José Reck

Proteomic analysis of cattle tick Rhipicephalus (Boophilus) microplus saliva: a comparison between partially and fully engorged females.

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.

First report of fluazuron resistance in Rhipicephalus microplus: A field tick population resistant to six classes of acaricides

The control of the cattle tick Rhipicephalus microplus is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick populations. Currently, there are six main classes of acaricides commercially available in Brazil to control cattle ticks, with fluazuron, a tick growth regulator with acaricidal properties, being the only active ingredient with no previous reports of resistance. Ticks (designated the Jaguar strain) were collected in a beef cattle ranch located at Rio Grande do Sul state, Southern Brazil, after a complaint of fluazuron treatment failure. To characterise the resistance of this strain against acaricides, larval tests were performed and showed that the Jaguar strain was resistant to all of the drugs tested: cypermethrin (resistance ratio, RR=31.242), chlorpyriphos (RR=103.926), fipronil (RR=4.441), amitraz (RR=11.907) and ivermectin (3.081). A field trial was conducted to evaluate the efficacy of fluazuron treatment in heifers that had been experimentally infested with the Jaguar or a susceptible strain. Between 14 and 28 days after treatment, the average efficacy in cattle experimentally infested with the susceptible strain was 96%, while for the Jaguar strain the efficacy was zero. Additionally, the Jaguar strain response to fluazuron was evaluated in vitro using a modified adult immersion test (AIT) and the artificial feeding assay (AFA). With the AIT, 50 ppm of fluazuron inhibited 99% of larvae hatching in the susceptible strain (POA) and less than 50% in the Jaguar strain. Results of the AFA showed a larval hatching rate of 67% at 2.5 ppm of fluazuron with the Jaguar strain; conversely, only 3% of larvae of the susceptible strain hatched at the same fluazuron concentration. The results showed here demonstrated the first case of fluazuron resistance in R. microplus and the first tick population resistant to six classes of acaricides in Brazil.

Systemic alterations of bovine hemostasis due to Rhipicephalus (Boophilus) microplus infestation.

The tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that causes considerable economic losses to cattle breeding. Although R. microplus saliva contains several molecules that interfere with the blood coagulation process, so far the systemic alterations in the host hemostatic system have not been described. This study aims to determine if R. microplus infestation induces any disturbance to the hosts hemostatic system. To address these questions, six calves were experimentally infested with 20,000 R. microplus larvae and their blood was collected before and 7, 14 and 21 days post-infestation. Collagen and ADP-induced platelet aggregation as well as coagulation (activated partial thromboplastin time and prothrombin time) decreased in infested bovines. Platelet blood count and fibrinogen increased during the course of infestation, probably as a compensatory response. These alterations may play a role in host health status, and show that the host cannot fully counteract the tick anti-hemostatic mechanisms.

Multi-antigenic vaccine against the cattle tick Rhipicephalus (Boophilus) microplus: a field evaluation.

The tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite of cattle that severely impairs livestock production. Studies on tick immunological control address mostly single-antigen vaccines. However, from the commercial standpoint, so far no single-antigen vaccine has afforded appropriate protection against all R. microplus populations. In this context, multi-antigen cocktails have emerged as a way to enhance vaccine efficacy. In this work, a multi-antigenic vaccine against R. microplus was analyzed under field conditions in naturally infested cattle. The vaccine was composed by three tick recombinant proteins from two tick species that in previous single-vaccination reports provided partial protection of confined cattle against R. microplus infestations: vitellin-degrading cysteine endopeptidase (VTDCE) and boophilus yolk pro-cathepsin (BYC) from R. microplus, and glutathione S-transferase from Haemaphysalis longicornis (GST-Hl). Increased antibody levels against three proteins were recorded after immunizations, with a distinct humoral immune response dynamics for each protein. Compared to the control group, a statistically significant lower number of semi-engorged female ticks were observed in vaccinated cattle after two inoculations. This reduction persisted for 3 months, ranging from 35.3 to 61.6%. Furthermore, cattle body weight gain was significantly higher in vaccinated animals when compared to control cattle. Compared to the single-antigen vaccines composed by VTDCE, BYC or GST-Hl, this three-antigen vaccine afforded higher protection levels against R. microplus infestations.

Lonomia obliqua caterpillar envenomation causes platelet hypoaggregation and blood incoagulability in rats

Envenomation caused by Lonomia obliqua is a public health hazard in Southern Brazil. Envenomed victims present severe hemorrhagic syndrome that can progress to intracranial hemorrhage and death. To understand the mechanisms that lead to hemorrhage, we investigated the platelet dysfunction and blood coagulation disturbances following experimental envenomation in rats. L. obliqua bristle extract was injected (s.c.) and blood collected at different times post-venom administration for determination of platelet response and analysis of blood coagulation. Rats presented hypofibrinogenemia and platelet hypoaggregation in platelet rich plasma (PRP). After addition of exogenous fibrinogen to PRP, platelet hypoaggregation was not corrected. Interestingly, normoaggregation was observed when platelets were separated from plasma. In addition, incubation of plasma from envenomed rats inhibits aggregation response of normal washed platelets. These results indicate that an aggregation inhibitor is generated in plasma during envenomation. Moreover, rats presented an increase in nitric oxide plasmatic levels which coincided with maximum inhibition in platelet aggregation. Animals also showed blood incoagulability and a significant increase in thrombin, plasmin and urokinase plasmatic activities. Despite this intravascular thrombin generation, only a slight decrease in platelet numbers was detected. Certainly, the platelet hypoaggregation and blood incoagulability described herein contribute to systemic bleeding observed in patients.

