Toxicon 88 (2014) 99e106

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Toxicon

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Experimentally induced toxicosis in rats bitten by brasiliensis (: ): A clinico-pathological characterization

* Jose Reck a, , Paulo Bandarra b, Saulo Pavarini c, Carlos Termignoni d, e, David Driemeier c,Joao~ Ricardo Martins a, Jorge A. Guimaraes~ d a Instituto de Pesquisas Veterinarias Desiderio Finamor (IPVDF), Fundaçao~ Estadual de Pesquisa Agropecuaria (FEPAGRO), Eldorado do Sul, RS, Brazil b Faculdade de Veterinaria, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brazil c Faculdade de Veterinaria, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil d Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil e Departamento de Bioquímica, UFRGS, Porto Alegre, RS, Brazil article info abstract

Article history: Ornithodoros brasiliensis, also known as the mouro tick, is an argasid tick only found in the Received 27 March 2014 highlands of Southern Brazil. O. brasiliensis is associated with severe reactions in Received in revised form 16 June 2014 its hosts ranging from local pruritus and pain to systemic disturbances. Recently, the re- Accepted 18 June 2014 emergence of O. brasiliensis parasitism in humans and dogs drew attention to the clinical Available online 26 June 2014 findings induced by its bite, which are poorly understood and described. Moreover, rare experimental data about tick bite effects under controlled conditions were available. Thus, Keywords: this study aimed to describe clinical and pathological findings induced by O. brasiliensis bites Argasidae Venomous in experimentally parasitized rats. feed for ~40 min in rats, and their weight increased fi Skin lesion by approximately four times after the blood meal. Rats bitten by ve adult ticks showed Hemorrhage hyperemia of the oral/ocular mucosa, piloerection, tachypnea, claudication, ocular and nasal discharge, pruritus, and swollen and erythemic lesions. A large hemorrhagic lesion was observed on rat skin in tick attachment sites, reaching ~17 mm in diameter 12 h after a bite. Bitten rats also presented an increased bleeding tendency (~50%) 6 h after a tick bite, eval- uated by the tail-cut rat model of bleeding. Blood samples of bitten rats were taken, and clinical pathology analysis showed significant alterations in the eosinophil and basophil counts, in creatine phosphokinase (CPK) and CPK MB fraction, and lactate dehydrogenase (LDH) activity, and fibrinogen level. Histopathological analysis revealed marked subcu- taneous hemorrhage, edema and slight muscle degeneration at the bite site. Also, muscle degeneration and necrosis were observed in the myocardium of bitten rats 72 h after bites by histopathology and immunohistochemistry against troponin C. This work showed the ability of O. brasiliensis to cause severe disturbances in experimentally parasitized rats, compatible with a tick toxicosis syndrome. This observation associated with the re-emergence of O. brasiliensis parasitism makes this parasite as a public health hazard in southern Brazil. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction * Corresponding author. Laboratorio de Parasitologia, Instituto de Pes- quisas Veterinarias Desiderio Finamor (IPVDF), Estrada do Conde, 6000, Ticks are hematophagous parasites distributed world- þ Eldorado do Sul, 92990-000, RS, Brazil. Tel./fax: 55 51 3481 3711. wide. Its parasitism could cause severe deleterious E-mail address: [email protected] (J. Reck). http://dx.doi.org/10.1016/j.toxicon.2014.06.017 0041-0101/© 2014 Elsevier Ltd. All rights reserved. 100 J. Reck et al. / Toxicon 88 (2014) 99e106 consequences both to humans and . These conse- guidelines for experimentation, and all procedures quences can be related to the transmission of tick-borne were approved by the local ethics committee (CEUA/IPVDF pathogens or directly associated with the tick bite. The 04/2011). All procedures were also in accordance with the non-infectious noxious consequences related to tick bite NIH Animal Care Guidelines. The study design was adjusted are generally referred to as tick toxicosis (Walker, 1998; to follow ethical guidelines, which involved reducing to the Sharifi et al., 2003; Mans et al., 2008). minimum the number of animals per group and at each Ticks of the Ornithodoros are well known because time interval. their bite can cause severe non-infectious disturbances in their hosts. The effect of Ornithodoros spp. bite may range 2.3. Study design from local pain and pruritus to severe toxicosis that can even progress to death (Hoogstraal and Gallagher, 1982; Rats were divided into control (18 animals) and exper- Mans et al., 2008; Venzal et al., 2007). imental groups (36 animals). Rats of the experimental Ornithodoros brasiliensis is a tick species only found