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Volume 1 Number 2 (Summer 2009) 26 - 31

26

COMPARATIVE STUDY OF HEMOLYTIC ACTIVITY OF BORDETELLA SPESIES

Comparative study of hemolytic activity of Bordetella species

Bodade R G , Khobragade C N* , Ahemad N

Biotechnology Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathawada University, Nanded, India

Received: December 2008 , Accepted: February 2009.

ABSTRACT

Background and objectives: Bordetella species colonize the respiratory tract of mammals and thereby cause the whooping cough. Most of the species produce adenylate cyclase - a toxin ( hemolysin ) responsible for increasing intracellular cyclic AMP (cAMP) levels in mammalian neutrophils and macrophages and as a consequence their phagocytic function get impaired . This study was carried out to isolate species of Bordetella and to study the hemolytic activity of each species on RBCs of sheep, human and poultry at varied culture conditions by altering the temperature, pH and cell age.

Materials and Methods: Three pathogenic Bordetella species were isolated from fifty suspected whooping cough patients on Bordet-Gengou agar and identified by their biochemical profiles. The hemolytic activity of B.pertussis, B. parapertussis and B. bronchiseptica was investigated in terms of cell bound and cell free hemolysin on human, poultry and sheep RBCs at variable pH, temperature and cell age in Stainer Scholt broth. The hemolysin activity was also determined qualitatively on blood agar containing different blood samples.

Results: All the species revealed optimum hemolytic activity in pH range 7.5-8.0 (in slight alkaline condition), temperature 37°C and cell age up to 20-24 hrs. The cell bound hemolytic activity was found to be maximum than cell free activity and varied with blood samples of different species. B.pertussis showed maximum hemolytic activity on human red blood cells followed by poultry and sheep RBCs. B. parapertussis and B. bronchiseptica showed maximum hemolytic activity on sheep and poultry RBCs respectively.

Conclusion: The findings of our study revealed that different determinants are involved in host interactions and virulence of Bordetella species.

Keywords: Animal RBC , s, Bordetella species, Hemolytic activity.

I

NTRODUCTION

Bordetella species cause upper respiratory tract

infection in a variety of hosts. B.pertussis is the

caus-ative agent of whooping cough that is particularly

serious in children. B. parapertussis infect both

hu-mans and sheep. B. bronchiseptica has a broad host

range causing chronic and often asymptomatic respi-ratory infections in a wide host range of animals but rarely in human beings (1,2). The vaccination against whooping cough leads to a dramatic decrease in mor-bidity and mortality among infants and children.

Cur-rently the disease is considered to be re-emerging due to short-lived immunity, rendering adolescents and adults susceptible to re-infection (3,4).

Many of the virulence factors play an important role in the pathogenesis of diseases associated with Bordetella species. These include adhesins like fila-mentous hemagglutinin (FHA), pertactin, tracheal colonization factor, fimbriae and toxins like Adeny-late Cyclase – hemolysin, dermonecrotic toxin and

tracheal cytotoxin (5). Bordetellae coordinate the

differential regulation and virulence gene expression through the two component regulatory systems Bvg AS . The inner membrane Bvg S sensor recognizes a stimulatory signal, initiating a cascade which results in phosphorylation of the cytoplasmic response regu-lator, Bvg A. This in turn, binds to DNA promoter * Corresponding Author: Dr. C.N. Khobragade

Tel: 91(2462)229242 Fax: 91(2462)229245

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26

region and activates the expression of specific viru-lence genes thereby helping to colonize the bacteria in upper respiratory tract by specific adhesion to the ciliated cells (6,7). Expression of virulence factors in B.pertussis is controlled by growth conditions or

environmental stimuli viz. temperature, osmolarity of

growth medium, anaerobiosis and metal ion concen-tration. Under certain stress conditions local changes in the superhelicity of DNA induce or repress genes. The stress induced proteins have a role in survival of bacteria as well as in the manifestation of

patho-genecity in vivo (6-8).

