Biochemical Properties of Bacterial Contaminants Isolated from Livestock Vaccines
Asghar Ali Kamboh,
In present study, 40 livestock vaccines were tested for bacterial contaminants. Four different bacterial species were identified from the vaccine samples. The species were Escherichia coli, Pasteurella multocida, Bacillus cereus and Bacillus subtilis. Of the 40 livestock vaccines studied, 1 Haemorrhagic septicaemia (H.S) and 2 Anthrax vaccines were found positive for bacterial contaminants, possessing batch numbers 057, 079 and 010 respectively, while 37 samples were observed without any bacterial growth. The percentage prevalence of positive vaccine samples was recorded as 7.5%. The pure contamination was recorded in 1 (33.33%) Anthrax vaccine sample with batch number 079, while 2 (66.67%) samples, 1 H.S and 1 Anthrax with batch numbers 057 and 010 respectively were recorded for mixed bacterial species. During investigating biochemical properties, it was observed that Escherichia coli show the positive reaction to catalase, and negative to oxidase, urease and indole. While Pasteurella multocida, Bacillus cereus and Bacillus subtilis were positive to catalase and oxidase, while negative to urease and methyl red.
Vaccination or active immunization is the artificial introduction of antigens from a microbe into an individual in a controlled way, leading to the stimulation of the immune system without the symptoms of the full-blown disease. This leads to the production of memory cells within the host, so that on a second encounter with the microbe the immune system can generate a rapid antibody response thereby preventing infection (Nicklin et al., 1999). There are several types of vaccines, which are used in veterinary practice like, Attenuated whole-agent vaccines (live vaccines), Inactivated whole-agent vaccines (dead vaccines), Toxoid vaccines, Subunit vaccines, Conjugated vaccines and Nucleic acid vaccines (Tortora et al., 2001).
During the vaccine manufacturing process, pathogens are cultivated on artificial or living media for to obtain their bulk amounts. For viral vaccines, viruses are grown in animals or chick embryo. The virulence of these microorganisms is reduced by passage through a series of animals other than the normal host species. Live viruses may be inactivated by phenol or ultraviolet rays, while bacteria usually by formalin (West, 1998).
The advantages of vaccines that contain dead organisms are that, they are safe with respect to residual virulence, since organisms are already dead. These are commonly available in liquid form along with formalin and have very little risk of alive contamination. While live vaccines may posses residual virulence and these are usually available in freeze-dried form and always run the risk of contamination with unwanted organisms. Out breaks of reticuloendotheliosis in chickens in Japan and Australia has been traced to contaminated Mareks disease vaccine (Tizard, 1995). Samad (2001) reported extraneous contaminants in different manufactured anthrax vaccines. He dected Bacillus megaterium, Bacillus cereus, Bacillus mycoids, and Bacillus subtilis from anthrax live-spore vaccines through the cultivation of vaccine batches on Brain Heart Infusion Agar (BHIA). Feeling the gravity of the situation, it is therefore planed to carry out study of the bacterial contaminants of livestock vaccines and their reorganization on the basis of biochemical properties.
MATERIALS AND METHODS
Collection of vaccine samples: Forty livestock vaccines (both live and
killed) were collected from the market and vaccine production centres of the
country and brought to the laboratory of the Department of Microbiology, Faculty
of Animal husbandry and Veterinary Sciences, Sindh Agriculture University Tando
Jam and Vaccine Production unit Tando Jam, in the thermo flask with ice and
then stored in the refrigerator at 4oC.
Isolation and identification: Vaccine samples were inoculated by streaking method on blood, nutrient, BHI (brain heart infusion) and MacConkeys agar media and incubated aerobically and anaerobically at 37oC for 24 h. Following 24 h of incubation, colonies from blood, MacConkeys and nutrient agars were picked-up by sterilized wire-loop and cultured on nutrient and MacConkeys agar plates. The process of sub-culturing continued until pure growths were obtained. Purity of the isolated bacterial strains was determined on the basis of their morphological and cultural characteristics. This was done by making the smear, stained with Grams stain and examined under microscope. The organisms were isolated and identified by adopting the method as prescribes by Khalil (1992). The species of the organisms were recognized by checking their biochemical properties.
The prevalence of bacterial contaminants in livestock vaccines: The
number and percentage prevalence of bacterial species as contaminants recognized
from livestock vaccines are presented in Fig. 1. A total of
40 livestock vaccines were examined (Table 1), from which
3 (7.5%) vaccines 1 Haemorrhagic Septicaemia (H.S) and 2 Anthrax possessing
batch numbers 057, 079 and 010 respectively, were found positive for various
bacterial isolates while 37 (92.5%) were exhibited no growth and recorded as
negative for any bacterial contaminants.