in group were submitted to an experimental protocol in southern Brazilian highlands. Since it was first reported, which they were bitten by five adult female specimens of O. this tick has been associated with severe host reactions brasiliensis (~10 mm length). Rats of the experimental after bites, particularly in humans. The O. brasiliensis bite in group were slight sedated with acepromazine (6 mg/kg, humans is frequently followed by pruritus, the develop- i.p.) to avoid tick removal, and 10 min later ticks were ment of a slow-healing lesion at the site of tick bite, edema, placed on the rat's skin (one tick on each hind-limb, and erythema and focal skin rash. In some cases, victims also three on the abdomen) to feed. Rats of control group only reported local pain, induction of blisters, limb edema, received acepromazine and were not submitted to any malaise, headaches and dyspnea (Martins et al., 2011; Reck other treatment. et al., 2013a, 2013b). In dogs, O. brasiliensis parasitism was From these animals cited above, nine rats of control associated with apathy, skin rash, petechiae, and mucosal group (three for each time interval) and 18 of experimental hyperemia (Reck et al., 2011, 2013a). This information group (six for each time interval) were used for determi- makes O. brasiliensis a public health concern in southern nation of bleeding effect (tail-cut bleeding model), as Brazil because of its potential to causes toxicosis. further described. Since the results of the control group are With the exception of one clinical case report about a quite homogeneous, they were presented as the dog bitten by O. brasiliensis (Reck et al., 2011), most infor- means ± SEM of all control animals (n ¼ 9) used for of tail- mation available concerning clinical effects of O. brasiliensis cut bleeding model. The tail cut was performed 6 (n ¼ 3), 24 bite are from epidemiological/retrospective analyses (Reck (n ¼ 3), or 72 (n ¼ 3) hours after acepromazine et al., 2013a) or dated anecdotal reports (Pinto and di administration. Primio, 1931; Aragao,~ 1936; di Primio, 1937). Moreover, All other animals (nine rats from control group and 18 experimental data about this tick bite effects under from experimental group) were utilized for the observation controlled conditions is rarely available (Reck et al., 2013b). of clinical findings, determination of clinical pathology Thus, the present study aims to describe the clinical and parameters, and histopathological analysis. Blood samples pathological effects induced in rats experimentally para- for clinical pathology tests were taken from rats of exper- sitized by O. brasiliensis. imental group at 6, 24 and 72 h after tick bites (six animals at each time). Blood samples were obtained by puncture of 2. Materials and methods the femoral veins (after inguinal incision) of previously anesthetized rats (xylazine/ketamine, 15/90 mg/kg, i.p.). 2.1. Ticks Blood samples of the control group were obtained 6 (n ¼ 3), 24 (n ¼ 3) or 72 (n ¼ 3) hours after acepromazine admin- The ticks used in this study were collected in the field in istration. After blood collection, rats were killed with a the municipality of Sao~ Francisco de Paula (29 200 0000 S, xylazine/ketamine overdose. 48 300 2100 W) in highlands region of southern Brazil, Rio Grande do Sul state, Brazil. They were collected in summer 2.4. Clinical findings season and were maintained under laboratory conditions (20 C, ±80% humidity) in Petri dishes with sterile sand Rats were observed at indicated times, and all clinical (1 mm depth) for three months until the experiment was findings were recorded. The main characteristics observed performed. Ticks used in this work had never been fed in rats were the traditional clinical signs used for rodent under laboratory conditions before this experiment. They clinical visual examination, as mucosa coloration, piloer- were identified as O. brasiliensis ticks according to Aragao's~ ection, respiratory frequency, dehambulation, presence of descriptions (1923, 1931). itching, self-grooming, aspect of lesion, and general con- ditions. Rats from the experimental group were inspected 2.2. Animals 6, 24 and 72 h after tick bites. Animals from the control group were inspected 6, 24 and 72 h after acepromazine Male Wistar rats (~300 g) were utilized in this work. The administration. The record of clinical findings was per- rats were maintained in temperature-controlled (21e24 C, formed by the inspection of the animals for 30 min. Rats in 12-h light/dark cycles) rooms and had access to water were maintained in their habitual cages and rooms during and food ad libitum. All experiments performed in this the observation, and inspections were always made by the work were carried out in accordance with ethical same observer. J. Reck et al. / Toxicon 88 (2014) 99e106 101

