Background and Aim: Brucellosis is a major constraint to livestock production in Egypt as well as many developing countries worldwide. Bovine brucellosis is an economically important disease with reproductive failure as a principal manifestation resulting in abortion, premature birth and decreased milk production in females, and orchitis and epididymitis in males. In spite of the efforts of Egyptian veterinary services to overcome brucellosis, the disease is still prevalent in both animals and humans and represents one of the most important public health hazards in Egypt. The aim of the present work was to investigate the efficacy of the control program implemented by the General Organization of Veterinary Services in Brucella infected buffalo farm on serological, molecular, cultural, and histopathological basis. Brucella melitensis biovar 3 was recovered from 6 buffalo-cows.

Materials and Methods: Blood samples were collected from a total of 750 non-vaccinated lactating buffalo-cows. These animals were proved positive for Brucella by the Egyptian brucellosis national program. Sera were tested using buffered acidified plate antigen test and rose Bengal test as screening tests and complement fixation test as a confirmatory test. Positive animals were separated for slaughtering under the supervision of the Egyptian veterinary authorities. Remaining animals were tested every 3 weeks with slaughtering of positive cases and this continued until the remaining animals revealed three successive negative serological tests. Different lymph nodes (prescapular, prefemoral, mediastinal, retropharyngeal, and supramammary) were collected from 11 Brucella seropositive buffalo-cows slaughtered after being confirmed serologically as Brucella infected cases. Samples were collected and processed for bacterial isolation and nucleic acid detection using polymerase chain reaction (PCR). Parts of these specimens were fixed in 10% neutral buffered formalin for 48 h then processed by paraffin embedding technique.

Results: "Test and slaughter" policy was applied on Brucella infected dairy buffalo farm. The program continued for 6 months with slaughtering of positive cases until the herd was proved Brucella free. B. melitensis biovar 3 could be recovered from six buffalo-cows. Universal PCR confirmed Brucella on genus level and Bruce-ladder multiplex, PCR confirmed the presence of B. melitensis on the species level. Histopathological examination of Brucella-infected lymph nodes revealed massive rarified and depleted lymphoid areas of both sub-capsular and deep cortical lymphoid follicles, macrophage cells granulomatous reaction, as well as fat, infiltrates, and chronic vasculitis. The chronic nature of Brucella lesions has been confirmed in this study as indicated by the chronic vasculitis and collagen deposition.

Conclusion: Freedom status from brucellosis in this study required 6 months which are considered long time allowing the spread of infection to other localities especially under unhygienic conditions, husbandry system favoring mixed populations of different ages, sex, aborted and pregnant, and lack of controlled movement of animals. Therefore, effective control of animal brucellosis requires surveillance to identify infected animal herds, elimination of the reservoirs, and vaccination of young heifers. B. melitensis biovar 3 is the cause of the Brucella outbreak in buffalo which still remains the prevalent type of Brucella in Egypt. The disease runs a chronic course allowing further spread of infection.

Keywords: bruce-ladder, brucellosis, buffalo, histopathology, polymerase chain reaction.

1. Corbel, M.J. (2006) Brucellosis: An overview. Emerg. Infect. Dis., 3: 213-221. [Crossref] [PubMed] [PMC]

2. Ackermann, M.R., Cheville, N.F. and Deyeoe, B.L. (1988) Bovine ileal dome lymphoepithelial cell: Endocytosis and transport of Brucella abortus strain 19. Vet. Pathol., 25: 28-35. [Crossref]

3. Nielsen, K.H. and Duncan, J.R. (1990) Animal Brucellosis. Florida, Boca Raton: CRC Press Int. p238.

4. Riley, L.K. and Robertson, D.C. (1984) Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes. Infect. Immun., 46: 224-230. [PubMed] [PMC]

5. Silva, F.L., Paixa o, T.A., Borges, A.M., Lage, A.P. and Santos, R.L. (2005) Bovine brucellosis. Cad. Tecn. Vet. Zootecnia, 47: 1-12.

