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Year : 2013  |  Volume : 31  |  Issue : 2  |  Page : 148--153

Intraperitoneal inoculation of Haemophilus influenzae local isolates in BALB/c mice model in the presence and absence of virulence enhancement agents

N Mojgani1, V Maldjae2, M Rahbar3, SM Mirafzali4, S Khoshnood1, A Hatami5,  
1 Biotechnology Department, Iran Reference Health Lab, Ministry of Health and Medical Education, and Pathology, Milad National Hospital, Tehran, Iran
2 Department of Quality Control, Razi Vaccine and Serum Research Institute, Karadj, Iran
3 Department of Microbiology, Iran Reference Health Lab, Ministry of Health and Medical Education, and Pathology, Milad National Hospital, Tehran, Iran
4 Department of Medical Vaccine Research, Razi Vaccine and Serum Research Institute, Karadj, Iran
5 Department of Pathology, Razi Vaccine and Serum Research Institute, Karadj, Iran

Correspondence Address:
N Mojgani
Biotechnology Department, Iran Reference Health Lab, Ministry of Health and Medical Education, and Pathology, Milad National Hospital, Tehran


Purpose:Haemophilus influenzae (Hi), predominantly type b accounts for approximately 4% of cases of community-acquired and nosocomial meningitis, in adults. The objective of this study was to evaluate the pathogenicity of local Hi isolates (type b, f and non-typable) in BALB/c mice in the presence of virulence enhancement agents. Materials and Methods: Three different concentrations of the Hi isolates were inoculated intraperitoneally in BALB/c mice in the presence of 2% hemoglobin and 4% mucin as virulence enhancing agents (VEA). The ability of the isolates to produce bacteremia, the percent survival and lethal dose (LD 50 ) were recorded in different challenge groups. Results: The 3 Haemophilus influenzae type b (Hib) isolates used in study were able to show virulence in BALB/c mice model only in the presence of VEA and their LD 50 decreased significantly when 2% hemoglobin and 4% mucin were used. All survived animals showed bacteremia within 4 h of inoculation which was cleared within 18 h. Significant differences ( P < 0.01) in the virulence and survival percentage of Hib challenge groups were observed based on their dose of inoculation and VEA. None of the isolates were able to induce infection in the absence of VEA. Non-type b isolates failed to produce disease in the mice models even at the highest inoculated dose (10 8 cfu) and in the presence of VEA. Conclusions: BALB/c mice appeared suitable for evaluating the virulence of Hib strains, and 2% hemoglobin with 4% mucin an appropriate concentration for inducing infection in this animal model.

How to cite this article:
Mojgani N, Maldjae V, Rahbar M, Mirafzali S M, Khoshnood S, Hatami A. Intraperitoneal inoculation of Haemophilus influenzae local isolates in BALB/c mice model in the presence and absence of virulence enhancement agents.Indian J Med Microbiol 2013;31:148-153

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Mojgani N, Maldjae V, Rahbar M, Mirafzali S M, Khoshnood S, Hatami A. Intraperitoneal inoculation of Haemophilus influenzae local isolates in BALB/c mice model in the presence and absence of virulence enhancement agents. Indian J Med Microbiol [serial online] 2013 [cited 2020 Jan 19 ];31:148-153
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Haemophilus influenzae type b (Hib) is the leading cause of invasive Haemophilus influenzae (Hi) in children younger than 5 years in unvaccinated populations. [1],[2],[3] Currently, the incidence of Hib invasive diseases has greatly decreased in many developed countries because of the widespread of the Hib conjugate vaccine while other serotypes of mainly non-typable Haemophilus influenzae (NTHi) are becoming more common cause of invasive disease in all age groups. [4],[5],[6] . However, in many developing countries where Hib vaccination is not routine, invasive Hib disease is still a significant cause of morbidity and mortality. [5],[7],[8]