Adenylate Cyclase Toxin (ACT) a large modular bifunctional protein encoded by a structural gene cya A of Cya operon and posses both adenylate Cyclase (AC) and hemolytic activity . It is located predomi-nantly in the periplasmic space of the bacteria and small portion of it is released into the medium dur-ing bacterial growth (9,10). It belongs to the RTX (Repeats in toxin) family of bacterial toxin, as the C- terminal hemolysin domain is homologous to the

hemolysin of E. coli (gene hlyA, hlyC) responsible

for extra intestinal infection, therefore acting

inde-pendently as hemolysin and causes lysis of RBC,

while the N terminal 400 amino acid contain the cata-lytic domain responsible for enzymatic activity (AC) (11,12). AC domain of ACT is activated by

calmod-ulin (CaM) a Ca2+ binding protein resulting in rapid

ATP depletion and uncontrolled level of cAMP ac-cumulation particularly in human macrophages, neu-trophills and erythrocytes therefore causing cell lysis. This strategy becomes a major way for the bacterium to evade the immune system of the infected host effi-ciently. Therefore, both enzyme (AC) and hemolytic

activities are involved in induction of cell lysis. In

vitro studies have revealed that hemolytic activity is affected by a number of environmental and cultural conditions (13).

In the present study we report the different factors affecting the AC-hemolysin production

by three potent human pathogens of Bordetella

species vizB.pertussis, B. parapertussis and B.

bronchiseptica in terms of cell bound and cell free hemolysin on human, poultry and sheep RBCs at variable pH, temperature and cell age using Stainer Scholte media, which is particularly interesting in the microbial pathogenecity study. Hemolytic activity determination is carried out either by growing the test organism on blood agar for 24 hrs or bacterial cell suspension is incubated with specific host red blood cell suspension.

MATERIALS AND METHODS

This study was carried out as per the guide lines of Helsinki Declaration on ethical principles for medical research involving human subjects.

Chemicals. Hemoglobin, Bordet-Gengou agar,

and Blood agar were purchased from HiMedia Labo-ratories Pvt. Ltd., Mumbai. All other chemicals used were of analytical grade.

Blood sample and specimen Collection. Blood

samples of sheep and poultry were collected from local slaughter houses and the local market Nanded, Maharashtra under the supervision of veterinary practitioners, while human blood samples were obtained from normal healthy volunteers by lab

technicians in sterile bulbs. Bordetella species were

isolated from the specimen (n=50) collected from nasopharynx region of suspected whooping cough patients using calcium alginate swabs during the period of April 2006 to April 2007. The swabs were immediately stored in Stainer Scholte transport media to maintain cell viability and used without delay. All the fifty samples were collected and processed for

bacterialisolation.

Bacteriological analysis. The samples were

primarily cultured on Bordet-Gengou agar and Blood agar [HiMedia Laboratories Pvt. Ltd] by spreading 0.1 ml of Stainer Scholte media (14). The plates

were incubated aerobically at 37°C for 3-5 days until

the appearance of colonies. The suspected colonies were selected and maintained on Nutrient agar slant

at 4°C throughout the work. The selected colonies

were then identified by their cultural and biochemical charecteristics in Urea broth, Nitrate reduction broth,

Simmon’s citrate broth and by motility test as B.

pertussis (n= 35), B. parapertussis (n=18) and B. bronchiseptica (n=4). For preservation of RBC’s, 1 ml of Alsever’s solution was used for 10 ml of blood samples to defibrinate the RBC’s and to prevent from clotting (15). Hemoglobin standard curve was prepared by dissolving hemoglobin powder (10mg/ ml) and diluted serially in a phosphate saline buffer (PBS). Absorbance of each dilution was recorded at 555 nm (Schimadzu type UV 1601).

Screening for hemolytic activity. Hemolytic

activityofBordetella species were qualitatively tested

on different blood agar plates containing human, sheep and poultry blood separately. Spot inoculated blood

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28

BODADE ET AL . IRAN. J. MICROBIOL. 1(2) : 26 - 31 COMPARATIVE STUDY OF HEMOLYTIC ACTIVITY OF BORDETELLA SPESIES

of clearance (in mm) confirms the hemolytic activity caused due to RBC cell lysis by hemolytic enzyme. Hemolytic activity was screened from 30 min to 180 min to determine the optimum time for reaction. For Assay of cell bound and cell free hemolytic activity, loop full culture of each bacterial isolate was inoculated aseptically in 100 ml of Stainer Scholte broth supplied

with 10 % blood and incubated at 37°C for 3 h. After

proper incubation, the broth was centrifuged at 8000 rpm for 20 min. The cell pellet and supernatant were collected separately and used for cell bound and cell free hemolytic activity, respectively. The cell pellets were resuspended in 100 ml of PBS till the absorbance value

of 0.2 at 555nm was attained which correspond to 2×109

CFU/ml. It was then tested for hemolytic activity.