6. Ko, J. and Splitter, G.A. (2003) Molecular host-pathogen interaction in brucellosis: Current understanding and future approaches to vaccine development for mice and humans. Clin. Microbiol. Rev., 6: 65-78. [Crossref]

7. Jubb, K.V.F., Kennedy, P.C. and Palmer, N. (2016) Pathology of Domestic Animals. 6th ed. Saunders, Elsevier, Philadelphia, PA.

8. Khoudair, R.M., Ibrahim, E.M., Saker, G.G. and Hafez, M.A. (2009) Clinico-diagnostic and pathological studies on cattle and buffaloes suffering from brucellosis and tuberculosis in Kafr El Sheikh Governorate. Egypt. J. Comp. Path. Clin. Pathol., 22(1): 148-174.

9. Radostits, O.M., Gay, C.C., Hinchcliff, K.W. and Constable, P.D. (2007) Diseases associated with Brucella species. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. Elsevier Limited., New York.

10. Olsen, S. and Tatum, F. (2010) Bovine brucellosis. The veterinary clinics of North America. Food Anim. Pract., 26: 15-27. [Crossref] [PubMed]

11. Hosein, H.I., Rouby, S., Menshawy, A. and Ghazy, N. (2016) Seroprevalence of camel brucellosis and molecular characterization of Brucella melitensis recovered from dromedary camels in Egypt. Res. J. Vet. Pract., 4(1): 17-24. [Crossref]

12. Hosein, H.I., Rouby, S.R., Menshawy, A. and AbdAl-Ghany, A.E. (2017) Sensitivity and specificity of the commonly used diagnostic procedures of bovine brucellosis. Vet. Sci. Res. Rev., 3(3): 45-52. [Crossref]

13. Affi, M.M., Abdul-Raouf, U.M., El-Bayoumy, E.M., Montasser, A.M. and Mohamad, H.A. (2015) Isolation and biotyping of Brucella melitensis from Upper Egypt. Report Opin., 7: 79-85.

14. Menshawy, A., Perez-Sancho, M., Garcia-Seco, T., Hosein, H.I., Garcia, N., Martinez, I., Sayour, A.E., Goyache, J., Azzam, R.A.A. and Dominguez, L. (2014) Assessment of genetic diversity of zoonotic Brucella spp. Recovered from livestock in Egypt using multiple locus VNTR analysis. BioMed. Res. Int. 2014: 1-7. [Crossref] [PubMed] [PMC]

15. Salem, A.A. and Hosein, H.I. (1990) Brucella strains prevalent in Egypt. Assuit. Vet. Med. J., 22: 160-163.

16. Pappas, G., Akritidis, N., Bosilkovski, M. and Tsianos, E. (2005) Brucellosis. N. Eng. J. Med., 352(22): 2325-2336. [Crossref] [PubMed]

17. Kaltungo, B., Saidu, S., Sackey, A. and Kazeem, H. (2014) A review on diagnostic techniques for brucellosis. Afr. J. Biotechnol., 13: 1-10. [Crossref]

18. Fensterbank, R. (1986) Brucellosis in cattle, sheep and goats: Diagnosis, control and vaccination. Rev. Sci. Tech. Off. Int. Epiz., 5(3): 605-618.

19. Abd-El Halim, M.H., Abeer, A.E., Shalaby, M.N.A. (2017) Prevalence of brucellosis in buffaloes and its control measures. J. Vet. Med. Res., 24(1): 161-170.

