High-frequency electroacupuncture versus carprofen in an incisional pain model in rats

The objective of the present study was to compare the effect of electroacupuncture (EA) and carprofen (CP) on postoperative incisional pain using the plantar incision (PI) model in rats. A 1-cm longitudinal incision was made through skin, fascia and muscles of a hind paw of male Wistar rats and the development of mechanical and thermal hypersensitivity was determined over 4 days using the von Frey and Hargreaves methods, respectively. Based on the experimental treatments received on the third postoperative day, the animals were divided into the following groups: PI+CP (CP, 2 mg/kg, po); PI+EAST36 (100-Hz EA applied bilaterally at the Zusanli point (ST36)); PI+EANP (EA applied to a non-acupoint region); PI+IMMO (immobilization only); PI (vehicle). In the von Frey test, the PI+EAST36 group had higher withdrawal force thresholds in response to mechanical stimuli than the PI, PI+IMMO and PI+EANP groups at several times studied. Furthermore, the PI+EAST36 group showed paw withdrawal thresholds in response to mechanical stimuli that were similar to those of the PI+CP group. In the Hargreaves test, all groups had latencies higher than those observed with PI. The PI+EAST36 group was similar to the PI+IMMO, PI+EANP and PI+CP groups. We conclude that 100-Hz EA at the ST36 point, but not at non-acupoints, can reduce mechanical nociception in the rat model of incisional pain, and its effectiveness is comparable to that of carprofen.

Conservação de inimigos naturais (Insecta) em tomateiro orgânico / Conservation of natural enemies (Insecta) in the organic tomato crop

A adoção de práticas culturais como o consórcio de culturas e o tipo de irrigação podem beneficiar a comunidade de inimigos naturais no agroecossistema ao disponibilizar micro-habitas mais favoráveis e recursos alternativos, principalmente em períodos de baixa precipitação pluviométrica. O objetivo deste trabalho foi avaliar como o consórcio tomate + coentro e o tipo de irrigação (gotejamento e aspersão) podem beneficiar a comunidade de inimigos naturais no agroecossistema do tomateiro. O trabalho foi realizado no campo experimental da Embrapa Hortaliças, Gama, DF, de setembro a novembro de 2008. Os tomateiros foram plantados em monocultura ou consorciados com o coentro e irrigados por gotejamento e por aspersão (três repetições por tratamento), formando dois conjuntos de parcelas experimentais de acordo com o tipo de irrigação. A comunidade de inimigos naturais foi amostrada por observações diretas em 20 plantas de tomate por parcela e nas plantas de coentro sacudindo-se as plantas em cima de uma bandeja onde eram coletados os insetos. A abundância, riqueza e diversidade das espécies de inimigos naturais foram maiores nas parcelas cultivadas com coentro, independente do sistema de irrigação. Nas parcelas plantadas em monocultura foram encontradas mais espécies quando o tomateiro foi irrigado por aspersão. No entanto, a resposta de cada espécie de inimigo natural ou grupo de espécies foi distinta para o consórcio ou o tipo de irrigação. Portanto, em períodos de baixa precipitação, o consórcio tomate + coentro e a irrigação por aspersão podem favorecer a conservação de inimigos naturais no agroecossistema do tomateiro orgânico.

The adoption of cultural practices such as companion plants and the kind of irrigation system can benefit the community of natural enemies in the agroecosystem due to the increase of more favorable microhabitats available and as a source of alternative resources, mainly in periods of low precipitation. This study evaluated how the tomato + coriander intercrop and the kind of irrigation (drip and sprinkler) could benefit the community of natural enemies in the tomato agroecosystem. This work was carried out in the experimental field of Embrapa Hortaliças, Gama, DF, from September to November 2008. The tomatoes were planted in monoculture or with coriander (companion plant) and irrigated by drip and sprinkler irrigation (three replicates per treatment), forming two groups of experimental plots according to the kind of irrigation. The community of natural enemies was sampled by the direct observation of specimens on 20 tomatoes plants per plot and in the treatments. Coriander plants were shaken over a plastic tray for collection of insects. The abundance, richness and diversity of natural enemies were higher in tomato + coriander plots, regardless of the irrigation system. We observed more species of natural enemies in tomato monoculture plots when it was irrigated by sprinkler irrigation. However, the cultural practices adopted had a different effect on each species or group of species. Therefore, in periods of low precipitation, the tomato + coriander consortium associated with sprinkler irrigation can enhance the conservation of natural enemies in the organic tomato agroecosystem.

