PROBIOTIC INCREASE THE ANTIRABIES HUMORAL IMMUNE RESPONSE IN BOVINE

1Universidade do Oeste Paulista. UNOESTE. Presidente Prudente. SP, Brazil. 2Universidade Norte do Paraná. Pólo de Apoio Presencial de Assis. UNOPAR VIRTUAL. Assis. SP, Brazil. 3Laboratory of Rabies. Instituto Butantan. São Paulo. SP, Brazil. *Correspondence: Rodovia Raposo Tavares, km 572. Bairro Limoeiro. CEP 19067-175. Presidente Prudente. SP, Brazil. centraldepesquisaepublicacoes@yahoo.com.br SHORT NOTE


INTRODUCTION
Rabies is an infection caused by a virus of the genus Lyssavirus, order Rhabdoviridae.It's a major animal disease in the world because causes fatal encephalitis in mammals, including humans, has wide geographical distribution and constitutes a serious health problem (Consales et al., 2006).The vaccination is the half most effective and economical instrument for the control of disease (Oliveira et al., 2000).
The use of probiotics is a strategy to increase the immunological response of animals vaccinated or infected by virus or bacteria (Hamilton-Miller, 2004).Havenaar and Huis In't Veld defined probiotics in 1992 as viable microorganisms, including lactic bacteria and yeast, like lyophilized cells or fermented products that have beneficial effects on host health after being ingested.These substances are used as growth pro-moters (Arenas et al., 2007).
In the present study was evaluated the effect of different concentrations of probiotic added to the mineral diet, in the immunological response of bovine immunized with rabies vaccine.

MATERIALS AND METHODS
Nellore cattle (Bos taurus indicus) aged 18 months, from a farm in Presidente Bernardes, SP, Brazil was utilized in this study.These animals were divided randomly into 5 groups (N= 15), animals from the control group (Gc) fed pasture and received mineral salt (Premix R ® ) ad libitum and the other groups were fed in a similar system, but each one received 2, 3, 4 or 8 g probiotic (Proenzime ® ) added to 70 g of mineral salt, groups G 2 , G 3 , G 4 and G 8 respectively.This probiotic/salt ratio was chosen because in observations taken for 20 days before the experiments the daily average salt intake was estimated at 70 g per bovine.All the animals were immunized with 1 dose of rabies vaccine on the first day of the experiment (day 0).
A commercial rabies vaccine with satisfactory antigenic values contains a suspension of fixed rabies PV (Pasteur virus) obtained in BHK-21 cells, inactivated by beta-propiolactone, adsorbed in aluminum hydroxide and preserved with thimerosol at 1:10 000.
On days 0, 30 and 60, blood (10 ml) was collected from the jugular vein in vacuum tubes with no anticoagulant.After the blood samples were clotted and centrifuged 2500 rpm, 10 min, serum samples were stored at -20 °C for further determination of rabiesneutralizing antibodies in BHK-21 cells.
The neutralizing antibodies were determined by serum neutralization in BHK-21 clone 13 cells.This test is based on the Rapid fluorescent focus inhibition test (RFFIT) (Smith et al., 1996) and the fluorescent inhibition microtest (FIMT RFFIT) (Zanlan et al., 1979).
To compare the frequency of animals immunized against rabies among the experimental groups as a function of observation day, the Goodman test was used.In all the analyses, probability of error was set at 5% (Zar, 1999).

RESULTS AND DISCUSSION
The World Health Organization (WHO) recommends rabies-neutralizing antibody titers of at least 0.50 UI/ml for effective prevention in humans against rabies virus contamination.Some studies argue that this neutralizing antibody titer is the minimal level required to protect cattle (Albas et al., 2005).
On day 0, the results from titration of rabies neutralizing antibodies, sampled from the serum of cattle under the different experimental treatments, were not reactive, indicating that none of the animals had either prior contact with the rabies virus or vaccination against rabies.Thus, the variations found among rabies antibody titers were induced by rabies vaccination as well as by the probiotic treatment.
Significant interaction was found between the probiotic level and vaccination time (F (4,63) = 2.58; p= 0.046).After rabies vaccination, all the cattle had rabiesantibody titers, irrespective of whether or not they had received probiotic added to the mineral mixture.However, animals from groups G 4 and G 8 had a significant increase (p<0.05) in the production of rabies antibodies 30 and 60 days after vaccination (figure 1), and also the frequency of immunized animals (antibody titers= 0.50 UI/ml) (figure 2) on both observation days.In this way, these supplementations caused a persistent immunostimulant effect.But their action mechanism is not completely known (Silva et al., 2006).These results agree with previous studies in swine (Ávila et al., 1998) and poultry (Kabir et al., 2004) which indicate that the immune response is improved when probiotic administration is associated with vaccination.
In group G 3 , there was a significant increase (p<0.05) in rabies antibody titers on day 30, but on day 60 the titers decreased significantly (figure 1).Thus, the immu- nostimulant action caused was not persistent.
In group G 2 , there was not a significant increase (p<0.05) in the titers of rabies antibodies (figure 1).Moreover, in this group, the frequency of immunized animals decreased significantly (p<0.05) 60 days after vaccination, similarly to group Gc (figure 2).The supplementation was not an immunostimulant.
In group control cattle had the protective rabies antibody titers in any of the two observation days, i.e., all the animals were exposed to the risk of rabies contamination after 30 and 60 days of vaccination (figure 2).In fact, Albas et al. (2005), and Reis et al. (2008) report that cattle which are firstvaccinated against rabies require a booster vaccination to achieve protective titers of rabies anti-bodies.
The results found in the present study lead to the conclusion that the probiotic (Proenzime ® ) increases both the humoral immune response and the frequency of cattle immunized by rabies vaccine.