Veterinary Serum and Vaccine Research Institute,

 Abbassia, Cairo,

Local immune response at respiratory tract of calves vaccinated with Pasteurella multocida vaccines

(With 4 Tables and 4 Figures)

By

A.M. Daoud; Hala A.Fadl; H. G.El-Din;

Mervat A.Elkofy and S.M. Aboul Saoud

(Received at 26/12/2004)

الاستجابة المناعية الموضعية في القناة التنفسية للعجول الصغيرة المحصنة بلقاح التسمم الدموي

 أحمد محمود داوود ، هالة أحمد فضل , حامد يوسف جمال الدين

مرفت عبد المنعم الکوفي , سيد ابو السعود

 تم في هذه الدراسة بحث مدي فاعلية تحصين العجول عن طريق الأنف بلقاح الباستريلا ملتوسيدا الحي وذلک بالمقارنة باللقاح المثبط. وقد تم تقييم الاستجابة المناعية عن طريق قياس المناعة الخلوية باختبار تحور الخلايا الليمفاوية , وقياس الاستجابة المناعية باستخدام اختنار التلازن الدموى وقياس الاستجابة المناعية السائلة باستخدام اختبار الأنزيم المرتبط ELISA  بالإضافة إلى قياس الأجسام المناعية الإفرازية من نوعIgA  باستخدام اختبار الأنزيم المرتبط ELISA . وقد ادي التحصين بلقاح الباستريلا مالتوسيدا الحي عن طريق الأنف إلى حدوث استجابة مناعية خلوية اعلي من اللقاح المثبط التقليدي, کما أدى إلى ارتفاع مستوي المناعة الموضوعية الإفرازية IgA وبشکل مبکر عنها في العجول المحصنة باللقاح المثبط بالإضافة إلى المناعة السائلة من نوع IgG.

SUMMARY

In this study we investigate theeffectiveness of intranasal vaccination of calves with live streptomycin-dependent mutant of P.multocida type B vaccine in comparison with inactivated one. Live P.multocida vaccine gave higher mitogenic response of the peripheral blood lymphocytes as measured by lymphocyte blastogenesis assay than inactivated one allover the period of the experiment. The haemagglutinating antibodies as measured by Passive haemagglutination test (HA) and the IgG as measured by ELISA rose from one day to reach the peak at 15 days, and then decline till 12 weeks post vaccination for live vaccine. However for inactivated vaccine it reached the peak at 8 weeks and then decline till 12 week post vaccination. Calves with live P.multocida vaccine gave higher and earlier secretory IgA in nasal secretions than inactivated one. In conclusion, the intranasal administration of live streptomycin-dependent serotype of P.multocida mutant vaccine was efficient and induced local cellular immune response, IgA antibodies and systemic IgG antibodies.

Key words: Respiratory tract, calves, Pasteurella multocida, vaccination.

Introduction

The protective immune response to a vaccine may be due to the presence of circulating antibody (humoral immunity), the actions of sensitized T-lymphocytes (cell-mediated immunity), the presence of secretory IgA on mucosal surfaces (mucosal immunity), and or a combination of these factors (Mestecky 1987). The most infectious agents enter via mucosal surfaces, thus, immunity at these sites would prevent initiation of infection (Babiuk et al, 1995). Since colonization of the organism on the respiratory mucosa seems to be a prerequisite for infection, antibodies in respiratory secretion may prevent or suppress the colonization of bacteria, and vaccinal immunity may be mediated by secretory antibodies. Secretory IgA is important in protecting against bacterial and viral diseases where the organism must attach to epithelial surfaces in order to produce disease (Roth, 1993). Gilmour et al, 1990, reported that locally produced humoral components, such as secretory IgA may played an active role in enhancing antibacterial defense of the lung and showed that exposure of the respiratory tract induces local and systemic immunity and hastens the elimination of inhaled bacteria in subsequent exposures. Stites et al., (1994) believed that IgA is the major immunoglobulin in nasal secretions. However, Brennan et al., (1998), found that IgG titers were higher than IgA titers in nasal secretions. Thus, the stimulation of secretory immune responses, which included mucosal IgA and IgG, the predominant antibodies in mucosal secretions are considered to be crucial to vaccine development (Outlaw et al 1990).

The nasal route for vaccination offers some important opportunities, especially for the prophylaxis of respiratory diseases (Babiuk, 1999). Live attenuated vaccines have the advantage as a natural routeof entry into the host. Aboul Saoud (1990) had previously developed a live streptomycin-dependent mutant of P.multocida type B, using N-methyl-N-nitro-N-nitrosoguandine that has been shown to be highly immunogenic in mice, rabbits and calves. This vaccinal strain offered protection in calves against challenge with the homologous virulent P.multocida. However, little is known about theefficacy of intranasal administration of some antigensin inducing protective mucosal immunity to P. multocida in calves. So the aim of this study was to investigate theeffectiveness ofintranasalvaccination of calves with live streptomycin-dependent mutant of P.multocida type B vaccine in comparison with inactivated onethroughevaluation of the cell mediated immune response using lympholcyte blastogeneesis assay, humoral and secretory IgA against P.multocida using passive HA and (ELISA).

Materials and Methods

3.1. Vaccines:

3.1.1. Live vaccine a live streptomycin-dependent mutant of P.multocida type B, developed previously by Aboul Saoud (1990) using N-methyl-N-nitro-N-nitrosoguandine was obtainedfrom

Dr. Aboul Saoud, Head of Aerobic Bacterial Vaccine Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo.

3.1.2. Dead vaccine: P.multocida strain (type B:6) a locally isolated strain from field cases of cattle with HS in Egypt (Geneidy and El-Affandy, 1963). Culture of P.multocida was prepared and standardized for vaccine production according to the method of Aboul Saoud et al., (2004).

Sterilitytests were done by culturing the vaccines onto fluid thioglycolate, soya bean casein digest and Sabouroud^,s to check  absence of bacteria before use. Also, safety tests were carried out for prepared vaccines by inoculation of a group of 10 healthy mice with 0.2 ml subcutaneously (double vaccinal dose) and observed for 7 days for any untoward effect as the procedure described in De Alwis, 1989).

3.2. Calves: Seven normal healthy Holstein-Friesian calves, six month age were used. These calves were tested to be free from nasal and systemic P.multocida antibodies. Calves were allotted to three groups: Group (1) contains 3 calves, they were vaccinated intranasally with 2 ml of a live streptomycin-dependent mutant of P.multocida vaccine (contain 10⁹ CFU) (Frank et al., 1987).Group (2) another 3 calves were vaccinated intramuscularly with 2 ml of oil adjuvants vaccine (contain 10⁹ CFU)(Kucera et al., 1981).The third group (one calf) was left as non vaccinated control.

3.3. Samples:

3.3.1. Blood samples: Blood was collected for estimation of lymphocyte transformation and serum separation before vaccination & at 1, 2, 4, 7, 10, 15, 21, 30, 45, 60, 75, 90 days post-vaccination. Sera were collected and stored at -20⁰C until assayed for serological assays for antibodies.

3.3.2. Nasal secretions:  Nasal secretion samples were also collected as described by Brennan et al., (1998) at the same time intervals of blood sampling was subjected for detection of IgG & IgA secretory anti- Pasteurella multocida antibodies.

3.4. Evaluation of cellular immunity: The mitogenic response of lymphocytes was determined by the delta of optical density (optical density) using phytohaemagglutinin as a T cell mitogen and 6– [3-(4,5-Dimethyl thiazol-2-YL) 2,5–diphenyl Tetrazolium Bromide] (MTT). This test was carried according to Momann (1983). Peripheral blood lymphocyte was isolated from heparinized blood samples collected from jugular vein of calves (2ml). The samples were overlaid carefully with equal amounts of ficol (Flow Laboratories, UK) centrifuged at 2400 rpm for 30 minute. The mononuclear leukocytes washed three times in HBSS. The viable lymphocytes were counted according to (Mayer et al., 1974) and a concentration of 5x10⁶ cells were suspended in 1 ml RPMI medium supplemented by 15% fetal calf serum (GIBCO). Tissue culture plates were used where 3 wells containing suspended lymphocytes only, 3 wells containing suspended lymphocytes and phytohemagglutinin solution and five wells containing growth medium only as control. The plates were incubated 48 hours at 37⁰C then 10μ MTT (Sigma)/well were added. Plates were incubated for 4 hours then 50μ /well SDS was added then incubated over night at 37^oC and read at 750 nm wave length.

3.5. Evaluation of humoral immune response: This was estimated   by passive HA test and (ELISA):

3.5.1. Passive haemagglutination test: Was carried out for measuring P.multocida antibodies according to Carter and Rappy (1962) in microtiter plates for measuring the passive haemagglutination antibodies by using formalized RBCs.

3.5.2. Enzyme linked immunosorbent assay (ELISA): For titration of antibody to P.multocida in serum of calves according to Marshall et al (1981) and performed in flat-bottom micro-titration plates (TPP, Switzerland). Preparation of ELISA antigen from mutant P.multocida, diluted with carbonate bicarbonate buffer pH 9.6. To the coated plates, 100 ml of tested serum, each serum sample was diluted 1:500 (best dilution titer) including the control positive (was obtainedfrom Dr. Aboul Saoud) and negative sera (Non vaccinated non infected calves) as well as the blank control (no serum, no conjugate and no substrate) and incubation at 37⁰C for one hour. Then 100 ml of diluted conjugate (horseradish peroxidase sheep anti-bovine IgG, Serotec Company, UK) 1:12,000 (best dilution titer) were added to all wells. 100 ml of substrate (OPD Sigma) was added for 15 minutes at 37⁰C. The reaction was stopped by adding 50 ml / well of 2 M. of sulphoric acid and absorbance (OD) values were read by using an ELISA reader at a wave length of 490 nm. The transformation of absorbance values into a single figure representing the antibody titer depends on using a positive serum predetermined end titer to calculate the titers of the test samples according to Williams (1987). All samples were calculated as according to the following formula:

OD serum sample   – OD negative control _{x end titer of positive serum}

OD positive serum – OD negative control

3.6. Titration of IgA in nasal secretions: Enzyme linked immunosorbent assay (ELISA) were used for detection of IgG & IgA secretory anti-P.multocida antibodies according to Brennan et al.,(1998). To the coated plates, 100 ml of tested nasal secretions, each nasal secretions sample was diluted 1:50 (best dilution titer) and incubation at 37⁰ C for one hour. Then 100 ml of diluted conjugate (horseradish peroxidase sheep anti-bovine IgA, Serotec, Company, UK) 1:2,000 (best dilution titer) were added to all wells and 1:12,000 for IgG and completed as IgG.

Results

The mitogenic response of the peripheral blood lymphocytes of live vaccine were elevated from one day to reach the peak at 7 days post vaccination and then decline till 4 weeks. The mitogenic response of the peripheral blood lymphocytes of inactivated vaccine were elevated from one day to reach the peak at two weeks post vaccination and then decline till 4 weeks the end of observation period (table and figure 1).

As shown in table and figure 2, the haemagglutinating antibody after vaccination with live vaccine was rose from one day to reach the peak at 15 days post vaccination and then decline till 4 weeks. the end of observation period. For inactivated vaccine it rose from one day to reach the peak at 8 weeks post vaccination and then decline till 12 weeks the end of observation period.

It can be shown from table and figure 3, that IgGin serum of calves after vaccination with live vaccine as measured by ELISA were rose from one day to reach its peak at 3 weeks post vaccination and then decline till 12 weeks. After vaccination with inactivated vaccine IgG was rose from one day to reach the peak at 8 weeks post vaccination and then decline till 12 weeks.  

It can be seen from (table and figure 4) that IgA in nasal secretions of calves after vaccination with live vaccine as measured by ELISA was rose from one day to reach the peak at 3 weeks post vaccination and then decline till 12 weeks. IgA in nasal secretions of calves after vaccination with dead vaccine was rose from one day to reach the peak at 8 weeks post vaccination and then decline till 12 weeks.

The haemagglutinating antibody did not rise in nasal secretions of calves after vaccination with live and dead vaccines as measured by passive haemagglutination test. Also, there was a tracing of antibodies of Anti-P.multocida IgG in nasal secretions of calves after vaccination with live vaccine and dead vaccines as measured by ELISA.

Discussion

Induction of mucosal immunity begins with the uptake of antigen by membranous (M) cells (specialized epithelial cells) on the mucosal surface. These cells either process and present antigen to underlying T cells or B cells themselves or transport antigen to parenchymal macrophages, dendritic and B cells. Once interactin of the antigen presenting cell (APC) with T and B lymphocytes has occurred, an immune response or mucosal tolerance may result. Immune responses generally involve antibody production with IgA the predominant antibody isotype. Antigen sensitized immune cells are then circulated to other systemic and mucosal sites for expansion of effector mechanisms (Mestecky 1987).

In the present study we investigated theeffectiveness ofintranasalvaccination with live streptomycin-dependent mutant of P.multocida type B vaccine in comparison with inactivated one.

The mitogenic response of the peripheral blood lymphocytes was estimated by phytohaemaggulutinin in the trial to evaluate the cellular immune response of calves vaccinated with P.multocida vaccines. The results shown in (Table and Figure 1) indicated that live P.multocida vaccine gave higher immune response than inactivated one allover the period of the test. These results were coincide with that of El-Kofy (1997) who proved that vaccination of ducks by live duck virus hepatitis gave higher ΔOD than that of inactivated one. Also, the same results were detected by Maheswaran and theis (1979) who reported that lymphocytes from cattle immunized with various strains of P.multocida showed higher stimulation indices when incubated with homologous antigen, which suggested an involvement of cell mediated immunity. Moreover, Local CMI response to IBR virus were greater in cattle vaccinated intranasally than in cattle vaccinated intramuscularly (Gerber et al., 1978).

As shown in table and figure 2&3, the haemagglutinating antibodies as measured by Passive haemagglutination test and IgG  as measured by ELISA after vaccination with live vaccine were elevated from one day to reach the peak at 15 days post vaccination and then decline till 12 weeks the end of observation period. The haemagglutinating antibody and IgG after vaccination with inactivated vaccine was rose from one day to reach the peak at 8 weeks post vaccination and then decline till 12 weeks.

Secretory IgA is important in protecting against bacterial and viral diseases where the organism must attach to epithelial surfaces in order to produce disease and against diseases induced by toxins produced at mucosal surfaces.

The findings in (Table and Figure 4) indicated that intranasal vaccination of calves with live P.multocida vaccine induced local IgA antibodies and gave higher and earlier P.multocida antibody (IgA) than inactivated one. These results were coincide with that of Brennan et al, (1998) who found that intranasal administration of P.haemolytica 1:A may be a better method for stimulating protective immune responses in the upper portion of the respiratory tract than lung administration. As such, local secretory antibodies (specific IgA) are produced due to intranasal vaccination with live P.multocida vaccine, which may be an advantage in preventing natural infection of haemorrhagic septicemia. Also, Tomoda et al. (1995) found that the IgA antibody was negligibly detected in the nasal wash specimens before vaccination, and was induced by vaccination.

Gilmour et al, (1990) reported that locally produced humoral components, such as secretory IgA and mucosal IgG, may have played an active role in the enhanced antibacterial defense of the lung and showed that exposure of the respiratory tract induces local and systemic immunity and hastens the elimination of inhaled bacteria in subsequent exposures.

The haemagglutinating antibody did not rise in nasal secretions of calves after vaccination with live and dead vaccines as measured by passive haemagglutination test. Also, there was a trace amount of antibodies of Anti-P.multocida IgG in nasal secretions of calves after vaccination with live vaccine and dead vaccines as measured by ELISA. In contrastto the results described previously by Brennan et al., (1998), who found that IgG titers were higher than IgA titers in nasal secretions, in this work IgG was present in trace amount in nasal secretions and this may be due to the ratios of IgG\IgA in lower respiratory tract secretion which should be greater than that in upper respiratory tract secretions (Quinn et al, 2002). However, Stites et al, (1994) believed that IgA is the major immunoglobulin in nasal secretions.

In addition, control calves remained seronegative for P.multocida antibodies till the end of experiment.

The above findings indicated that the intranasal administration of live streptomycin-dependent serotype of P.multocida mutant vaccine was efficient and induced cellular immune response, local IgA antibodies and systemic IgG antibodies. These results were coinciding that of Ellis, (1999) who reported that live vaccines usually elicit both humoral immunity as well as cellular immunity. Also, Gerber et al, (1978) whom found that intranasal or intramuscular vaccination with Infectious bovine Rhino-tracheitis virus (IBR) stimulated local and systemic antibody but the local immune responses to IBR  in IM vaccinated calves were not as strong as in I/N vaccinated calves.

In conclusion, for prevention and recovery from P.multocida infection, intranasal vaccination is the best route as it induced cellular immune response, local IgA antibodies and systemic IgG antibodies.

References

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Table 1: Evaluation of cell mediated immune response of cattle vaccinated with P.multocida vaccines, using lymphocyte blastogenesis.

* ΔOD : Delta Optical Density Values.

** ND : Not detected.

Figure (1) Evaluation of cell mediated immune response of cattle vaccinated with P.multocida vaccines, using lymphocyte blastogenesis.

Table (2) Haemagglutinating antibodies of P.multocida in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by Passive haemagglutination test.

Figure (2) Haemagglutinating antibodies of P.multocida in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by Passive haemagglutination test.

Table (3) Antibodies of P.multocida (IgG)in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

Figure (3) Antibodies of P.multocida (IgG)in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

Table (4) IgA in nasal secretion of calves following vaccination with

live and inactivated P.multocida vaccine as measured by ELISA.

Figure (4) IgA in nasal secretion of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

   معهد بحوث اللأمصال و اللقاحات البيطرية – العباسية

Table 1: Evaluation of cell mediated immune response of cattle vaccinated with P.multocida vaccines, using lymphocyte blastogenesis.

Figure 1: Evaluation of cell mediated immune response of cattle vaccinated with P.multocida vaccines, using lymphocyte

Table 2: Haemagglutinating antibodies of P.multocida in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by Passive haemagglutination test.

Figure 2: Haemagglutinating antibodies of P.multocida in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by Passive haemagglutination test.

Table 3: Antibodies of P.multocida (IgG)in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

Figure 3: Antibodies of P.multocida (IgG)in serum of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

Table 4: IgA in nasal secretion of calves following vaccination with               live and inactivated P.multocida vaccine as measured by ELISA.

Figure 4: IgA in nasal secretion of calves following vaccination with live and inactivated P.multocida vaccine as measured by ELISA.

* ΔOD : Delta Optical Density Values.

** ND : Not detected.