Usually the efficacy of a vaccine developed is evaluated in a laboratory animal model which is susceptible to the infection. Numerous have indicated lack of an appropriate and suitable animal model for studying the exact mechanism of virulence of different Hi strains. A number of animal models including mice, rats, rabbits used for studying the pattern of meningococcal disease had limitations in that a large number of bacteria were often required for inducing lethal infection. In 1985, Brodeur et al., [9] infected mice intraperitoneally in the presence of enhancement vehicles in an effort to find an appropriate animal model with uniform and high susceptibility to virulent strains by developing a progressive and fatal bacteremia. Later, a number of virulence enhancing agents (VEA) were proposed which could enhance the susceptibility of the animals to Hi infection and reduce the infective dose. Virulence-enhancement agents studied includes ferric ammonium citrate, capsicum, hematin, hemoglobin, iron dextran, iron sorbitol, lysed red blood cells, mineral oil, mucin, transferin, trypsin, and hemoglobin. [2],[9],[10],[11] In a study, combination of mucin-hemoglobin was shown to be significantly more effective in increasing the susceptibility of mice to bacterial infections, and hence suggested that mucin and hemoglobin interferes with the phagocytic process of the macrophages in the peritoneal cavity or systemic clearance or both, and thus allowing organism to enter the blood circulation. [12],[13],[14] The main objective of this study was to analyze the virulence of locally isolated Haemophilus influenzae typeb (Hib), type f (Hif) and an uncapsulated (NTHi) strain. The virulence and lethal dose (LD 50 ) of the selected isolates was studied by intraperitonial injection of different concentrations of the bacterial suspension in BALB/c mice with 2 different enhancement agents.

 Materials and Methods

Bacterial strains and growth conditions

Previously isolated clinical strains of Hib (three isolates), Hif (one isolate) and NTHi (one) were used in this study. The culture samples were grown on gonococcus media (GC) (Hi Media, India) and Chocolate Agar (5% sterile sheep or horse defibrinated blood), Brain Heart Infusion Broth supplemented with 3 and 1.5 mg/ml of Hemin and nicotinamide adenine dinucleotide (Sigma, USA), respectively. All strains were grown overnight at 37°C in 5% CO 2 incubator.

All the isolates were maintained for more than a week at 4°C in chocolate agar slants. Strains were also maintained at 4°C by weekly transfer on agar media. Long-term storage was best accomplished by storing the freshly grown suspension of the culture in skim milk with 10% glycerol in Liquid Nitrogen tanks.

Bacterial suspension

The strains were grown for 10-12 h (to reach logarithmic phase) in supplemented Brain Heart Infusion broth (1.5 mg/ml ofbeta Nicotinamide adenine dinucleotide β-NAD and 3.0 mg/ml of Hemin) at 37°C under shaking (200 rpm), in the presence of 5% CO 2 . After incubation the culture broth were centrifuged at 4°C for 10 min and pellet washed twice with Phosphate buffer saline (PBS). The samples were diluted in PBS to achieve different concentrations in colony forming unit cfu/ml adjusted spectrophotometrically and based on McFarland 3 standard.

Virulence enhancement factors

Purified bovine hemoglobin (Oxoid, England) and porcine mucin (Sigma Chemical Co, USA) were used as VEA. Hemoglobin was diluted to 2% in 0.9% saline and sterilized by autoclaving. Mucin was used at concentrations of 4% and sterilized by filtration (0.2 μm filters; Sartorius, Germany). 2% hemoglobin individually and in combination with 4% mucin was used with different concentrations of bacterial suspension for inoculation into animal models as described previously. [12]

Animal model

Inbred male BALB/c mice were obtained from the Department of Laboratory Animal Research, Razi Vaccine and Serum Research Inst. Iran. Groups of eight mice, 4-5 weeks and weighing 17-22 g were housed under standard conditions of temperature and relative humidity with a 12 h lighting schedule. Mice Food and water were available ad libitum.

All mice were injected intraperitoneally with different doses of bacterial suspensions (1 ml, 0.5 ml and 0.25 ml) with and without the enhancement agents. Criteria of disease included clinical observations, and blood culture. Percentage survival was recorded in each group of mice by observing the survival and death rate for 72 h.


Bacteremia was monitored by quantitative culture of blood removed aseptically from the tail after different time intervals. 100 μl of the collected blood were plated on chocolate agar plates and incubated at 37°C in presence of 5% CO 2 for 24 h. The lower limit of presence of bacteremia for each group of animals was considered 100 cfu/ml.

