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Fulltext Occupational exposure and challenges in tackling M. bovis at human-animal interface: a narrative review

Devi KR, Lee LJ, Yan LT, Syafinaz AN, Rosnah I, Chin VK

Int Arch Occup Environ Health, 2021 08;94(6):1147-1171.
PMID: 33725176 DOI: 10.1007/s00420-021-01677-z

Zoonotic tuberculosis caused by Mycobacterium bovis (M. bovis), a member of Mycobacterium tuberculosis complex (MTBC) has increasingly gathered attention as a public health risk, particularly in developing countries with higher disease prevalence. M. bovis is capable of infecting multiple hosts encompassing a number of domestic animals, in particular cattle as well as a broad range of wildlife reservoirs. Humans are the incidental hosts of M. bovis whereby its transmission to humans is primarily through the consumption of cattle products such as unpasteurized milk or raw meat products that have been contaminated with M. bovis or the transmission could be due to close contact with infected cattle. Also, the transmission could occur through aerosol inhalation of infective droplets or infected body fluids or tissues in the presence of wound from infected animals. The zoonotic risk of M. bovis in humans exemplified by miscellaneous studies across different countries suggested the risk of occupational exposure towards M. bovis infection, especially those animal handlers that have close and unreserved contact with cattle and wildlife populations These animal handlers comprising of livestock farmers, abattoir workers, veterinarians and their assistants, hunters, wildlife workers as well as other animal handlers are at different risk of contracting M. bovis infection, depending on the nature of their jobs and how close is their interaction with infected animals. It is crucial to identify the underlying transmission risk factors and probable transmission pathways involved in the zoonotic transmission of M. bovis from animals to humans for better designation and development of specific preventive measures and guidelines that could reduce the risk of transmission and to protect these different occupational-related/populations at risk. Effective control and disease management of zoonotic tuberculosis caused by M. bovis in humans are also hindered by various challenges and factors involved at animal-human interface. A closer look into factors affecting proper disease control and management of M. bovis are therefore warranted. Hence, in this narrative review, we have gathered a number of different studies to highlight the risk of occupational exposure to M. bovis infection and addressed the limitations and challenges underlying this context. This review also shed lights on various components and approaches in tackling M. bovis infection at animal-human interface.

Matched MeSH terms: Mycobacterium bovis*
Fulltext Development of vaccination strategies: from BCG to new vaccine candidates

Nadiya T. Al-alusi, Mahmood A. Abdullah

Pertanika Journal of Science & Technology, 2014;22(2):387-400.
MyJurnal

During recent years, extensive development has been made to improving vaccines for tuberculosis. This is due to the presence of genome sequences of diverse mycobacterial species and Mycobacteroum tuberculosis (M. tuberculosis) isolates which has led to advances in the characterization of genes and antigens of M. tb and better realization of protective immune responses to the disease in both animals and humans. This review summarizes vaccine types, reasons for variable efficacy of BCG, latest advances in tuberculosis vaccine development and major vaccine design strategies.

Matched MeSH terms: Mycobacterium bovis
TUBERCULOSIS IN MALAYA

SODHY JS

Med J Malaysia, 1964 Jun;18:239-50.
PMID: 14199442
Matched MeSH terms: Mycobacterium bovis*
BCG Vaccine-associated Complications in a Large Cohort of Children With Combined Immunodeficiencies Affecting Cellular and Humoral Immunity

Al-Herz W, Husain EH, Adeli M, Al Farsi T, Al-Hammadi S, Al Kuwaiti AA, et al.

Pediatr Infect Dis J, 2022 11 01;41(11):933-937.
PMID: 36102730 DOI: 10.1097/INF.0000000000003678

