Background and Aim: Recently, cases of mastitis refractory to treatment have been reported frequently. There are limited routine laboratory investigations on Camelidae infections. Mastitis has been estimated to affect more than 25% of lactating she-camel with up to 70% milk loss. The details of Bacillus spp. pathogenesis in mastitis are not yet fully described. The present study is the first detailed phenotypic and genotypic characterization of Bacillus licheniformis isolates from recurrent mastitic she-camels with sepsis in Egypt.

Materials and Methods: The udders of 100 she-camels were investigated, samples collected from smallholders' farmers in 10 localities within three governorates in Egypt: Marsa Matrouh, Giza, and Sharkia governorates. The pathogens ascend from udder inducing abortion at different trimesters of pregnancy. Polymerase chain reactions-mediated proofs of identity were applied for diagnostic and taxonomic purposes, where the 16S rRNA gene sequence and the β subunit of RNA polymerase encoding gene rpoB are the molecular targets.

Results: The genetic elements classified the subspecies to B. licheniformis 61.4%, in addition to, Corynebacterium bovis 29.8%. The somatic cell count (≤1x10⁷ cells/ml) and California mastitis test reactivity (+3 or +4) of milk clinically classified the she-camels population (n=100) under investigation into 50, 20, and 30 as healthy, subclinical, and clinical mastitic she-camels, respectively. During bacterial isolation, 80 species were noticed, of which 71.25% (57/80) and 28.75% (23/80) were Gram-positive and negative, respectively, in two clinical forms: Single (40%, n=16/40) and mixed (60%, n=34/40) bacterial infections. In vitro, 100% sensitivity for gentamycin (10 μg) and ofloxacin (5 μg) was noted; however, it was reduced to 50%. Moreover, during in vivo treatments cloxacillin (5 μg) upraised as the most effective alternative with 90% sensitivity.

Conclusion: Neither recurrent mastitis nor Bacillus species are thoroughly investigated with regard to reproduction performance in Egypt and the usefulness of these strains as antimastitis probiotics. Both persistent bacteremia and dormant endospores were formed but unaffected by standard schemes of antimicrobials injections which proposed the risk of pathogenic bacilli contaminating row milk from apparently healthy she-camel. The discrepancies between treatment results were induced by the resistance that started to develop by the organisms due to frequent and/or faulty use of applied antibiotics.

Keywords: 16S rRNA gene, antimicrobial resistance, Bacillus species, Camelidae, Egypt, probiotics, recurrent mastitis, rpoB gene.

1. Levy, S.B. and Marshal, B. (2004) Antibacterial resistance worldwide: Causes, challenges and responses. Nat. Med., 10: 122-129. [Crossref]

2. Tibary, A., Fite, C., Anouassi, A. and Sghiri, A (2006) Infectious causes of reproductive loss in Camelids. Theriogenology, 66(3): 633-647. [Crossref] [PubMed]

3. Osman, K.M., Samir, A., Orabi, A. and Zolnikov, T.R. (2014) Confirmed low prevalence of Listeria mastitis in she-camel milk delivers a safe, alternative milk for human consumption. Acta Trop., 130: 1-6.

4. Sadashiv, S.O. and Kaliwal, B.B. (2014) Isolation, characterization and antibiotic resistance of Bacillus sps. From bovine mastitis in the region of north Karnataka, India. Int. J. Curr. Microbiol. App. Sci., 3(4): 360-373.

5. Saleh, S.K. and Faye, B. (2011) Detection of subclinical mastitis in dromedary camels (Camelus dromedaries) using somatic cell counts, California mastitis test and udder pathogen. Emir. J. Food Agric., 23(1): 48-58.

6. Alamin, M.A., Alqurashi, A.M., Elsheikh, A.S. and Yasin T.E. (2013) Mastitis incidence and bacterial causative agents isolated from lactating she-camel (Camelus dromedaries). IOSR J. Agric. Vet. Sci., 2(3): 7-10. [Crossref]

7. Fazlani, S.A., Khan, S.A., Faraz, S. and Awan, M.S. (2011) Antimicrobial susceptibility of bacterial species identified from mastitis milk samples of camel. Afr. J. Biotechnol., 10(15): 2959-2964.