Determination of median LD 50 and percentage survival

The median LD was calculated by the method of Reed and Muench. [15] The median lethal inoculum was calculated by Combistats software. Comparison of the mortality rates was performed with the Chi-square test analysis. Values of P < 0.05 were considered significant.


The virulence of previously isolated clinical isolates of Hib, Hif and NTHi were studied by intraperitoneal inoculation of three different concentrations of the cultures in BALB/c mice models in the presence of 2% hemoglobin and 4% mucin. The mentioned concentrations of the VEA used in study were selected based on their low toxicity in mice, as higher concentrations of mucin (5%) resulted in severe diarrhea and weakness in the animals (unpublished data).

[Table 1] and [Table 2], shows the effect of intraperitoneal inoculation of different doses of Hi isolates in the presence of 2% hemoglobin during different time intervals. According to results the frequency of the infection caused by all Hib isolates increased with the inoculum size. High doses (2 × 10 8 cfu/ml) of the Hib strains NM3 and NM2 proved lethal and killed the challenged mice within 24 h of post infection. Although both the mentioned Hib isolates were able to kill the mice within 24 h at the same dose but the survival percentage was different for both the isolates. Approximately 10 8 cfu/ml of Hib NM2 with 2% hemoglobin were sufficient enough to kill 50% of the animals, while 100% of the mice died within 72 h after injected with 4.9 × 10 8 of Hib isolate NM3. No death was seen in mice groups infected with the lowest inoculum dose of 1.2 × 10 7 cfu/ml. The mice groups challenged with NM4 in the presence of 2% hemoglobin showed transient bacteremia with no death and 100% recovery within 24 h, and thus appeared to be the least virulent strain. None of the non-type b isolates tested in study were able to cause infection in the BALB/c mice groups and were considered non virulent in this animal model.{Table 1}{Table 2}

The virulence of the selected Hib isolates in the presence of 2% hemoglobin and 4% mucin is seen in [Table 3]. The lethality of the Hib isolates in study were significantly enhanced in the presence of hemoglobin and mucin in combination rather than hemoglobin alone. According to results, the virulence of the mice groups challenged with Hib isolates in the presence of 2% hemoglobin alone was significantly lower and high doses of bacterial inoculations were required to induce infection. In comparison, in the groups challenged with 2% hemoglobin in combination with 4% mucin a significant decrease in the LD 50 was observed (almost half the LD 50 of the groups inoculated with 2% hemoglobin alone). Hib NM3 appeared to be the highest virulent strain and approximately 100 bacteria were able to give 100% death of the challenged mice groups. Low concentrations of the Hib strains were able to induce infection and bacteremia was observed in these animals, with higher survival percentage [Table 4].{Table 3}{Table 4}

In all survived animals challenged with the Hib isolates with VEA, bacteremia was usually observed within 4 h of inoculation which was cleared completely within 18 h of post-infection. All the infected animals showed weakness and loss of appetite during the initial hours of inoculation and were clinically normal thereafter.

In the absence of enhancement agents, all the Hib isolates appeared non-infective and were not able to kill the challenged mice even at high doses of 10 8 cfu/ml. However, a transient bacteremia was observed in few of the animals injected with high doses of Hib NM3 without hemoglobin and or mucin.

Intraperitoneal inoculation of Hif strain (NM7) and uncapsulated strain (NM9) in BALB/c mice models appeared an ineffective way for inducing infection as neither of the strains were able to induce bacteremia or death in the injected mice models even at the highest dose and in the presence of mucin and hemoglobin.

During statistical analysis the two way analysis of variance was done with independent values taken as cfu and the strains while LD 50 was the dependent value. The results indicated the significance of both the dependent values with P < 0.05. The virulence of the strains was also compared based on their percentage survival (mortality rate) in the group of mice treated with combination of the enhancing agents. The difference between NM2 versus NM3 appeared non-significant (P < 0.73) while there was significant difference in the virulence of these two isolates with NM4 isolate (NM2 vs. NM4 P < 0.03; NM3 vs. NM4 P < 0.05).