Assay of cell bound hemolytic activity. 100 ml

of each bacterial cell suspension was mixed thoroughly with 2 ml of RBC cell suspension (1% v/v in PBS) along with 0.5 ml of Alsever’s solution. The sample flasks were

incubated for 3 h at 37°C with occasional shaking. After

3 hrs., the erythrocytes were recentrifuged at 3000 rpm for 15 min. The absorbance of cell free supernatant was recorded at 555 nm against a proper blank and amount of hemoglobin released was determined from a standard hemoglobin curve. Flasks without inoculums were treated as control.

Assay of cell free Hemolytic activity: The cell free

hemolytic activity assay was identical to cell bound hemolytic assay in which instead of bacterial cell suspension, cell free culture supernatant was mixed with RBC suspension.

Factors affecting Hemolytic activity: Effect of

temperature on hemolytic activity of both cell bound and cell free hemolysin under stationary condition was studied by growing bacterial species in Stainer Scholte

media at various temperatures ranging from 20-60°C

for 28 h. The hemolytic activity was calculated from hemoglobin standard graph. The effect of pH was determined by adjusting the broth pH ranging from 6-9

and incubated at 37°C for 28 h. Effect of cell age was

studied in Stainer Scholte broth at 37 °C for 28 h in a

test flask. Every 4 h of incubation, 10 ml of broth was removed aseptically and used for calculating the cell free (Table1) and cell bound (Table 2) hemolytic activity.

RESULTS

All three isolates of Bordetella species were

isolated after five days of incubation using Gengou

Bordet agar at 37°C and identified as B. pertussis,B.

parapertussis and B.bronchiseptica using the standard methods (14). All three isolates showed maximum hemolytic activity on blood agar measured in terms

of zone of clearance by B. pertussis (7mm on human

RBC’s), B. parapertussis (6 mm on sheep RBC’s)

and B. bronchiseptica (5.5 mm on poultry RBC’s)

at 37°C. Both cell bound and cell free hemolysin

activity was observed for B. pertussis followed by B.

parapertussis and B. bronchiseptica on human, sheep and poultry RBCs respectively. Cell bound hemolytic activity was found to be higher than that for cell free hemolytic activity (Table 1, 2). Hemolytic activity of all isolates was also screened from 30 min to 180 min and found optimum at 3 h. Temperature effect on hemolytic activity was assayed in the range of

20 to 60°C and was revealed optimum at 37°C for

all species and then declined with respect to rise in temperature.At optimum pH 7.6-8.0 (alkaline condition) all species showed maximum hemolytic activity, while reduced activity was recorded above the optimum pH. The hemolysin production was found to be increased with respect to cell age and time. The cells grown up to 20 to 24 h revealed maximum hemolytic activity and later on its production was stabilized. Within 4 to 8 hrs., a two fold production of hemolysin was recorded .

DISCUSSION

A clear zone on different blood agars confirms the beta hemolysin production by all the three Bordetella species. Variability in activity may be due to the distribution of membrane receptors on the RBCs of these animals (16,17). Results revealed that the cell bound activity was found always higher than the cell free hemolytic activity and thereby confirms the intracellular behavior of the enzyme (9). B. pertussis showed maximum cell bound activity

on human RBCs followed by sheep and poultry. B.

parapertussis showed maximum hemolytic activity

on sheep followed by human and poultry, while B.

bronchiseptica demonstrated maximum hemolytic

activity on poultry, human and sheep RBCs. The

variation in hemolytic activity may be due to host

specificity (18). This result is in contrast to the Arie

Rogel et al., who reported that, the human erythrocytes

are resistant to the AC lysis in case of B. pertussis (19).

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giving maximum activity at 37°C and decreasing with

increases in temperature irrespective of the species.

This can be attributed to the fact that virulence genes may be activated at this temperature and remain repressed at very low and high temperatures (20,21). This result also coincides with the result of other species, confirming ACT virulent expression to be dependant on environmental factors (22,23). The hemolysin expression at optimum pH appeared to be a strain specific feature and was found maximum in the pH range 7.5- 8.0 with respect to blood samples

(24). B. pertussis showed maximum activity at 7.5, B.

parapertussis and B. bronchiseptica showed activity at 7.5-8.0. For the 24-48 h of incubation, the hemolytic activity was increased on human RBC while all the isolates demonstrated increased activity up to 20 h

on sheep and poultry agar. The variability pattern of hemolytic activity with respect to cell age may be due to the fact that up to 20-24 h, old cultures may be in a transition phase where the hemolysin synthesis and extracellular excretion of hemolysin was decreased, while during cell age of 4-16 h cells were in active phase giving maximum hemolytic activity (22). Thus, we concluded that pH, temperature and cell age affect

the hemolytic activity of Bordetella species and are

responsible for expression of ACT virulence gene.