20. Refai, M. (2002) Incidence and control of brucellosis in the Near East region. Vet Microbiol., 90: 81-110. [Crossref]

21. Wareth, G., Hikal, A., Refai, M., Melzer, F., Roesler, U. and Neubauer, H. (2014) Animal brucellosis in Egypt. J. Infect. Dev. Ctries, 8(11): 1365-1373. [Crossref]

22. OIE. (2016) Brucellosis: Brucella abortus, B. melitensis and B. suis. Chapter 2.1.4. Paris, France: OIE.

23. Alton, G.G., Jones, L.M., Angus, R.D. and Verger, J.M. (1988) Techniques for Brucellosis Laboratory Institute. 3rd ed., Vol. 1. Paris: Institut National de la Recherche Agronomique (INRA).

24. Ewalt, D.R., Packer, R.A. and Harris, S.K. (1983) An improved selective medium for isolating Brucella sp. from bovine milk. In: Proceeding of the Process International Symposium Veterinary Laboratory Diagnosticians. p577-589.

25. Ouahrani-Bettache, S., Soubrier, M.P. and Liautard, J.P. (1996) IS6501-anchored PCR for the detection and identification of Brucella species and strains. J. Appl. Bacteriol., 81: 154-160. [Crossref] [PubMed]

26. Garcia-Yoldi, D., Marin, C.M., De Miguel, M.J., Munoz, P.M., Vizmanos, J.L. and Lopez-Goni, I. (2006) Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin. Chem., 52: 779-781. [Crossref]

27. Bancroft, J.D. and Gamble, M. (2008) Theory and Practice of Histological Techniques. 6th ed. North Hollywood, USA.

28. Petrrie, A. and Watson, P. (2013) Statistics for Veterinary and Animal Science. 3rd ed. Wiley-Blackwell, London, UK.

29. Bricker, B.J. (2002) PCR as a diagnostic tool for brucellosis. Vet. Microbiol., 90: 435-446. [Crossref]

30. Cloeckaert, A., Vizcaino, N., Paquet, J.Y., Bowden, R.A. and Elzer, P.H. (2002) Major outer membrane proteins of Brucella spp.: Past, present and future. Vet. Microbiol., 90: 229-247. [Crossref]

31. Holt, H., Eltholth, M., Hegazy, Y., El-Tras, W., Tayel, A. and Guitian, J. (2011) Brucella spp. infection in large ruminants in an endemic area of Egypt: Cross-sectional study investigating seroprevalence, risk factors and livestock owner's knowledge, attitudes and practices (KAPs). BMC Public Health, 11: 341-350. [Crossref] [PubMed] [PMC]

32. Godfroid, J., Scholz, H., Barbier, T., Nicolas, C., Wattiau, P., Fretin, D., Whatmore, A., Cloeckaert, A., Blasco, J., Moriyon, I., Saegerman, C., Muma, J.B., AL Dahouk, S., Neubauer, H. and Letesson, J.J. (2011) Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev. Vet. Med., 102: 118-131. [Crossref] [PubMed]

33. Seleem, M.N., Boyle, S.M. and Sriranganathan, N. (2010) Brucellosis: A re-emerging zoonosis. Vet. Microbiol., 140: 392-398. [Crossref] [PubMed]

34. Rodriguez, M.C., Viadas, C., Seoane, A., Sangari, F.J., Lopez-Go-i, I. and Garcia-Lobo, J.M. (2012) Evaluation of the effects of erythritol on gene expression in Brucella abortus. PLoS One, 7(12): e50876. [Crossref]

35. Sangari, F.J., Aguero, J. and Garcia-Lobo, J.M (2000) The genes for erythritol catabolism are organized as an inducible operon in Brucella abortus. Microbiology, 146: 487-495. [Crossref] [PubMed]

36. Lopez-Goni, I., Garcia-Yoldi, D., Marin, C.M., de Miguel, M.J., Munoz, P.M., Blasco, J.M., Jacques, I., Grayon, M., Cloeckaert, A., Ferreira, A.C., Cardoso, R., Correa de Sa, M.I., Walravens, K., Albert, D. and Garin-Bastuji, B. (2008) Evaluation of multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species including the vaccine strains. J. Clin. Microbiol., 46(10): 3484-3487. [Crossref] [PubMed] [PMC]