2.5. Skin hemorrhagic focus diameter methanol. Contra-staining was carried out using Harris hematoxylin. The troponin C IHC technique stains normal/ Skin hemorrhage at the site of tick bite was measured at healthy myocardial cells, and a diminished or absent indicated times using a high-precision pachymeter (Mitu- staining was observed in injured cells (Ortmann et al., toyo, Sao~ Paulo, Brazil). The hemorrhagic focus was 2000). measured considering the larger hemorrhagic diameter (mm) of the skin lesions. This measurement was performed 2.8. Tail-cut bleeding model (bleeding effect) after the record of clinical findings, to avoid alterations in animal behavior due to handling. The bleeding effect, i.e., systemic hemorrhagic tendency was evaluated using a classical rat tail-cut model. The 2.6. Clinical pathology methodology was performed according to a previously described protocol (Nazareth et al., 2006), with minor Blood was drawn from rats using EDTA and sodium modifications. Briefly, rats were anesthetized with sodium citrate as anticoagulants. The following parameters were thiopental (85 mg/kg, i.p.), and their tails were put in a determined: complete blood cell examination (erythrocyte warm bath (37 C) for 5 min. A segment of 5 mm of the tail and platelets count; differential leukocytes count; hemo- was excised using a scalpel, and then the tail was placed globin content; hematocrit; mean corpuscular hemoglobin inside a beaker with distilled water (40 mL) at 37 C for concentration, MCHC; mean corpuscular volume, MCV), 30 min. After that rats were killed with a sodium thiopental platelet count, creatine phosphokinase (CPK) activity, cre- overdose. The blood loss, as an index of bleeding effect, was atine phosphokinase MB fraction (CPK-MB) activity, total evaluated by spectrophotometry to determine the solution lactate dehydrogenase (LDH) activity, aspartate amino- absorbance at a wavelength of 540 nm (Abs540). For data transferase (AST) activity, alanine aminotransferase (ALT) comparison, the Abs values of the experimental group fi 540 activity, plasma urea and creatinine levels, brinogen, were expressed as fold of control. prothrombin time (PT), and thromboplastin time (aPTT). CPK, CPK-MB, LDH, AST, and ALT activities were determined by spectrophotometry using commercial kits (Labtest 2.9. Statistical analysis Diagnostica, Lagoa Santa, Brazil). Plasma urea and creati- ± nine levels were also determined by commercial kits The results were expressed as mean S.E.M. of n sam- ples. The statistical analyses were carried out using a one- (Labtest Diagnostica, Lagoa Santa, Brazil). Fibrinogen level, PT and aPTT were evaluated as previously described (Reck way analysis of variance (ANOVA) followed by the Dun- nett post hoc test. Values of P less than 0.05 were consid- et al., 2009). Other mentioned blood tests (erythrocytes, fi platelets and leukocytes count, hemoglobin content, he- ered statistically signi cant. The statistical analyses were conducted using the GraphPad Prism 3.0 software matocrit, MCHC, and MCV) were done in an automated ® veterinary hematology analyzer (Bio-1800 Vet , Bioeasy (GraphPad Software Inc., San Diego, CA, USA). Diagnostica S/A, Belo Horizonte, Brazil). Samples were kept at 20 C until determination of plasma parameters. The 3. Results blood cell count was performed immediately after blood collection. Rat blood samples were also analyzed for the 3.1. Tick feeding characteristics presence of hemoparasites by Giemsa staining. Since the results of the control group were quite homogeneous, they Immediately after the contact with rat skin, ticks were presented as the mean ± SEM of all control animals attached and started to feed. The average time to complete (n ¼ 9). a blood meal and spontaneous detachment (as an engorged tick) from rat was ~40 min (ranging from 21 to 110 min). 2.7. Histopathology and immunohistochemistry During feeding (~10 min after the blood meal starts), ticks start the production of a large amount of coxal fluid, a Histopathological investigation consisted of colorless liquid excreted by coxal glands. Most ticks fl hematoxylin-eosin standard (HE) staining in thin sections excreted coxal uid for an additional 60 min after detach- fl of rat organs embedded in paraffin blocks. The organs were ment. The average volume of coxal uid excreted was m m collected immediately after rat death and were placed in ~120 L per tick (ranging from 30 to 200 L). After a blood 10% buffered formalin solutions until use. meal, a tick increased to about four times its original Heart tissue of experimental and control rats underwent weight. The estimated blood intake average per tick was immunohistochemistry (IHC) analysis against troponin C. ~245 mg, calculated based on the following equation: fl þ fi e For IHC, thin sections of heart tissue were prepared using (coxal uid weight nal tick weight) initial tick weight. ® ImmunoSlide slide glass (EasyPath, Sao~ Paulo, Brazil). For antigen detection a monoclonal mouse antibody against 3.2. Clinical findings troponin C (Novocastra Reagents, Newcastle, UK) diluted to 1:40 was employed. IHC was performed using the strep- The main clinical findings observed in bitten rats were tavidinebiotineperoxidase complex technique (DakoCy- oral/ocular mucosa hyperemia, piloerection, tachypnea, tomation Inc., Carpinteria, USA) and revealed using claudication, ocular and nasal discharge, pruritus, and diaminobenzidine (DakoCytomation Inc, Carpinteria, USA). swollen and erythemic lesion. The relative frequency of Endogenous peroxidase was blocked using 10% H2O2 in clinical findings is summarized in Table 1. 102 J. Reck et al. / Toxicon 88 (2014) 99e106

