evaluation of cellular and humoral immune response in ... (1-16).pdfmahmoud h. abd el-haleem*,...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 3 (September) 2008; 1 – 16

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Referred byReferred by Prof. Dr. Rawhia Dogium Professor of Pathology, Fac. Vet. Med.,

Cairo University Prof. Dr. M.O. El-Shazly Professor of Pathology, Fac. Vet. Med.,

Cairo University

Evaluation of cellular and humoral immune response in heifers vacci-nated with strain 19

By Mahmoud H. Abd El-Haleem*, Ramadan M. Khodeer* and Ahmed

A. Ramadan** Animal Health Research Institute

*Brucella Department, **Immunology Department

SUMMARY

C ellular and humoral immune responses of heifers vaccinated with Br. abortus strain 19 vaccine was evaluated in the current study.

Two weeks post vaccination whole blood and serum samples were col-lected from 30 heifers chosen randomly. Serum was saved for serologi-cal testes and monocytes were isolated from whole blood and cultured in vitro to obtain macrophages. After 6 days incubation, macrophages were treated with opsonized Brucella melitensis biovar 3 local strain isolated from cattle within the same farm at dose of 109 cfu in vitro. Survival of Brucella organisms two hours post incubation with macrophages was checked by culturing 0.1 ml of the supernatant on Brucella agar and in-cubated for 4 days. Control plates contained 109 Brucella organisms not incubated with macrophages. The number of bacteria survived phagocy-tosis of professional macrophages significantly reduced (P=0.001) in comparison with control. Nitric oxide and lysozyme concentrations sig-nificantly increased (P= 0.001) in macrophages treated with Brucella vaccine in comparison with non treated control ones. A positive correla-tion (r=0.83839) was found between numbers of colony forming units killed by macrophages and titer of tube agglutination test (TAT) of vac-cinated cows (P< 0.0001).

INTRODUCTION

B rucellosis is a zoonotic dis-ease transmitted primarily

from infected animals to humans through direct and indirect routes

and of major economic importance in developing countries. Economic losses include production loss as-sociated with infection in animals, preventive programs, and human


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diseases (Radostits et al., 2000). In dairy animals, the organism lo-calizes in the supra-mammary lymph nodes and mammary glands of 80% of infected animals, and these can continue to excrete the pathogen in milk throughout their lives so that infected animals are considered the main source of hu-man infection (Morgan and Mackinnon, 1979).

Brucella melitensis is en-

demic in Egypt and is the most prevalent infection in humans and a wide range of animals. EL-Taweel., (1998) indicated that brucellosis causes annual eco-nomic losses in livestock of about 24 million Egyptian pound, beside serious losses in human labor force. Br. melitensis biovar 3 was proved to be the prevalent strain among cattle in Egypt and in Mediterranean area and in Middle east which demanding mass vacci-nation of sheep and goats which act as a sole source for infection among cattle. We advise to use Rev-1 vaccine for vaccination of sheep and goat Khoudier (2000) and Khoudier (2004). Moreover, Br. melitensis biovar 3 isolated from serologically positive cattle from EL-Menofeia, El-Behera, Beni Suef, and EL Minia gover-norates proved to be the prevalent strain among cattle as the causative agent of brucellosis in Egypt (Torky, et al. 2001).

In case of brucellosis, many gram-negative bacteria including, Salmonella, and Yersinia can pro-duce antibodies in infected ani-mals, which cross-react with dif-ferent Brucella antigens leading to misdiagnosis (Corbel, 1985 and Alton et al, 1988). Most of the ap-plied serological tests used for de-tection of Brucellosis in infected animals were proved to be either too specific (give false negative results) or too sensitive (give false positive results) (Nicoletti and Muraschi, 1966; Farina, 1985). In addition, up till now, no single serological test can discriminate between infected and vaccinated animals. Although direct detection of this pathogen through bacterial isolation from contaminated sam-ples, is the golden-mark of diagno-sis.