AIMS: To present the details of Bacillus Calmette-Guérin (BCG)-vaccine associated complications (VACs) in combined immunodeficiencies (CID) patients.
METHODS: Five centers participated in this retrospective study and completed a data form, which included general patients' information, clinical and laboratory data.
RESULTS: Among 236 CID patients, 127 were BCG vaccinated. 41.9% of patients with family history of CID and 17.1% who were diagnosed by screening were BCG vaccinated. Twenty-three patients (18.1%) developed BCG-VACs. The median age of VACs was 6 months and the median time from vaccination to complications was 6 months. The highest rate of BCG-VACs was recorded in patients receiving the Russian BCG strain compared to the Tokyo and Danish strains. Univariate analysis of T-lymphocyte subsets showed increased odds of BCG complications in patients with CD3+, CD4+, and CD8+ counts of ≤250 cells/µL. Only CD8 + count ≤250 cells/µL had increased such odds on multivariate analysis. VACs were disseminated in 13 and localized in 10 patients. Localized complication occurred earlier after vaccination (median: 4 months) compared with disseminated ones (median: 7 months). There were no significant associations between sex, administered vaccine strain, serum immunoglobulins levels, lymphocyte subsets counts, and the chance of having either localized or disseminated BCG-related complications.
COCLUSIONS: Although contraindicated, many patients with CID continue to be vaccinated with BCG. Low CD8 + count is a risk factor for BCG-related complications and localized complications occurred earlier than disseminated ones. Considerations should be undertaken by health care authorities especially in countries with high incidence of CID to implement newborn screening, delay the time of BCG vaccine administration beyond 6 months of age and to use the relatively safer strains like the Danish and Tokyo ones.

Matched MeSH terms: Mycobacterium bovis*
Fulltext Role of Mantoux test in the diagnosis of tuberculosis

Loh KY

Malays Fam Physician, 2011;6(2):85-86.
PMID: 25606232 MyJurnal

Mantoux test is a sensitive but non-specific in the diagnosis of active tuberculosis. The positive cut-off of 10 mm in a person without BCG and 15 mm with previous BCG is appropriate. The interpretation of Mantoux needs to be correlated to the patient’s clinical context. Mantoux test may have a role in assisting extrapulmonary tuberculosis and latent tuberculosis in children.

Matched MeSH terms: Mycobacterium bovis
Serum antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans

Kilbourn AM, Godfrey HP, Cook RA, Calle PP, Bosi EJ, Bentley-Hibbert SI, et al.

J. Wildl. Dis., 2001 Jan;37(1):65-71.
PMID: 11272506

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.

Matched MeSH terms: Mycobacterium bovis/immunology*; Mycobacterium bovis/isolation & purification
Fulltext Plasmodium falciparum 19 kDa of merozoite surface protein-1 (MSP-1(19)) expressed in Mycobacterium bovis bacille Calmette Guerin (BCG) is reactive with an inhibitory but not a blocking monoclonal antibody

Nurul AA, Rapeah S, Norazmi MN

Trop Biomed, 2010 Apr;27(1):60-7.
PMID: 20562815

Proteins on the surface of Plasmodium falciparum merozoites are good targets for vaccine development against malaria because they are accessible to antibodies in the plasma. The 19 kDa C-terminus of merozoite surface protein-1 (MSP-1(19)) has been shown to induce both inhibitory as well as blocking antibodies, the latter blocking the protective effects of the former. Inhibitory antibodies bind to MSP-1(19) and inhibit merozoite invasion of red blood cells (RBC) but the binding of blocking antibodies can prevent binding of inhibitory antibodies thereby allowing the parasite to invade RBC. We constructed a synthetic version of the MSP-1(19) of the P. falciparum using mycobacterium codon usage by assembly PCR. The synthetic MSP-1(19) was mutated at various sites to promote the production of inhibitory but not blocking antibodies as previously reported. The native and mutated MSP-1(19) were cloned and expressed in Mycobacterium bovis bacille Calmette-Guerin (BCG) and the expressions of the recombinant proteins were detected by specific monoclonal antibodies (mAbs) namely, 12.10 and 1E1 against MSP-1(19) using Western blotting. The mutated MSP-1(19) protein reacted with the inhibitory mAb, 12.10, but not the blocking mAb, 1E1, paving the way for the construction of a potential recombinant BCG (rBCG) blood stage vaccine against malaria.

Matched MeSH terms: Mycobacterium bovis/genetics; Mycobacterium bovis/metabolism*
Fulltext Phagocytic activity and pro-inflammatory cytokines production by the murine macrophage cell line J774A.1 stimulated by a recombinant BCG (rBCG) expressing the MSP1-C of Plasmodium falciparum

Rapeah S, Dhaniah M, Nurul AA, Norazmi MN

Trop Biomed, 2010 Dec;27(3):461-9.
PMID: 21399587 MyJurnal

Macrophages are involved in innate immunity against malaria due to their ability to phagocytose infected erythrocytes and produce inflammatory cytokines, which are important for controlling parasite growth during malaria infection. In this study, the ability of a recombinant BCG (rBCG) vaccine expressing the 19-kDa C-terminus of merozoite surface protein-1 (MSP1-C) of Plasmodium falciparum, to stimulate the phagocytic activity and secretion of pro-inflammatory cytokines by the macrophage cell line J774A.1 was measured at varying times. The results demonstrate the ability of the rBCG construct to activate the inflammatory action of macrophages, which is important as a first-line of defence in clearing malaria infections.