Effect of L-5-Hydroxytryptophan on drinking behavior in Coturnix japonica (Temminck and Schlegel, 1849) (Galliformes: Aves): involvement of renin-angiotensin system

The purpose of this study was to explore the role of L-5-hydroxytryptophan (L-HTP) and its relationship with the renin-angiotensin system (RAS) on the drinking behavior in Japanese quails. Normally-hydrated quails that received injections of L-HTP (12.5; 25 and 50 mg.kg-1) by the intracoelomic route (ic) expressed an increase in water intake, which was inhibited by captopril, an angiotensin converting enzyme (ACE) inhibitor. In addition, captopril also induced such a response in birds under previous fluid deprivation. High doses of captopril (35-70 mg.kg-1, sc) in normally-hydrated quails decreased the spontaneous water intake while low doses of captopril (2-5 mg.kg-1, sc) did not prompt water intake after L-HTP administration. Losartan, an AT1 receptor antagonist in mammals, did not change the water intake levels in normally-hydrated or water-deprivated birds. Serotonin (5-HT) injections did not provoke its known dipsogenic response.

O objetivo deste estudo foi investigar a influência do L-5-hidroxitriptofano (L-HTP) e sua relação com o sistema renina-angiotensina (SRA) no comportamento dipsogênico de codornas. Codornas normohidratadas que receberam L-HTP em diferentes doses (12,5; 25 e 50 mg.kg-1) por via intracelomática (ic) expressaram um aumento na ingestão de água, o qual foi suprimido pela administração prévia de captopril (inibidor da ECA-enzima conversora de angiotensina). Esta ação inibitória do captopril, em menor intensidade, foi também evidenciada em aves previamente submetidas ao jejum hídrico. O tratamento isolado com captopril (35-70 mg.kg-1) reduziu consideravelmente a ingestão espontânea de água em codornas normohidratadas, enquanto baixas doses (2-5 mg.kg-1) não provocaram aumento na ingestão de água induzida pelo L-HTP. Losartan, um antagonista de receptores AT1 em mamíferos, não foi capaz de modificar os níveis de ingestão hídrica, tanto em aves normohidratadas quanto em aves privadas de água. Serotonina aplicada perifericamente não promoveu a conhecida resposta dipsogênica de mamíferos.

Nocturnal plasma levels of melatonin in quails (Coturnix japonica) injected with l-5-hydroxy-tryptophan

This study aimed to demonstrate the influence of the systemic administration of l-5-hydroxy-tryptophan (L-HTP) on the plasma levels of melatonin during the dark period in quails. Throughout daylight, the plasma levels of melatonin did not differ significantly, oscillating between 110.2 ± 15.8 pg.mL-1 and 157.4 ± 34.8 pg.mL-1, from 8 to 16 hours. L-HTP (25 mg.kg-1, through the intracelomic route) administered at 18 hours lessened significantly the nocturnal increase of the plasma levels of melatonin (controls, 327.3 ± 20.1 and 315.8 ± 20.9 pg.mL-1 vs. 242.1 ± 24.8 and 217.5 ± 21 pg.mL-1, respectively, at 20 and 24 hours, P < 0.05). The results obtained showed that the administration of LHTP reduced the nocturnal melatonin release, possibly by bringing about an increase in serotonin synthesis and synaptic release in the pineal. Therefore, the serotoninergic transmission from the raphe towards the pineal would constitute a mechanism of modulation of the synthesis and melatonin release in quails.

Este trabalho objetivou demonstrar a influência da administração sistêmica de l-5-hidroxi-triptofano (L-HTP) sobre os níveis plasmáticos de melatonina durante o período noturno em codornas. Ao longo do período claro, os níveis plasmáticos de melatonina não diferiram significativamente, oscilando entre 110,2 ± 15,8 pg.mL-1 e 157,4 ± 34,8 pg.mL-1, de 8 às 16 horas. L-HTP (25 mg.kg-1, via intracelomática), administrado às 18 horas atenuou significativamente a elevação noturna dos níveis plasmáticos de melatonina (controles, 327,3 ± 20,1 e 315,8 ± 20,9 pg.mL-1 vs. 242,1 ± 24,8 e 217,5 ± 21 pg.mL-1, respectivamente, às 20 e 24 horas, P < 0,05). Os resultados obtidos mostraram que a administração de L-HTP reduziu a liberação noturna de melatonina, possivelmente por suscitar um aumento da síntese e liberação sináptica de serotonina na pineal. Portanto, a transmissão serotoninérgica da rafe para a pineal constituiria um mecanismo de modulação da síntese e/ou liberação de melatonina em codornas.