The incidence of pure and combined bacterial contaminants in local and imported livestock vaccines: The bacterial contaminants identified from local livestock vaccines are given in Fig. 2 and 3. A total of 40 samples of livestock vaccines were examined, 1 (33.33%) with batch number 079 and 2 (66.67%) with batch numbers 057 and 010 were determined having pure and combined bacterial species respectively. The pure bacterial contaminant was Bacillus cereus from Anthrax Vaccine with batch number 079, while the mixed bacterial contaminants in H.S vaccine sample with batch number 057 were Pasteurella multocida + Escherichia coli and Anthrax vaccine with batch number 010 were Bacillus cereus + Bacillus subtilis (Fig. 3).
Biochemical properties of bacterial contaminants isolated from livestock vaccines: After isolation bacterial organisms were checked for their biochemical properties (Table 2). During investigating biochemical properties, it was observed that Escherichia coli show the positive reaction to catalase and negative to oxidase, urease and indole. While Pasteurella multocida, Bacillus cereus and Bacillus subtilis were reacted positively to enzymes catalase and oxidase, while negative to urease and methyl red. For Voges proskauer Escherichia coli and Pasteurella multocida were negative, while Bacillus cereus and Bacillus subtilis were positive. For gelatin liquefaction and citrate utilization Escherichia coli was negative, while Bacillus subtilis was positive.
During present investigation, a total of 40 livestock vaccine samples were
examined, out of which 3 (7.5%) were found positive for various bacterial contaminants
possessing batch numbers 057, 079 and 010, while 37(92.5%) were found free from
bacterial species (Fig. 1 and 3).
and percentage prevalence of bacterial contaminants in livestock vaccines
and percentage prevalence of pure and mixed bacterial contaminants in
The bacterial contaminants identified from livestock vaccines during this study
are given in Figure 3. A total of 40 samples of livestock
vaccines were examined, 1 (33.33%) and 2 (66.67%) were determined having pure
and combined bacterial species respectively (Fig. 2). The
pure bacterial contaminant was Bacillus cereus from Anthrax Vaccine,
while the mixed bacterial contaminants were in H.S vaccine and Anthrax vaccine
of bacterial contaminants in livestock vaccines
incidence of individual bacterial species in livestock vaccines
The bacterial species isolated from anthrax live-spore vaccines by Samad (2001) as mixed contaminants were Bacillus megaterium, Bacillus cereus, Bacillus mycoids, and Bacillus subtilis. Whereas, Kojima et al. (1997) reported the contamination of avian Mycoplasma DNA in the avian live virus vaccines. The specificity of the primers showed 34 strains belonging to nine species of avian Mycoplasma, from which Mycoplasma synoviae and Mycoplasma gallisepticum were predominant. Mbulu et al. (2004) reported Mycoplasma mycoides subsp. mycoides in cattle due to use of contaminated Contagious Bovine Pleuropneumonia (CBPP) vaccine. Landman et al. (2000) examined Mareks disease vaccine and observed the pure contamination by Enterococcus faecalis.
Although, the findings of some workers are not in close agreement to the bacterial
contaminants identified during the present study. They also determined some
other bacterial species and viruses that had contaminated livestock and human
vaccines. The bacterial species recognized in our study were more or less same
as recorded by Samad (2001). While Mbulu et al. (2004), Landman et
al. (2000) and Kojima et al. (1997) reported the different organisms
which were not recorded during the present study. The presence of the organisms
in the livestock vaccines was due to the several practical reasons. It could
be due to use of poor quality preservative, use of poor instruments and old
techniques, use of poor sterilized packing material, unhygienic condition at
laboratory, poor management at laboratory especially in the culture room and
unsound technical staff at vaccine production units/centers.
properties of bacterial species recognized from livestock vaccines
-ve = negative- = not done+ve = positive
K/A = alkaline slant and acidic butt
TSI = triple sugar iron
A/A = acidic slant and acidic butt
H2S = Hydrogen sulphide gas
MR = methyl red
VP = Voges Proskauer
GL = gelatin liquefaction
NR = Nitrate reduction
The findings of biochemical properties of isolated organisms are presented
in Table 2. According to these cells of Escherichia coli
were found catalase positive but oxidase negative. It exhibited A/A but did
not produce H2S in TSI agar. It interacted positively with methyl red and nitrate
reduction but negatively to indole, urea, Voges-Proskauer, gelatin and citrate.