The variations in hemolytic effect with respect to pH, temperature and cell age help to explain the difference in host range and pathogenesis between these closely related isolates.

Table 1. Effect of pH on cell free hemolytic activity (Hb released in mg) of Bordetella species on different RBCs

B. bronchiseptica (N= 4) B. parapertussis (N= 18) B. pertussis(N= 35)

Sheep

RBC Poultry RBC Human RBC Sheep RBC Poultry RBC Human RBC Sheep RBC Poultry RBC Human RBC pH

1 6 1.52±0.02 1.5±0.01 1.08±0.02 1.56±0.05 1.20±0.02 1.20±0.01 1.56±0.02 1.74±0.02 2.04±0.02 2 6.5 1.60±0.03 1.56±0.03 1.44±0.03 1.68±0.03 1.18±0.02 1.32±0.01 1.60±0.03 1.82±0.02 2.28±0.02 3 7 1.80±0.04 1.74±0.04 1.52±0.03 1.76±0.03 1.44±0.03 1.44±0.02 1.74±0.03 1.86±0.02 3.06±0.02 4 7.5 1.85±0.05 1.80±0.07 1.62±0.04 2.04±0.02 1.50±0.04 1.50±0.02 1.95±0.04 2.04±0.03 3.30±0.03 5 8 1.84±0.05 1.86±0.08 1.40±0.05 1.80±0.02 1.28±0.05 1.74±0.03 1.76±0.05 1.80±0.03 2.22±0.04 6 8.5 1.74±0.32 1.70±0.03 1.15±0.02 1.52±0.03 1.24±0.06 1.17±0.03 1.52±0.03 1.68±0.03 1.86±0.04 7 9 1.48±0.02 1.50±0.02 1.08±0.04 1.30±0.03 1.02±0.02 1.11±0.02 1.40±0.02 1.56±0.01 1.80±0.03

Temperature (C)

1 20 1.80±0.04 2.28±0.03 1.80±0.02 1.74±0.03 1.38±0.04 1.80±0.03 1.90±0.02 2.46±0.04 1.68±0.03 2 30 1.86±0.04 2.40±0.02 1.87±0.03 1.78±0.04 1.50±0.03 1.84±0.01 2.00±0.02 2.76±0.03 2.15±0.02 3 37 2.16±0.03 2.58±0.02 2.16±0.03 2.89±0.05 1.70±0.03 2.04±0.01 2.46±0.03 2.88±0.02 2.22±0.03 4 50 1.20±0.02 2.34±0.01 1.15±0.02 1.70±0.03 1.48±0.03 1.15±0.02 1.97±0.03 2.22±0.03 1.38±0.03 5 60 1.14±0.02 2.22±0.02 1.08±0.02 1.52±0.03 1.40±0.02 1.08±0.02 1.84±0.03 2.10±0.03 1.32±0.04

Cell Age (hrs.)

1 4 0.24±0.02 0.63±0.02 0.42±0.03 0.33±0.03 0.75±0.02 0.48±0.04 0.42±0.03 0.78±0.03 0.50±0.03 2 8 1.34±0.03 0.93±0.02 0.93±0.02 1.32±0.02 1.08±0.01 1.02±0.03 1.56±0.03 1.14±0.04 1.08±0.02 3 12 1.74±0.03 1.50±0.02 1.82±0.04 1.65±0.01 1.62±0.01 1.84±0.02 1.76±0.03 1.68±0.03 1.95±0.01 4 16 1.79±0.04 1.83±0.01 1.86±0.02 1.74±0.01 1.86±0.02 2.12±0.01 1.82±0.03 2.10±0.01 2.16±0.03 5 20 1.80±0.04 2.56±0.01 2.09±0.04 1.80±0.02 2.52±0.03 2.16±0.02 2.16±0.01 2.40±0.02 2.28±0.04 6 24 1.84±0.04 2.22±0.03 2.04±0.03 1.80±0.02 2.40±0.03 2.22±0.02 2.04±0.02 2.46±0.02 2.28±0.03 7 28 1.82±0.04 1.86±0.03 1.86±0.05 1.78±0.02 1.95±0.04 2.12±0.03 1.86±0.03 1.50±0.02 2.15±0.02

Sr.No. Factor

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30

BODADE ET AL . IRAN. J. MICROBIOL. 1(2) : 26 - 31 COMPARATIVE STUDY OF HEMOLYTIC ACTIVITY OF BORDETELLA SPESIES

B. bronchiseptica(N= 4) B. parapertussis(N= 18) B. pertussis(N= 35) Sheep

RBC Poultry RBC Human RBC Sheep RBC Poultry RBC Human RBC Sheep RBC Poultry RBC Human RBC pH