Behavioral and neuropharmacological evidence that serotonin crosses the blood-brain barrier in Coturnix japonica (Galliformes; Aves)

This study was carried out aiming to reach behavioral and neuropharmacological evidence of the permeability of the blood-brain barrier (BBB) to serotonin systemically administered in quails. Serotonin injected by a parenteral route (250-1000 æg.kg-1, sc) elicited a sequence of behavioral events concerned with a sleeping-like state. Sleeping-like behaviors began with feather bristling, rapid oral movements, blinking and finally crouching and closure of the eyes. Previous administration of 5-HT2C antagonist, LY53857 (3 mg.kg-1, sc) reduced the episodes of feather bristling and rapid oral movements significantly but without altering the frequency of blinking and closure of the eyes. Treatment with the 5-HT2A/2C antagonist, ketanserin (3 mg.kg-1, sc) did not affect any of the responses evoked by the serotonin. Quipazine (5 mg.kg-1, sc) a 5-HT2A/2C/3 agonist induced intense hypomotility, long periods of yawning-like and sleeping-like states. Previous ketanserin suppressed gaping responses and reduced hypomotility, rapid oral movements and bristling but was ineffective for remaining responses induced by quipazine. Results showed that unlike mammals, serotonin permeates the BBB and activates hypnogenic mechanisms in quails. Studies using serotoninergic agonist and antagonists have disclosed that among the actions of the serotonin, feather bristling, rapid oral movements and yawning-like state originated from activation of 5-HT2 receptors while blinking and closure of the eyes possibly require other subtypes of receptors.

Este estudo foi desenvolvido objetivando ampliar as evidências comportamentais e neurofarmacológicas da permeabilidade da barreira hematoencefálica (BHE) à serotonina administrada sistemicamente em codornas. A serotonina injetada por via parenteral (250-1000 æg.kg-1, sc) produziu uma seqüência de eventos relacionados com um estado semelhante ao sono. Comportamentos semelhantes ao sono começaram com o eriçamento das penas, movimentos orais rápidos, piscadelas e finalmente agachamento e fechamento dos olhos. A administração prévia do antagonista do receptor 5-HT2C, LY53857 (3 mg.kg-1, sc) reduziu significativamente os episódios de eriçamento das penas e movimentos orais rápidos, mas não alterou a freqüência de piscadelas e fechamento dos olhos. Tratamento com o antagonista do receptor 5-HT2A/2C, quetanserina (3 mg.kg-1, sc) não afetou nenhuma das respostas evocadas pela serotonina. A quipazina (5 mg.kg-1, sc), um agonista dos receptores 5-HT2A/2C/3, induziu intensa hipomotilidade e longos períodos de comportamentos semelhantes ao bocejo e ao sono. O tratamento prévio com quetanserina suprimiu as reações de bocejo e reduziu a hipomotilidade, os movimentos orais rápidos e as piscadelas, mas foi sem efeito para as demais respostas induzidas pela quipazina. Os resultados mostraram que, diferentemente dos mamíferos, a serotonina atravessa a BHE e ativa mecanismos hipnogênicos em codornas. Estudos com agonistas serotoninérgicos e antagonistas revelaram que, entre as ações da serotonina, o eriçamento das penas, os movimentos orais rápidos e o comportamento semelhante ao bocejo foram originados pela ativação de receptores 5-HT2, enquanto o piscar e o fechamento dos olhos possivelmente requereu outros subtipos de receptores.

Influence of serotonergic transmission and postsynaptic 5-HT2C action on the feeding behavior of Coturnix japonica (Galliformes: Aves)

