Effects of spraying different dietary killed- Tsukamurella inchonensis levels on growth performance, small intestine morphometry and immune responses in Newcastle disease vaccinated Japanese quails

Introduction: Irrational use of antibiotics as growth promoter and as therapeutic agents in livestock and poultry is a major issue under discussion. Inappropriate use of antibiotics is not only responsible for an increase in microbial resistance to antibiotics but also the presence of antibiotic residues in animal products is a topic of public health importance. The International Study Group on Antimicrobial Strategies (ISGNAS) has also mentioned the increased microbial resistance to antibiotic as a serious problem. Newcastle Disease (ND) is one of the most devastating diseases of the domestic fowl, which can cause high level mortality of these animals. ND is caused by Newcastle Diseases Virus (NDV), an avian Paramyxovirus type 1 (APMV-1) that belongs to the genus Avulavirus, family Paramyxoviridae (Silva et al., 2010). Chickens are the natural host of the virus, but it can infect a variety of avian species causing severe disease (Carrasco et al., 2016). The commercial production of Japanese quails (Coturnix coturnix japonica) is extensively distributed in several countries around the world and many studies showed that this species can easily adapt to commercial management conditions, with good performance in terms of meat and egg production (Lima et al., 2004). The purpose of this study was to evaluate the effects of heat-killed Tsukamurella inchonensis (T.inchonensis) in diet as an alternative on growth performance, intestinal morphology and immune responses of Japanese quail.
 Material and methods: Animal care and experimental designUnsexed Japanese quail (C. cotumix japonica) were obtained at one day of age acclimated to laboratory surroundings for one week before immunizations and measurements were begun. Quail were housed nine cages measuring 61 × 56 × 81 cm. Temperature in the animal room was maintained at 23˚C. Food and water were available ad libitum. Quail were fed bird food containing approximately 20% crude protein that contained no coccidiostat or other medications. Fluoresent lights provided a photoperiod of12 h light and 12 h dark. The University of Tabriz Animal care and Use Committee approved all protocols.
Seventeen Japanese quail were assigned randomly to each of nine groups in a 3 × 3 factorial design with three bacterial treatments. The quails were randomly assigned to each treatment, because sexing could not be done until later development. Quail were sometimes moved between cages if pecking started to occur. The bacterial treatments consisted of (1) a control or no bacteria group, (2) a low dose group, and (3) a high dose group. The low- and high dose groups received 10⁵ or 10⁶ CFU per bird per day in food, respectively from the first day of age.
Growth performance and sample collection Body weight gain (BWG) and feed consumtionwas recorded during grower period and finisher period. On d 14, 28 and 42, two birds closet to the median weight from each pen (6 per treatment) were randomly selected, weighed, stunned and slaughtered by exsanguination.  The parameters of investigation included hemagglutination inhibition and ELISA titer of antibodies against NDV, intestinal morphology development and IgA and cytokines titer. Feed intake were recorded daily on cage basis. Body weight was determined every week. At days 1, 7, 14, 21, 28, 35 and 42 of the experiment (42 days), antibodies against Newcastle disease were measured. At the end of days 14, 28 and 42, two quails from each cage were randomly selected and killed by cervical dislocation for histopathological and histomorphometrical studies.
Results and discussion: In the present study, we determined whether an Actinomycetales species which had previously been shown to enhance treatment of asthma and sweet-itch (Stanford and Stanford, 2012), could affect the development of antibodies in sera of chickens.  FI and FCR were not affected by different doses of T.inchonensis supplementation. The high dose in feed tended to give higher BWG than the control group (Table 2). Interferones were so named due to their anti-viral properties. They consist of two classes: Type I interferons (IFN-α and IFN-β) that have well-defined anti-viral activity, and type II interferon or IFN-γ which plays a vital role in macrophage activation and modulation of the immune system, in addition to its anti-viral activity (Wigley and Kaiser, 2003).There was a significant difference in the measurement of IL-4 and INF-α in the serum of different experimental groups on days 7 and 42. In the measurement of IgA, the high dose recipient group, had significant differences in day 42 concerning other two groups. T.inchonensis caused a statistically significant difference in antibody titer against Newcastle disease at 42 days of age in low dose group. At microscopically examination, there was no hepatotoxicity, nephrotoxicity, cardiac toxicity and neurotoxicity or other side effects in liver, kidney, heart and brain, respectively (Fig 4-5).  The histomorphometrical measurement results of different parts of small intestine and lymphoid tissue of caecal tonsil are shown in Tables 6-8 and figure 6. T.inchonensis treatments, affected (p < 0.05) lengh and thickness of villi, crypt depth, crypt number and number of goblet cells.
Conclusion: According to macroscopic and immunological findings, it can be concluded that supplementation of T.inchonensis can improve intestinal morphology and immune responses in Japanese quail.