There is evidence to show that Matricaria chamomilla extract has immunomodulatory [2,5] and allogeneic properties on lymphocyte proliferation and activation of T cells, although further elucidation is needed [2]. The aim of this work was to evaluate the effect of Matricaria chamomilla CH₁₂ as well as the number of doses of rabies vaccine, on the humoral immune response in cattle.

Sixty Nelore calves (Bos taurus indicus), about 12 months old, belonging to a farm situated in Lutecia, SP, Brazil, were studied. These calves were fed on Brachiaria decumbens from an extensive pasture system and supplemented with commercial mineral salt in an ad libitum regime. The Matricaria chamomilla CH₁₂, was produced by the homeopathic Veterinary Laboratory (Arenales Fauna & Flora, Brazil). This product was composed of Matricaria chamomilla CH₁₂ , milk CH₁₂, Bixa orellana (0.75 g) and sucrose (100 g).

The experimental animals were randomly divided into four groups, FEV₁, FEV₂, V₁, and V₂ (15 animals per group). Cattle from FEV₁ and FEV₂ groups received Matricaria chamomilla CH₁₂ mixed with mineral salt (Fosbov 15; Tortuga Cia Zootécnica Agrária, Brazil) for 90 days, and animals from groups V₁ and V₂ received only mineral salt. In the first 30 days, cattle were held for adaptation to pasture conditions and determination of the consumption of Matricaria chamomilla CH₁₂ ingested with the mineral salt.

The determination of Matricaria chamomilla CH₁₂ consumption per animal was performed in the first month of the experiment as follows: the mineral salt supplemented with Matricaria chamomilla CH₁₂ was weighed, put in the feeder and, after 24 h, removed to be weighed again. The difference between the first and the second weighing divided by the number of animals that fed at the feeder was considered the average salt consumption per calf in 24 h. From these calculations, the amount of Matricaria chamomile consumed by each cattle was determined to be about 2 g a day.

We used a commercial liquid rabies vaccine (Rai-Vac; Fort Dodge Saúde Animal, Brazil) containing a suspension of fixed rabies Pasteur Virus cultured on baby hamster kidney (BHK)-21 cells, inactivated by beta-propiolactone, adsorbed to an adjuvant aluminum hydroxide and preserved with thimerosol at 1 : 10,000. The vaccine had antigen levels within the range recommended to reach an efficient immunological response, which was approved by the Brazilian Ministry of Agriculture, Livestock and Supply (MAPA). All the animals (groups FEV₁, FEV₂, V₁ and V₂) were vaccinated on day 0; the cattle from groups FEV₂ and V₂ received a second dose on day 30.

For blood sampling on days 0, 30 and 60, cattle were taken to the corral in the morning and restrained individually in a Brete chute. Blood (10 ml) was collected from the jugular vein in vacuum tubes with no anticoagulant. After the blood samples were clotted and centrifuged at 2,500 rpm for 10 min, the serum samples were stored at -20℃ for further determination of rabies-neutralizing 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 [13] and the Fluorescent Inhibition Microtest [14].

Analysis of variance followed by the Tukey-Kramer method was used to compare serum titers among the 4 groups on days 30 and 60 [3]. To compare the serum titers between days 30 and 60 within each group (groups FEV₁, FEV₂, V₁ and V₂), the Student t-test for paired samples was used. For all the analyses, the significance level was set at 5% [3].

Rabies neutralizing antibody titers are typically used to evaluate the humoral immune response in cattle after rabies vaccination [1,8,11,12]. Moreover, it is recommended by the Centers for Disease Control and Prevention (USA). The first serum samples collected from all cattle tested on day 0 (FEV₁, FEV₂, V₁ and V₂) were not reactive for rabies, indicating that these animals had no prior contact with rabies virus or the vaccine. Thus, all the antibody titers found here were induced by the rabies vaccination during the study. In the present study, Matricaria chamomilla CH₁₂ did not stimulate the production of rabies neutralizing antibodies (Fig. 1). On day 30 the antibody titers were similar between cattle that received Matricaria chamomilla CH₁₂ and the respective treatment without supplementation (FEV₁ × V₁; FEV₂ × V₂). This suggested that the immunomodulatory effect of Matricaria chamomilla, found by Amirghofram et al. [2] and Gharagozloo and Ghaderi [5] in humans, did not occur in cattle for rabies immunization.

The World Health Organization recommends rabiesneutralizing antibody titers of at least 0.5 IU/ml for effective prevention in humans against rabies virus contamination. Some studies have stated that this neutralizing antibody titer is the minimal level required to protect cattle [1,6] against rabies. However, a descriptive analysis showed that 93.3% of the cattle that received a single vaccine dose (FEV₁ and V₁) had antibody titers under 0.5 IU/ml after 60 days of vaccination, independent of the treatment with Matricaria chamomilla CH₁₂. This agrees with the report by Queiroz da Silva et al. [11], Albas et al. [1] showing that the humoral response induced by a single rabies vaccination is inefficient in protecting cattle against rabies virus because the antibodies are not produced in high quantities.

Still concerning imunization, cattle from groups FEV₂ and V₂, which were injected with rabies vaccine on days 0 and 30, had higher rabies-neutralizing antibody titers on day 60 compared to day 30 and to the groups vaccinated only once (Fig. 1). These animals had rabies-netralizing antibody titers above 0.5 UI/ml, i.e., 100% of the cattle were immunized against rabies on day 60. Indeed, other studies on cattle [1,11] show a significant increase in rabies-neutralizing antibodies after the second dose of rabies vaccine inactivated.

The results found in the present study lead to the conclusion that the use of Matricaria chamomilla CH₁₂ added to mineral salt did not affect the humoral immune response. In addition, two doses of rabies vaccine were shown to be required for rabies protection (≥ 0.5 UI/ml) in cattle.