Matched MeSH terms: Mycobacterium bovis/genetics; Mycobacterium bovis/immunology*
Fulltext BCG adenitis - Need for increased awareness

Govindarajan KK, Chai FY

Malays J Med Sci, 2011 Apr;18(2):66-9.
PMID: 22135589

Bacille Calmette-Guerin (BCG) vaccination for protection against tuberculosis has been in use for long. Although the vaccine is safe, its administration can result in complications such as BCG adenitis. We report here a series of children with BCG adenitis with a view to recognise and manage this condition. It is hoped that this case series would encourage the increased identification of this condition.
Study site: Paediatric Surgical Unit, Department of Surgery, Hospital Tengku Ampuan Afzan, Kuantan, Pahang, Malaysia

Matched MeSH terms: Mycobacterium bovis
Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110

Fomukong NG, Tang TH, al-Maamary S, Ibrahim WA, Ramayah S, Yates M, et al.

Tuber. Lung Dis., 1994 Dec;75(6):435-40.
PMID: 7718832 DOI: 10.1016/0962-8479(94)90117-1

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Matched MeSH terms: Mycobacterium bovis/genetics
Fulltext Antibodies against testicular germinal cells in lepromatous leprosy

Wall JR, Wright DJ

Clin Exp Immunol, 1974 May;17(1):51-9.
PMID: 4619358

Testicular germinal cell antibodies were found in forty-four out of the fifty-nine patients with lepromatous leprosy and in four out of ten patients with tuberculoid disease. A similar pattern was found in twelve out of 262 control patients and
normal subjects. The antibody was found to be of the IgG class and forty out of forty-nine of these antibodies were shown to be complement fixing. Spermatozoal antibodies were detected in twelve patients, but no ovarian antibodies were found in any specimen. There was no close correlation between erythema nodosum leprosum (ENL) and testicular antibodies. It was found that the characteristic of the testicular antibody in leprosy was its ability to be absorbed by Mycobacterium BCG suspension suggesting that this is another antibody induced by infection. A similar fluorescent pattern was seen in some patients who did not have leprosy, but in these cases it could not be abolished with BCG. It is concluded that autoimmunity may be one of the factors involved in the pathogenesis of orchitis in leprosy.
Study site: MRC Leprosy Research Unit, Sungei Buloh, Selangor, Malaysia.

Matched MeSH terms: Mycobacterium bovis/immunology
Effect of disaccharide-polyol systems on the thermal stability of freeze-dried Mycobacterium bovis

Tan YZ, Chong YQ, Khong E, Liew YK, Chieng N

Int J Pharm, 2019 Jul 20;566:400-409.
PMID: 31136777 DOI: 10.1016/j.ijpharm.2019.05.063

Live attenuated Mycobacterium bovis (M. bovis), marketed as Bacille Calmette-Guérin is the only FDA-approved vaccine against tuberculosis. The prerequisite of cold chain storage between 2 and 8 °C hinders the global vaccination effort. The study aims to investigate the effect of trehalose, sucrose and glycerol combinations in enhancing the stability of M. bovis. The bacilli were formulated in various ratios of trehalose-glycerol, sucrose-glycerol, trehalose-sucrose-glycerol systems (test samples) and sodium glutamate (control), freeze-dried and stored for 28 days at 4 °C, 25 °C and 37 °C. Bacteria viability at pre-, post-freeze-drying and after storage were quantified by its density in colony-forming unit per milliliter (CFU/mL) as obtained through the pour plate method. Formulations were characterized using differential scanning calorimetry. Structural collapsed cakes were found on all freeze-dried formulations because of the low Tg'. Comparing between binary and ternary formulations, trehalose-sucrose-glycerol was found to be a superior lyoprotectant. Upon storage, the viability of bacteria in disaccharide-polyol formulations was highest when stored at 4 °C followed by 25 °C. The lowest viability was found after storage at 37 °C. While the ternary disaccharide-polyol system may be used as a thermoprotectant up to 25 °C, sodium glutamate has a superior thermoprotective effect at temperature above 25 °C.

