Brucellosis is a severe zoonosis, that affects many animal species, with most important the domestic ruminants, such as sheep, goats and cattle. Both breeders and the National Economy deal with grade economic losses such as the reduction of milk production, the trade restrictions, the culling of seropositive animals (and in some cases seronegative, as well), the abortions and stillbirths, the cost of veterinary services, and miscellaneous factors arising from brucellosis on farms (Rossetti, et al., 2017).
Rose Bengal test (RBT) and Complement Fixation Test (CFT) have been used for many years, in brucellosis eradication programs (Garin-Bastuji, et al., 1998, Nagati, et al., 2016). The diagnosis of brucellosis in small ruminants and cattle requires the use of more than one serological test and RBT and CFT are among the most useful tests for routine diagnosis (Baum, et al., 1995). Even if serological cross-reactions have been demonstrated between smooth Brucella species and Escherichia coli serotypes 0:116 and 0:157, Salmonella serotypes of Kaufmann-White group N (0:30 antigen, Pseudomonas maltophilia and Yersinia enterocolitica serotype 0:9, tend to be of a little significance (FAO/Brucellosis in human and animals., 2006).
The results of our study using the above PCR protocol indicates that detection of Brucella spp. DNA directly from testicles and spleen of seropositive male animals was not a convenient procedure confronting with the results derived from serology tests (RBT/CFT) in the first group of 83 seropositive animals. In fact, only 14,45% of the seropositive animals were found positive in PCR analysis. Testes and spleen as biological samples-target for the detection of brucellosis via PCR is mostly rare. According to the literature, the most frequently tested tissues from male animals in order to detect Brucella spp. are serum, semen, nasal secretions, feces, lymph nodes and whole blood (Bricker, 2002, Ilhan, et al., 2008, Yu, et al., 2010).
Respectively, in the second group of 108 suspected animals, 16.67% of the female animals and fetuses were found positive in PCR analysis. The relatively few Brucella PCR positive samples from female ruminants with clinical symptoms from the reproductive system in the present study might also suggest the existence of other causes of abortion than brucellosis and it warrants further studies (Petridou, et al., 2018).
PCR-based tests are proving to be faster and more sensitive than conventional methods. Several PCR assays have been described for the detection of Brucella DNA using pure cultures or animal or human clinical samples. However, the sensitivity and specificity of the PCR for Brucella spp. varies between laboratories, standardization of sample preparation, PCR inhibitors on tissues, target genes, selected primers and clinical status of the animal (acute/chronic) (Amin, et al., 2001, Tekle, et al., 2019). The challenge lies in simplifying the procedure for detecting Brucella spp. at the species level in specialized laboratories (Navarro, et al., 2004, Ghorbani, et al., 2012,).
Τhe modified PCR protocol used in our study was time consuming in terms of samples preparation resulting to a minimum of three days for the results to be delivered while the cost was much higher than the one required for serology.
An ideal diagnostic method should be specific, sensitive, rapid, not expensive and easy to perform (Geresu, et al., 2016). According to the presented study, it seems that for the time being this PCR protocol described does not fulfil all these requirements. Finally, the purchase of reagents is fairly expensive compared with RBT/CFT, but cost may decrease as more PCR diagnostic assays are developed.
In both groups, the prevalence of the Brucella strains detected in samples derived from goats (♂12,5% and ♀50%) and bovines (♂71,43% and ♀25,56%) was much higher than the prevalence of the same detected bacterium in sheep (♂3,03% and ♀8,22%). Actually, goats are approximately four times more likely to be infected than sheep (Sintayehu, et al., 2015). Generally, sheep are less susceptible to Brucella infection than goats, and this could be partly explained because sheep excrete the bacteria for shorter periods. This may reduce the potential for spread of the disease within and between sheep flocks (Radostits, et al., 2007).
In the first group, preferred tissue for detecting the bacterium in rams and billy goats is testicle (8,7% and 19,23%) instead of spleen (0% and 4,76%), even if it is commonly known that spleen is the most heavily colonized organ, during brucellosis (Silva, et al., 2011). The stage of infection may very well influence the number and location of Brucella organisms in white blood cells and lymphoid tissue glands (Gupta, et al., 2006). Concerning bulls, it seems that inguinal lymph nodes give encouraging results (71%), while no other kind of tissues were provided in this study.
The fact that vaccine strain Rev1 was detected in non-vaccinated rams and billy goats, indicates that possibly errors during the vaccine administration in females may occurred resulting in accidental vaccination of the male animals which were found seropositive during the surveillance program. Finally, due to the above vaccine administration errors male animals were slaughtered while the farm was quarantined for a period of at least 2 to 6 weeks. The development of a rapid and cost-effective laboratory method that will discriminate the vaccine antibodies from those caused by wild-type strains is of paramount importance. Alternatively, the development of a new vaccine, like the RB51 strain (Dorneles, et al., 2015), which is implemented in cattle and the produced antibodies are not detectable by RBT/CFT may solve this inconvenience equally.
Among female small ruminants and cattle from the second group which aborted, we suggest as preferred tissues to examine and reach to a positive PCR result tissues from fetal liver or/and stomach content from fetuses than other fetal tissues (fetal membranes, fetal spleens, placenta and posterior mammary lymph nodes).