While, Pasteurella multocida was found positive to catalase, oxidase,
indole and nitrate reduction, but negative to urea, methyl red and citrate.
It exhibited A/A without production of H2S in TSI agar. Khalil and Gabbar (1992);
Nizamani (1999); Devrajani (2005) and Dewani (2000) who recorded similar biochemical
properties of Escherichia coli as demonstrated in the present investigation.
Similarly, Khan and Rind (2001) and Fazlani (2005) also recorded similar biochemical
properties of Pasteurella multocida as recorded in the present study.
The findings about biochemical properties of Bacillus cereus observed
in this survey are also in line to the other workers (Fazlani, 2005; Dewani,
2000 and Devrajani, 2005). While results about Bacillus subtilis were
in close agreement as observed by Samad (2001), Merchant and Packer (1999).Therefore
one should say that these are the same species identified in their investigations,
also recognized in the present study.
Conclusion: From the present study, it is concluded that some livestock vaccines possess extraneous bacterial contaminants. It is further observed that anthrax (live spore) vaccines contain extraneous bacilli along with actual vaccinal organisms. The bacterial organisms isolated from vaccines are same in biochemical properties as they have originally.
1: Devrajani, K., 2005. Bacteriological study on camel wounds. M.Sc. Thesis, Department of Microbiology, Sindh Agriculture University Tandojam.
2: Dewani, P., 2000. Bacteriological studies on mastitis in ewes and goats. M.Sc. Thesis, Department of Microbiology, Sindh Agriculture University Tandojam.
3: Fazlani, S.A., 2005. Bacteriological study on clinical mastitis in camel. M.Sc. Thesis, Department of Microbiology, Sindh Agriculture University Tandojam.
4: Khalil, M.A. and A. Gabbar, 1992. Procedures in Veterinary Microbiology. 2nd Edn., CVDL, Tandojam, Sindh, Pakistan, pp: 6-93
5: Khan, T.S. and R. Rind, 2001. Isolation and characterization of bacterial species from surgical and non-surgical wounds located on body surface of buffaloes, cattles, sheep and goats. Pak. J. Biol. Sci., 4: 696-702.
CrossRef | Direct Link |
6: Kojima, A., T. Takahashi, M. Kijima, Y. Ogikubo and M. Nishimura et al., 1997. Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction. Biologicals, 25: 365-371.
CrossRef | PubMed | Direct Link |
7: Landman, W.J.M., K.T. Veldman, D.J. Mevius and P. Doornenbal, 2000. Contamination of Marek`s disease vaccine suspensions with enterococcus faecalis and its possible role in amyloid arthropathy. Avain Pathol., 29: 21-25.
8: Mbulu, R.S., G. Tjipura-Zaire, R. Lelli, J. Frev and P. Pilo et al., 2004. Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp. Mycoides SC. Vet. Microbiol., 98: 229-234.
Direct Link |
9: Merchant, I.A. and R.A. Packer, 1999. Veterinary Bacteriology and Virology. 8th Edn., CBS Pubilishers and Distributers, New Delhi, India, pp: 386-387
10: Nicklin, J., K. Graeme-Cook, T. Paget and R.A. Killington, 1999. Control of Bacterial Infection. In: Instant Notes in Microbiology. Bios scientific publishers, London, pp: 176-179
11: Nizamani, A.W., 1999. Studies on the Bacterial flora of uteri of slaughter sheep. M.Sc. Thesis, Dept. of Microbiology, Sindh Agriculture University Tandojam.
12: Samad, A., 2001. Use of antimicrobial susceptibility pattern as an identification marker of vaccinal and wild type strains of Bacillus anthracis in vaccine manufacturing process. M.Sc Thesis, Department of Veterinary Microbiology, Sindh Agriculture University, Tandojam.
13: Tizard, I.R., 1995. Immunoprophylaxis: General Principles of Vaccination and Vaccines: An Introduction to Veterinary Immunology. 4th Edn., W.B. Saunders, Philadelphia, pp: 178-191
14: Tortora, J.G., R.B. Funke and L.C. Case, 2001. Microbiology: An Introduction. 7th Edn., Benjamin Cummings, San Francisco, pp: 501-505
15: West, P.G., 1998. Black`s Veterinary Dictionary. 18th Edn., Jaypee Brothers, New Delhi, India, pp: 581