Study on protective effect of specific igy antibody on cholera toxin intoxicated suckling mice

Objectives: To evaluate the protective effect of anti-choleratoxin IgY antibody on choleratoxin-intoxicated animal. Subjects and methods: Suckling mice (3-5 days old) were intoxicated with choleratoxin, then treated with anti-choleratoxin IgY.

Journal of military pharmaco-medicine no1-2018 STUDY ON PROTECTIVE EFFECT OF SPECIFIC IgY ANTIBODY ON CHOLERA TOXIN-INTOXICATED SUCKLING MICE Hoang Trung Kien*; Nguyen Anh Tuan* Le Thu Hong**; Nguyen Dang Dung* SUMMARY Objectives: To evaluate the protective effect of anti-choleratoxin IgY antibody on choleratoxin-intoxicated animal Subjects and methods: Suckling mice (3 - days old) were intoxicated with choleratoxin, then treated with anti-choleratoxin IgY The protective effect of IgY antibody was evaluated by measuring survival time of choleratoxin-intoxicated suckling mice treated with anti-choleratoxin IgY and by histopathological analysis of epithelial cells of the mice's intestinal mucosa Results: After 66 hours of choleratoxin infection, 65% of mice treated with anti-choleratoxin IgY were still alive (compared to 0% in control group); pathohistological images of the mice's intestinal mucosa showed less damages in those from mice treated with anti-choleratoxin IgY antibody compared to controls Conclusions: IgY antibody against cholera toxin helps protect suckling mice from toxic effect of choleratoxin * Keywords: Vibrio cholerae; IgY antibody; Sucking mice INTRODUCTION Cholera is a serious diarrhea disease caused by gastrointestinal infection of Vibrio cholerae In 2016, 172,454 cholera cases were reported by WHO, including 1,304 cases of deaths in 42 countries in the world with fatality rate of 0.8%, and the total number of cholera cases and deaths has not decreased in the last five years, but the fatality rate for cholera is till high Following the gastrointestinal infection, the bacteria secretes choleratoxin (CT), which consists of one A (active) and five B (binding) subunits The B subunits of CT (CTB) can bind to GM1 ganglioside expressing on epithelial cells of the intestinal mucosa Once bound, the cleavage between subunit A1 and A2 segments is facilitated and A1 portion moves into the cells The A1 component stimulates the production of adenylate cyclase, leading to increased production of cyclic AMP inside the cells This increased intracellular level of cyclic AMP results in a disruption of the active transport of electrolyte across the cell membrane, which leads to fluid secretion into small intestine The diarrhea occurs when the volume of fluid entering the colon from intestine is over the reabsorptive capability of the colon * Vietnam Military Medical University ** 103 Military Hospital Corresponding author: Nguyen Dang Dung (dzungmd@yahoo.com) Date received: 20/10/2017 Date accepted: 18/12/2017 151 Journal of military pharmaco-medicine no1-2018 The utilization of antibodies specific to V cholerae exerted preventive and therapeutic effects against cholera on animal orally infected with live V cholerae However, whether IgY antibody specific to CT can protect animal from cholera-like disease caused by oral CT infection has not yet been completely elucidated In this study, we developed an animal model of CT infection and evaluate the protective effect of specific IgY antibody on CT-intoxicated suckling mice SUBJECTS AND METHODS Subjects - Suckling mice (3 - days old), regardless of gender, provided by the Animal House of Military Medical University - C0852 CT freeze-dried powder provided by Sigma - IgY isolated from egg yolk laid of hens immunized with CT by method described previously [1, 3] - Reagents used for immunohistochemical (IHC) staining Methods * Establishing choleratoxin-intoxicated animal model: Table1: Choleratoxin intoxication model on suckling mice Group Group Group Group Group Group (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) Mice given Mice given Mice given Mice given Mice given with Mice given with with 50 µL of with 50 µL of with 50 µL of with 50 µL of 50 µL of 50 µL of choleratoxin choleratoxin choleratoxin choleratoxin choleratoxin 0.008 mg/mL 0.04 mg/mL 0.2 mg/mL mg/mL mg/mL NaCl 0.9% The experimental animal was suckling mice (3 - days old), separated from their mother hours before the start of experiment A total of 30 mice were devided into groups, with mice in each group In group (control group): mice were given with 50 µL of NaCl 0.9% through esophageal intubation using a plastic flexible needle; in the remaining groups (from group to 6), mice were given with 50 µL of CT at concentrations of 0.008; 0,04; 0,2; and mg/mL in NaCl 0.9%, respectively Thereafter, all the mice were inspected every hours for diarrhea and death, of which number of mice died in each group was recorded at 2-hour time intervals until all mice in the CT-intoxicated groups died 152 Journal of military pharmaco-medicine no1-2018 * Investigation of the protective effect of specific IgY antibody against CT on CTintoxicated suckling mice: Table 2: Group Intervention Choleratoxin intoxication (0 hour) Treatment (3 hours after choleratoxin intoxication) (n = 20) (n = 20) (n = 20) (n = 20) 50 µL NaCl 0.9% 50 µL choleratoxin mg/mL 50 µL choleratoxin mg/mL 50 µL choleratoxin mg/mL 50 µL NaCl 0.9% 50 µL NaCl 0.9% 50 µL 50 µL IgY against Freund adjuvant IgY against choleratoxin groups of suckling mice (3 - days old), with 20 mice in each group, were adopted for this experiment Suckling mice were separated from their mother hours before testing number of mice died in each group recorded at 2-hour time intervals until all mice in group (treated with NaCl 0.9%, served as negative control-1 group) died In group (biological control group): mice were given with 50 µL of NaCl 0.9% through esophageal intubation; in the groups 2, 3, and 4, mice were given with 50 µL of CT at mg/mL Three hours after CT injection, mice in the groups and were given with 50 µL of NaCl 0.9%; mice in group were administered with 50 µL of IgY isolated from egg yolk laid of hens immunized with Freund adjuvant only (served as negative control2 group), meanwhile mice in group were administered with 50 µL of IgY antibody isolated from egg yolk laid of hens immunized with a mixture of CT antigen and Freund adjuvant Immunohistochemistry (IHC) technique All the mice were then inspected every hours for diarrhea and death, with * Pathohistological analysis: was adopted to determine CT on intestinal mucosal epithelium, and to evaluate the intestinal epithelial injuries of the experimental CT-intoxicated suckling mice Briefly, a mouse with anti-CT IgG antibody (the primary antibody) was incubated with intestinal specimen's slide After being washed, the slide was incubated with the secondary antibody (a biotynated rabbitanti-mouse IgG antibody) The slide was washed, and then incubated with an avidin-peroxidase conjugate Finally, diaminobenzidine substrate was added for slide's microscopic visualization and analysis 153 Journal of military pharmaco-medicine no1-2018 RESULTS AND DISCUSSION Establishment of choleratoxin intoxication model on suckling mice Graph 1: Survival time of mice after choleratoxin intoxication In an attemp to establish an animal model for CT infection, we have chosen suckling mice, since the animal was previously reported to be susceptible to cholera caused by gastrointestinal infection with live V cholerae In the present study, we investigated the susceptibility of the mice with different doses of CT infection Mice in group served as biological control group, all of which were given NaCl 0.9% only, to ensure that an the esophageal intubation and injection of the solutions did not unexpectedly kill the mice Graph presented the results of establishment of the CT-infection animal model, with a single injection (through esophageal intubation) of 50 µL of CT at 0.008 mg/mL; 0.04 mg/mL; 0.2 mg/mL; mg/mL; mg/mL for each suckling 154 mice, respectively CT infection could only kill the experimental animals significantly when using a CT solution at concentration of mg/mL and mg/mL (group and group 6), with all mice died at 58 and 66 hours following CT intubation, respectively No significant difference in survival rate and time of CT-intoxicated mice in the two groups (intoxicated with mg/mL and mg/mL of CT, respectively) was observed The survival rate of mice in group (non-CT infection group) and in groups 2, and (intoxicated with CT at concentrations of 0.008, 0.04 and 0.2 mg/mL, respectively) at 66 hours following CT infection ranged from 60 to 80% The survival time of mice in group compared to those in groups and was significantly different (p < 0.01) Journal of military pharmaco-medicine no1-2018 Analysis on pathohistological images of intestinal mucosa from CT-intoxicated mice revealed obvious presence of choleratoxin on intestinal mucosa (figure 2B), and epithelial cells' damages (figure 2A) were not observed in specimens from (A) non-CT-intoxicated mice (figure 1A) which showed negative choleratoxin staining by immuno-histochemical staining method (figure 1B); this is in accordance with the role of CT in pathogenesis of V cholerae infection (B) Figure 1: Normal intestinal mucosa of mice; (A): HE staining, X400; (B): immuno-histochemical staining, using anti-choleratoxin antibody, X400: choleratoxin (-) (A) (B) Figure 2: Intestinal mucosa of mice intoxicated with CT: (A) CT-intoxicated, no anti-CT IgY treatment; HE staining, X400; (B) CT-intoxicated, no anti-CT IgY treatment; immuno-histochemical staining with anti-choleratoxin antibody, 400X: choleratoxin (+) It was therefore revealed by the results that oral CT intubation can cause death to suckling mice (3 - days old), and the death was likely due to CT-induced cholera-like disease 155 Journal of military pharmaco-medicine no1-2018 Protective effect of anti-choleratoxin IgY on choleratoxin-intoxicated mice likely due to the specificity of the IgY to CT; in other words, it was very likely that the CT-specific IgY bound to the CT molecule and therefore neutralized it, thus exerting the therapeutic effect as observed Graph 2: Survival time of choleratoxinintoxicated mice treated with anti-choleratoxin IgY antibody Figure 3: Intestinal mucosa of CTintoxicated mice treated with anti-CT IgY; immuno-histochemical staining with anti-choleratoxin antibody, X400: choleratoxin (+) The results presented in graph showed that survival time of mice in group (CT-intoxicated mice treated with NaCl 0.9%) and in group (CT-intoxicated mice treated with IgY against Freund adjuvant, i.e IgY isolated from egg's yolk laid of hens immunized with Freund adjuvant only) was similar; additionally, the survival rates of mice in both groups at 66 hours following CT infection were 0% These results indicated that a "normal" IgY (i.e non-specifically CT-immunized IgY) had no therapeutic effect on CT infection Meanwhile, in group (CY-intoxicated mice, treated with anti-CT IgY), survival time of mice was significantly higher compared to those in groups and (p < 0.001) The results indicated that IgY specific to CT administered gastrointestinally possessed a therapeutic effect on CTintoxicated suckling mice This effect was 156 Importantly, pathohistological images of intestinal mucosa from CT-intubated mice treated with anti-CT IgY showed little epithelial damages compared to that of control (figure 3) Hirai K (2010) demonstrated that IgY against choleratoxin B (CT-B) subunit exerted therapeutic effect on suckling mice infected with live V cholerae Together with our findings in this study, it was suggested that anti-CT IgY used in our study bound to CT-B subunit on the CT molecule, therefore inhibited the binding of CT (or CT-B subunit) to its GM1 receptor on intestinal mucosa's epithelial cell Further investigation is needed to elucidate the mechanism of action of anti-CT IgY in protecting mice intoxicated with CT Journal of military pharmaco-medicine no1-2018 CONCLUSIONS Single injection of 0.05 mL choleratoxin (at concentration of mg/mL) through esophageal intubation caused cholera-like disease in suckling mice (3 - days old) In choleratoxin-intoxicated suckling mice, a single administration of anti-choleratoxin IgY gastrointestinally after infection can protect choleratoxin-intoxicated suckling mice from the toxic effect REFERENCES Hoang Trung Kien; 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