With the wide spread use of Hib vaccination especially in developed countries there are reports of decline in Hib disease. However, other serotypes of Hi besides b have been evolving as an important cause of Hi disease in these countries.

In this study, the virulence attributes of previously isolated Hi cultures mainly type b, f and an uncapsulated strain was studied by intraperitoneal inoculation of different doses of the bacterial culture in the presence of mucin and hemoglobin in BALB/c mice model. All three type b cultures used in this study were clinical isolates, isolated from cerebro spinal fluid (CSF) of new born with meningitis. [16]

Different animal models have been used for studying the kinetics of Hi disease including rats, guinea pigs, rabbits, mice etc., Each of these models provides certain advantages over others, and each simulates to a limited extent some aspect of the diseases. [17] Based on the report of Rodriguez et al. [14] which stated that for testing the mechanism of active immunization mice with mature immune system are required, we selected 4-5 weeks BALB/c mice for our studies. In our studies, natural lack of susceptibility of BALB/c mice to Hib infection was evident and mice groups exposed to 10 8 Cfu/ml of Hib without the enhancement agents had a 100% survival rate, indicating the natural resistance of adult BALB/c mice to Hib infection. Hence, the infectivity of these isolates was evaluated in the presence of enhancement factors.

In accordance with the results of other researchers, [2],[11],[14] mucin and hemoglobin used in combination with different doses of bacterial suspensions appeared to be able to significantly enhance the virulence of the Hib isolates in study. The survival percentage of the challenged mice groups varied with the strains, VEA used and dose of inoculation. The LD 50 of Hib NM3 in the presence of combination of 2% hemoglobin and 4% mucin was significantly reduced (2.2 × 10 4 ) compared to (1.8 × 10 8 ) when 2% hemoglobin was inoculated alone. Based on our results, high infectivity of Hib isolates could be achieved by using combination of 2% hemoglobin and 4% mucin as virulence enhancing factors.

Data were statistically compared by an analysis of proportions, using the Chi-square analysis [18] and values of P < 0.05 were considered significant. Significant difference between the mice groups inoculated with hemoglobin alone and the groups treated with combinations (hemoglobin and mucin) were observed (P < 0.01). Similarly, the three Hib isolates in study showed significant differences (P < 0.0001) in their virulence attribute with NM3 as the highest virulent strain and NM 4 the least virulent.

Reports are available which indicates the infectivity of other types of Hi besides type b. Hif sepsis has been reported in a fully immunized, immunocompetent, and previously healthy 9-month-old and a 3-year-old. [19],[20] In a report, Hif was identified as the cause of meningitis in an 8-year-old girl with congenital humoral immunodeficiency. Similarly, NTHi has also been reported to be involved in cases of invasive infections in normal hosts. Lower respiratory tract infections associated with NTHi, are a major cause of mortality in both infants and children in developing countries. [4],[9],[18],[21] The two non-type b isolates used in this study (Hif and NTHi) were isolated from nasopharynx secretions of a 7-year-old and an adult with high fever, sore throat, and pneumonia symptoms, respectively. However, in this study we were unable to prove their [16] infectivity in BALB/c animal models, as both the isolates failed to produce bacteremia or death in the challenged animal even at high doses and in the presence of virulence enhancement agents. As suggested by other researchers, [17],[22] NTHi is not a natural colonizer or pathogen in any of the animal models used and thus there are significant limitations to the usefulness of these models when attempting to make comparisons with human infections. To better understand and confirm the infectivity of non-type b Hi isolates we at present are testing other animal models like rats with intranasal inoculations.

In conclusion, BALB/c mice models used in this study were essentially susceptible to Hib strains in combination with hemoglobin and mucin and hence this animal could be considered suitable for testing the virulence and affectivity of Hib vaccines in future.


This work was supported by Ministry of Jihad-e-agriculture Iran (project No 2-18-18-88038). The authors wish to thank Ms. Mohammadzadeh at Microbiology laboratory, Pathology Department, Milad National Hospital Tehran, for providing the Haemophilus influenza clinical strains. We also wish to thank the laboratory animal research department at Razi Research Institute for providing the animal facilities.


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