ResearchersLinks
Pakistan Journal of ZoologyA ResearchersLinks journal
Submit manuscriptReview for this journal

Short Communication · Volume 53, Issue 3, 4 April 2021 · Open Access

Prevalence and Antimicrobial Susceptibility Profile of Mycobacterium bovis Isolated from Egyptian Buffaloes’ Mastitic Milk

· ·

Affiliations & Notes ⌄
  1. 1Department of Microbiology, Faculty of Veterinary Medicine, Matrouh University, Matrouh, Egypt. 2Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. 3Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. , * Corresponding author: khalifa.eman@alexu.edu.eg
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
4 April 2021
Download PDF ePub eFLIP Set Alert Get Rights Reprints

From the article

  • Out of 100 buffaloes’ mastitic milk sample, the isolation and phenotypic and biochemical characterization revealed 7 samples positive for Mycobacterium bovis.
  • The antimicrobial profile of the bacterial isolates from buffaloes’ mastitic milk is summarized in Table I. M. bovis showed resistance to ciprofloxacin; gentamycin; sulphamethoxazole-trimethoprim.
  • It can be concluded that Mycobacterium bovis is the causative agent of mastitis in Egyptian buffaloes, which in return is a great hazard for public health.

Selected from the article's own text.

Abstract

From the world’s highlight on dangers of tuberculosis as a zoonotic and debilitating disease threatening animal and human health beside economy, this research aimed to determine the prevalence of Mycobacterium bovis from buffaloes’ mastitic milk by isolation on Lowenstein-Jensen medium (LJ medium), Ziehl-Neelsen staining; identification by different biochemical tests and polymerase chain reaction (PCR) at 500bp diagnostic for M. bovis, as well as (2) to determine the therapeutic efficacy of the various antimicrobials (9 types) on the isolated M. bovis by using different antimicrobial sensitivity plate method. A total number of 100 samples of mastitic milk from native lactating buffaloes’ breed were collected aseptically from 4 private farms in Cairo-Alexandria desert road in the north part of the Egypt and subjected to bacteriological and molecular examination for M. bovis followed by their antibiogram profile. Seven (7%) isolates were identified as M. bovis from totally examined 100 milk samplesand isolated M. bovis showed resistance to ciprofloxacin; gentamycin and sulphamethoxazole-trimethoprim while were intermediate sensitive to both of erythromycin and norfloxacin but on the contrary were sensitive to amikacin; cefotaxime; clindamycin and streptomycin. It can be concluded that M. bovis can be the causative agent of mastitis in Egyptian Buffaloes and could be a potential risk for zoonotic transmission to man as well as economic losses. So, strict hygienic regulations and novel diagnostic tools should be used for prevention and detection of tuberculous mastitis in buffaloes’ farms. The general public health should also be intensely warned from consuming raw or unpasteurized milk. All these prophylactic measures will eventually lead to a positive impact on public health.

Materials and methods

A total number of 100 samples of mastitic milk from native breeds lactating buffalos from four private farms were collected aseptically according to guidelines of National Mastitis Council (1990). After discarding the first three milking streams, approximately 50 ml of milk was collected aseptically from each mastitic milk in sterile test tube. The milk samples were transported on ice to private lab at Alexandria city where they were immediately cultured or stored at 4°C for a maximum of 48h until cultured on standard bacteriological media.

Five mL of milk was centrifuged at 3000 rpm for 20 min inoculated in duplicate in Lowenstein-Jensen media (Sigma-Aldrich), and incubated at 37°C for 3 months. Colonies suggestive of Mycobacteria were stained by Ziehl Neelsen stain . Samples identified as acid-fast bacilli (AFB) by Ziehl Neelsen stain were subjected to molecular identification.

The thermal cycler for PCR comprised initial denaturation at 94oC for 4 min 30 cycles of denaturation at 94oC for 1 min, annealing at 55oC for 1 min, Extension at 73oC for 1 min, and final elongation at 74oC for 10 min. A 500bp product was visible on 1.5% agarose gel.

