Proposal of abolition of the skin sensitivity test before equine rabies immune globulin application

An epizootic outbreak of rabies occurred in 1995 in Ribeirão Preto, SP, with 58 cases of animal rabies (54 dogs, 3 cats and 1 bat) confirmed by the Pasteur Institute of São Paulo, and one human death. The need to provide care to a large number of people for the application of equine rabies immune globulin (ERIG) prevented the execution of the skin sensitivity test (SST) and often also the execution of desensitization, procedures routinely used up to that time at the Emergency Unit of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (EU-UHFMRP-USP), a reference hospital for the application of heterologous sera. In view of our positive experience of several years with the abolition of SST and of the use of premedication before the application of antivenom sera, we used a similar schedule for ERIG application. Of the 1489 victims of animal bites, 1054 (71%) received ERIG; no patient was submitted to SST and all received intravenously anti-histamines (anti-H1 + anti-H2) and corticosteroids before the procedure. The patients were kept under observation for 60 to 180 minutes and no adverse reaction was observed. On the basis of these results, since December 1995 ERIG application has been decentralized in Ribeirão Preto and has become the responsibility of the Emergency Unit of the University Hospital and the Central Basic Health Unit, where the same routine is used. Since then, 4216 patients have received ERIG (1818 at the Basic Health Unit and 2398 at the EU-UHFMRP), with no problems. The ideal would be the routine use of human rabies immune globulin (HRIG) in public health programs, but this is problematic, because of their high cost. However, while this does not occur, the use of SST is no longer justified at the time of application of ERIG, in view of the clinical evidence of low predictive value and low sensitivity of SST involving the application of heterologous sera. It is very important to point out that a negative SST result may lead the health team to a feeling of false safety that no adverse reaction will occur, but this is not true for the anaphylactoid reactions. The decision to use premedication, which is based on knowledge about anaphylaxis and on the pharmacology of the medication used, is left to the judgment of health professionals, who should always be prepared for eventual untoward events.

Seroprevalence of antibodies to Venezuelan equine encephalitis complex (subtypes IAB and VI) in humans from General Belgrano Island, Formosa, Argentina

This work presents the results of the detection of antibodies (immunoglobulin G) for subtypes I and VI of VEE viruses complex (Togaviridae family) in people from the General Belgrano island, Formosa province (Argentina). The prevalence of neutralizing (NT) antibodies for subtype VI was from 30% to 70% and the prevalence of antibodies inhibitory of hemagglutination (HI) was of 0% in the first and second inquiry respectively. For the subtype IAB the prevalence of NT antibodies was from 13% to 3.6%, similar to the prevalence total for both subtypes. HI antibodies were not detected in any inquiries for any subtype. It was observed that both subtypes circulate simultaneously, while subtype VI remains constant with some peaks, subtype I was found in low level.

Fisiología de la Transferencia Pasiva de Inmunidad en Equinos. Physiology of the Transfer of Passive Immunity in Equine.