Table 1 Clinical findings induced by O. brasiliensis tick bite in Wistar rats.

Clinical finding Controla Time after tick bites

6hb 24 hc 72 hd

Hypermia of oral/ocular mucosa 0/9 61% (7/18) 42% (5/12) 17% (1/6) Piloerection 0/9 72% (13/18) 33% (4/12) 17% (1/6) Tachypnea 0/9 28% (5/18) 17% (2/12) 0% (0/6) Claudication 0/9 17% (3/18) 8% (1/12) 0% (0/6) Ocular and nasal dischargee 0/9 0% (0/18) 42% (5/12) 0% (0/6) Pruritusf 0/9 72% (13/18) 50% (6/12) 33% (2/6) Swollen lesion 0/9 89% (16/18) 75% (9/12) 33% (2/6) Erythemic lesion 0/9 100% (18/18) 100% (12/12) 67% (4/6)

a Control animals were observed at 6, 24 and 72 h. b 18 animals were observed at 6 h after tick bite. c 12 animals were observed at 24 h after tick bite, since six were killed at 6 h. d Six animals were observed at 72 h after tick bite, since six were killed at 6 h, and other six at 24 h. e Reddish mucous discharge. f Animals itching and licking the lesion site.

3.3. Skin hemorrhagic focus diameter and tail-cut bleeding 3.5. Histopathology and IHC against troponin C

The hemorrhagic focus at the tick bite site in rat skin Histopathological analysis revealed an extensive sub- progressively increased until 12 h post-bite. At this time, cutaneous hemorrhage and edema at the site of the tick the lesion's mean diameter (n ¼ 6) was ~17 mm. Fig. 1 bite at all times (6, 24 and 72 h). At 72 h after tick bite, in shows the progression of hemorrhagic focus diameter addition to marked subcutaneous hemorrhage and edema, after tick bite. Fig. 2 shows the aspect of typical tick bite mild muscle fiber degeneration was also observed. Muscle lesions in a rat's hind limb. degeneration was characterized by increased cytoplasmic The tail-cut bleeding time significantly increased (~50%) eosinophilia and reduction of characteristic muscle stria- at 6 h after tick bite, compared to control animals (Fig. 3). tions. Also, intra-epidermal hemorrhage with slight No statistical differences in tail bleeding were observed at epidermal detachment was observed. Fig. 4 shows the any other tested time. typical histological findings of a tick bite lesion at 72 h after bite. Polymorphonuclear infiltration was rarely seen or 3.4. Clinical pathology absent at the site of tick bite. When present, infiltrated cells were predominantly eosinophils. The clinical pathology analysis showed significant Morphological alterations were also seen in heart tissue alteration (at least at one tested time) of the following of bitten rats 72 h after tick bites (Fig. 5). The main alter- blood parameters: eosinophil and basophil count, CPK, ation observed was myocardium degeneration and necro- CPK-MB and LDH activity, and fibrinogen level. All other sis, characterized by cell retraction, enhanced cytoplasmic measured clinical pathology parameters were not signifi- eosinophilia, lack of cytoplasmic striations, nuclear pyk- cantly altered. Clinical pathology parameters significantly nosis and occasional nuclear karyorrhexis and karyolysis. altered in experimental group are summarized in Table 2. The lesions were found mainly in papillary heart muscles No hemoparasites or tick-borne microorganisms were and in areas near the inner (left) ventricle wall. Several found in rat blood smears stained with Giemsa. incidents of subendocardial hemorrhage, particularly in left ventricle, were also observed in heart tissue (data not shown). Other organs and tissues presented no alterations. Myocardial injury was confirmed by IHC against cardiac troponin C. Areas of myocardial necrosis and degeneration in HE sections co-localized with non-stained areas in IHC sections (Fig. 5). In heart tissue from all control rats, a marked and homogenous staining for troponin C was observed (data not shown).

4. Discussion

O. brasiliensis parasitism has been frequently associated with severe reactions to tick bite, particularly in humans (Reck et al., 2013a). These noxious reactions to tick bite are also frequently observed after the bite of other Ornithodoros species. The bite of the Arabian tick Ornithodoros muese- Fig. 1. Hemorrhagic focus diameter: skin hemorrhage at the site of tick bite was measured at indicated times using a high-precision pachymeter. The becki may lead to blisters, pruritus, fever and headache in results are expressed as mean ± SEM of six animals per group. humans (Hoogstraal and Gallagher, 1982). In laboratory and J. Reck et al. / Toxicon 88 (2014) 99e106 103

Fig. 2. O. brasiliensis bite lesions: typical aspect of tick bite induced lesions in rat hind limbs. Left panel, skin lesion, 1 h after tick bite. Right panel, skin lesion, 12 h after tick bite.

