Control ofbrucellosis in livestock depends on the reliability and sensitivity of themethods used for detection and identification of bacteria which would create aconsequential control of the disease in wildlife and also in humans (Poester etal., 2010). However, putting into consideration that the incubation period ofbrucellosis varies considerably and is affected by several factors whichinclude; gestation, exposure dose, age, vaccination, and other unknownhost-resistance influence (Thomsen A.
, 1950) diagnosis would be cumbersome.Most protection against infection by B.abortusis largely a result of cell-mediated immunity (Cheers et al., 1983) but mostdiagnostic tests developed rely on detecting a response in humoral immunesystem (Stemshorn., 1983). Th1 cells in the spleen plays an important role inconferring protective immunity to B.
melitensisinfection (Vitry et al.,2014). In this presentstudy, Brucella abortus specific histopathological changes were observed. Therecruitment of lymphocytes in the liver was evident forming micro-granulomaswhich corresponded to a study done by Mariana et al., 2013; who showed inIL-1Oflox/CD4cre mice exhibiting an inflammatory response in the liver as wellas the spleen. In addition, there was distinct evidence in architecturalchanges of the spleen histology. However, Mariana et al marked an influx ofneutrophils and histiocytes in the spleen at an acute stage of infection, laterdepicting that in chronic infections particularly in B.
abortus infection, andalso granuloma formation comprised of mononuclear cells not depicted in this study. The Brucella organism’s predilection fororgans rich in reticuloendothelial cells (spleen, liver, bone marrow, lymphnodes) and its intracellular location are responsible for the chronicity of thedisease, which can last for months or even years (Bothwell, 1963; Spink, 1964;Robbins, 1968). The spleen and liver are organs that contain many bacterialcells after Brucella invasion. (Hortet al.
, 2003). Although bacteriological diagnosisbeing a gold standard diagnostic method for brucellosis, culture of the speciesis challenging as it is a fastidious bacterium requiring rich media for primarycultures (Hadush., 2013; Bricker., 2002). In the present study, results didshow that there was a high prevalence of Brucellaabortus strain 544 isolated in the vector control group than in theimmunized groups with a slight effectivity of the ST system given at a higherdosage 5 X 1010 CFU/ml per animal over the animals immunizedusing ST system 5 X 109 CFU/ml per animal.
In addition, results suggest that thebacterium may be readily isolated in lymph tissue rather than in thereticuloendothelial tissue samples. IHC allows in situ localization of the organismwithin Brucella induced lesion (Poesteret al.,2006). Immunostaining does not require viable bacteria and thus a betteroption to bacterial culture in those regards. In this study, it was noticedthat immunohistochemistry could not detect bacterial antigens exactlycorresponding to bacterial culture results also suggesting that the sectioningof the tissue reduces the chances of locating the positive cells.The presentstudy also explored the use of real-timePCR approach using the SYBR Green I (double-strandedDNA intercalating dye (Newby, 2003), the advantages being that it was faster asthey was no electrophoretic analysis and does not require post amplication handling of PCR products. However,it was thus noticed that almost all DNA templates examined showed a fluorescentsignal. Even though SYBR GREEN I assays may not require probe design, the lackof additional discrimination that can be gained by incorporating of one or moreprobes may result in false positives.
To overcomesuch errors a melt analysis/ or gel analysis can be done but then what would bethe need of using it as a diagnostic tool (Newby et al). Real-time PCR can be used as a confirmatorydiagnostic alternative for problematic culturing of Brucella sp. The high detection sensitivity of real-time PCR can beexplained by the fact that it detects DNA from bacteria that are damaged andnon-inviable and therefore impossible to isolate by conventional cultures.
Inthe present study IS711 real-time PCR wasable to detect positive animals which were negative by bacterial isolation anddetect additional animals that were immunohistochemically negative. Therefore,the importance of using more than one type of diagnosis for detection ofbrucellosis in animals (Elfaki et al., 2005: Ilhan et al., 2008).