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Short Communications
Short Communications Effects of levamisole and ranitidine on antibodyforming responses induced by killed Mycoplasma vaccine antigens in Saanen goats E. M. Temizel, K. Onat, Z. Mecitoglu, S. C. Kasap, H. Gocmen, M. Ulgen Contagious agalactiae (CA), caused by Mycoplasma agalactiae, is among the most significant diseases affecting small ruminants. CA is described as an endemic disease in the Mediterranean region, where it causes significant economic impact due to reduced milk production and increased lamb mortality rates (Nicholas 1995, Bergonier and others 1997). Killed M agalactiae vaccine is used for the control of CA, but the level of acquired immunity is relatively low (Leon and others 1995). Immunotherapy as an adjunct to vaccine application may improve the resulting immune response. Various immunostimulant drugs, such as recombinant growth factors, cytokines, levamisole and beta-glucan, have been tested and used in veterinary and human medicine (Suzuki and others 1990, Qureshi and others 2000, Senturk and others 2003). Levamisole has been used for the treatment of many nematode infections in animals. The immunostimulating activity of levamisole has been well documented in several experimental and clinical studies (Neveu 1970, Symoens and Rosenthal 1977, Sharma and others 1990, Obminska and Calkosinski 1994, Senturk and others 2003, Senturk and others 2009). It is hypothesised that levamisole restores cell-mediated immune function in peripheral T lymphocytes, and stimulates phagocytosis by monocytes (Barragy 1994). Sharma and others (1990) reported that haemorrhagic septicemia vaccination combined with the administration of levamisole hydrochloride resulted in higher serum antibody titres and longer-lasting immunity (Sharma and others 1990). One mode of action of levamisole is the promotion of T-cell activity and restoration of the activities of T-helper and T-suppressor cells (Gonsette and others 1982). Some studies have also indicated that the mode of action of levamisole as an immunomodulator is achieved by boosting cell-mediated immunity via the induction Veterinary Record (2012) E. M. Temizel, Assoc. Prof., Department of Internal Medicine, K. Onat, DVM, PhD H. Gocmen, DVM, M. Ulgen, Prof., Department of Microbiology, Z. Mecitoglu, DVM, PhD S. C. Kasap, DVM, PhD Department of Internal Medicine, Faculty of Veterinary Medicine, University of Uludag, Gorukle, Bursa 16059, Turkey
doi: 10.1136/vr.101098 E-mail for correspondence:
[email protected] Provenance: not commissioned; externally peer reviewed Accepted September 21, 2012
of type 1 cytokines (Szeto and others 2000). Additionally, Olusi and others (1979) reported that levamisole significantly restored the cellmediated immune response in malnourished rats. Various animal and human studies have shown that levamisole affects the existing levels of serum immunoglobulin, or specific antibody production (Symoens and Rosenthal 1977, Sharma and others 1990, Argani and Akhtarishojaie 2006). In anergic patients, levamisole has been shown to increase the number of circulating B cells, and to enhance the antibody response to typhoid but not to influenza vaccine or to diphtheria toxoid (Lods and others 1975, Hirshaut and others 1976, Delespesse and others 1977). The effect of levamisole on the humoral immune system can be explained through the activation of T cells, the reduction of suppressor T-cell function and probably macrophage function, especially in responses requiring T- and B-cell cooperation (Hersey and Werkmeister 1981, Babiuk and Misra 1982). Ranitidine is a synthetic H2-receptor antagonist that has been used extensively to treat gastric ulcers in many species (Wallace and others 1994, Ahmed and others 2001). Oral administrations of high doses of ranitidine (45 mg/kg, orally) have been found to increase the abomasal pH in sheep for 24–48 hours (Duran and others 1993). The results of studies conducted in ruminants have also suggested that ranitidine (10–50 mg/kg) increases the abomasal pH in milk-fed calves. In practice, oral preparation of ranitidine is cheaper than parenteral preparation. Ranitidine is 3–4 times more potent than cimetidine, and the required oral doses of ranitidine are thus smaller than the required oral doses of cimetidine (Ahmed and others 2001). In human medicine, the pathophysiological significance of the histaminergic receptors located on the membranes of immunocompetent cells has been shown in various studies (Moharana and others 2000, Jutel and others 2009). Histamine affects the acquired and innate immune response by suppressing the IgG production of both B cells and T cells, and also by causing changes in some cytokines, produced by cytotoxic T lymphocytes involving both Th1 and Th2 receptors. Cytokines, such as interferon-γ (IFN-γ), are natural mediators of the immune system. Their immune-stimulating activities are well known, and their adjuvant effects have been shown in various animal species (Heath and others 1989, Cao and