The effect of fermented wheat germ extract (FWGE, Immunovet-HBM) was studied in chickens challenged with Mycoplasma gallisepticum. Ninety M. gallisepticum- and M. synoviae-free 3-wk-old chickens were exposed to aerosol infection of M. gallisepticum. One group (30 birds) was treated with FWGE, a second group with tiamulin, and a third group was untreated. The fourth group was exposed to PBS aerosol as a negative control. On d 9, all chickens were slaughtered and examined for the presence of gross and histological lesions, the presence of the challenge strain in the organs and specific antibodies in the serum. Body weight gains and feed conversion rates were recorded. In the groups treated with FWGE and with tiamulin, the chickens remained clinically healthy: their BW gains were 441.7 g and 446.8 g, respectively. Feed conversion ratios were 1.72 and 1.71 for FWGE- and tiamulin-treated birds, respectively. Control birds had BW gain of 480.8 g, and feed conversion ratio of 1.78. The numbers of birds with gross lesions (15 and 11, respectively) and lesion scores (25 and 25, respectively) of the FWGE- and tiamulin-treated groups were significantly lower than in the infected untreated group (25 birds, lesion score of 190). No mycoplasma was reisolated from brain, liver, spleen, heart, or kidneys of the FWGE-treated birds, and the number of mycoplasma isolations from the respiratory tract samples was less frequent (10) than from the infected untreated group (64). In addition, 35 samples from other internal organs were also positive. Twenty percent of the birds treated with FWGE showed serological response with a 5.0% reaction score, whereas in the infected untreated group, 83.3% of birds were reactors, with a 62.5% reaction score.

 In another experiment, FWGE inhibited tumor progression and metastasis in the in vivo models 3 LL-HH, B16, and HCR-25 (Hidve´gi et al., 1999b). Pretreatment of mice with FWGE increased the blastic transformation of peripheral T lymphocytes induced by Concanavalin A. When used in the treatment of thymectomized mice (C57 BI10), FWGE significantly decreased the graft survival time, showing an immunostimulatory effect (Hidve´gi et al., 1999a). Treatment with FWGE resulted in a significant decrease in the number of metastases in various tumor models, including Lewis lung carcinoma (3 LL-HH), HCR25 human colon carcinoma, and B16 melanoma. It synergistically enhanced the metastasis inhibiting effect of antineoplastic agents in C38 mouse colon carcinoma and B16 mouse melanoma models (Hidve´gi et al., 1999b). Co-administration of vitamin C with FWGE gave the strongest metastasis inhibitory effect in most of the tumor models (Hidve´gi et al., 1998). Administration of FWGE proved to be effective as supportive therapy to surgery plus chemotherapy for a colorectal cancer patient (Jakab et al., 2000). Furthermore, it did not show any toxic effects (Hidve´gi et al., 1999b). Considering these results, we believe that the effect of FWGE on chickens experimentally infected with M. gallisepticum should be investigated. MATERIALS AND METHODS Experimental Birds Arbor Acres chicks, free of M. gallisepticum (n = 140), were transported to our facility upon hatching and were reared in isolated conditions. 

The parent flock of chickens had been tested throughout the rearing and laying period by regular serological tests including rapid plate agglutination (RPA) with stained Nobilis M. gallisepticum and Nobilis M. synoviae antigens, and blocking ELISA (MyGa and MySy tests2 ) (Czifra et al., 1993, Ne´meth et al., 1993) for antibodies against M. gallisepticum and M. synoviae infections, with negative results. The negative status of experimental chickens was tested serologically by ELISA with sera collected from 20-d-old slaughtered chicks to exclude the presence of maternal antibodies. No maternal antibodies could be detected. No mycoplasma was detected in the nasal cavities, trachea, lungs, or air sacs of these chicks on medium B (Ernø and Stipkovits, 1973). The chicks were fed a standard diet containing diclazuril as an anticoccidial. Challenge At the age of 3 wk, the chickens were marked individually, weighed, and distributed into 4 groups of 30 chickens each. The groups were organized so that the average BW of the groups did not differ by Student’s t-test. Chickens were placed into a 200-L volume box. The groups were 2 MyGa and MySa tests, Diagnosticum Co. Ltd., Budapest, Hungary. 3 Biromedicin Co. Ltd., Budapest, Hungary. 4 Tiamulin, Biochemie GmbH, Kundl, Austria. treated as follows. Group 1 was not infected. Ten milliliters of sterile medium was sprayed into the box; the birds were exposed for 20 min, and then transferred to a separate room. These chickens were considered the negative (control) group. 