Brucellae are facultative intra-

cellular bacterial pathogens of both humans and animals. The bacteria penetrate the mucosa of the nasal, oral, or pharyngeal cavities and are phagocytized by host macro-phages, where survival and repli-cation occurs. They have the abil-ity to survive and multiply in pro-fessional and non-professional phagocytes, and cause abortion in domestic animals.

Brucella melitensis biovar 3 is

the most pathogenic strain among Brucellae and biovar 3 is the domi-nant strain in Egypt (Sayour,


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2004). It survives and multiplicats in the host macrophages hence, macrophages play an important role in influencing the acquired re-sistance of the host after vaccina-tion. Strain 19 is the most com-monly used in vaccination pro-grams against bovine brucellosis in Egypt and all over the world (Crawford et al., 1991 and Abd El-Haleem, 2007). The main ad-vantage of strain 19 is that it gives a considerable humoral and cellu-lar protection against different types of Brucellae even when used at a reduced dose (3-8 X 109 cfu). Yet its main disadvantage is the production of smooth antibodies which interferes with the diagnosis of diseases using conventional se-rological tests (Alton et al., 1984). As no vaccine has been proven to provide 100% protection against brucellosis, vaccination alone is effective in reducing disease prevalence but is unlikely effective in production losses and reducing transmission and shedding of the organism.

The aim of the current study

is to evaluate the phagocytic abil-ity of macrophages cultured from vaccinated heifers two weeks post vaccination and pulsed with Brucella melitensis biovar 3 and to evaluate their humoral response. Out of hundred 3-8 months old vaccinated heifers with reduced dose of strain 19 (3-8 X 109 cfu)

from Touch Tanbisha Farm, thirty heifers were chosen randomly for the evaluation of both cellular and humoral immune responses two weeks post vaccination. Humoral immune response was evaluated using the three serological tests namely, tube agglutination test (TAT) which detect mainly IgM, Rose Bengal plate test (RBPT) which detects partially IgM, and complement fixation test (CFT) which detects mainly IgG.

MATERIAL AND METHODS

Samples

B lood samples were collected from 30 out of 100 vacci-

nated Friesian heifers 3-8 months age with S19 from Touch Tanbisha farm and divided in two parts: one tube coated with heparin and other part in test tube which was left un-til complete coagulation. The sam-ples were taken under strict hygi-enic conditions, kept on ice and sent to the laboratory within two hours. The serum samples which collected were tested by the fol-lowing serological techniques: 1-standard Tube Agglutination test (SAT) as described by Alton et al. (1988). 2-Rose Bengal Plate Test (RBPT); as described by Alton et al. (1988). 3-CFT: by microtiter plate technique as described by Al-ton et al. (1988). Strain 19 Vac-cine obtained from Abassyia Se-rum and Vaccine Institute. Vacci-nation was done at dose of 3-8 X


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109 cfu (OIE, 2004). Brucella me-litensis biovar 3 Field strain was isolated and prepared in Depart-ment of Brucellosis Research, Ani-mal Health Research Institute (AHRI) Dokki, Egypt which we isolated and identified as described by (Alton et al., 1988) as follow; Brucella melitensis biovar 3 Field strain was isolated from tissue of slaughtered reactor cattle was cul-tured on plates of tryptose soy agar medium supplemented with differ-ent antibiotics. The following con-centration of antibiotics were added per liter of media; cyclo-heximide (100 mg), bacitracin (25 000 units), polyxin B sulphate (5000 units), vancomycin (20 mg), nalidixic acid (5 mg) and nystatin (100,000 units) (Oxoid). The plates were then incubated in a 10% CO2 incubator at 37ºC for at least seven days; suspected colo-nies were identified according to the methods adopted by Alton et al. (1988). Typing of Brucella iso-lates was done according to CO2 requirement, H2S production, growth in the presence of dyes (thionin and basic fuchsin –20 µg/mL), reaction with mono-specific sera (A&M), and lyses by Tblizi and Iz phages (Alton et al., 1988). Macrophage culture: macro-phages were cultured as described according to Paul et al. (2007) heparinized blood was obtained from cows two weeks after vacci-nation. Mononuclear cells were

