A comparative study of direct fluorescent antibody, mouse inoculation, and tissue culture infection testing for rabies diagnoses.

The laboratory diagnosis of rabies is of fundamental importance to the evaluation of suspected cases of rabies virus (RABV) infection. Confirmation of direct fluorescent antibody test (DFAT) results via viral isolation (VI) is recommended, and the mouse inoculation test (MIT) is being replaced by the rabies tissue culture infection (RTCIT) test for ethical reasons. We evaluated 6.514 results from central nervous system (CNS) samples of different animals analyzed at the Pasteur Institute between 2008 and 2016 using the DFAT, RTCIT and MIT techniques and evaluated their concordance, sensitivity, specificity, and accuracy indices. The DFAT technique presented the best sensitivity (93.58 %), specificity (95.90 %), and accuracy (95.67 %) results. The RTCIT values of sensitivity, specificity and accuracy (70.42 %, 86.16 % and 84.62 % respectively) were lower than those of DFAT. The concordance between RTCIT and DFAT was moderate, with a kappa quotient k = 0.341. The MIT values of sensitivity, specificity, and accuracy were 89.58 %, 100 % and 98.97 % respectively. The concordance between MIT and DFAT was substantial, with a k value of 0.720. DFAT, considered the "gold standard", was effective in all animals except horses. Our analyses evidenced that DFAT presents satisfactory results, although RTCIT did not appear favorable as a confirmatory technique.

Suplementação de vacas leiteiras com homeopatia: células somáticas do leite, cortisol e imunidade / Supplementation of dairy cows with homeopathy: milk somatic cells, cortisol and immunity

Avaliou-se o efeito da suplementação de uma combinação homeopática sobre a contagem de células somáticas do leite (CCS), o teor sanguíneo de cortisol e a resposta de anticorpos neutralizantes antivírus da raiva de vacas leiteiras. Trinta e duas vacas Holandesas em lactação foram blocadas em pares e aleatoriamente alocadas a um de dois tratamentos por 63 dias, posterior a um período de padronização de 14 dias. A CCS mensurada no final da padronização ajustou os valores semanais de CCS no modelo de análise estatística. Os tratamentos foram: 150 gramas de uma combinação homeopática (Hypothalamus, 10-30; Colibacilinum, 10-30; Streptococus Beta Hemolyticum, 10-60; Streptococus Uberis, 10-60; Phytolacca, 10-60; Calcium Phosphoricum, 10-30; Natrum Muriaticum, 10-60; Urtica Urens, 10-30; Silicea Terra, 10-400) em veículo mineral, ou 150 gramas do mesmo veículo mineral (controle). A homeopatia tendeu a aumentar a CCS de 124 para 222 x1.000 células mL-1 (P=0,09) e a CCS linearizada (P=0,08). Não foram detectados efeitos de tratamento sobre a concentração sérica de cortisol após estresse induzido por aspiração percutânea do saco ventral do rúmen (P=0,59) ou sobre o título de anticorpos neutralizantes em resposta à vacinação antivírus da raiva (P=0,40). A suplementação com homeopatia tendeu a aumentar a CCS de vacas com baixa CCS.

The effect of supplementing a homeopathic combination on milk somatic cell count (SCC), blood cortisol content and the antibody response to rabies vaccination of dairy cows was evaluated. Thirty-two lactating Holstein cows were paired blocked and randomly assigned to one of two treatments for 63 days, following a 14-day standardization period. The SCC measured at the end of standardization period adjusted weekly SCC values in the statistical analysis model. Treatments were: 150 grams of a homeopathic combination (Hypothalamus, 10-30; Colibacilinum, 10-30; Streptococcus Beta Hemolyticum, 10-60, Streptococcus Uberis, 10-60; Phytolacca, 10-60; Calcium Phosphoricum, 10-30; Natrum Muriaticum, 10-60; Urtica Urens, 10-30, Silicea Terra, 10-400) in mineral vehicle, or 150 grams of the same mineral vehicle (Control). Homeopathy tended to increase SCC from 124 to 222 x1,000 cells mL-1 (P=0.09) and linear SCC (P=0.08). There were no detectable treatment effects upon serum cortisol concentration following stress induced by percutaneous aspiration of the ventral rumen (P=0.59) and upon serum antibody title in response to rabies vaccination (P=0.40). The supplementation with homeopathy tented to increase the SCC of low SCC cows.

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle.

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.

Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines.

Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac) and an inactivated-adjuvanted PV (IPVvac) vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87% of the sera showed virus-neutralizing antibody titers (VNA) higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.