8. Hamouda, R.H., Allam, H.A. and Allam, N.A.T. (2014) Molecular identification of bovine recurrent mastitis status with regards to Arcanobacerium pyogenes (Trueperella pyogenes). J. Egypt. Vet. Med. Assoc., 74(4): 739-754.

9. Fo, W., Okoth, M.W. and Wangoh, J. (2012) Survey of post-harvest handling, preservation, and processing practices along the camel milk chain in Isiolo district, Kenya. Afr. J. Food Agric. Nutr. Dev., 12: 6897-6912.

10. Salih, R.R.M., Basit, A. and Ahmed, O. (2011) Bovine mastitis caused by Bacillus spp. in Khartoum state, Sudan. U. K. J. Vet. Med. Anim. Prod., 2(2): 25-32.

11. Tumini, M., Nagel, O., Molina, M.P. and Althaus, R. (2017) Microbiological assay with Bacillus licheniformis for the easy detection of quinolones in milk. Int. Dairy J., 64: 9-13.

12. Kosciuczuk, E.M., Lisowski, P., Jarczak, J., Krzyzewski, J., Zwierzchowski, L. and Bagnicka, E. (2014) Expression patterns of β-defensin and cathelicidin genes in parenchyma of bovine mammary gland infected with coagulase-positive or coagulase-negative Staphylococci. BMC Vet. Res., 10(1): 246. [Crossref]

13. Gupta, D.K. and Vyas, M. (1989) Efficacy of Bacillus subtilis against mosquito larvae (Anophelis culicfacies). Zeitschrift fuer Angewandte Zoologie, 76(1): 85-91.

14. Abdelgadir, A.E. (2014) Mastitis in camels (Camelus dromedaries): Past and recent research in pastoral production system of both East Africa and Middle East. J. Vet. Med. Anim. Health, 6(7): 208-216.

15. Alqurashi, A.M., Alamin, M.A., Elsheikh, A.S. and Yasin, T.E. (2013) Sensitivity of bacterial isolates from mastitic she-camel (Camelus dromedaries) to antibiotics. J. Am. Sci., 9(4): 47-52.

16. Al-Tarazi, Y., Hijazin, M., Alber, J., Lammler, C., Hassan, A.A., Timke, M., Kostrzewa, M., Prenger-Berninghoff, E. and Zschock, M. (2012) Phenotypic and genotypic characteristics of Trueperella (Arcanobacterium) pyogenes isolated from lung abscesses of one-humped camels (Camelus dromedarius) in Jordan. J. Camelid Sci., 5: 99-104.

17. Guliye, A.Y., Creveld, C.V. and Yagil, R. (2002) Detection of subclinical mastitis in dromedary camels (Camelus dromedarius) using somatic cell counts and the N-acetyl-beta-D-glucosaminidase test. Trop. Anim. Health Prod., 34(2): 95-104. [Crossref]

18. De Jong, A., Thomas, V., Simjee, S., Godinho, K., Schiess, B., Klein, U., Butty, P., Valle, M., Marion, H. and Shryock, T.R. (2012) Pan-European monitoring of susceptibility to human-use antimicrobial agents in enteric bacteria isolated from healthy food-producing animals. J. Antimicrob. Chemother., 67(3): 638-651. [Crossref] [PubMed]

19. Oxoid Manual. (2000) The Oxoid Manual of Culture Media Ingredients and Other Laboratory Services. 10th ed. Wade Road Basingstoke, Oxoid Limited, Hampshire, England.

20. Barrow, G.I. and Feltham, R.K.A. (2003) Cowan and Steel's Manual for Identification of Medical Bacteria. 3rd ed. Cambridge Press, New York.

21. Hussein, W., Awad, H. and Fahim, S. (2016) Systemic resistance induction of tomato plants against ToMV virus by surfactin produced from Bacillus subtilis BMG02. Am. J. Microbiol. Res., 4(5): 153-158.