Matched MeSH terms: Mycobacterium bovis/drug effects*
Is targeted removal a suitable means for tuberculosis control in wild boar?

Che'Amat A, Armenteros JA, González-Barrio D, Lima JF, Díez-Delgado I, Barasona JA, et al.

Prev Vet Med, 2016 Dec 01;135:132-135.
PMID: 27843020 DOI: 10.1016/j.prevetmed.2016.11.002

We assessed the suitability of targeted removal as a means for tuberculosis (TB) control on an intensely managed Eurasian wild boar (Sus scrofa) hunting estate. The 60km(2) large study area included one capture (treatment) site, one control site, and one release site. Each site was fenced. In the summers of 2012, 2013 and 2014, 929 wild boar were live-captured on the treatment site. All wild boar were micro-chipped and tested using an animal side lateral flow test immediately after capture in order to detect antibodies to the Mycobacterium tuberculosis complex (MTC). The wild boar were released according to their TB status: Seropositive individuals onto the release site (hunted after summer), and seronegative individuals back onto the treatment site. The annual summer seroprevalence of antibodies to the MTC declined significantly in live-captured wild boar piglets from the treatment site, from 44% in 2012 to 27% in 2013 (a reduction of 39%). However, no significant further reduction was recorded in 2014, during the third capture season. Fall-winter MTC infection prevalence was calculated on the basis of the culture results obtained for hunter-harvested wild boar. No significant changes between hunting seasons were recorded on either the treatment site or the control site, and prevalence trends over time were similar on both sites. The fall-winter MTC infection prevalence on the release site increased significantly from 40% in 2011-2012 to 64% in 2012-2013 and 2013-2014 (60% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted removal, failed to reduce TB prevalence when compared to the control site. However, it generated valuable knowledge on infection pressure and on the consequences of translocating TB-infected wild boar.

Matched MeSH terms: Mycobacterium bovis/physiology*
Priorities in tuberculosis control measures in developing countries

Sodhy JS

Med J Malaysia, 1963 Sep;18:38-41.
PMID: 14064295
Matched MeSH terms: Mycobacterium bovis*
COMPARATIVE TUBERCULIN TESTING IN MALAYA

CHIN J

Tubercle, 1964 Jun;45:114-24.
PMID: 14161910
Matched MeSH terms: Mycobacterium bovis*
Fulltext Detection of Mycobacterium tuberculosis complex antibodies in free-ranged wild boar and wild macaques in selected districts in Selangor and reevaluation of tuberculosis serodetection in captive Asian elephants in Pahang, Peninsular Malaysia

Lekko YM, Che-Amat A, Ooi PT, Omar S, Mohd-Hamdan DT, Linazah LS, et al.

J Vet Med Sci, 2021 Oct 31;83(11):1702-1707.
PMID: 34544936 DOI: 10.1292/jvms.21-0144

Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, affecting several domestic animals, wildlife species and humans. The preliminary investigation was aimed to detect antibody against MTBC among indigenous wildlife which are free-ranged wild boar, free-ranged wild macaques and captive Asian elephants in selected areas of Selangor and elephant conservation centre in Pahang, respectively. The results indicate that MTBC serodetection rate in wild boar was 16.7% (7.3-33.5 at 95% confidence interval (CI)) using an in-house ELISA bPPD IgG and 10% (3.5-25.6 at 95% CI) by DPP®VetTB assay, while the wild macaques and Asian elephant were seronegative. The univariate analysis indicates no statistically significant difference in risk factors for sex and age of wild boar but there was a significant positive correlation (P<0.05) between bovine TB in dairy cattle and wild boar seropositivity in the Sepang district.

Matched MeSH terms: Mycobacterium bovis*
Fulltext Inherited human IFN-γ deficiency underlies mycobacterial disease

Kerner G, Rosain J, Guérin A, Al-Khabaz A, Oleaga-Quintas C, Rapaport F, et al.