The antimicrobial susceptibilities of Mycobacterium bovis were determined according to CLSI, 2006. The antimicrobial agents tested were: Amikacin (AMK) (30 µg); cefotaxime (CEF) (30 µg); ciprofloxacin (CIP) (5 µg); clindamycin (CLI) (2 µg); erythromycin (E) (15 µg); gentamycin (GN) (10 µg); norfloxacin (NOR) (100 µg); streptomycin (SM) (1µg); and sulphamethoxazole-trimethoprim (SXT) (23.7 + 1.25 mg). The results of sensitivity of microorganisms to different antibiotic discs were measured by the diameter of inhibitory zones and compared with antibiotic susceptibility testing sheet to get the result (sensitive, intermediate or resistant) and interpreted in accor­dance with the recommendations of Clinical and Laboratory Standards Institute (2008).

Results and discussion

Out of 100 buffaloes’ mastitic milk sample, the isolation and phenotypic and biochemical characterization revealed 7 samples positive for Mycobacterium bovis.

This prevalence is lower than the data published by Jha et al. (2007) who isolated 6 (17%) M. bovis from 36 buffaloes milk samples in Nepal. Basit et al. (2018) reported 4 (6.2%) isolates identified as Mycobacterium bovis out of 64 milk samples collected from lactating buffaloes in north east of Pakistan. On the contrary, Shaukat et al. (2014) reported that all the mastitis positive animals when tested for TB by SITT (Single intradermal tuberculin test), Z.N staining and PCR were found to be negative. Therefore, they assumed that the incidence of TB in mastitis is not very common in screened population of bovines around Lahore. This is parallel to the studies of Al-Soub and Chako (1996) who reported tuberculous mastitis as a rare disease, which may be due to the hygienic measures, vaccination program and public health education.

The antimicrobial profile of the bacterial isolates from buffaloes’ mastitic milk is summarized in Table I. M. bovis showed resistance to ciprofloxacin; gentamycin; sulphamethoxazole-trimethoprim. The bacteria showed ntermediate sensitivity to erythromycin and norfloxacin; and high sensitivity to amikacin, cefotaxime, clindamycin and streptomycin. The use of the most effective antibiotic is very important for controlling the intramammary infection ; ; .

Our results suggested risk of infection to humans through consumption of raw milk, which is similar to data recorded by Basit et al. (2018). Also, more efforts are needed in the prevention and control of clinical mastitis in buffaloes (Bhutia et al., 2019).

Table I. The antimicrobial profile of isolated M. bovis from buffaloes’ mastitic milk samples.

Antimicrobial disc

Zone around discs

Indication of sensitivity

AMK (30 µg)

23 mm

++++

CEF (30 µg)

20 mm

+++

CIP (5 µg)

0 mm

-

CLI (2 µg)

19 mm

+++

GN (10 µg)

0 mm

-

E (15 µg)

9 mm

++

NOR (100 µg)

8 mm

++

SM (1µg)

27 mm

++++

SXT (23.7 + 1.25 mg)

0 mm

-

AMK, Amikacin; CEF, Cefotaxime; CIP, Ciprofloxacin; CLI, Clindamycin; GN, Gentamycin; E, Erythromycin; NOR, Norfloxacin; SM, Streptomycin; SXT, Sulphamethoxazole-Trimethoprim; -, Resistant; +, weakly sensitive; ++, moderately sensitive; +++, quite sensitive; ++++, highly sensitive.

Conclusion

It can be concluded that Mycobacterium bovis is the causative agent of mastitis in Egyptian buffaloes, which in return is a great hazard for public health.

Statement of conflict of interest

The authors have declared no conflict of interest.