Multiple resistance to acaricides in field populations of Rhipicephalus microplus from Rio Grande do Sul state, Southern Brazil

Acaricide resistance is a major obstacle to the control of Rhipicephalus microplus. Historically, the indiscriminate use of chemical compounds has contributed to the selection of populations resistant to different classes of acaricides. Therefore, multiple acaricide resistance is an important threat to the chemical control of the cattle tick. To investigate the occurrence and extent of multiple resistance to acaricides in Southern Brazil we performed larval tests with cypermethrin, chlorpyriphos, amitraz, fipronil and ivermectin on 104 cattle tick field samples from different ranches in Rio Grande do Sul, between the years 2013 and 2015. Adult immersion tests with a commercial formulation mixture of chlorpyriphos and cypermethrin were performed on 75 samples. Four levels of resistance were established according to the mortality of larvae: Level I: mortality between 82% and 95%; Level II: mortality between 57% and 82%; Level III: mortality between 25% and 57%; and Level IV: mortality lower than 25%. Resistance to cypermethrin was detected in 98.08% of the samples evaluated, mostly at resistance level IV. The frequency of samples resistant to amitraz, chlorpyriphos, ivermectin and fipronil was 76.92%, 60.58%, 60.58% and 53.85% respectively. Multiple resistance to three or more compounds was found in 78.85% of the samples. The results obtained in this study are alarming and reveal a new scenario for the challenge of tick control using chemicals. This is an issue of high importance to cattle production systems where this tick is responsible for a high economic impact.

Spotted Fever Group Rickettsia in the Pampa Biome, Brazil, 2015–2016

To the Editor: Several cases of tickborne rickettsiosis have been reported in South America in recent years (1,2). In Brazil, 2 spotted fever group (SFG) Rickettsia species, R. rickettsii and Rickettsia sp. strain Atlantic Rainforest, have been identified as causes of human disease. Rio Grande do Sul is the southernmost state of Brazil and the only part of the country located in the Pampa biome. Despite confirmed cases of human spotted fever in that state since 2005, little information is available regarding Rickettsia species. We report an eco-epidemiologic investigation of R. parkeri in Amblyomma tigrinum ticks on dogs from a household (and neighborhood) where a case of human spotted fever was diagnosed.

Does Rhipicephalus microplus tick infestation increase the risk for myiasis caused by Cochliomyia hominivorax in cattle

The larval phase of Cochliomyia hominivorax (screwworm) is an obligate parasite of vertebrate animals, particularly mammals, and widespread in South America, where it remains one of the most important parasitic diseases of domestic animals. The skin of cattle highly infested by ticks, with cutaneous lesions, exudation of tissue fluid and blood scent seems to produce the ideal environment for fly attraction. However, an association between these parasites was never investigated. The aim of this work was to verify if there is an association between Rhipicephalus microplus tick load and the occurrence of C. hominivorax myiasis in cattle, and to quantify the risk. Sixty bovine (Bos taurus taurus, Angus breed) under field conditions were observed for 24 weeks, during which weekly tick counts and examination for the presence of myiasis were performed. There was a significant association between a high tick burden (24-week mean above 50 ticks per animal) and myiasis occurrence (P=0.0102). The calculated relative risk (RR) for C. hominivorax myiasis occurrence in cattle with high tick burden was 3.85 (CI95%=1.23-12.13); indicating that cattle highly parasitized by R. microplus have about four times more risk of myiasis than those with a low parasite load. As far as we aware, this is the first statistically based evidence of the relationship between R. microplus parasitic load and occurrence of myiasis by C. hominivorax. This result could be useful for the design of integrated control strategies for these parasites and to provide more information for the understanding of cattle tick parasitism in cattle production.

Lonomia obliqua venom: In vivo effects and molecular aspects associated with the hemorrhagic syndrome

Caterpillar envenomation has been an emergent health issue. Lonomia obliqua is a medically important animal that causes a hemorrhagic syndrome that can progress to acute renal failure, intracranial hemorrhage and death. In the past few years the molecular characterization of L. obliqua venom in addition to experimental models has provided fundamental information to the understanding of the envenomation syndrome. Herein studies from several authors which characterized the complex toxic-pharmacological actions of whole venom are reviewed.

Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation

The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.