1. 6.0 2.58±0.002 2.46±0.04 3.20±0.02 2.76±0.02 2.74 0.04 2.70±0.04 2.70±0.01 2.82±0.04 3.46±0.02

2. 6.5 3.24±0.03 3.12±0..01 3.32±0..03 3.08±0.02 3.00 0.02 2.94±0.02 2.84±0.02 2.84±0.02 4.50±0.02 3. 7.0 3.47±0.02 3.18±0.02 3.60±0.004 3.24±0.01 3.54 0.03 3.24±0.01 2.97±0.04 3.18±0.002 4.76±0.02

4. 7.5 3.68±0.01 4.26±0.02 3.44±0.02 3.90±0.01 3.72 0.04 3.30±003 3.50±0.02 3.36±0.02 5.04±0.01

5. 8.0 3.78±0.03 4.05±0.04 3.28±0.02 3.72±0.02 3.68 0.02 3.72±0.02 3.00±0.01 3.30±0.01 3.00±0.002

6. 8.5 3.14±0.03 2.82±0.03 3.14±0.02 3.40±0.02 3.60 0.02 3.18±0.02 2.80±0.01 3.50±0.02 2.94±0.04

7. 9.0 2.84±0.02 2.52±0.03 3.08±0.01 2.86±0.02 3.18 0.02 3.08±0.01 2.42±0.04 3.24±0.02 2.72±0.04

Temperature (°C)

1. 20 3.00±0.04 1.94±0.02 3.06±0.02 3.42±0.01 3.06±0.02 3.78±0.02 3.56±0.02 3.24±00.02 3.06±0.02

2. 30 3.24±0.04 3.18±0.03 3.60±0.02 3.54±0.02 3.40±0.03 4.02±0.02 3.72±0.01 3.45±0.03 3.24±0.04

3. 37 3.60±0.03 3.78±0.03 3.78±0.02 4.19±0.03 3.50±0.02 4.14±0.003 4.08±0.02 3.66±0.05 4.20±0.04

4. 50 2.70±0.02 2.65±0.04 2.75±0.01 3.30±0.03 2.22±0.01 2.82±0.02 3.64±0.02 2.88±0.05 2.60±0.04

5. 60 2.64±0.02 2.52±0.04 2.60±0.02 2.97±0.02 1.92±0.01 2.70±0.02 3.47±0.02 2.46±0.05 2.22±0.02

Cell Age (hrs.)

1. 4 2.16±0.02 1.83±0.04 1.89±0.02 2.18±0.05 1.89±0.02 1.92±0.03 2.24±0.06 1.92±0.04 1.98±0.02

2. 8 3.24±0.02 2.40±0.03 3.54±0.02 3.36±0..03 2.58±0.04 3.60±0.04 3.52±0.02 2.64±0.04 3.90±0.02

3. 12 4.19±0.04 2.84±0.03 4.08±0.03 4.34±0.03 2.97±0.02 4.14±0.05 4.42±0.01 3.00±0.04 4.19±0.03

4. 16 4.42±0.03 3.45±0.02 4.4±0.03 4.59±0.03 3.54±0.04 4.74±0.05 4.70±0.02 3.69±0.02 4.86±0.04

5. 20 4.52±0.03 4.44±0.04 4.55±0.04 4.72±0.02 4.34±0.003 4.77±0.05 4.80±0.04 4.19±0.02 4.97±0.05

6. 24 4.50±0.02 3.96±0.02 4.48±0.05 4.74±0.02 4.08±0.03 4.82±0.05 4.78±0.03 4.14±0.02 4.88±0.01

7. 28 4.48±0.01 3.93±0.01 4.40±0.05 4.68±0.02 3.96±0.02 4.78±0.06 4.59±0.02 4.08±0.02 4.80±0.02

Sr.No. Factor

Table 2. Effect of pH, Temperature & cell Age on Cell bound hemolytic activity (Hb released in mg) of Bordetella species on different RBCs

Data represented is mean of three replicates

ACKNOWLEDGMENTS

The authors are thankful to the Head of the government medical College and Hospital Department of Microbiology Nanded for excellent help.

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Figure

Table 1. Effect of pH on cell free hemolytic activity (Hb released in mg) of Bordetella species on different RBCs
Table 2. Effect of pH, Temperature & cell Age on Cell bound hemolytic activity (Hb released in mg) of Bordetella species on different RBCs

References

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