domestic animals, the bite of the African sand tampan tick even quite similar to those associated with the bite of other induces intense local pain, dyspnea, Ornithodoros species (Venzal et al., 2007; Mans et al., 2008). hemorrhages and death, probably associated with Compared with the main clinical findings described for anaphylactic shock or cardiac arrest (Mans et al., 2008). bitten humans, there are some similarities, particularly in Ornithodoros aff. puertoricensis produced marked delete- local symptoms, such as pruritus and erythemic and rious effects in laboratory mice, such as hyperemia in both edematous lesions. The occurrence of dyspnea, despite nasal and ocular mucosa, dyspnea, motor incoordination, being rarely observed in experimental animals, was also and death (Venzal et al., 2007). reported by a few bitten humans. In bitten humans, Despite the evident public health importance of O. discomfort and malaise is frequently reported. Despite the brasiliensis and its re-emergence in southern Brazil (Reck fact that these characteristics cannot be directly recorded in et al., 2013a), there are no comprehensive experimental rodents, some findings, such as piloerection, indicate that data describing the clinical-pathological effects of O. bra- rats are in a stressful situation (Carstens and Moberg, siliensis feeding on controlled conditions. In this study, we 2000). The occurrence of mucosa hyperemia seems to be addressed this issue, characterizing the clinical and path- a common finding in animals bitten by O. savignyi and O. aff. ological effects induced by O. brasiliensis parasitism in rats. puertoricencis (Venzal et al., 2007; Mans et al., 2008) and Clinical findings observed in the first 72 h after tick bite was also observed in a dog bitten by O. brasiliensis (Reck were both local (pruritus, swollen and erythemic lesion, et al., 2011). Ocular and nasal discharge observed in claudication) and systemic (oral/ocular mucosa hyperemia, bitten rats was characterized by a reddish mucous secre- ocular and nasal discharge, piloerection, tachy/dyspnea). tion, a clinical condition never described for animals These symptoms were compatible/similar with those naturally bitten by O. brasiliensis. This finding may be observed after parasitism by O. brasiliensis in humans and associated with an increase in nasal/ocular mucous dogs (Martins et al., 2011; Reck et al., 2011, 2013a), and are permeability, which is in accordance with mucosa hyper- emia. Alternatively, this finding may be associated with a hemorrhagic tendency. However, the most reasonable cause of nasal and ocular discharge was the increased secretion of Harder's gland, a common finding in rats under highly stressful situations or after contact with irritating agents (Quinton, 2003). The occurrence of nasal and ocular discharge was also frequently observed in mice bitten by O. aff. puertoricencis (Venzal et al., 2007) and domestic ani- mals bitten by Hyalomma truncatum, the agent of a tick toxicosis syndrome called sweating sickness (Dolan and Newson, 1980). This set of observed symptoms in bitten rats is compatible with the definition of a tick toxicosis syndrome. A marked finding observed in both humans and animals bitten by O. brasiliensis is the extensive skin hemorrhagic lesion induced by tick bite. Here we registered the evolu- tion of the hemorrhagic lesion, showing it increased until Fig. 3. Tail-cut bleeding assay: blood loss after tail cut was evaluated as 12 h after tick bite, when it reached approximately 17 mm index of bleeding tendency. For data comparison, the bleeding was diameter. This hemorrhagic focus can be considered an expressed as fold-change compared to control. The results are expressed as means ± SEM of nine animals in control group and six in each experimental extensive hemorrhagic lesion and usually bitten rats take group (**P < 0.01 compared with control group). 7e10 days to heal. Just for comparison purposes, 104 J. Reck et al. / Toxicon 88 (2014) 99e106

Table 2 Blood parameters of Wistar rats bitten by O. brasiliensis ticks.

Blood parametersa Control Time after tick bite

6h 24h 72h

Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM

Eosinophil count (/mL) 380 ± 36 1100 ± 150** 980 ± 130* 1020 ± 180* Basophil count (/mL) 90 ± 25 440 ± 110* 320 ± 90 290 ± 70 CPK (U/L) 135 ± 37 149 ± 94 864 ± 156*** 729 ± 116** CPK-MB (U/L) 18 ± 627± 991± 30* 31 ± 11 Total LDH (U/L) 111 ± 39 109 ± 28 326 ± 63* 319 ± 57* Fibrinogen (g/L) 2.8 ± 0.1 1.5 ± 0.1* 2.2 ± 0.14 5.1 ± 0.5***