El reconocimiento temprano de anormalidades en la transferencia pasiva de inmunidad en equinos es importante para un manejo satisfactorio de los potrillos. La placenta de la yegua, epiteliocorial, no permite el pasaje de inmunoglobulinas.(Igs). La ingesta de calostro es vital ya que provee las Igs necesarias para alcanzar una concentración sérica de IgG mayor a 800 mg por ciento. Se considerara falla parcial con niveles de IgG entre 400 y 800 mgpor ciento; y total con niveles menores a 400 mg por ciento. La absorción de las Igs es máxima hasta 8 hs después del nacimiento y disminuye hasta hacerse nula a las 24 hs posparto. Los objetivos son: a) estudiar la cinética de la transferencia pasiva de Igs determinando la concentración de IgG sérica en potrillos en el primer trimestre de vida. b) relacionar la concentración de IgG del suero y calostro de la yegua con la concentración sérica de IgG en el potrillo. c) Relacionar en calostro la concentración de inmunoglobulina G con la densidad específica y la determinación semicuantitativa de inmunoglobulina G. d) Relacionar en el suero del potrillo a las 18 - 24 hs posparto la concentración de inmunoglobulina G con la densidad específica y la determinación semicuantitativa de inmunoglobulina G. Material y método: Diseño de estudio: de cohorte, observacional, descriptivo. Animales: 70 yeguas y 70 potrillos de raza Puro Polo. Calostros: 70 muestras Toma de muestras: Yeguas: se tomará una muestra de sangre en el periparto y una muestra de calostro posparto, antes del calostrado del potrillo. Potrillos: se tomarán muestras de sangre seriadas: al nacimiento (precalostrado), 6 hs posparto, 12 hs, 18 hs y 24 hs posparto y a los 21, 60 y 90 días posparto. Determinación de IgG (Suero y calostro): a) Técnica de inmunodifusión radial simple, los resultados se expresará en mg por ciento, en muestras seriadas en intervalos de tiempo preestablecidos. b)Refractometría (con refractómetro Modelo RHC-200/ATC- Arcano). c) Test de gluteralehído, Inmuno -G test. Análisis estadístico: Comparaciones de medias con prueba t apareada o de diferencia de medias, Se considera p significativa < 0,05. Se realizará un análisis de componentes principales. Se correlacionará la concentración de Ig G de suero y calostro de la yegua con la concentración en suero de potrillo. Con los resultados de este trabajo se determinarán los valores de inmunoglobulina en las yeguas y potrillos y su comportamiento en el tiempo, y se validará la sensibilidad y especificidad de las técnicas diagnósticas utilizadas. Los resultados permitirán obtener conocimientos para un manejo racional, desde la perspectiva inmunológica, de los potrillos, al establecer mediante técnicas cuantitativas y semicuantitativas los niveles de Igs séricos alcanzados, favoreciendo un diagnóstico precoz de inmunodeficiencia por fracaso de la transferencia de anticuerpos que pondría en riesgo la vida del potrillo.

Transmission and diagnosis of equine babesiosis in South Africa

The transmission and prevalence of Babesia equi and B. caballi are being studied. Rhipicephalus evertsi mimeticus an ixodid tick from Namibia was identified as a new vector of B. equi, however, R. turanicus, previously reported to be a vector, failed to transmit both B. equi and B. caballi in the laboratory. The accurate diagnosis of B. caballi is being investigated because the nature of its low level parasitaemia does not allow easy detection in thin blood smears, routinely used for diagnosis, by clinicians. Consequently its role as a pathogen remains obscure. The importance of identifying infected horses, destined for export to Babesia-free coutries, is also stressed. Thock and thin blood smears, serology (IFAT) and DNA probes are currently employed to study disease prevalence. To date 293 healthy, adult, throughbred horses have been screened by all three methods. The percentage positives are as follows: B. equi 4.4%, 70.6%, 13% and B. caballi 0.7%, 37%, 18.4% respectively. The DNA probes were more sensitive than blood smear examination for diagnosing carrier infections but are probably not sensitive enough to identify all carrier infections. A poor correlation was found between detection of the parasites' DNA and seropositivity. However, polymerase chain reaction could be used to amplify parasite DNA in a particular sample and its could result in more accurate diagnosis.

The Seroprevalence of Equine Trypanosomosis in the Pantanal

Since little information is available on the epizootiological status of Trypanosoma evansi in South America and particularly Brazil, we evaluated equine serum samples collected in 1993, 1994, 1995 and 1997 for the presence of antibodies against this trypanosome species. Our study shows corroborative evidence about the correlation among high T. evansi seroprevalence and the rainy season in the Pantanal, Brazil. The higher seroprevalence was 79.2% in horses from a ranch located in the Nhecolândia sub-region in 1994 and the lower 5.8% in animals from the same ranch in 1997. No seroprevalence was found in 1993. The possible re-introduction of T. evansi in the region as well as the relationship among our results with the outbreaks reported in 1994, are briefly discussed.

What is the most relevant standard of success in assisted reproduction?: The cumulated singleton/twin delivery rates per oocyte pick-up: the CUSIDERA and CUTWIDERA.

National and international registries are essential tools for establishing new standards and comparing success rates, but they do not take into account the total pregnancy/delivery rate per oocyte recovery. In Switzerland and Germany, because of legal constraints, a maximum of three two-pronuclear zygotes are allocated for transfer whereas all the supernumerary pronuclear zygotes are immediately cryopreserved, preventing selection of the transferred embryos. We report on a 10 years' experience (1993-2002) of our centre which performs transfers of unselected embryos and cryopreservation at the two-pronuclear zygote stage. As approximately 30% of all deliveries are from cryo cycles, it is essential to take into account the contribution of the cryo transfers, and we propose therefore to evaluate, as a measure of IVF performance, the cumulated delivery rate per oocyte pick-up. This delivery rate is broken down further into the cumulated singleton delivery rate (CUSIDERA) and the cumulated twin delivery rate (CUTWIDERA). The sum (S) of these two rates is a measure of efficacy while the ratio CUTWIDERA/S as a percentage is a measure of safety of IVF treatments. Using these new indexes, the average 10 year efficacy and safety of our IVF programme were 26 and 19%, respectively. Both CUSIDERA and CUTWIDERA can be calculated easily in any clinical situation and yield useful parameters for patient counselling and internal/external benchmarking purposes.

Equine secretory IgA and secretory component.

A complete secretory immunologie system has been identified in the equine species. It is characterised by the presence of a secretory component either bound to secretory IgA (SigA) or remaining in the free form (FSC). The mean molecular weights of SigA, serum lgA and FSC have been estimated. The homology of the equine and human IgA classes have been demonstrated by cross-reaction with anti-human lgA antisera. A quantit ative study of equine immunoglobulins in various fluids have shown that SlgA is predominant in saliva, mature milk, nasal and lacrimal secretions, but not in colostrum. In vitro binding of human and bovine FSC is found to occur mostly with the polymerie form of equine serum lgA.

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for SLEV in equines regardless of vaccine status, and 36.4% for WEEV and 47.7% for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Development of a well-defined medium for the in vitro maturation of immature bovine cumulus-oocyte complexes.

PURPOSE: Our purpose was to develop a well-defined medium for the in vitro maturation (IVM) of immature bovine cumulus-oocyte complexes (COC). METHODS: The COC were cultured in the presence of three protein supplementations: fetal bovine serum (FBS), bovine serum albumin, and Synthetic Serum Substitute. The embryos obtained after in vitro fertilization of IVM oocytes were cocultured with Vero cells and their development to the morula and blastocyst stages was studied. RESULTS: When FBS was absent from the IVM medium, a significantly lower fertilization rate was observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. When FBS was replaced by a defined protein supplementation, the best results were obtained with Synthetic Serum Substitute. CONCLUSIONS: Adequate protein supplementation of the IVM medium optimizes the fertilization rate and the development of bovine IVM oocytes. The implication of these results in the human field is discussed.

Ejaculation failure on the day of oocyte retrieval for IVF: case report.

Unexpected ejaculation failure on the day of oocyte retrieval for IVF occurs once or twice a year in our Reproductive Medicine Unit, where approximately 500 oocyte retrievals are performed each year. Two clinical situations which occurred in 2001 are presented. In the first case, sperm were finally obtained by epididymal aspiration and resulted in the fertilization of five oocytes by ICSI. The transfer of two fresh embryos did not result in a pregnancy and the three supernumerary zygotes were cryopreserved. The male patient presented an anxio-depressive episode necessitating psychiatric hospitalization 1 week after the oocyte retrieval. In the second case, no sperm were obtained and the four oocytes were therefore lost. The couple went through a crisis in their relationship and tried another cycle of IVF 10 months later, after the preventive cryopreservation of a sperm sample. On the day of oocyte retrieval the patient was unable to produce a fresh sample but three zygotes were obtained through ICSI using the back-up cryopreserved sperm. Two embryos were transferred but no pregnancy ensued. The clinical decision-making processes for these two cases are described, as well as the measures employed to help prevent these unfortunate situations.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.