References

  1. Ali, S., Akhtar, R., Younu, M., Saleem, G., Nisa, Q. U. and Zahid, B. 2014. Eur. J. environ. Ecol., 1: 7-11.
  2. Al-Soub, H., Chacko, K., 1996. Br. J. clin. Pract., 50: 50-51. https://doi.org/10.1016/S0026-0657(96)94156-9[CrossRef]
  3. Araujo, K.B.S., Rangel, A.H.H., Fonseca, F.C.E., Aguiar, E.M., Simplicio, A.A., Novaes, L.P. and Lima-Junior, D.M., 2012. Ital. J. Anim. Sci., 11: 87-91.
  4. Basit, A., Hussain, M., Shahid, M., Ayaz, S., Rahim, K., Ahmad, I., Rehman, A. U., Hassan, M. F. and Ali, T., 2018. Pak. Vet. J., 38: 199-203.
  5. Bhutia, P.S., Bansal, B.K., Gupta, D.K., Singh, R.S. and Uppal, S.K., 2019. Trop. Anim. Hth. Prod., 51: 1551-1557.
  6. Bobadilla-Del Valle, M., Torres-Gonzalez, P., Cervera-Hernandez, M.E., Martinez-Gamboa, A., Crabtree-Ramirez, B., Chavez-Mazari, B., Ortiz-Conchi, N., Rodríguez-Cruz, L., Cervantes-Sánchez, A., Gudiño-Enríquez, T., Cinta-Severo, C., Sifuentes-Osornio, J. and Ponce de León, A. 2015. PLoS. Negl. Trop. Dis., 9: e0004124. https://doi.org/10.1371/journal.pntd.0004124[CrossRef]
  7. Cadey, R.A., Shah, S.K., Schermerhorn, E.C. and Mcdowell, R.E., 1983. J. Dairy Sci., 66: 578-586. https://doi.org/10.3168/jds.S0022-0302(83)81828-1[CrossRef]
  8. Chakrabortya, S., Dhamab, K., Tiwaric, R., Yatood, M.I., Khuranae, S.K., Khandiaf, R., Munjalf, A., Munuswamyb, P., Kumarb, M.A., Singhg, M., Singhb, R., Guptah, V.K. and Chaicumpai, W., 2019. Vet. Quart., 39: 76–94.
  9. Clinical and Laboratory Standards Institute (CLSI), 2008. Detection of methicillin resistant staphylococci, 3rd ed. Approved standard M31-A3. CLSI, Wayne, PA.
  10. Cormican, P., Meade, K.G., Cahalane, S., Narciandi, F., Chapwanya, A., Lloyd, A.T. and O’farrelly, C., 2008. Immunogenetics, 60: 147-156. https://doi.org/10.1007/s00251-007-0269-8[CrossRef]
  11. Du Preez, J.H. and Giesecke, W.H., 1994. In: Infectious diseases of livestock (eds. J.A.W. Coetzer and G.R. Thomson). Oxford University Press, Edit. 2: 1564-1595.
  12. Jha, V.C., Moryta, Y., Dhakal, M. and Besnet, B., 2007. J. Vet. Med. Sci., 69: 819-825. https://doi.org/10.1292/jvms.69.819[CrossRef]
  13. Jones, G.M., 1986. J. Dairy Sci., 69: 1699-1707. https://doi.org/10.3168/jds.S0022-0302(86)80589-6[CrossRef]
  14. Khan, A.Z. and Muhammad, G., 2005. Pak. Vet. J., 25: 9-12.
  15. Linda, N., Christine, J., Misha, R., Dhakalc, I.P., Bettb, B., Pramod, D. and Rupak Khadkhac, R., 2010. Prev. Vet. Med., 1, 96: 179–185.
  16. Quinn, P.J., Carter, M.E. and Markey, B.K., 1994. Clinical veterinary microbiology. Wolfe, London. pp. 144-55.
  17. Radostits, O., Gay, C.C., Blood, D.C. and Hinchcliff, K.W., 2000. Vet. Med., 9: 603-700.
  18. Shaukat, A., Akhtar, R., Younus, M., Saleem, G., Nisa, Q.U. and Zahid, B., 2014. Eur. J. environ. Ecol., 1: 7-11.
  19. Xiong, H., Guo, B., Gan, Z., Song, D., Lu, Z., Yi, H., Wu, Y., Wang, Y. and Du, H., 2016. Sci. Rep., 6: 27070. https://doi.org/10.1038/srep27070[CrossRef]
  20. Yeung, A.T.Y., Shaan, L.G. and Hancock, R.E.W., 2011. Cell. Mol. Life Sci., 68: 2161-2176. 63. https://doi.org/10.1007/s00018-011-0710-x[CrossRef]

Related articles

Services

Article usage statistics

Loading…

Prevalence and Antimicrobial Susceptibility Profile of Mycobacterium bovis Isolated from Egyptian Buffaloes’ Mastitic Milk · ResearchersLinks