*P < 0.05; **P < 0.01; ***P < 0.001. a Other clinical pathology parameters revealed no significant alterations between control and experimental groups. researchers working with snake venoms created the findings could be explained by the differences among these concept of minimal hemorrhagic dose (MHD), which is methodologies; while PT and aPTT tests evaluate only the useful to define and compare hemorrhagic snake venoms. activity and levels of coagulation factors (Parry, 1989), the The MHD is the minimum dose required to induce a tail-cut bleeding model evaluates the hemostasis in vivo, hemorrhagic lesion of 10 mm in diameter 24 h after which includes all components of this system, such as experimental venom injection, a kind of threshold for coagulation and anti-coagulation factors, platelets and hemorrhagic lesions induced by venoms (Gutierrez et al., endothelium. 1985). In this case, one tick bite is able to induce a hem- Clinical pathology information in victims of tick toxi- orrhagic lesion over the MHD threshold for hemorrhagic cosis, particularly in humans/animals bitten by Ornitho- venoms. doros ticks are quite rare (Reck et al., 2013a). In this work, The hemorrhagic lesions induced by O. brasiliensis bites we conducted a complete blood cell examination and and the finding that a dog bitten by this tick has a slight examined the most important clinical pathology delay in blood coagulation (Reck et al., 2011) suggests that biochemical markers. Increases in eosinophil and basophil O. brasiliensis secretes potent anti-hemostatics. To verify counts have frequently been found in parasitic diseases and whether O. brasiliensis bites are able to disrupt the host's in non-infectious inflammatory skin lesions, such as hemostatic system, we determined the bleeding tendency allergic reactions (Costa et al., 1997). This finding was also of bitten animals using the rat tail-cut bleeding model and present in a dog bitten by O. brasiliensis,aswellasthe also measured the coagulation parameters PT and aPTT increased CPK activity (Reck et al., 2011) and in some ex vivo. The increased bleeding time at 6 h after tick bite human cases of tick toxicosis caused by other tick species indicates a systemic hemorrhagic tendency. Intriguingly, no (Boffey and Paterson, 1973). CPK increase can be attributed alterations in PT and aPTT tests were observed. These to potential tick saliva toxicity to skeletal muscle or cardiac tissue, since damage to these tissues are the main causes of CPK increase (van der Veen and Willebrands, 1966). Lesions in cardiomyocytes were frequently related to the increase of specific CPK isoforms, particularly, the MB fraction (van der Veen and Willebrands, 1966). In bitten rats, a major increase in CPK-MB was observed 24 h after tick bite, suggesting a cardiac injury in bitten animals. In some cases, the increase in CPK-MB activity could be associated with a sub-clinical cardiopathy condition (Gautam et al., 2004). Cardiac injury is the most severe clinical consequence of tick toxicosis induced by O. savignyi, which contains potent cardiotoxins in its saliva (Mans et al., 2002). Heart inter- stitial edema and degeneration of myocytes were observed in mice bitten by O. aff. puertoricencis (Venzal et al., 2007). Despite the correlation between increased LDH activity and heart injury, this biochemical marker could be better described as a non-specific marker of cytotoxicity and tis- sue injury, since several pathological conditions can lead to an LDH activity increase (Sobel and Shell, 1972). Therefore, Fig. 4. Histological analysis of local lesions induced by O. brasiliensis bite: in bitten rats the increased LDH activity is possibly both a Histological (HE staining) findings of tick bite lesion (in rat hind-limb) at molecular marker of the extensive tissue damage at the site 72 h after bite. Note the marked subcutaneous hemorrhage (triangles) and of tick bite and also myocardium injury. edema (asterisks); muscle degeneration (filled arrows), evinced by increased The fibrinogen level showed a dual profile. At first, 6 h cytoplasmic eosinophilia and reduction of characteristic striations; and intraepidermal hemorrhage with epidermal detachment (open arrows). Bar, after tick bite, animals showed a reduction in fibrinogen 210 mm. levels, which coincides with the time of increased bleeding J. Reck et al. / Toxicon 88 (2014) 99e106 105

Fig. 5. Histological analysis and IHC against troponin C in heart tissue of rats bitten by O. brasiliensis: Serial sections of heart papillary muscle of rats 72 h after tick bites. Panel A and B, HE staining showing myocardium degeneration; note retraction of cells, enhanced cytoplasmic eosinophilia and reduction/lack of muscle characteristic striations. Panel C and D, IHC against troponin C; note the diminished or absent staining for troponin C, which indicates cell injury (Panel A and C, bar, 350 mm; Panel B and D, bar, 210 mm).

