The small nuclear ribonucleoprotein U1A interacts with NS5 from yellow fever virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

RNA interference inhibits yellow fever virus replication in vitro and in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Esterase profile in a pyrethroid-resistant Brazilian strain of the cattle tick Boophilus microplus (Acari, Ixodidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of macrocyclic lactone acaricides (MLs). Technical and commercial MLs were used in larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT). Tn LIT and AIT the toxicity of MLs was much higher than for LPT. In the AIT, diluting the injectable formulation of MLs in water was as effective as dilution in ethanol + Triton X-100. LC50, LC99.9 and 95% confidence limits were determined so that a discriminating dose (DD) could be set for larval and adult tests in order to diagnose potential resistance to MLs in field samples of the tick. These DDs are for Australian strains of B. microplus and may not be suitable for other strains until further work is carried out.The value of these diagnostic tests can only be verified if or when resistance to MLs emerges in ticks. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Histopathology of tick-bite lesions in naturally infested capybaras (Hydrochoerus hydrochaeris) in Brazil

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 transmission as already shown for the equivalent cell type, the neutrophil, in the reaction of the dog against the Rhipicephalus sanguineus tick.

EFFECTS OF ENDOTOXIN, INTERLEUKIN-1-BETA AND PROSTAGLANDIN INJECTIONS ON FEVER RESPONSE IN BROILERS

1. The effect of endotoxin, interleukin-1 beta and prostaglandin on fever response was studied in 80 broilers (Hubbard strain). Endotoxin (E. coli, LPS) was injected iv (1.5 mu g/kg) and icv (1.5 mu g/bird); interleukin-1 (human recombinant IL-1 beta, 80 pg/bird) and prostaglandin E(2) (5 mu g/bird) were injected icv. Indomethacin (10 mg/kg, iv) pretreatment was also used before iv endotoxin injection. 2. The results showed that indomethacin was able to block the fever response induced by iv endotoxin injection, and IL-1 beta and PGE(2) were both effective in producing fever when injected icv. These data suggest a prostaglandin-mediated fever response by broilers, and also a strong evidence of the involvement of endogenous pyrogen (interleukin-1) in fever response in birds.

Hepatocyte lesions and cellular immune response in yellow fever infection

The study of the in-situ cellular immune response is very important for the understanding of different liver infections. In the present study, 53 liver samples obtained by viscerotomy from patients who died during the course of jungle yellow fever were analyzed. The diagnosis was confirmed by serology, viral isolation and virus-specific immunohistochemistry. The specimens were analyzed by immunohistochemistry using specific antibodies for apoptosis, CD45RO, CD4, CD8, CD20, S100, CD57 and CD68. Quantitative analysis of the labeling pattern showed a clear predominance of the different phenotypes in the portal tract and midzone region of the acini. There was a predominance of T CD4+ lymphocytes, accompanied by the presence of T CD8+ lymphocytes, natural killer cells (CD57), macrophages and antigen-presenting cells (S100). The disproportion between the intensity of inflammation and the degree of hepatic injury was probably due to the intense apoptotic component, which classically does not induce an inflammatory response. The present study demonstrates that, despite the disproportion between injury and inflammation, the cellular immune response plays an important role in the pathogenesis of the hepatocytic injury observed in yellow fever, probably as a result of cytolytic actions through mechanisms involving MHC II and the activation of Fas receptors and granzymes/perforins. (C) 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

CROSS-REACTIVITY BETWEEN HARD TICK ANTIGENS

1. The present study was carried out to determine the target cells and tissues for anti-tick immunoglobulins using an indirect immunohistochemical technique.2. Sections in triplicate prepared from unfed ticks Rhipicephalus appendiculatus, R. evertsi and Amblyomma variegatum were used to assess the cross-reactivity of serum from guineapigs naturally infested with these tick species or immunized against them.3. The sections showed slight (+) to strong (++++) labelling of several structures in the tick body, e.g. salivary gland, gut lumen and malpighian tubules, depending on the serum used.4. The immune serum resulting from the immunization of guinea pigs with an extract of unfed nymphs of R. appendiculatus ticks showed the most intense cross-reactivity with the sections examined.

Dermal mast cell counts in F2 Holstein x Gir crossbred cattle artificially infested with the tick Boophilus microplus (Acari : Ixodidae)

The role of dermal mast cells (DMC) in the host resistance to ticks has been studied but it is not totally explained yet. Studies have proposed that zebuine cattle breeds, known as highly resistant to ticks, have more DMC than taurine breeds. In the present study, we compared the number of adult female ticks Boophilus microplus and the mast cells' countings in the skin of F-2 crossbred Gir x Holstein cattle, before and after tick infestation. F-2 crossbred cattle (n = 148) were divided into seven groups and artificially infested with 1.0 x 10(4) B. nticroplus larvae and, 21 days afterwards, adult female-fed ticks attached to the skin were counted. Skin biopsies were taken and examined under light microscopy with a square-lined ocular reticulum in a total area of 0.0625 mm(2) in both the superficial and deep dermis. Results demonstrated that infested F-2 crossbred cattle acquired resistance against the cattle-tick B. microplus probably associated to an increase in the dermal mast cell number. It is concluded that the tick infestation may lead to an environmental modification in the dermis of parasitized hosts due to the massive migration of mast cells or their local proliferation.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari : Ixodidae)

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Amblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 mug extract in 0.03 rut of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo(R) device before and 10 min, 1, 2,4,18, 24,48,72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60 % increase in ear thickness) and a delayed reaction (18 h) (85 % increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60 % increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

5-HT2A serotoninergic receptor in the locus coeruleus participates in the first phase of lipopolysaccharide-induced fever

This study was aimed at testing the hypothesis that serotoninergic receptors in the locus coeruleus (LC) play a role in bacterial lipopolysaccharide-induced fever. To this end, 5-HT1A (WAY-100635; 3 mu g/100 nL) and 5-HT2A (ketanserin; 2 mu g/100 nL) antagonists were microinjected into the LC and body temperature was monitored by biotelemetry. Intra-LC microinjections of ketanserin or WAY-100635 caused no change in body temperature of euthermic animals. 5-HT2A antagonism abolished the first phase of the lipopolysaccharide-induced fever. Taken together, these results indicate that serotonin acting on 5-HT2A receptors in the LC mediates the first phase of the febrile response, whereas 5-HT1A receptors are not involved in the lipopolysaccharide-induced fever.

Gene discovery in Boophilus microplus, the cattle tick - the transcriptomes of ovaries, salivary glands, and hemocytes

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus

Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome.We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium-mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro.The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.