37. Vemulapalli, R., McQuiston, J.R., Schurig, G.G., Sriranganathan, N., Halling, S.M. and Boyle, S.M. (1999) Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains. Clin. Diagn. Lab. Immunol., 6: 760-764. [PubMed] [PMC]

38. Bricker, B.J. and Halling, S.M (1994) Differentiation of Brucella abortus 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis biovar 1 by PCR. J. Clin. Microbiol., 32(11): 2660-2666. [PubMed] [PMC]

39. Ewalt, D.R. and Bricker, B.J (2000) Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains 19 and RB51. J. Clin. Microbiol., 38(8): 3085-3086. [PubMed] [PMC]

40. Ahmed, Y.F., Sokkar, S.M., Desouky, H.M. and Madbouly, A.A. (2012) Studies on buffalo-cows naturally infected with Brucella melitensis. Glob. Vet., 9(6): 663-668.

41. Maria-Jesus, G., Blasco, J.M., Gorvel, J.P., Moriyon, I. and Moreno, E. (2012) What have we learned from brucellosis in the mouse model? Vet Res., 43(1): 29. [Crossref] [PubMed] [PMC]

42. Barrionuevo, P., Delpino, M.V., Velasquez LN, Samartino, C.G., Coria, L.M., Ibanez, A.E., Rodriguez, M.E., Cassataro, J. and Giambartolomei, G.H. (2011) Brucella abortus inhibits IFN-gamma-induced FcgammaRI expression and FcgammaRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages. Microbes Infect, 13: 239-250. [Crossref] [PubMed]

43. Pollak, C.N., Delpino, M.V., Fossati, C.A. and Baldi, P.C. (2012) Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response. PLoS One, 7: e50214. [Crossref]

44. Xavier, M.N., Paixa o, T.A., Poester, F.P., Lage, A.P. and Santos, R.L. (2009) Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. J. Comp. Pathol., 140: 149-157. [Crossref] [PubMed]

45. Meador, V.P., Deyoea. L. and Chevill, N.D.N.F. (1989) Pathogenesis of Brucella abortus infection of the mammary gland and supramammary lymph node of the goat. Vet. Pathol., 26: 357-368. [Crossref] [PubMed]

46. Neta C., Alcina V., MoAl, J.P.S., Xavier, M.N., Paixao, T.A., Lage, A.P. and Santos, R.L. (2010) Pathogenesis of bovine brucellosis. Vet J., 184(2010): 146-155. [Crossref] [PubMed]

47. Hosein, H.I., EL-Nahass, E.L.S., Rouby, S.R. and El-Nesr, K.A. (2018) Detection of Brucella in tissues and in formalin-fixed paraffin-embedded (FFPE) specimens using PCR. Adv. Anim. Vet. Sci., 6(2): 55-62. [Crossref]

48. Cheville, N.F., Olsen, S.C., Jensen, M.G. and Stevens, A.M. (1996) Bacterial persistence and immunity in goats vaccinated with a pur E deletion mutant or the parenteral 16M strain of Brucella melitensis. Infect. Immun., 64: 2431-2439. [PubMed] [PMC]

49. Benitez, p.C.A., Serantes, D.R., Herrmann, C.K., Viglietti, A.I.P., Vanzulli, S., Giambartolomeia, G.H., Comercib, D.J. and Delpino, M.V. (2016) The effector protein bpe005 from Brucella abortus induces collagen deposition and matrix metalloproteinase 9 downmodulation via transforming growth factor β1 in hepatic stellate cells. Infect Immunol., 84: 598-606. [Crossref] [PubMed] [PMC]

50. Gonzalez-Peramato, P., Jimenez-Heffernan, J.A., Sabater, C. and Vicandi, B. (2002) Lipogranulomatous lymphadenopathy as a potential source of error in fine needle aspiration cytology. A case report. Acta Cytol., 46(4): 772-775. [Crossref] [PubMed]

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