22. Sambrook, J., Fritscgh, E.F. and Meniates, A. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

23. Hamouda, R.H. and Allam, N.A.T. (2011) Use of multiplex PCR-based molecular typing of genes encoding virulence determinants in coliform mastitis in dairy cattle. J. Egypt. Vet. Med. Assoc., 11(1): 245-256.

24. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X. and Wilson, A.C. (1989) Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA, 86: 6196-6200. [Crossref]

25. Woo, P.C.Y., Lau, S.K.P., Chan, K.M., Fung, A.M.Y., Tang, B.S.F. and Yuen, K.Y. (2005) Clostridium bacteraemia characterized by 16S ribosomal RNA gene sequencing. J. Clin. Pathol., 58(3): 301-307. [Crossref] [PubMed] [PMC]

26. Khamis, A., Raoult, D. and La Scola B. (2004) rpoβ gene sequencing for identification of Corynebacterium species. J. Clin. Microbiol., 42(9): 3925-3931. [Crossref] [PubMed] [PMC]

27. Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N., Wagner, M. and Fach, P. (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol., 42(5): 1863-1868. [Crossref] [PMC]

28. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22(22): 4673-4680. [Crossref]

29. Ramadan, R.O., El-Hassan, A.M., Abdin-Bey, R., Al Gasnawi, Y.A., Abdalla, E.M. and Fayed, A.A. (1987) Chronic obstructive mastitis in the camel a clinic pathological study. Corn. Vet., 77(2): 132-150.

30. Al-Qumber, M. and Tagg, J.R. (2006) Commensal bacilli inhibitory to mastitis pathogens isolated from the udder micro biota of healthy cows. J. Appl. Microbiol., 101(10): 1152-1160. [Crossref] [PubMed]

31. Suheir, I.A., Salim, M.O. and Yasin, T.E. (2005) Bacteria, mycoplasma, and fungi associated with subclinical mastitis in camel. Sudan J. Vet. Res., 20: 23-31.

32. Hafez, A.M., Fazig, S.A., El Amrousi, S. and Ramadan, R.O. (1987) Studies on mastitis on farm animals in Al Hassa: I-Analytical studies. Assuit Vet. Med. J., 19(37): 140-145.

33. Hoa, N.T., Baccigalupi, L., Huxham, A., Smertenko, A., Van, P.H., Ammendola, S., Ricca, E. and Cutting, S.M. (2000) Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl. Environ. Microbiol., 66(12): 5241-5247. [Crossref]

34. Le Duc, H., Hong, H.A., Barbosa, T.M., Henriques, A.O. and Cutting, S.M. (2004) Characterization of Bacillus probiotics available for human use. Appl. Environ. Microbiol., 70(4): 2161-2171. [Crossref] [PMC]

35. Yan, L., Boyd, K.G., Adams, D.R. and Burgess, J.G. (2003) Biofilm-specific cross-species induction of antimicrobial compounds in bacilli. Appl. Environ. Microbiol., 69(7): 3719-3727. [Crossref] [PMC]

36. Dewani, P. (2000) Bacteriological Studies on Mastitis in Ewes and Goats. M.Sc. (Hons) Thesis, Department of Microbiology, Sindh Agriculture University, Tando Jam.

37. Rind, R. and Khan, T.S. (2000) Antibiogram sensitivity of bacterial organisms identified from surgical and non-surgical wounds of Animals. Palest. J. Biolog. Sci., 3(10): 1719-1723.

38. Hammer, P. and Heeschen, W. (1995) Antibody-capture immunoassay for the detection of norfloxacin in raw milk. Milchwissenschaft, 50: 513-520.

39. Nagel, O., Molina, P. and Althaus, R. (2013) Microbiological system in microtitre plates for detection and classification of antibiotic residues in milk. Int. Dairy J., 32: 150-155.

40. Riley, M.A. and Wertz, J.E. (2002) Bacteriocins: Evolution, ecology, and application. Ann. Rev. Microbiol., 56: 117-137.