J Clin Invest, 2020 Jun 01;130(6):3158-3171.
PMID: 32163377 DOI: 10.1172/JCI135460

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients' herpesvirus Saimiri-immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

Matched MeSH terms: Mycobacterium bovis/immunology
Fulltext Immunomodulatory effects of recombinant BCG expressing MSP-1C of Plasmodium falciparum on LPS- or LPS+IFN-γ-stimulated J774A.1 cells

Mohamad D, Suppian R, Mohd Nor N

Hum Vaccin Immunother, 2014;10(7):1880-6.
PMID: 25424796 DOI: 10.4161/hv.28695

Macrophage phagocytosis is the first line of defense of the innate immune system against malaria parasite infection. This study evaluated the immunomodulatory effects of BCG and recombinant BCG (rBCG) strains expressing the C-terminus of the merozoite surface protein-1 (MSP-1C) of Plasmodium falciparum on mouse macrophage cell line J774A.1 in the presence or absence of lipopolysaccharide (LPS) or LPS + IFN-γ. The rBCG strain significantly enhanced phagocytic activity, production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and inducible nitric oxide synthase (iNOS) as compared with parental BCG strain, and these activities increased in the presence of LPS and LPS+IFN-γ. Furthermore, the rBCG strain also significantly reduced the macrophage viability as well as the rBCG growth suggesting the involvement of macrophage apoptosis. Taken together, these data indicate that the rBCG strain has an immunomodulatory effect on macrophages, thus strengthen the rational use of rBCG to control malaria infection.

Matched MeSH terms: Mycobacterium bovis/genetics; Mycobacterium bovis/immunology*
Fulltext Dataset of complete genome assembly and analysis of mycobacterium tuberculosis strain SIT745/EAI1-MYS

Abdullah M, Suraiya S, Mohamad S, Harun A

Data Brief, 2020 Aug;31:105949.
PMID: 32671154 DOI: 10.1016/j.dib.2020.105949

In this dataset, we report the genome assembly and data analysis of Mycobacterium tuberculosis strain SIT745/EAI1-MYS. Previously, this strain was isolated from a Malaysian patient with extra-pulmonary tuberculosis, and identification of this strain is done by spoligotype patterns with fifteen known Shared International Type (SITs). Further analysis showed that this strain has a remarkable phylogeographical specificity for Malaysia. Based on the National Center for Biotechnology Information (NCBI) nucleotide database information, the complete genome consists of 150 contigs with various sequence lengths and was not assembled. In this assembly, the aforementioned contigs along with reference sequence from Mycobacterium tuberculosis strain H37Rv and Mycobacterium bovis strain AF2122/97 was used for gap closures, were assembled into a single circular chromosome length of approximately 4.42 Mega bases (Mb) with an average GC content of 65.6%. The single circular chromosome was shown to contain 4,009 protein-coding sequences, 3 ribosomal RNAs, 45 transfer RNAs, and 12 superclasses distributed with 277 subsystems which constitute nearly 1900 genes, respectively. The genome information will provide fundamental knowledge of this organism as well as insight for understanding genomic and proteomic profiling, phylogenetic relationship.

Matched MeSH terms: Mycobacterium bovis
Universal child immunization coverage in Kuala Lumpur

Sinniah, D., Rajeswari, B., Koh, S., George, J., Sundari, J., Sosapillai, J.N., et al.

Malaysian Journal of Child Health, 1991;3(1):23-28.
MyJurnal

To verify the actual immunisation coverage in Kuala Lumpur, City Hall Health Department and the Malaysian Paediatric Association (NGO ) carried out a survey. The survey revealed that the immunisation coverage determined at the child's first birthday for BCG was 95%, DPT 3 94%, OPV 3 94%, and measles = 27% (59% at 2 years). These figures correspond closer to City Hall's estimated coverage rather than the rates projected by the Ministry of Health. The main reasons for immunisation failure were, child ill 31.8% (not brought = 20.1%, brought but not given vaccine 11.7%), lack of information 28.6%, lack of motivation 9.1%, mother too busy 9.1%. Measles immunisation coverage at 1 year was low because of wrong information on schedules. Tetanus toxoid immunisation coverage of pregnant women was low. Only 27% of children were protected against neonatal tetanus although 97% of pregnant women received antenatal care and 50% had attended other health facilities as well during pregnancy. Private medical practitioners were responsible for more than 40% of all immunisations but were not submitting returns to the Health Department. Recommendations to improve immunisation coverage include education and motivation of the public and also doctors and health personnel on prevention of missed opportunities, contraindictions to immunisation and correct schedules. (Copied from article).

Matched MeSH terms: Mycobacterium bovis

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