In ovo inoculation of duck embryos with different strains of Bacillus cereus to analyse their synergistic post‐hatch anti‐allergic potentialities

Abstract Background Bacillus cereus is a Gram‐positive, facultative anaerobic bacteria with few strains reported to be used as probiotics for animals and birds in recent times if the doses are formulated properly. Objectives To analyse the synergistic anti‐allergic potentiality of different Bacillus cereus strains on experimental in ovo and in vitro duck model, as probiotic immune stimulant. Materials and methods Different strains of Bacillus cereus from 29 isolates were identified through 16S rRNA gene sequencing from the milk samples of buffalo breeds of South Asia. The probiotic properties were tested in aspects of gram staining, catalase test, coagulase, test, bile salt tolerance, pH tolerance and phenol tolerance test. MIC50 and MIC90 levels were profiled using nine different antibiotics, and antimicrobial activity against eight different enteric pathogens was assessed. Finally, the test strains of Bacillus cereus (Colony Forming Unit [CFU] 30X1011) were combined‐infused at different concentrations in embryonated duck eggs to assess the post‐hatch anti‐allergic effects against histamine‐induced allergic reaction and their immunoglobulin E (IgE) level was tested. Results Molecular identification confirmed the test strains as B. cereus HKS 1–1, B. cereus LOCK 1,002 and B. cereus BF2, which were all motile, spore‐forming, catalase‐positive and rod‐shaped. All were 0.3% bile salt, 0.4% phenol and pH tolerant. Two‐way ANOVA test P values revealed that B. cereus BF2 was statistically significant (p < .0014) in bile salt tolerance test. B. cereus HKS 1–1 was significant in phenol and pH tolerance at p < .0002 and p < .0489, respectively. Besides, the test strains showed antibiotic sensitivity and antimicrobial activity to different enteric pathogens. In vivo model referred the test strains as effective in partial allergy reduction at same CFU but at different concentrations with p < .0001 among the groups. Conclusion The isolated and characterized strains of B. cereus showed partial immune‐stimulating potentiality against experimentally induced allergic reaction.


| INTRODUC TI ON
Hypersensitivity reaction is instigated by allergens which can make touching base with immune systems through inhalation, ingestion, insect bite or skin contact; the phenomenon is then known as allergy. In response to ordinary exposure to allergens (usually proteins), immunoglobulin E (IgE) antibodies are formed and typical symptoms such as asthma, rhino conjunctivitis, eczema/dermatitis etc. may be disclosed (Tang, Chang, & Chen, 2015). For decades, different types and doses of antihistamine drugs have been used to suppress unwanted allergies for human, but things are quite different for animals and birds. Along with the conventional antihistamine treatments, distinguishable natural probiotic feed supplements for chicken, ducks, geese and other birds are suggested. Bacillus subtilis is commonly referred as a probiotic immune-stabilizer against diseases for ducks (Rajput, Li, Li, Jian, & Wang, 2013), while the probiotic potentiality of Lactobacillus has already been established for duck immunization (Vasai et al., 2014).
Several studies were found successful using Bacillus subtilis in search of immunizing broilers worldwide (Jeong & Kim, 2014). In contrast, the safety of 15 commercial probiotic B. cereus isolates has already evaluated in China but the safety remains questionable in terms of mislabeling, toxin production, and transferable antimicrobial resistance (Zhu et al., 2016). Bacillus cereus is a Gram-positive, facultative aerobic bacteria and structurally spore forming rod shaped, having a ubiquitous nature of sources such as soil, decaying organic matter, vegetation, fresh and marine waters, and the invertebrate gut, along with dirt, air, and stools are various sources of the bacterium. It is also found in pasteurized as well as in raw milk of different sources (Bottone, 2010;Gherardi, 2016;Te Giffel, Beumer, Granum, & Rombouts, 1997). Due to the adhesive nature of its endospores, the bacterium spreads to all kinds of food, and thus is associated with food poisoning. Nausea, vomiting, and diarrhea with abdominal cramping can occur within 1 to 6 hours of ingestion of contaminated food (Stenfors Arnesen, Fagerlund, & Granum, 2008).
In recent times, nonpathogenic spores of Bacillus cereus have been used as animal feed supplements in diversified ways and options (Mietke, Beer, Schleif, Schabert, & Reissbrodt, 2010). The bacteriocins synthesized by B. cereus have found strong therapeutic properties against gut enteric pathogens (Naclerio, Ricca, Sacco, & Felice, 1993), and even the newly identified 'Cerein' is considered as the novel bacteriocin (Sebei, Zendo, Boudabous, Nakayama, & Sonomoto, 2007). Though there are seldom research approaches directly mentioning B. cereus-based allergy reduction in domestic animals and birds, B. cereus have been found very proactive in suppressing the allergy-stimulating mites in animal feeds via typical mite-B. cereus symbiosis formation, which newly increased the veterinary and medical importance of B. cereus in allergen detection (Erban, Rybanska, Harant, Hortova, & Hubert, 2016).
So, our focus in this study convoluted to the identified strains of B. cereus to analyse whether they are functionally probiotic in nature against different types of enteric pathogens and induced hypersensitivity reaction in experimental ducks.

