Discussion
Brucellosis is a worldwide re-emerging transboundary anthropozoonosis associated with huge economic losses and public health problems (McDermott et al., 2013). B. melitensis is endemic in ruminants in Middle Eastern countries and previous studies have shown that current national brucellosis control programs are not effective in eradicating or reducing disease prevalence in either animals or humans (Eltholth et al., 2017; Abdel-Hamid et al., 2020; Sayour et al., 2020).
In the current study, B. melitensis bv3 was the predominant strain isolated from the typical (small ruminants) and atypical hosts (large ruminants). This finding indicates the potential cross-species transmission of B. melitensis bv3 from the original hosts to large ruminant species in the country and this may be attributed to the uncontrolled movement of animals in infected areas, as well as the type of animal husbandry practiced (Wareth et al., 2020). In Egypt, part of the national control program of brucellosis includes the voluntary annual vaccination of female calves with B. abortus S19, while adult native cows are vaccinated with B. abortus RB51 vaccine and female kids and lambs with B. melitensis Rev 1 vaccine (Refai, 2002). The vaccination programme for both small and large ruminants is limited and does not cover all animals (Eltholth et al., 2017). Issues around uptake could be because of the unsustainability of vaccination programs due to insufficient budget, uncontrolled animal movement within infected areas, and the lack of control on open animal markets (infected animals mixed and in contact with non-infected ones). This was confirmed by the failure of isolation and genotyping of Rev 1 vaccine strain in any specimen (García-Yoldi et al., 2007). These factors may all contribute to the widespread prevelance of B. melitensis among small and large ruminants.
Our results indicated that B. melitensis could be circulated and spread among cattle populations without the presence of sheep and goats (preferential host). These findings are in agreement with Godfroid et al. (2017) and Wareth et al. (2020) who reported that cattle could be a reservoir for B. melitensis and could transmit it to other cattle in the absence of small ruminants (spill over the infection). Thus, the risk of B. melitensis transmission is increased in production systems where ewes, goats, and cattle are kept together in the same flock and can’t be isolated during parturition or abortion . Consequently, some B. melitensis strains may cross the species barrier and may be sustainably transmitted among cattle, without the persistent influx from the preferential host (Godfroid et al., 2014). This was also concluded by other researchers who isolated B. melitensis from cattle, even in the absence of sheep, suggesting a possible role of cattle in maintaining and transmiting this pathogen (Musallam et al. 2016).
The 19 genotypes of B. melitensis bv3 based on the MLVA-16 analysis obtained in this study regardless of the high genetic similarities indicate the longterm, widespread prevelance of the disease in Egypt (El-Sayed and Awad, 2018). Also, the result could be from a small mutation that occurs in the Brucella spp. genome (Abdel-Hamid et al., 2020). This requires further investigation to confirm the reproducibility (Bricker, 2002).
Interestingly, in this study, we found more than one genotype in isolates obtained from the same animal and this was also found by others for B.  abortus (Mathew et al. 2015). Detection of bothB. abortus and B. melitensis DNA in ovine has been observed in Egypt (Wareth et al., 2015) and this means that depending on the production system, preferential hosts can be infected with two different species or biovars of Brucella at the same time (Martirosyan et al., 2011). The gathering of small ruminants from infected mobile flocks that permits several sources of infection in one place or the introduction of replacement rams purchased from herds with unknown brucellosis status may play a vital role in this finding. Another reason for this finding, may be the lack of cross protection between genotypes against each other. This may reflect the absence of biosafety measures in flocks where different animal species are kept together and in close contact with humans (Hassell et al., 2017). The high seroprevalence and spread of the disease may also stand behind these criteria. The genetic diversity of the B. melitensisgenotypes identified in this study is in part related to the high discriminatory power of panel 2B markers.
The geographic distribution of the 19 genotypes obtained in the current study showed the close similarity of some of these genotypes with other genotypes from governorates outside of the study area. Also, 100% genetic similarity was recorded between two Brucella genotypes (61-Egy-Bm3-Sharq and 18-Egy-Bm3-Kshkh) recovered from Sharqia and Kafrelsheikh governorates (outside of the study area) with M17_K.E and M11_K.E genotypes found inside the study area. These findings re-ascertain the trans-species transmission of B. melitensisacross different geographical regions of Egypt. This may be attributed to the lack of the animal movement control in the country. Furthermore, the existence of a large live animal market in close proximity to the study area—Kotor market in Gharbia governorate—may be a risk factor for the distribution of the infection with different genotypes.
Globally, the genotypes identified in the current study are closely related to the Western clonal lineage, with one strain from the African lineage (Tunisia). Egypt does not import animals from African countries for breeding but animals are imported from European countries, North America and Australia. Our findings suggests that the importation of live animals for breeding from European countries, especially from France and Italy, is a risk factor for the introduction and spread of different B. melitensis genotypes in Egypt (Mugizi et al., 2015) . This importation of live animals is also a risk factor for the diversity of brucella genotypes more generally (Wareth et al., 2020). These findings could be attributed to the illegal introduction of animals from Libya, a country that suffers from political instability. This allows animals to be smuggled into Egypt through the country’s Western borders. Therefore, a risk analysis is required to identify whether the importation and smuggling of live cattle and sheep through country borders for breeding, contributes to the existence and spread of the different Brucella genotypes obtained.