Group 2 was infected with an aerosol of M. gallisepticum strain 1226, cloned 3 times and filtered through a 450-nm Millipore filter. The strain was lyophilized and stored at −20°C. Before challenge, the strain was propagated in medium B to a concentration of 9.5 × 108 cfu/mL. Chickens were placed in the same box as Group 1. Ten milliliters of M. gallisepticum broth culture was sprayed into the box; after exposure for 20 min, the birds were transferred to a separate room. This group received no treatment. Group 3 was challenged in the same way as group 2 and placed in another separate room. The chickens were treated with FWGE3 via the feed, at a concentration of 3.0 g/kg of feed for 9 d. Based on preliminary studies, the optimal level of FWGE was in the range of 0.3 to 3.0 g/kg of feed. Group 4 was infected as group 2, and was transferred into a separate room and treated with 80% tiamulin4 at a concentration of 200 mg of active substance (tiamulin hydrogen fumarate) per kg of feed for 9 d. To avoid cross-contamination, different persons attended each of the groups. Feeding and management of all groups were the same. On d 9 of the experiment, chickens were slaughtered and examined for gross and histopathological lesions as well as for the presence of M. gallisepticum antibodies and mycoplasmas in the respiratory tract and inner organs. Testing the Efficacy of Treatment The chickens were examined daily for the development of clinical signs. Body weights of individual chickens were obtained before challenge and on d 9. Average BW of the groups were compared by Student’s t-test. In each group, feed consumption was recorded and divided by the weight gain of the group. 

All chickens were slaughtered on d 9 after infection. They were examined for the presence of pathological lesions induced by M. gallisepticum. The pathological lesions of left and right air sacs and peritoneum were scored from 0 to 4 as follows: 0 = no lesions; 1 = cloudy air sac wall; 2 = small quantity of serous exudate in air sac or peritoneum; 3 = serousfibrinous exudate, thickened air sac wall; and 4 = large quantity of fibrinous mass in air sacs and peritoneum. The numbers of birds with lesions and the sum of lesion scores in the groups were compared statistically by χ2 - test. Lungs, spleen, and bursa of Fabricius were examined histologically. Tissue slides were stained with hematoxylin and eosin. Lungs were examined for the presence of foci of interstitial pneumonia, lymphohistiocytic bronchitis, catarrhal pneumonia, or pleurisy. The severity of lesions was scored as follows: 0 = no lesions; 1 = slight; 2 = medium; and 3 = severe lesions. The spleen and the bursa of Fabricius were also examined for the presence of follicles rich in lymphocytes, and for the severity of lymphocyte depletion.Test for Mycoplasma Infection Swab samples from the organs placed on solid medium B were used to reisolate M. gallisepticum from the trachea, air sacs, lung, brain, liver, spleen, kidney, and heart of the chickens. Isolates were identified by epifluorescence examination (Bradbury, 1998) of colonies using conjugated anti-M. gallisepticum hyperimmune serum and species-specific PCR (Kempf et al., 1993). The proportion of reisolation in groups was compared by χ2 -test. Sera of chickens collected at slaughter were examined for the presence of specific antibodies against M. gallisepticum by the RPA test. The intensity of reactions was scored from 0 to 4.