others 1992, Heath and Playfair 1992, Cobbold and others 1994). Histamine has a marked inhibitory effect on the production of IFN-γ via the H2 receptors (Horvath and others 1999). However, ranitidine, as an H2-receptor antagonist, completely reversed the inhibition of INF-γ production (Lagier and others 1997). The aim of the present study was to evaluate the effects of levamisole and ranitidine on the antibody response induced by killed M agalactiae vaccine in clinically healthy goats. The present study involved 48 non-pregnant female Saanen goats which were 3–5 years old (mean 3.4 years), and had body weights of 40–55 kg (mean 49 kg), which were vaccinated using an inactive killed M agalactiae vaccine. The goats included in this study were housed at a special farm located in Bursa in northwestern Turkey. All the goats were kept in different compartments within the same pen. All the animals were fed with alfalfa as forage, wheat bran, sunflower meal and soybean meal, as well as a commercial concentrate mixture for lambs, which consists of ground corn, barley, calcium carbonate and vitamin premix. The goats had free access to water. Before beginning the immunisation cycle, all the goats were clinically examined to exclude past or ongoing pathologies, especially those involving the mammary glands. In addition, to exclude any previous contact with M agalactiae, sera samples were analysed. The 48 goats were randomly divided into four groups. An inactivated monovalent vaccine (Agalaxipra, Hipra Laboratorios SA, Spain) was administrated to all the groups via two subcutaneous injections (2 ml), the first on day 0 and the second on day 21 of the experiment. Two different dose regimens of ranitidine were as follows: group A 15 mg/kg, and group B 45 mg/kg, orally (Ahmed and others 2001). Ranitidine doses were administrated once per day for one week December 8, 2012 | Veterinary Record
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Short Communications after both the first and second immunisations. Levamisole (group C 2.5 mg/kg, subcutaneous) was administrated once per day for one week after both the first and second immunisations. Unmedicated capsules (group D, placebo, orally) were administered once per day for one week after both the first and second immunisations. All applications were performed under the control and approval of the University of Uludag Ethical Committee in accordance with the Animal Welfare Guidelines (2010–01/01). Blood samples were obtained from all the goats on three different days: prior to beginning the vaccination cycle, on day 21 and on day 42. The blood samples were obtained through jugular venipuncture into empty vacuum tubes (10 ml) (Hema&Tube, Turkey) for serologic analysis. The serum samples were separated by centrifugation (3000 rpm for 20 minutes at 20°C). These samples were then tested for specific IgG antibodies against M agalactiae by indirect ELISA (ELISA M agalactiae verification, Institut Purquier, France) according to the manufacturer’s protocol, and the optical densities (OD) of the samples were evaluated at 450 nm using an ELISA reader (ELx800, Biotek Instruments, Winooski, Vermont, USA). The per cent values of the samples were calculated as described below: Sample %=Corrected OD.450*value of sample/ Mean corrected OD.450 value of positvie control×100 *Only the even-numbered column wells of the microplate are coated with the specific antigen; the odd-numbered column wells are uncoated. The corrected OD.450 values of the samples and the positive controls were calculated by subtracting the OD.450 value obtained from the uncoated well from the OD.450 value obtained from the coated well. All the data passed normality and homogeneity tests. The data were analysed using one-way repeated measurements of ANOVA, and a Tukey test was used as a post-hoc test. P value of less than 0.05 was considered as significant. All statistical analyses were performed using the Sigma Stat 3.1 for Windows statistical package (Systat Software, Inc, Point Richmond, California, USA) None of the vaccinated goats died, and the goats that received two doses of the vaccines showed no reaction after vaccination. The mean clinical parameters in all the groups were observed to be normal. The serologic results that indicate production of specific IgG antibodies against M agalactiae in all the groups of goats vaccinated by the killed Mycoplasma vaccine are shown in Table 1 as mean values. All the groups were seronegative at the beginning of the experiment, with similar antibody percentages. A similar rise in the percentages of antibodies against the killed vaccine was seen in all the groups on day 21. However, at day 42 following the first vaccination (day 21 following the second vaccination), the percentages of the antibody were almost equivalent to the values at day 21 in groups A, B and C, compared with the control group (Table 1). In the control group, in contrast with groups A, B and C, the percentage of the antibody was significantly reduced at day 42 after the first vaccination (P