Investigamos nesse estudo o papel dos receptores 5-HT2C e da transmissão serotonérgica no controle do comportamento alimentar em codornas. Em grupo de aves em jejum, a administração do liberador de serotonina, fenfluramina (FEN) e dos agonistas 5-HT2C, mCPP e MK212, nas doses de 1,0 e 3,3 mg/Kg induziu a uma redução significativa da ingestão alimentar (0,71 ± 0,18 g e 0,47 ± 0,2 g; 0,49 ± 0,22 g e 0,48 ± 0,29 g; 0,82 ± 0,13 g e 0,71 ± 0,16 g; respectivamente). A ingestão de alimento nos grupos controles variou de 2,89 ± 0,21 g a 2,97 ± 0,22 g, 60 min após a reapresentação de alimento, P < 0,0001). Resultados similares foram obtidos com as codornas normoalimentadas. Tanto o liberador de serotonina, FEN, quanto os agonistas 5-HT2C, mCPP e MK212 em doses de 3,3 mg/Kg induziram resposta hipofágica (FEN, 0,78 ± 0,08 g; mCPP, 0,89 ± 0,07 g; MK212, 1,25 ± 0,17 g vs. controles, 2,05 ± 0,12 g, 120 min após a oferta de alimento, P < 0.0001 a P < 0.01). A administração prévia do antagonista 5-HT2C, LY53857 (5,0 mg/Kg) bloqueou a resposta hipofágica induzida pelos agonistas 5-HT2C, 60 min após a apresentação de alimento. Os resultados obtidos demonstram o papel modulatório da liberação de serotonina e dos receptores pós-sinápticos 5-HT2C, no controle do comportamento alimentar de codornas.

Chronic excitotoxic lesion of the dorsal raphe nucleus induces sodium appetite

We determined if the dorsal raphe nucleus (DRN) exerts tonic control of basal and stimulated sodium and water intake. Male Wistar rats weighing 300-350 g were microinjected with phosphate buffer (PB-DRN, N = 11) or 1 æg/0.2 æl, in a single dose, ibotenic acid (IBO-DRN, N = 9 to 10) through a guide cannula into the DRN and were observed for 21 days in order to measure basal sodium appetite and water intake and in the following situations: furosemide-induced sodium depletion (20 mg/kg, sc, 24 h before the experiment) and a low dose of dietary captopril (1 mg/g chow). From the 6th day after ibotenic acid injection IBO-DRN rats showed an increase in sodium appetite (12.0 ± 2.3 to 22.3 ± 4.6 ml 0.3 M NaCl intake) whereas PB-DRN did not exceed 2 ml (P < 0.001). Water intake was comparable in both groups. In addition to a higher dipsogenic response, sodium-depleted IBO-DRN animals displayed an increase of 0.3 M NaCl intake compared to PB-DRN (37.4 ± 3.8 vs 21.6 ± 3.9 ml 300 min after fluid offer, P < 0.001). Captopril added to chow caused an increase of 0.3 M NaCl intake during the first 2 days (IBO-DRN, 33.8 ± 4.3 and 32.5 ± 3.4 ml on day 1 and day 2, respectively, vs 20.2 ± 2.8 ml on day 0, P < 0.001). These data support the view that DRN, probably via ascending serotonergic system, tonically modulates sodium appetite under basal and sodium depletion conditions and/or after an increase in peripheral or brain angiotensin II.

c-Fos expression induced by electroacupuncture at the Zusanli point in rats submitted to repeated immobilization

In laboratory animals, acupuncture needs to be performed on either anesthetized or, if unanesthetized, restrained subjects. Both procedures up-regulate c-Fos expression in several areas of the central nervous system, representing therefore a major pitfall for the assessment of c-Fos expression induced by electroacupuncture. Thus, in order to reduce the effect of acute restraint we used a protocol of repeated restraint for the assessment of the brain areas activated by electroacupuncture in adult male Wistar rats weighing 180-230 g. Repeated immobilization protocols (6 days, 1 h/day and 13 days, 2 h/day) were used to reduce the effect of acute immobilization stress on the c-Fos expression induced by electroacupuncture at the Zusanli point (EA36S). Animals submitted to immobilization alone or to electroacupuncture (100 Hz, 2-4 V, faradic wave) in a non-point region were compared to animals submitted to electroacupuncture at EA36S (4 animals/subgroup). c-Fos expression was measured in 41 brain areas by simple counting of cells and the results are reported as number of c-Fos-immunoreactive cells/10,000 æm². The protocols of repeated immobilization significantly reduced the immobilization-induced c-Fos expression in most of the brain areas analyzed (P < 0.05). Animals of the EA36S groups had significantly higher levels of c-Fos expression in the dorsal raphe nucleus, locus coeruleus, posterior hypothalamus and central medial nucleus of the thalamus. Furthermore, the repeated immobilization protocols intensified the differences between the effects of 36S and non-point stimulation in the dorsal raphe nucleus (P < 0.05). These data suggest that high levels of stress can interact with and mask the evaluation of specific effects of acupuncture in unanesthetized animals.