| Isolation and primary identification
Milk samples of three buffalo breeds namely Nili-Ravi (Indian), Nili-Deshi (Indo-Bangladesh hybrid) and Murrah (Pakistani) were collected from Government Buffalo Farm, Bagerhat, Khulna, Bangladesh. From each sample, primary culture was prepared at dilution up to 10 -11 times and was inoculated into Bacillus cereus selective Polymyxin pyruvate egg-yolk mannitol-bromothymol blue agar (PEMBA) medium (Holbrook & Anderson, 1980) at 37°C for 24 to 72 hr separately to obtain individual colonies. A total of 29 isolates were prepared from the seventh subculture plate of each sample and three most viable isolates were finally taken for further steps.

Conclusion:
The isolated and characterized strains of B. cereus showed partial immune-stimulating potentiality against experimentally induced allergic reaction.

K E Y W O R D S
16S rRNA gene sequencing, anti-allergic potentialities, antimicrobial activity, Bacillus cereus, in ovo inoculation, probiotic properties accession numbers corresponding the bacterial strains were registered and the evolutionary relationships of the strains were analysed by MEGA5.

| Characterization for probiotic properties
The morphological (size, shape and motility); biochemical (gram staining and catalase test) and physiological (pH tolerance, bile salt tolerance and phenol tolerance) characterizations were carried out following Barai, Hossain, Rahman, Al Mazid, and Gazi (2018).

| Antimicrobial activity test
The antimicrobial activity of the isolated strains was observed with the methodology of El-Banna and Qaddoumi (2016) against clinically pre-isolated selective enteric pathogens.

| Isolation and molecular identification
The isolates were Bacillus cereus due to their survivability in B.
Cereus Selective Agar based on highly specific PEMBA medium hav-

| Biochemical tests of the isolated B. cereus strains
Bile salt tolerance test revealed that B. cereus HKS 1-1, B. cereus LOCK 1002 and B. cereus BF2 were tolerant at 0.3% concentration while, B.
cereus BF2 (p < .0014) were statistically significant ( Table 1). All of the strains were sensitive to grow at low pH (acidic conditions) but their survivability increased with the increase of pH level (Table 1), where B. cereus HKS 1-1 (p < .0489) was statistically significant and all of the test strains were found tolerant to phenol at 0.4% (Table 1), when B. cereus HKS 1-1 was highly significant (p < .0002) among the test strains.

| Antibiotic sensitivity test
The strains of B. cereus were found susceptible to different antibiotics (

| Antimicrobial activity test
Antimicrobial activity of the isolated B. cereus strains was phenomenal with a wide range of inhibition patterns (Table 3). B. cereus HKS TA B L E 1 Physiological characterization of the isolated B. cereus HKS 1-1 ( †), B. cereus LOCK 1,002 ( ‡) and B. cereus BF2 ( §) is mentioned at 24 hr of incubation, with all significant (a) Two-way ANOVA test p values (in scale of significance p < .05). The p values are .0014* ( §), .0489** ( †) and .0002*** ( †) (which are all significant values for bile salt tolerance, pH tolerance and phenol tolerance test, respectively). In all cases, ' ‡' was statistically insignificant showed minimum antagonistic traits against the selective pathogens (Table 3).

| In vivo sampling and data analysis
In this research, all the experimental ducks showed differential antiallergic response considering their in ovo belonging groups and their corresponding treatment agents during the post-hatch (in vivo) period of histamine-induced allergic reaction. TG2 executed slightly more immune stimulation against allergy than TG1 (Figure 3), which mentioned the dose effects of the combined testing of B. cereus strains.
The result of PCG was the most significant in boosting anti-allergic action as histacin was used for standardization. In contrast, NCG was the worst to response against histamine with a high pick of IgE level ( Figure 3).

| D ISCUSS I ON
Bacterial growth on PEMBA medium preliminary identified them as Bacillus cereus due to their survivability which was previously reported by Holbrook and Anderson (1980) and properties like Grampositive, rod-shaped, coagulase-positive, catalase-positive and motility which directly matched with the findings of Bottone (2010).  The evolutionary history was inferred using the Neighbour-Joining method as referred by Saitou and Nei (1987). Following Felsenstein (1985), the bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analysed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed.
The test strains were tolerant to 0.3% even after 24 hr of incubation and B. cereus BF2 was the most significant survival among the others ( Table 1). The same bile salt tolerance capabilities have already been reported to happen for other B. cereus strains like B. cereus BF2 by Kristoffersen et al. (2007) and even for some established probiotics like Lactobacillus spp. and Bifidobacterium spp. as explained by Barai et al. (2018) and cereus (Table 1) according to Singh, Singh, and Chandra (2009)  cereus has also reported by Banerjee and Ghoshal (2017).
The strains of B. cereus were found susceptible to different antibiotics (Table 2) in the Epsilometer test (E-test) method, which was referred as an authentic way of high-throughput sensitivity detection by Turnbull et al. (2004). Besides, the result of our research directly reflects the response of Bacillus spp. to different antibiotics similar to the approach of Coonrod, Leadley, and Eickhoff (1971).
The two-way ANOVA report of IgE testing among the in vivo groups was highly significant (p < .0001) and even the value remained same when all groups were compared with the NCG, except CG. The P value from t test is <0.0067 and < 0.0034 between the TG1 and TG2 and between the NCG and TG1, respectively ( Figure 3). shows the two-way ANOVA*** among the groups presented p < .0001 which is highly significant. p value from t test is <.0067* and <.0034** between the Treatment Groups 1 and 2 and between the Negative Control Group and Treatment Group 1, respectively; except CG, p < .0001 a when others are compared with the NCG; p < .0001 b when TG 1 and TG 2 are compared with CG, except the NCG and PCG (which are all significant values, in scale p < .05) newly identified and characterized B. cereus strains can be used as biocompatible and nutritious supplements with either basal diets or therapeutics for ducks as alternative preventives to suppress duck allergy allowing chemical hazard-free immunization and reproduction.

ACK N OWLED G EM ENTS
The authors extend their gratitude to the authority of Regional Duck Farm and Hatchery, Khulna and Buffalo Breeding and Development Farm, Bagerhat for providing all types of logistic supports and suggestions.

CO N FLI C T O F I NTE R E S T
The authors have no competing interest.

E TH I C A L S TATEM ENTS FO R A N I M A L R E S E A RCH
The care and handling were according to the ethical guidelines ap-