Lymphohistiocytic bronchitis or foci of interstitial pneumonia in the lungs of noninfected chickens were found very rarely (in 6 and 2 cases, respectively). Catarrhal pneumonia and pleurisy were not detected. The incidence of lymphohistiocytic bronchitis, interstitial pneumonia, and catarrhal pneumonia in the infected untreated group was high (43, 33, and 12, respectively), whereas incidences in the FWGE- and tiamulin-treated groups was lower (P ≤ 0.001) (12, 14, and 0, respectively, in group 3 and 17, 8, and 0, respectively, in group 4), and were not different from the noninfected control group. There was no difference between the treated groups. Lymphocyte depletion in the bursa of Fabricius and spleen occurred very rarely in the noninfected group, and in the groups infected and treated with FWGE or tiamulin. In all birds, follicles rich in lymphocytes were observed. Because of the infection, the number of follicles decreased (P ≤ 0.001) in the infected untreated group. Mycoplasma Infection No mycoplasmas were reisolated from birds of the noninfected group. In contrast, mycoplasmas were isolated from most of the birds of the infected untreated groups, mainly from the trachea, lungs, and air sacs. Mycoplasmas were found relatively often (35 cases) even in other inner organs, such as the liver, spleen, heart, and kidneys. The mycoplasma reisolation rates from respiratory tract were lower ( P≤ 0.001) in the FWGE- and the tiamulintreated groups (10 and 3, respectively) than in the infected untreated group. The tiamulin-treated group had fewer mycoplasma reisolates than the FWGE-treated group (P ≤ 0.05). 

Reisolation from other inner organs was not successful in these groups. Birds in the noninfected group remained serologically negative. In the infected untreated group, 25 birds became seropositive with a reaction score of 75 (Table 3). In the treated groups, lower (P ≤ 0.001) numbers of birds (6 and 8) showed serological reaction with scores of 6 and 11 for groups 3 and 4, respectively. There was no difference between the treated groups. In the experiment presented in this paper, chickens exposed for 20 min to 9.5 × 108 cfu/L of M. gallisepticum became infected and showed clinical signs of respiratory disease, which was of fatal outcome in some birds. In the field, birds are often exposed to such conditions for several weeks. In the study birds following infection, gross and histopathological lesions were found that were characteristic of M. gallisepticum infection. The birds became serologically positive. These data confirmed the observations of other authors (Jordan et al., 1989, Kleven et al., 1991; Jordan, 1996). It is well known that tiamulin is one of most effective antimycoplasma drugs (Jordan et al., 1989). After treatment with tiamulin, infected birds remained clinically healthy: gross and histopathological lesions developed in fewer birds, and lesions were much less severe than those in the infected untreated group. The mycoplasma reisolation rate was much lower and mycoplasmas could be reisolated from the trachea only, whereas in the infected untreated group, mycoplasma was isolated from the trachea, lungs, air sacs, liver, spleen, kidney, heart, and brains. Serological responses were observed in a few birds only in the tiamulin- and FWGE-treated groups. 

Based on the experimental data obtained, FWGE, like tiamulin, was able to protect birds from M. gallisepticum infection. Infected birds treated with FWGE did not develop clinical signs, and no mortality was recorded. In the slaughtered birds, pathological lesions were mild and rarely seen. According to the results of histological examination, catarrhal pneumonia and pleurisy did not occur; lymphohistiocytic bronchitis and foci of interstitial pneumonia were recorded rarely and in a mild form.The rate of mycoplasma reisolation and detection of serological response were very low. These parameters were similar to the tiamulin-treated group. In conclusion, these findings have demonstrated the positive effects of the immunomodulator, FWGE. The effects are probably due to activation of macrophages, the induction of transcription of cytokine genes, the release of inflammatory cytokines, the increase of blastic transformation of lymphocytes (Hidve´gi et al., 1998, 1999a,b), and the reduction of MHC class I expression, thus exposing mycoplasmas to natural killer cell activity, which has been demonstrated in various tumor experiments. These findings are of considerable importance as they show that immunomodulators such as FWGE are effective against M. gallisepticum infection, similar to the wellknown antimycoplasma drug, tiamulin. Mycoplasma gallisepticum infection is widespread throughout the world, and FWGE could be used to reduce the economic losses caused by mycoplasma infection. This is especially relevant as the agriculture industry seeks to reduce its use of use of antibiotics.