The Profile of Immunoglobulin A and Immunoglobulin G Subclasses in Sprague Dawley Rats Experimentally Infected with Brucella abortus Biotype 1

Abstract

This study measured total serum immunoglobulin A (IgA), immunoglobulin G (IgG)1, IgG2a response against whole cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), cytoplasmic protein (CP), and crude Brucella protein (CBP) of Brucella abortus in experimental brucellosis induced with B. abortus biotype 1 in Sprague Dawley (SD) rats during a 17-week infection period. Six- to 8-week-old SD rats (n = 44) were experimentally infected with 1 × 10⁹ colony forming unit of B. abortus biotype 1 through the intraperitoneal route. Serial serum samples were collected from the rat at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after inoculation. The sera were tested by enzyme linked immunosorbent assay. We have noticed a very low level and short persistence of IgA antibody in our experiment. The low level and short persistence of IgA antibody suggest that this antibody isotype might not be protective against brucellosis in rats. Both Th1 and Th2 specific immune responses were recorded in our study with the production of IgG1 and IgG2a antibody isotopes, respectively. We noticed significant dominant IgG2a antibody responses over IgG1 responses throughout the experiment (p < 0.001) against WCA and OMP. The mixed Th1 and Th2 dominant immune responses mediated by IgG2a and IgG1 antibody isotypes were observed against CP, PP, and CBP. Data of our study suggest that IgG2a dominant responses in the early stages of disease play the main role in conferring protection against brucellosis and with the progress of disease IgG1 dominant responses were elicited.

Introduction

Brucellosis is a zoonosis caused by Brucella species (Grilló et al. 2012), a Gram-negative facultative intracellular pathogen that affects humans and animals, leading to significant impact on public health and animal industry (Silva et al. 2011). In humans, the disease causes high clinical morbidity and protean clinical manifestations as any organ can be affected (Berbari and Wilson 2001). Brucellosis can occur in several forms: acute/subacute, focal, and chronic (Jimenez de Bagues et al. 2005). Despite the early diagnosis and efficacious treatment, ∼10% of patients develop chronic disease, which is characterized by atypical clinical picture and relapses (Irmak et al. 2003).

Certain immunoglobulin G (IgG) subclasses such as IgG2a in mice and IgG3 in humans are preferentially generated in humoral immune responses against intracellular pathogens (Finkelman et al. 1988, Papadea et al. 1989). Presumably these antibodies have greater facilities than other isotypes to recognize microbial antigens on the surface of infected cells and killed those (Golding et al. 2001). Immunoglobulin A (IgA) provides protection against foreign antigens at body surfaces, including the respiratory epithelium and skin (Hill et al. 1995).

Rats are known to be harbor of Brucella organism in many parts of the world (Oliakova and Antoniuk 1989, Silva et al. 2011). Rats harbor places are commonly in human's houses and cattle farms. Infected rats could excrete Brucella organisms through feces (Colby 1997) and transmit brucellosis to humans and animals through direct contact. Immune responses to Brucella spp. have been studied mainly in mouse models (Baldwin and Parent 2002, Grilló et al. 2012). Our previous study measured the profiles of IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide (LPS) of Brucella abortus biotype 1 by enzyme linked immunosorbent assay (ELISA) (Khatun et al., 2009). No information is available about IgA and IgG subclasses related to B. abortus infection in rats. In the present study, the IgA and IgG subclasses were investigated using whole cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), cytoplasmic protein (CP), and crude Brucella protein (CBP) of B. abortus biotype 1 by ELISA.

Materials and Methods

Experimental animals

Ten- to 12-week old (n = 44) Sprague Dawley (SD) rats weighing 250–300 g were used. The parent stock of rats was purchased from a SPF laboratory animal company (Kotech, Pyeongtaek, Korea). The rats were housed in a stringently hygienic, climate-controlled environment and provided with commercial food and water ad libitum.

Bacterial strain

Pathogenic B. abortus biotype 1 originating from cattle in Korea was used for experimental infection in SD rats. B. abortus biotype I lyophilized stock culture was obtained from the laboratory stock Brucella, which was inoculated into the Brucella agar media (Difco, Kansas City) and incubated at 37°C for 5–7 days under 5% CO₂. The grown bacteria were harvested in normal saline.

Experimental infection

Rats (n = 40) were inoculated intraperitoneally with 0.1 mL saline containing 1 × 10¹⁰ colony forming unit/mL of B. abortus biotype 1. Four rats were inoculated intraperitoneally with sterile saline as uninfected control.

Collection of specimens

At 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection, four rats randomly selected in each time point were anesthetized by intraperitoneal administration of 7–25 mg/kg of Tiletamine and Zolazepam (Zoletil 50^®; Virbac Laboratories, Carros, France). Blood samples were collected from the infected and control rats by aseptic cardiac puncture. Sera were collected from blood samples and stored in aliquots at −80°C until used.

Preparation of WCA from B. abortus biotype 1

B. abortus biotype 1 (Korean bovine isolate) whole cell antigen (WCAs) were prepared as described previously (Colby 1997). Briefly, B. abortus biotype 1 was cultured onto Brucella agar medium at 37°C for 7 days under 5% CO_2. The bacteria were harvested in 10 mM Tris, pH 8.0., and killed by acetone. The bacteria were then washed twice with 10 mM Tris-HCL buffer and pelleted by centrifugation at ∼6000 × g at 4°C for 10 min. The pellet was resuspended with 10 mM Tris-HCL buffer and stored at −20°C.

Preparation of periplasmic, cytoplasmic, and OMPs of B. abortus biotype 1

The PP, CP, and outer membrane proteins (OMPs) were prepared by a modification of the lysozyme-osmotic shock method (Witholt et al. 1976). Briefly culture of B. abortus biotype 1 was harvested in phosphate-buffered saline (PBS) and centrifuged at 7000 × g for 10 min. The cell pellets were resuspended in 100 mM Tris-HCl buffer (pH 8.6) containing 500 mM sucrose and 0.5 mM ethylenediaminetetraaceticacid (EDTA). Hen egg white lysozyme (4-mg/mL stock solution) was added, followed immediately by the addition of 50 mM Tris-HCl buffer (pH 8.6) containing 250 mM sucrose, 0.25 mM EDTA, and 2.5 mM MgCl₂. After gentle agitation, the suspension was incubated for 15 min in an ice bath. Cells were removed by centrifugation at 7000 × g for 6 min followed by filtration of the supernatant through a 0.45 μm pore size filter. The filtered supernatant fluid served as the PP. Cells resuspended in 20 mM Tris-HCl (pH 8.6) were disrupted by two passages through a French pressure cell (American Instrument Company, Silver Spring, MD) and centrifuged at 7000 × g at 4°C for 6 min. The supernatant fluid was then centrifuged at 132,000 × g at 4°C for 1 h. The soluble fraction contained the CP. To isolate the OMP, total envelope pellets were resuspended in 20 mM Tris-HCl (pH 8.6) containing 1% sarkosyl and incubated for 30 min on ice. The OMP was obtained as a pellet after centrifugation at 132,000 × g at 4°C for 1 h. The pellet was resuspended in 20 mM Tris-HCl buffer (pH 8.6).

Preparation of CBP

The crude Brucella protein (CBP) was prepared from B. abortus biotype 1 according to the previously described methods (Onate et al. 2003) with modifications. Briefly, B. abortus biotype 1 was inoculated onto Brucella agar and incubated at 37°C for 5–7 days under 5% CO₂, harvested in sterile PBS after 7 days of incubation. Bacteria were washed thrice with the sterile PBS at 8000 × g for 10 min at 4°C and resuspended in 50 mL of sterile PBS and inactivated by heating at 60°C for 1 h. The inactivated culture was sonicated at melting ice temperature, applying six cycles at 100 W; each cycle was of 1 min duration and centrifuged at 1200 × g for 20 min at 4°C. The supernatant was collected in 2 mL aliquots and stored at −20°C until tested. The protein concentration of the antigen was measured using Bradford Kit (Bio-Rad, Hercules).

Determination of isotype-specific immunoglobulin by ELISA

Total IgA, IgG1, and IgG2a titers against WCA, OMP, PP, CP, and CBP were assessed by ELISA as follows. Flat-bottomed 96-well polystyrene microtiter plates (Nunc, Denmark) were coated overnight at 4°C with 100 μL of WCA, OMP, PP, CP, and CBP at a final concentration of 10 μg/mL suspended in 0.05 mM sodium bicarbonate buffer (pH 9.6). Affinity purified rat IgA, IgG1, and IgG2a (Bethyl Laboratories, Inc.) were used to coat the 96-well plate starting from 500 to 7.8 ng/well for generation of standard curve, respectively. Plates were washed thrice with wash solution (PBST: PBS, pH 7.4) with 0.05% (v/v) Tween 20. Each well of the antigen-coated plates was blocked with 200 μL of blocking solution of 1% (w/v) bovine serum albumin (Sigma Aldrich, Inc., St. Louis, MO) in PBS and incubated at 37°C for 30 min. After three washes with phosphate buffered saline with tween 20 (PBST), 100 μL of control and test sera samples, diluted 1:100 in sample diluent (50 mM Tris, 0.14 M Nacl, 1% bovine serum albumin [BSA], 0.05% Tween 20, pH 8.0), were added to each well in duplicate. The plates were sealed and incubated at 37°C for 1 h. After five washing cycles with PBST, each well was incubated with 100 μL of 1:100,000 dilution of goat anti-rat IgA, IgG1, and IgG2a antibodies conjugated to horseradish peroxidase (Bethyl Laboratories, Inc.) diluted in conjugate diluent (50 mM tris, 0.14 M Nacl, 1% BSA, 0.05% Tween 20, pH 8.0) and the plates incubated at 37°C for 1 h. After five washings as described above, the color reaction was developed by adding 200 μL/well of a solution containing 1.0 mg/mL of O-phenylenediamine dihydrochloride (Sigma, St. Louis) in 0.05 M citrate buffer (pH 4.0) with 0.04% (v/v) H₂O₂. The plates were incubated in the dark for 30 min at room temperature. The colorimetric reaction was stopped by the addition of 50 μL/well of 3 M H₂SO_4. The absorbance measurements were made at 492 nm, using an automatic ELISA reader (Tecan, Austria).

Statistical analysis

The IgA, IgG1, and IgG2a responses in the infected rats at different days of infection were analyzed for statistical significance by the Student's t-test using Microsoft Excel 2000. A p value of <0.05 was considered to be significant.

Results

Production of total serum IgA, IgG1, and IgG2a against WCA of B. abortus

The IgA specific antibody response against whole cell of B. abortus was detected in the sera of infected rats at 3 days after infection (5.45 ± 0.04 ng/mL). The highest level of IgA was recorded at 21 days after infection (10.56 ± 0.02 ng/mL). B. abortus specific IgG1 antibody response started at 3 days after infection (7.15 ± 0.36 ng/mL) and reached the peak at 28 days after infection (136.92 ± 2.83 ng/mL). The IgG2a level gradually increased from 3 days after infection and reached the peak at 42 days after infection (112.43 ± 3.26 ng/mL). The results of total serum IgA, IgG1, and IgG2a level in the rats against WCA of B. abortus during acute and subacute infection are shown in Fig. 1.

FIG. 1.

Total serum IgA (A), IgG1 (B), and IgG2a (C) against whole cell antigen of Brucella abortus biotype 1 infected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection. Results are expressed as mean value of four rats ± SD. Statistically significant differences of antibody titers between control and infected rats at different time points of infection are indicated by asterisks (*p < 0.05 and **p < 0.001). SD, stantard deviation; IgA, immunoglobulin A; IgG, immunoglobulin G.

Production of total serum IgA, IgG1, and IgG2a against OMP of B. abortus

B. abortus specific IgA antibody response at 0, 3, 7, and 14 days after infection was found to be almost at the same level (5.55 ± 0.02, 6.07 ± 0.03, 6.56 ± 0.04, and 6.57 ± 0.03 ng/mL, respectively). At 21 days after infection, IgA level was 8.8 ± 0.03 ng/mL, and the peak level was recorded at 28 days after infection (12.28 ± 0.02 ng/mL). The IgG1 level was detected at 7 days after infection (4.43 ± 0.12 ng/mL). Then the IgG1 level gradually increased and reached a peak at 42 days after infection (57.37±0.29ng/mL).The IgG2a specific antibody responses were detectable at 7 days after infection (22.40 ± 0.48 ng/mL) compared to uninfected control rats (17.70 ± 0.33 ng/mL). Then the IgG2a level gradually increased throughout the study, and the highest value was recorded at 90 days after infection (120.63 ± 3.51 ng/mL) followed by a decreased production at 120 days after infection (107.38±3.10ng/mL).The results of total serum IgA, IgG1, and IgG2a level in the rats against OMP of B. abortus during acute and subacute infection are shown in Fig. 2.

FIG. 2.

Total serum IgA (A), IgG1 (B), and IgG2a (C) against outer membrane protein of B. abortus biotype 1 infected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection. Results are expressed as mean value of four rats ± SD. Statistically significant differences of antibody titers between control and infected rats at different time points of infection are indicated by asterisks (*p < 0.05 and **p < 0.001).

Production of total serum IgA, IgG1, and IgG2a against PP of B. abortus

B. abortus specific IgA antibody response started at 7 days after infection (6.56 ± 0.04 ng/mL). The highest serum IgA level was recorded at 14 days after infection (28.88 ± 0.03 ng/mL) followed by a slight decrease at 21 days after infection (26.1 ± 0.03 ng/mL). Then the IgA level sharply declined at 28 days after infection (6.56 ± 0.04 ng/mL) and remained almost same level until the end of the experiment except 42 days after infection. The serum IgG1 level gradually increased from 7 days after infection and reached a peak at 42 days after infection (140.22 ± 2.89 ng/mL). The IgG2a specific antibody response was detected at 3 days after infection (24.02 ± 0.53 ng/mL). The IgG2a gradually increased after production and reached the highest level at 28 days after infection (119.88 ± 3.49 ng/mL). The IgG2a was found to be almost same level at 90 and 120 days after infection. The results of total serum IgA, IgG1, and IgG2a level in the rats against PP of B. abortus during acute and subacute infection are shown in Fig. 3.

FIG. 3.

Total serum IgA (A), IgG1 (B), and IgG2a (C) against periplasmic protein of B. abortus biotype 1 infected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection. Results are expressed as mean value of four rats ± SD. Statistically significant differences of antibody titers between control and infected rats at different time points of infection are indicated by asterisks (*p < 0.05 and **p < 0.001).

Production of total serum IgA, IgG1, and IgG2a against CP of B. abortus

Serum IgA responses were noticed at 7 days after infection (8.34 ± 0.03 ng/mL) and reached peak at 14 days after infection (31.93 ± 0.02 ng/mL). The IgA level was sharply declined at 28 days after infection (6.48 ± 0.04 ng/mL). The IgG1 specific antibody response was noticed at 3 days after infection (8.4 ± 0.37 ng/mL). The IgG1 level gradually increased from 3 days after infection and reached the peak at 42 days after infection (174.93 ± 0.46 ng/mL). Twofold decrease of IgG1 level was recorded at 120 days after infection (72.47 ± 0.34 ng/mL) compared to IgG1 level recorded at 90 days after infection. The IgG2a response was recorded at 3 days after infection (33.16 ± 0.71 ng/mL) compared to uninfected control rats (23.46 ± 0.37 ng/mL). Then the IgG2a level gradually increased and reached the peak at 35 days after infection (123.01 ± 0.73 ng/mL). The IgG2a level remained almost same level at 42, 60, 90, and 120 days after infection. The results of total serum IgA, IgG1, and IgG2a level in the rats against CP of B. abortus during acute and subacute infection are shown in Fig. 4.

FIG. 4.

Total serum IgA (A), IgG1 (B), and IgG2a (C) against cytoplasmic protein of B. abortus biotype 1 infected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection. Results are expressed as mean value of four rats ± SD. Statistically significant differences of antibody titers between control and infected rats at different time points of infection are indicated by asterisks (*p < 0.05 and **p < 0.001).

Production of total serum IgA, IgG1, and IgG2a against CBP of B. abortus

The highest serum IgA responses were recorded at 14 days after infection (23.24 ± 0.03 ng/mL) followed by a slight decrease at 21 days after infection (19.41 ± 0.03 ng/mL). B. abortus specific IgG1 antibody response started at 3 days after infection (9.56 ± 0.19 ng/mL) and gradually increased upto 42 days after infection (150.55 ± 0.26 ng/mL). The peak IgG1 value was recorded at 90 days after infection (154.27 ± 0.43 ng/mL). The IgG2a levels recorded at 0, 3, 7, and 14 days after infection were found to be almost same levels (48.27 ± 0.1, 53.83 ± 0.57, 55.62 ± 0.34, and 56.62 ± 0.34 ng/mL), respectively. The IgG2a level increased from 21 days after infection (86.08 ± 0.41 ng/mL) and reached a peak at 28 days after infection (117.73 ± 0.42 ng/mL). The results of total serum IgA, IgG1, and IgG2a level in the rats against CBP of B. abortus during acute and subacute infection are shown in Fig. 5.

FIG. 5.

Total serum IgA (A), IgG1 (B), and IgG2a (C) against crude Brucella protein of B. abortus biotype 1 infected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after infection. Results are expressed as mean value of four rats ± SD. Statistically significant differences of antibody titers between control and infected rats at different time points of infection are indicated by asterisks (*p < 0.05 and **p < 0.001).

Discussion

In this study, IgA specific antibody responses were mounted in the sera of the B. abortus infected rats against different protein antigens mainly in acute stages of infection between 14 and 42 days after infection. A very low level and short persistence of IgA antibody were recorded in this experiment. Serum IgA response was not found in mice after inoculation of LPS of B. abortus, but very low titers of serum IgA were detected in guinea pigs (Van De Varg et al. 1996). B. abortus infection induces the production of IgA antibody isotype detectable in both milk and sera of cattle (Nielsen et al. 1988). However, the role of IgA in conferring protection against brucellosis is not known. The low level and short persistence of IgA antibody in this study suggest that this antibody isotype might not be protective against brucellosis in rats.

ELISA is very sensitive, highly specific and detects all the isotypes of IgG in serum (Nielsen et al. 1988). In this experiment IgG subclass (IgG1 and IgG2a) responses were examined against different antigens of B. abortus as a possible reflection of differences in T cell activity. Th1 cells induce antigen-specific B cells to secret IgG2a, and Th2 cells induce the cells to secret IgG1 (Stevens et al. 1988). A significant dominant IgG2a antibody over IgG1 responses was noticed throughout the experiment (p < 0.001) against OMP antigens of B. abortus used in ELISA. Khatun et al. (2009) also noticed IgG2a dominant antibody response over IgG1 against LPS of B. abortus in ELISA during acute stages of brucellosis in SD rats. Dominant IgG2a response was observed in a mouse model after infection with B. abortus compared to IgG1 response (Stevens et al. 1988). The highest IgG2a response was also observed in mice at 21 and 35 days after B. abortus infection (High et al. 2007).

In this study, a mixed Th1 and Th2 dominant immune responses mediated by IgG2a and IgG1 antibody isotypes were observed against WCA, PP, CP, and CBP. In case of WCA, IgG2a dominant response was recorded up to 21 days after infection. Then IgG1 dominant response was recorded from 28 days after infection until 90 days after infection. When IgG subclass antibody responses were measured against PP of B. abortus, IgG2a dominant antibody responses were observed until 28 days after infection compared to IgG1 response (p < 0.001). Then IgG2a dominant responses were again noticed from 90 days after infection compared to IgG1 response until the end of the experiment (p < 0.001). A significantly dominant IgG2a response was noted in the sera of rats until 14 days after infection against CP compared to the IgG1 response (p < 0.001). Then IgG1 dominant response was recorded from 21 days after infection until 90 days after infection (p < 0.001). In case of CBP, IgG2a dominant response was recorded up to 7 days after infection compared to IgG1 responses (p < 0.001). In contrast, IgG1 dominant immune response compared to IgG2a response was noticed from 14 days after infection until the end of the study.

IgG1 is consistently produced at high levels in Brucella-exposed cattle sera. The predominance of IgG1 subclass compared to IgG2a subclass was observed in mice following repeated immunization with only CBP (Onate et al. 2000). IgG2a antibodies are preferentially generated in humoral responses against intracellular microorganisms (Papadea et al. 1989). The IgG2a isotype is complement fixing and associated with protection against intracellular bacteria (Golding et al. 2001). IgG1 isotype is mainly responsible for protection against extracellular pathogens (Romagnani 1997). Brucella spp. are intracellular organisms, and Th1 response is essential in clearing the infection (Mielke et al. 1998). IgG2a dominant responses in the early stages of disease play the main role in conferring protection against brucellosis, and with the progress of disease IgG1 dominant responses were elicited.

Conclusion

Data of the current study suggest that WCA, OMP, PP, CP, and CBP antigens could be used as serological diagnostic antigens in ELISA for the detection of acute and subacute brucellosis in free ranging wildlife such as rats. The findings of this study would be helpful for better understanding of antibody-mediated protective immunity to B. abortus biotype 1 infection in humans.

Footnotes

Acknowledgment

The authors acknowledge Dr. Stephen M. Boyle (Virginia Tech, United States) for editorial comments.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

The authors thank the Islamic Development Bank, Saudi Arabia for providing the fund to carry out the doctoral research.

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