(evaluated by tail-cut model). The causes of fibrinogen microorganism. It is important to note that, recently, reduction are not clear to date, but may be associated with studying a case of a dog showing a condition compatible a fibrinolytic activity, already identified in some tick species with tick toxicosis after natural parasitism by O. brasiliensis, (Francischetti et al., 2003). The presence of a fibrinolytic the possibility of infection by Babesia spp., Hepatozoon spp., component in O. brasiliensis saliva remains as a subject for Coxiella spp., spp., Ehrlichia spp., Anaplasma spp., further investigations. Later, at 72 h after tick bite, fibrin- and six Rickettsia species were excluded (Reck et al., 2011). ogen levels increase. Although this fibrinogen increase In this sense, O. brasiliensis can cause severe non-infectious seems paradoxical, it is a frequent finding in several in- disturbances directly associated with its bite, which is flammatory situations since this protein is also considered partially reproduced in experimental conditions. Moreover, one of the most sensitive acute phase inflammation this conclusion does not rule out the possibility that O. markers (Geiger et al., 1988). brasiliensis could be vector of a tick-borne disease. Indeed, The histopathological analysis showed a local lesion this possibility in under investigation. mainly characterized by severe hemorrhage, edema, and This work showed that O. brasiliensis causes severe slight cell degeneration. The occurrence of myocardial disturbances to its hosts. In spite of slight differences be- injury is compatible with the CPK-MB increase and similar tween the condition observed in humans, dogs and rats, in to what is observed for other Ornithodoros species, such as all cases the tick bite could lead to both local and systemic O. savignyi and O. aff. puertoricencis (Venzal et al., 2007; disturbances. The finding that O. brasiliensis causes tick Mans et al., 2008). These findings support the hypothesis toxicosis together with recent epidemiological data (Reck of a cardiac injury associated with O. brasiliensis tick toxi- et al., 2013a) makes this parasite a serious public health cosis and reinforces the severity of this condition. It is hazard in southern Brazil. Also, this work reveals infor- important to note that histopathological analysis of animals mation to aid our understanding of the complex syndrome bitten by Ornithodoros species are only rarely reported in known as tick toxicosis. literature. All findings described here are compatible with an Ethical statement experimentally induced condition of tick toxicosis. The occurrence of disturbances and lesions immediately after On behalf of, and having obtained permission from all tick bite and early on after bite (24e72 h) reinforces the the authors, Dr. Jose Reck declares that: (a) this manuscript hypothesis of a non-infectious condition. Usually, in tick- has not been published in whole or in part elsewhere; (b) borne diseases, the onset of symptoms begins 5e10 days the paper is not currently being considered for publication after tick bite (Bratton and Corey, 2005). In bitten rats, elsewhere; (c) all authors have been personally and actively blood smears did not allow the identification of any blood involved in substantive work leading to the report, and will 106 J. Reck et al. / Toxicon 88 (2014) 99e106 hold themselves jointly and individually responsible for its comparaçao~ entre anestesia subaracnoidea e peridural. Rev. Bras. e content; (d) all relevant ethical safeguards have been met Anestesiol. 54 (4), 491 500. Geiger, T., Andus, T., Klapproth, J., Hirano, T., Kishimoto, T., Heinrich, P.C., in relation to animal experimentation. Dr. Jose Reck testifies 1988. Induction of rat acute-phase proteins by interleukin 6 in vivo. to the accuracy of the above on behalf of all the authors. Eur. J. Immunol. 18 (5), 717e721. Gutierrez, J.M., Gene, J.A., Rojas, G., Cerdas, L., 1985. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms Acknowledgments by a polyvalent antivenom. Toxicon 23 (6), 887e893. Hoogstraal, H., Gallagher, M.D., 1982. Blisters, pruritus, and fever after We thank Coordenacao~ de Aperfeiçoamento de Pessoal bites by the Arabian tick Ornithodoros (Alectorobius) muesebecki. ~ Lancet 320, 288e289. de Nível Superior (CAPES), Fundaçao Estadual de Pesquisa Mans, B.J., Gothe, R., Neitz, A.W., 2008. Tick toxins: perspectives on pa- Agropecuaria (FEPAGRO), Fundaçao~ Estadual de Amparo a ralysis and other forms of toxicoses caused by ticks. In: Bowman, A.S., Pesquisa do Rio Grande do Sul (FAPERGS) and Conselho Nutall, P.A. (Eds.), Ticks e Biology, Disease and Control. Cambridge fi University Press, New York, NY, pp. 108e126. Nacional de Desenvolvimento Cientí co e Tecnologico Mans, B.J., Steinmann, C.M., Venter, J.D., Louw, A.I., Neitz, A.W., 2002. (CNPq) for financial support to this work. Special thanks to Pathogenic mechanisms of sand tampan toxicoses induced by the Inspetoria Veterinaria (IVZ) from Sao~ Francisco de Paula, tick, Ornithodoros savignyi. Toxicon 40 (7), 1007e1016. ^ Martins, J.R., Doyle, R., Barros-Battesti, D.M., Onofrio, V.C., namely to Mr. Angelo Valim, for the valuable technical Guglielmone, A.A., 2011. On the occurrence of Ornithodoros brasiliensis ~ support; to Dr. Renata Terra (UFRGS) for critical review of ARAGAO (: Argasidae) in Sao~ Francisco de Paula, RS, southern this manuscript; to Dr. Fernanda Marks and Dr. Markus Brazil. Neotrop. Entomol. 