isolated by density gradient cen-trifugation on Histopaque (density 1.077). Briefly, blood was centri-fuged at low speed and the red cell/plasma interface was mixed with an equal volume of complete RPMI-1640 tissue culture medium containing 2 mM L-glutamine, 10% heat-inactivated fetal calf se-rum, 20 mM HEPES, and 40 µg/ml gentamicin. Samples were lay-ered over 5 ml Histopaque in tis-sue culture tubes. After centrifuga-tion at 400 X g for 25 min at room temperature, the cell interface layer was carefully aspirated using sterilized pasture pipette and washed twice with HBSS and once with complete medium. Cell vi-ability was determined by 0.2% Trypan blue exclusion. Monocytes were counted using neutral red and were resuspended in complete me-dium at 1x106 monocyte/ml. Ex-actly 1 ml from the monocyte sus-pension was dispensed in 24-well flat-bottomed plates. After 2 hr in-cubation at 37°C under 5% CO2, non-adherent cells were removed by aspiration and each well was rinsed twice with complete me-dium and received 1 ml of com-plete medium and kept at 37°C and 5% CO2 for 6 days. Medium was changed once during the 6 days incubation period. At end of incu-bation, wells were checked for the heavy growth of macrophages us-ing phase contrast inverted micro-scope.


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Killing index of Brucella: exactly 1 ml of Brucella abortus organisms at concentration of 109 cell/ml was added to each well and incubated for two hours. At the end of incu-bation, two loads of 0.1 ml from each well was layered over two Brucella agar plates and distrib-uted on the plate using spreader. Plates were incubated for four days at 37°C and 5% CO2, and then bacterial count was made for each plate at end of incubation. The rest of the supernatants was collected from each well separately in eppin-dorf tubes and kept at -40° C until assayed for nitric oxide and ly-sozyme concentration. Nitric oxide assay: Measurement of nitric oxide was assessed ac-cording to the assay described by Ramadan and Attia (2003). Briefly, 100 µl from each sample was transferred into flat-bottom 96-well ELISA plate and 100 µl of Griess reagent (0.5% sulfanila-mide; Sigma Chemical Co.) in 2.5% phosphoric acid (Merck Co.) and 0.05% N-(1-naphthyl) ethyl-enediamine dihydrochloride (Sig-ma Co.). The mixture was incu-bated at 21°C for 10 minutes. Ab-sorbency of the samples and stan-dards was measured at 570 nm us-ing ELISA reader (Dynatech MR7000; Dynatech Laboratories Inc.). Absorbency of test samples was converted to micromolar of nitrite by comparison with absorb-ance values of sodium nitrite

(Sigma Co.) standard curve within a linear curve fit. Lysozyme concentration: Assay-ing for lysozyme was done accord-ing to Ramadan and Attia (2003). Briefly, lysoplates were prepared by dissolving 1% agarose in 0.067 M PBS at pH 6.3 and heated to 100°C for complete dis-solve, then cooled down to 60-70°C, 500 mg uniform suspension of Micrococcus lysodeikticus in 5 ml saline was added to 1 liter of aga-rose and mixed well. Plates were poured at thickness of 4 mm depth and left to cool down and wells 2 mm in diameter in 4 X 4 rows, 15 mm apart were cut in agarose. At time of assay, plates, serum sam-ples, and standard lysozyme solu-tions were brought at room tem-perature. Exactly 25 µl of each se-rum sample were put in 2 wells, the same was done with standard lysozyme solutions. Plates were incubated at room temperature for 12-18 hours. The diameter of clearance zone around each well was measured to the nearest 0.1 mm. A standard curve was devel-oped for lysozyme standard solu-tion at concentrations of 3, 15, 30, 60 and 120 µg/ml. Log of concen-trations were plotted against di-ameter of corresponding cleared zone on a semi-logarithmic graph.