A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing oaminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid rsidues, Abz-Darg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8muM, Kcat = 5.3 min-1, Kcat/Km = 2 min-1 muM-1 and Km = 5.0 muM, Kcat = 7.0 min-1, Kcat/Km = 1.4 min-1 muM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE;EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11)

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGdFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 µM,Kcat = 127 / min and Kcat / Km = 42 / min µM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demostrated by incubating Abz-GGdFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragments Abz-GGdFLRRnp accounted for more than 95 percent of the products wich were totally inhibited by 1 µM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.

A simple method using the light microscope to visualize spermatozoon tail swelling in the hypo-osmotic test

The functional integrity of the sperm (SPTZ) membrane is believed to be an important factor in fertilization. This function was assessed by Jeyendran et al. (Journal of Reproduction and Fertility, 70:219-228, 1984), who concluded that when SPTZ from normal fertile men are exposed to a hypo-osmotic solution with an ionic strength of 0.15 mol/l, 60 percent or more will exhibit tail swelling. Essentially no changes have occurred in the test procedure since it was first published, except that SPTZ could be fixed after exposure to the hypo-osmotic solution and observed at a later time using a phase-contrast microscope. We describe here a simple test which does not require phase-contrast microscopy to a read a stained preparation after the hypo-osmotic test. A drop of semen preincubated in the hypo-osmotic medium of Jeyendran et al. and fixed with 18.5 percent formalin is placed on an albumin-coated slide. A second (spreader) slide is placed on the first as a coverslip and pulled forward at moderate speed until all the sperm has been spread into a moderately thin film. The preparation is then air dried and submitted to Papanicolaou staining. The slide can be read at any time after staining with a light microscope and provides permanent documentation

The dot-ELISA test for the detection of anti-PGL-1 IgM in leprosy patients and their contacts

Since dot-ELISA has recently been reported to be a sensitive, simple and method, we have compared it with the conventional microplate ELISA method. Sera of 124 leprosy patients, 136 household and professional contacts, and 92 controls were tested for a antibodies against a Mycobacterium leprae antigen using dot-ELISA on nitrocellulose membrane filters and microplate ELISA. The sensitive of the techniques was similar for multibacillary patients, but dot-ELISA was less sensitive for paucibacillary patients although it was more specific (100%) than ELISA (93,4%). Of 21 household contacts that gave a response by ELISA, 3 were also positive by dot-ELISA; one of these 3 developed indeterminate leprosy 12 months later and the other was diagnosed as borderline lepromatous after 28 months. These data indicate that dot-ELISA has a high spedificity and can be a useful tool in field evaluation

Use of the anti-PGL-I antibody ELISA and the Mitsuda reaction in early diagnosis of leprosy

Cutaneous reaction to the Mitsuda antigen and anti-PGL-I IgM antibodies (ELISA) were determined in 134 leprosy patients, 290 household contacts, 52 healthy controls and 43 tuberculous individuals. The multibacillary patients did nort developed cell-mediated immunity (CMI), although they presented high levels of IgM (absorbance at 492 m > 0.5). The paucibacillatry patients presented CMI, although in varying degrees, and IgM levels did not exceed 0.5 absorbance units. Most of the contacts (107) showed a Mitsuda-postive test, and 25 of then were anti-PGL-I IgM seropositive (absorbance < 0.5 but > 0.22), although none became ill during the twoyear follow-up. Of the 17 Mitsuda-negative contacts, two wxhibited an immunological status of lepromatous leprosy (negative Mitsuda test and positive serology; absorvance > 0.05) and became ill (one borderline lepromatous and one indeterminate leprosy). These results show that the immunological status of lepromatous leprosy can appear prior to clinical symptoms, and thus serology for anti-PGL-I IgM together with the Mitsuda test can be useful in an activbe search for new preclinical cases among high risk populations

IgM immunoglobulins reacting with the phenolic glycolipid-1 antigen from Mycobacterium leprae in sera of leprosy patients and their contacts

For the first time in Brazil it was investigated the occurrence of IgM anti-PGL-1 in the sera of household contacts of leprozy patients using the ELISA methodology. The sera of the multipatients. It was observed a high subclinical infection incidence among household contacts (19.4%). The percentage of leprosy development was 5% (1/21) among the seropositive contact group. This finding suggests that serology could be useful as prognostic test, but for better definition is necessary to tet a population from endemic area for long period time