.

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infestations models that guinea pigs do develop a strong resistance against ticksfollowing repeated infestations and that tick feeding sites in this host are rich inbasophils but also in eosinophils (Allen 1973; Brown et al. 1984; Szabo andBechara 1999). On the other hand, several infestations with Rhipicephalussanguineus ticks do not induce resistance in its usual host, the dog and in thiscase tick attachment sites are characterized by a neutrophil rich exudate(Tatchell 1970; Theis and Budwiser 1974; Szabo and Bechara 1999). Lack ofresistance and a predominantly neutrophilic infiltration at the tick attachmentsite was also observed during the first and third infestations of C3H/HeJ micewith R. sanguineus (Ferreira et al. 2003). At the same time, neutrophils areknown to have reduced activities under the modulation of tick saliva (Ribeiroet al. 1990; Inokuma et al. 1997; Montgomery et al. 2004).

The role of heterophils in the reaction of capybaras against Amblyommaticks is not known but their overwhelming presence at lesions where ticks hadbeen attached for several days is uncommon. This observation indicates thatheterophils have a prolonged survival at, or are specifically attracted to thetick-attachment sites. Considering the current knowledge obtained fromexperimental data, it is tempting to speculate that capybaras by reacting mostlywith heterophils to their habitual tick species, are displaying a similar role ofneutrophils in dogs against R. sanguineus. This reaction, characterized by eitherneutrophil or heterophil infiltration, thus seem to allow for the proper devel-opment of ticks and might explain the intense infestations observed both indogs and capybaras. Such a reaction is also permissive for the transmission ofinfectious agents as shown by the vectoring capacity of R. sanguineus to dogs(Walker et al. 2000) but its significance in capybaras is unknown. Consideringthe capybara-tick-spotted fever association in Brazil, it is worthwhile to studythe matter further.

Acknowledgement

The authors would like to acknowledge Fundacao de Amparo a Pesquisa doEstado de Sao Paulo (FAPESP) for grants.

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