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RESULTS

Figure (1) Shows the average number of survived Brucella melitensis bio-var 3 organisms after 2 hours incubation in vitro with profes-sional macrophages obtained from vaccinated cows two weeks post vaccination.

Figure (2) Shows the average concentration of nitric oxide, in μM/ml, in the supernatant of professional macrophages, cultured from vac-cinated cows 2 weeks post vaccination and treated with Brucella melitensis biovar 3 for 2 hours.


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Figure (3) Shows the average concentration of lysozyme, in μg/ml, in the supernatant of professional macrophages, cultured from vacci-nated 30 cows 2 weeks post vaccination and treated with Brucella melitensis biovar 3 for 2 hours.


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Table (1): Results of the threes serological tests performed on sera of 30 cows two weeks after vaccination with strain 19 vaccine.

Se-rial

Rose Bengal plate test

Complement fixation test

Tube agglutination test

1 + - 80

2 ++ 1/16 186

3 + - 46.5

4 ++ 1/8 80

5 ++ 1/8 93

6 ++ 1/32 134

7 ++ - 80

8 ++ 1/8 67

9 ++ 1/32 134

10 ++ 1/16 160

11 + - 33.5

12 ++ 1/32 134

13 + - 33.5

14 ++ 1/16 186

15 ++ 1/32 134

16 + 1/8 67

17 + 1/8 67

18 + - 33.5

19 ++ 1/32 134

20 ++ 1/8 67

21 + - 67

22 ++ 1/16 160

23 + - 33.5

24 + 1/8 80

25 + - 33.5

26 + 1/8 93

27 + 1/8 80

28 ++ 1/16 160

29 + - 80

30 + 1/8 67


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Table (2) Correlation between numbers of colony forming units killed by macrophages and titer of tube agglutination test (TAT) of vac-cinated cows.

Parameters Titer of TAT

Number of cfu

r P

0.83839 0.0001

Figure (4) Lysoplate of the supernatants of macrophages incubated with Brucella melitensis organisms. Diameters of lyses zones were measured and the concentrations of lysozyme were calculated according to the standard curve.


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DISCUSSION

B rucella species are facultative intracellular pathogens that

have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. Resis-tance to facultative intracellular bacterial pathogens such as Brucellae depends on acquired cell-mediated resistance and acti-vation of macrophages. Macro-phages play a central role in host immune responses against patho-gens by acting as both professional phagocytic cells and as fully com-petent antigen presenting cells. Phagocytosis of Brucella by macrophages increases the cell res-piration and initiates oxidative burst inside the macrophages. Con-sequently, different killing mole-cules are formed and directed to-ward the phagosome where the or-ganism resides. Brucella species are able to survive and replicate within the phagocytic vacuole of macrophages that induce chronic infection in humans and domestic animals. The activation of oxida-tive bactericidal activity is one of the defense systems which protect the host from the toxic effects of pathogens.

In current study, macrophages cultured from the peripheral blood of cows vaccinated with Brucella

strain 19 were potent in killing bacteria about 3.5 times more than control (Figure 1). It is not clear that how the killing process oc-curred, but apparently the killing molecules secreted by the macro-phages played major part in the killing process. Also vaccination, in current study, enhanced the macrophages to eliminate infec-tion.