40 (1), 143e144. Berger (UFRGS) for their valuable suggestions; and to Zelma Nazareth, R.A., Tomaz, L.S., Ortiz-Costa, S., Atella, G.C., Ribeiro, J.M., Francischetti, I.M., Monteiro, R.Q., 2006. Antithrombotic properties of Regina de Almeida for help in animal handling. Ixolaris, a potent inhibitor of the extrinsic pathway of the coagulation cascade. Thromb. Haemost. 96 (1), 7e13. Conflict of interest Ortmann, C., Pfeiffer, H., Brinkmann, B., 2000. A comparative study on the immunohistochemical detection of early myocardial damage. Int. J. Leg. Med. 113, 215e220. The authors declare that there are no conflicts of Parry, B.W., 1989. Laboratory evaluation of hemorrhagic coagulopathies in interest. small animal practice. Vet. Clin. N. Am. Small Anim. Pract. 19, 729e742. Pinto, C., di Primio, R., 1931. Contribuiçao~ para a biologia dos do Transparency document Estado do Rio Grande do Sul (Brasil). Rev. Med. Cir. Bras. 34, 5e6. Quinton, J.F., 2003. Nouveaux animaux de compagnie: petits mammiferes. Elsevier Masson, Issy les Moulineaux, p. 208. Transparency document related to this article can be Reck Jr., J., Berger, M., Terra, R.M., Marks, F.S., da Silva Vaz Jr., I., found online at http://dx.doi.org/10.1016/j.toxicon.2014.06. Guimaraes,~ J.A., Termignoni, C., 2009. Systemic alterations of bovine 017. hemostasis due to Rhipicephalus (Boophilus) microplus infestation. Res. Vet. Sci. 86 (1), 56e62. Reck, J., Marks, F.S., Guimaraes,~ J.A., Termignoni, C., Martins, J.R., 2013a. References Epidemiology of Ornithodoros brasiliensis (mouro tick) in the southern Brazilian highlands and the description of human and animal retro- Aragao,~ H.B., 1923. Ornithodoros brasiliensis n. sp. Bras. Med. 37, 20. spective cases of tick parasitism. Ticks Tick. Borne Dis. 4 (1e2), Aragao,~ H.B., 1931. Notas sobre os Ornithodoros brasiliensis, rostratus e 101e109. turicata. Mem. Inst. Oswaldo Cruz 25, 227e231. Reck, J., Marks, F.S., Termignoni, C., Guimaraes,~ J.A., Martins, J.R., 2013b. Aragao,~ H.B., 1936. Ixodidas brasileiros e de alguns paizes limitrophes. Ornithodoros brasiliensis (mouro tick) salivary gland homogenates Mem. Inst. Oswaldo Cruz 31, 759e844. inhibit in vivo wound healing and in vitro endothelial cell prolifera- Boffey, G.C., Paterson, D.C., 1973. Creatine phosphokinase elevation in a tion. Parasitol. Res. 112 (4), 1749e1753. case of . Can. Med. Assoc. J. 108, 866e868. Reck, J., Soares, J.F., Termignoni, C., Labruna, M.B., Martins, J.R., 2011. Tick Bratton, R.L., Corey, R., 2005. Tick-borne disease. Am. Fam. Physician 71 toxicosis in a dog due to parasitism by Ornithodoros brasiliensis (12), 2323e2330. (mouro tick). Vet. Clin. Pathol. 40 (3), 356e360. Carstens, E., Moberg, G.P., 2000. Recognizing pain and distress in labo- Sharifi, K., Mohammadi, G.R., Tafti, A.K., 2003. Outbreak of tick paralysis in ratory animals. ILAR J. 41 (2), 62e71. a nomadic sheep flock in Iran. Vet. Rec. 153, 631e632. Costa, J.J., Weller, P.F., Galli, S.J., 1997. The cells of the allergic response: Sobel, B.E., Shell, W.E., 1972. Serum enzyme determinations in the diag- mast cells, basophils, and eosinophils. JAMA 278, 1815e1822. nosis and assessment of myocardial infarction. Circulation 45 (2), di Primio, R., 1937. Transmissores de doenças no Rio Grande do Sul. Arq. 471e482. Riogrand. Med. 306, 406e408. van der Veen, K.J., Willebrands, A.F., 1966. Isoenzymes of creatine phos- Dolan, T.T., Newson, R.M., 1980. Sweating sickness in adult cattle. Trop. phokinase in tissue extracts and in normal and pathological sera. Clin. Anim. Health Prod. 12 (2), 119e124. Chim. Acta 13 (3), 312e316. Francischetti, I.M., Mather, T.N., Ribeiro, J.M., 2003. Cloning of a salivary Venzal, J.M., Estrada-Pena,~ A., Fernandez de Luco, D., 2007. Effects pro- gland metalloprotease and characterization of gelatinase and duced by the feeding of larvae of Ornithodoros aff. puertoricensis fibrin(ogen)lytic activities in the saliva of the tick vector (Acari: Argasidae) on laboratory mice. Exp. Appl. Acarol. 42, 217e223. . Biochem Biophys. Res. Commun. 305 (4), 869e875. Walker, D.H., 1998. Tick-transmitted infectious diseases in the United Gautam, P.L., Katyal, S., Wander, G.S., Kaur, H., 2004. Isquemia miocardica States. Annu Rev. Public Health 19, 237e269. silenciosa em pacientes submetidos a prostatectomia transuretral: