Rabies virus in a pregnant naturally infected southern yellow bat (Lasiurus ega)

Current knowledge on bat lyssavirus infections in their native hosts is limited and little is known about the virulence, virus dissemination and transmission among free-living insectivorous bats. The present study is a brief description of rabies virus (RABV) dissemination in tissues of a naturally infected pregnant southern yellow bat (Lasiurus ega) and its fetuses, obtained by reverse-transcriptase polymerase chain reaction (RT-PCR). The RT-PCR was positive in samples from the brain, salivary gland, tongue, lungs, heart, kidneys and liver. On the other hand, the placenta, three fetuses, spleen, intestine and brown fat tissue tested negative. This research demonstrated the absence of rabies virus in the fetuses, thus, in this specific case, the transplacentary transmission was not observed.

Chicken embryo related (CER) cell line for quantification of rabies neutralizing antibody by fluorescent focus inhibition test.

Levels of rabies virus neutralization antibody in sera from vaccinated dogs and cattle were either measured by mouse neutralization test (MNT) or by rapid fluorescent focus inhibition test (RFFIT), performed on CER monolayers. The two tests were compared for their ability to detect the 0.5 International Units/ml (I.U.) recommended by the World Health Organization (WHO) as the minimum response for proof of rabies immunization. A significant correlation was found between the two tests (n=211; r=0.9949 in dogs and 0.9307 in cows, p<0.001), good sensitivity (87.5%), specificity (94.7%) and agreement (96.6%) as well. RFFIT method standardized on CER cell system for neutralizing antibodies detection turns the diagnosis easier and less expensive, specially when a great number of samples must be tested from endemic areas as commonly found in Brazil.

Interval between first dose and booster affected antibody production in cattle vaccinated against rabies

In this study, we compared the levels of neutralizing antibodies induced by inactivated rabies vaccine in cattle by using three alternative immunization procedures. Forty-five bovines (breed nelore) were then organized in three groups (A, B and C, with 15 animals/group). Group A received only one vaccine dose at day zero and Group B received the first dose at day zero and then another dose at day 30 (early booster). Group C was also immunized with two doses; however, the booster was postponed until day 180 after the first dose (delayed booster). Blood samples were withdrawn at days zero (before the first dose) and 30, 210, 390, and 540 after the beginning of immunization and the antibody titers were evaluated by mouse neutralization test. The protocol used to immunize Group C (booster at day 180) was clearly more efficient. In this group, antibody levels were higher and also remained higher for longer periods in comparison with the other two groups. These results show that booster timing significantly affected antibody levels. Therefore, programs addressed to control this disease in cattle should consider not only the use of a booster but also its administration time

[Effect of freezing on immunogenicity of the rabies vaccine produced in suckling mouse brain]. / Efeito do congelamento sobre a imunogenicidade da vacina contra a raiva produzida em tecido cerebral de camundongo.

The immune humoral response induced by the rabies vaccine produced in suckling mouse brain was studied in 23 dogs. The mouse neutralization test (MNT) was used to evaluate the level of rabies antibodies. Ten dogs received vaccine stored at 2 to 8 degrees C, showing the following results: 30 days after vaccination, six samples (60%) responded to the MNT; 180 days after vaccination, 4 samples (40%); and 360 days after vaccination, only one sample (10%). The remaining 13 dogs received previously frozen vaccine and 30 days after vaccination, only two samples (l5.4%) responded to the MNT. No titers were detected 180 and 360 days after vaccination. Statistical analysis of each variable used Tuckey analysis of variance, which showed statistically significant differences between the two groups.

Efeito do congelamento sobre a imunogenicidade da vacina contra a raiva produzida em tecido cerebral de camundongo / Effect of freezing on immunogenicity of the rabies vaccine produced in suckling mouse brain

The immune humoral response induced by the rabies vaccine produced in suckling mouse brain was studied in 23 dogs. The mouse neutralization test (MNT) was used to evaluate the level of rabies antibodies. Ten dogs received vaccine stored at 2 to 8 degrees C, showing the following results: 30 days after vaccination, six samples (60%) responded to the MNT; 180 days after vaccination, 4 samples (40%); and 360 days after vaccination, only one sample (10%). The remaining 13 dogs received previously frozen vaccine and 30 days after vaccination, only two samples (l5.4%) responded to the MNT. No titers were detected 180 and 360 days after vaccination. Statistical analysis of each variable used Tuckey analysis of variance, which showed statistically significant differences between the two groups.

A resposta imune humoral induzida pela vacina contra a raiva produzida em cérebros de camundongos recém-nascidos foi estudada em 23 cães e o teste de soroneutralização em camundongos foi usado para avaliação dos níveis de anticorpos rábicos. Um grupo com 10 animais recebeu vacina conservada de 2 a 8oC e apresentou os seguintes resultados: após 30 dias da vacinação 6 (60%) amostras responderam ao teste; após 180 dias 4 (40%) e após 360 dias apenas 1 (10%). O outro grupo com 13 cães recebeu vacina previamente congelada e somente 2 (15,4%) amostras no dia 30 apresentaram resposta satisfatória; os demais períodos (180 e 360) após a vacinação, não foi encontrado título. A análise estatística dos dados referentes a cada uma das variáveis consideradas no estudo foi efetuada segundo a técnica de análise de variância seguida por Tuckey e indicaram diferenças estatisticamente significativas entre os grupos.

Influence of canine brain decomposition on laboratory diagnosis of rabies.

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29 degrees C for up to 168 h. At 24 h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72 h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48 h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.

Effect of a booster-dose of rabies vaccine on the duration of virus neutralizing antibody titers in bovines.

Humoral immune response using inactivated rabies vaccine was studied in 35 nelore cross-bred bovines of western region of São Paulo state. Ninety days after vaccination, 13 (92.8%) animals presented titers > or = 0.5 IU/ml, through mouse neutralization test. After 180 days, 9 (64.3%) sera showed titers > or = 0.5 IU/ml, after 270 days, only one (7.1%) showed a titer of 0.51 IU/ml, and after 360 days, all animals showed titers < 0.5 IU/ml. Group of animals receiving booster dose 30 days after vaccination presented, two months after, all with titers > 0.5 IU/ml. At 180 days, 17 (80.9%) sera presented titers > 0.5 IU/ml; at 270 days, 15 (71.4%), with titers > or = 0.5 IU/ml and at 360 days, 4 (19.0%), with titers > or = 0.5 IU/ml. Booster-dose ensured high levels of neutralizing antibodies for at least three months, and 240 days after revaccination, 71.4% of animals were found with titers > or = 0.5 IU/ml.

Hyperimmune antirabies sera titration by standard mouse neutralization and counterimmunoelectrophoresis tests, comparing results of different laboratories.

To determine the rabies antibody level of twenty-four hyperimmune equine sera, Standard Mouse Neutralization (SMN) and Couterimmunoelectrophoresis (CIE) tests were carried out, both at the Instituto Butantan (IB) and Instituto Panamericano de Proteccíon de Alimentos y Zoonosis (INPPAZ). Statistical analysis has shown a correlation (r) of 0.9317 between the SMN and CIE performed at the IB, while at the INPPAZ it scored 0.974. Comparison of CIE data of both laboratories yielded a correlation of 0.845. The CIE technique has shown to be a sensitive and efficient as the SMN in titrating antirabies hyperimmune equine sera. Based on CIE results, a simple, rapid and inexpensive technique, titers of sera antibody can be rellably estimated in SMN test.

A comparison of two transparent film-type dressings in central venous therapy.

A prospective randomized clinical trial has been conducted to compare the clinical performance, with prolonged use, of two film-type transparent dressings used over subclavian and jugular single-lumen venous catheters. 'OpSite', a traditional dressing with a moderate moisture vapour permeability was compared with a new dressing of high moisture vapour permeability, 'OpSite IV3000'. Information was collected daily to assess the nature and incidence of complications, dressing durability and the ease of application and removal. One hundred and one patients provided two well-matched populations receiving a total of 153 dressings for a total of 780 catheter-days. No differences between the two dressings were noted with respect to the incidence of complications, such as moisture accumulation or lifting, and dressing durability. The low incidence of catheter-related sepsis ('OpSite' group three episodes and 'OpSite IV3000' group one episode) suggests that transparent dressings do not increase this risk. This clinical study demonstrated the new 'OpSite IV3000' to be easier to handle, leading to better application, improved catheter fixation and easy removal.

[Thermostability of rabies vaccine, type Fuenzalida & Palacios, for human use]. / Termoestabilidade da vacina contra a raiva, tipo Fuenzalida & Palacios, uso humano.

Ten lots of Fuenzalida & Palacios type antirabies vaccine for human use, produced at the Instituto Butantan (São Paulo, Brazil) were stored at temperatures of 45, 37, 28 and 2-8 degrees C. The potency of each lot was determined in samples taken at varied time intervals using the NIH method and lots presenting antigenic values > or = 0,3 were considered satisfactory for use. After 2 hours at 45 degrees C the antigenic value of one out of 10 lots tested was found to be less than the minimum required value. At 37 degrees C all lots maintained satisfactory antigenic values until the third day of storage, whilst at 28 and 2-8 degrees C the potency was fully maintained, respectively for 10 and 360 days. At the ideal temperature of 2-8 degrees C, 100% of the tested vaccines maintained the minimum required antigenicity for a longer period (16 months) than the expiration time of 6-12 months usually recommended for this type of biological produced in Latin American and Caribbean countries. Thus, the obtained data suggested that in countries still producing Fuenzalida & Palacios type vaccine, the expiration tim could be extended to 16 months, what could prevent the unnecessary discarding of products still in useful condition.

Imunofluorescencia realizada em cerebros de camundongos infectados com virus rabico-cepa CVS, em diferentes estagios de decomposicao / Immunofluorescence performed in brain of rabies virus infected mice - CVS cepa, at different stages of decomposition

O teste de imunofluorescencia (IF) foi avaliado na deteccao de virus rabico presente em cerebros de carcacas de camundongos infectados com virus da cepa CVS, os quais foram conseguidos atraves de uma combinacao de tratamentos, em que se variaram as temperaturas (4,25 e -20 graus Celsius) e o tempo de armazenamento. No teste de IF realizado com impressoes cerebrais de carcacas que haviam sido submetidas a temperatura de 25 graus Celsius por 12-18h, houve maior dificuldade de visualizacao imediata dos corpusculos de inclusao, enquanto que nos materiais conservados a 4 graus Celsius por ate 48h, as inclusoes foram facilmente reconhecidas. Carcacas mantidas a -20 graus Celsius mantiveram-se viaveis a identificacao pela IF mesmo apos terem sido armazenadas por 720h quando foram feitas as ultimas observacoes. Em carcacas mantidas a 25 graus Celsius por 10h, com tratamento posterior a 4 e -20 graus Celsius, o antigeno rabico nao pode ser identificado atraves da IF, em consequencia da decomposicao das carcacas que ocorrem, respectivamente, apos 10 e 24h. Recomenda-se, portanto, empregar o teste de IF, em carater de rotina, no controle de qualidade da vacina contra a Raiva, no que diz respeito a prova de virus residual (teste de verificacao da inativacao viral), de vez que ele permite esclarecer mortes assintomaticas...

[Immunofluorescence performed in brain of mice, infected with the CVS strain of the rabies virus, in different stages of decomposition]. / Imunofluorescência realizada em cérebros de camundongos infectados com vírus rábico--cepa CVS, em diferentes estágios de decomposição.

The efficiency of the fluorescent antibody (FA) test in detecting rabies virus antigen in decomposed specimens was evaluated in simulated conditions of the safety test recommended for the assessment of residual virus in inactivated rabies vaccines. The CVS-infected mice were submitted to different treatments combining time and temperature in order to cause different stages of carcass decomposition and, the FA test was carried out sequentially at pre-determined time intervals. For the materials stored at 25 degrees C, greater difficulties for prompt recognition of the inclusion bodies were found after 12-18h, whilst the specimens maintained at 4 degrees C, the inclusions were easily visualized for up to 48h. Brain smears of carcasses kept at -20 degrees C were suitable for adequate identification after 720 h of storage. In carcasses that had been maintained at 25 degrees C for 10 h with additional storage at 4 or -20 degrees C, rabies antigenicity could not be detected, respectively after 10 and 24 h, due to tissue decomposition. The authors recommend that the FA test, when applied as an additional tool for the control of the safety test of inactivated rabies vaccine using mice, care must be taken in order to avoid the use of decomposed materials.

Evaluacion de la calidad de los reactivos que se utilizan en la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos / Evaluation of the quality of the reagents employed in the counterimmunoelectrophoresis technique for the determination of rabies antibodies

En este estudio se comprobo que el Instituto Butantan produce antigenos y sueros indicadores que se pueden utilizar con exito en la prueba de contrainmunoelectroforesis para titular anticuerpos antirrabicos en personas inmunizadas. No se pudieron demostrar diferencias estadisticamente significativas entre los resultados de las pruebas de estandarizacion realizadas en el Instituto Butantan y las pruebas de control de referencia llevadas a cabo en el Centro Panamericano de Zoonosis. Se propone que el Instituto Butantan produzca y distribuya a nivel nacional los reactivos para que los laboratorios de diagnostico apliquen la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos.

[Quality of the reagents used in counterimmunoelectrophoresis technique for the determination of antirabies antibodies]. / Evaluación de la calidad de los reactivos que se utilizan en la tecnica de contrainmunoelectroforesis para la determinación de anticuerpos antirrábicos.

This study demonstrated that the antigens and indicator sera produced by the Butantan Institute may be employed with success in the counterimmunoelectrophoresis technique for the titration of rabies antibodies in sera from immunized individuals. No statistically significant differences were demonstrated between the results obtained in the standardization tests carried out at the Butantan Institute and the reference control tests performed at the Pan American Zoonoses Center. It is proposed that the Butantan Institute be in charge of the production and distribution of these reagents at the national level.

Reduced schedule of human antirabies immunization with Fuenzalida & Palacios vaccine.

The present study evaluates the humoral and cellular immune responses in 35 volunteers submitted to short antirabies vaccination schedules with the Fuenzalida & Palacios vaccine based on the administration of doses on non consecutive days. The volunteers were divided into two groups. The first group received a total number of five doses given on days 0, 4, 7, 20 and 35. The other group received four doses, the first one being a double dose given on day 0 and than three other single doses on days 7, 20 and 35. The evaluation of humoral immune response was carried out by serum neutralization (SN) and indirect immunofluorescence (IIF) tests, while the cellular immune response was evaluated by lymphoblastic transformation assay (LTA) and skin test (ST). According to our results these reduced schedules elicited early and effective humoral and cellular immune responses to rabies antigen suggesting that new reduced schedules should be extensively studied in order to give the proper bases to the proposition of changes in the current long-term schedule.

The preparation of cultured rabies virus and the production of antiserum for human use.

In this paper we describe a methodology for the preparation of the Pasteur strain of fixed rabies virus in BHK-21 clone 13 cells and also its use for the production of antisera in horses. The methodology showed here is simple, rapid, facilitates the attainment of high protective titers, and the antisera produced are of high quality.