Nitric oxide (NO) is an im-portant molecule which is a gen-eral characteristic of activated macrophages (Stuehr, and Mar-letta, 1987). Indeed, NO is a cru-cial factor in the elimination of Brucella infection. Immediately after engulfment of the bacteria, inducible nitric oxide synthase en-zyme is expressed in the phago-cytic cells (Antoine et al. 2004) and the levels of NO sharply in-crease. In fact, Antoine et al. (2004) reported Impairment of Brucella growth in human macro-phages that produce nitric oxide. It was hypothesized that in humans, the inability of human macro-phages to produce NO during Brucella infection contributes to the chronicity of infection (Gross et al., 1998 and Lopez-Urrutia et al., 2000).

In current study, in vitro NO production by infected macro-phages obtained from vaccinated cows increased nearly 3 times


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more than control non-vaccinated cows. Apparently, vaccination primed macrophages so that when they encountered the organism again, they secreted abundance of NO which played central role in elimination of the organism in vi-tro. Toxic effects of NO occur through the formation of peroxyni-trite, a powerful oxidant that causes chemical reactions in bio-logical systems, including protein and DNA nitrosylation as well as lipid peroxidation (Maeda and Akaike, 1998; Murphy, 1999). Yifan and Christina (1995) re-ported an increase amount of ni-trite in supernatants of spleen cells of mice infected with Brucella abortus compared with control mice. Moreover, they found that inhibition of inducible nitric oxide synthesase (iNOS), the enzyme re-quired for synthesis of nitric oxide, in activated peritoneal macro-phages increased survival of B. abortus in vitro; showing that nitric oxide limits bacterial replication in these cells. Erdogana et al. (2007) found that B. melitensis signifi-cantly increased NO levels in rat liver and spleen, and when NO suppressed artificially, bacterial survival increased. Also, Yifan and Cheers (1998) reported that in mice, NO production is a crucial component in the elimination of Brucella during in vivo infections.

Lysozyme (muramidase) is a stable polypeptide enzyme widely

distributed in mammalian tissues. Lysozyme was discovered in 1921 by Sir Alexander Fleming, who was then trying to understand the inhibitory property of his own na-sal mucus on the growth of Staphy-lococcus cultures. It is important host constitutive defense system, possesses wide range of biological activities among which, its prop-erty as antibacterial (Silvia Her-bert, 2007) and anticancer (Inouye, 1987). Bactericidal prop-erties of lysozyme are attributed to its N-acetylmuramidase activity (Patrick Veiga et al., 2007). Ly-sozyme targets the peptidoglycan moiety of bacterial cell walls (Chantal Abergel et al., 2007). Lysozyme activity, in current study, increased in macrophages cultured from vaccinated cows in response to Brucella challenge. The increase was about 3.5 times greater than the control. James and Edward (1981) reported an increase in lysozyme concentration in sera of infected mice twice the values of non-infected ones 14 days post infection. They hypothe-sized that lysozyme stimulates phagocytosis by macrophages.

Strain 19 is the most com-monly used in vaccination pro-grams against bovine brucellosis in Egypt and all over the world. The main advantage of this vaccine is that it initiates a considerable hu-moral and cellular protection against brucellosis even at small


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doses. In current study, three sero-logical tests were used to evaluate the humoral immune response of vaccinated cows 14 days post vac-cination (Table 1). Tube agglutina-tion test (TAT) mainly detects IgM; complement fixation test mainly detects IgG1, while Rose Bengale Plate Test (RPPT) detects IgM. In the initial phase of vacci-nation IgM is the predominant iso-type of immunoglobulins in the sera of animals. Indeed, TAT gave the highest result among the three serological tests. Moreover, there was a s t rong s ign i f ican t (P<0.0001) positive correlation (r=0.83839) between the number of cfu digested by macrophages and the titer of TAT. In other words, when the titer of TAT was high, the numbers of survived Brucella organisms were low indi-cating that the vaccine gave high level of protection on both the cel-lular and humoral sides.

I n conclusion, strain 19 vaccine given to cows enhanced both

humoral and cellular immune re-sponses in the form of increased phagocytic and killing activities of macrophages and increased titer of IgM in sera of cows.

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