Introduction
Brucellosis is a common anthropozoonosis caused by members of the genus Brucella . Due to its prevalence, it is associated with a major economic burden worldwide (Corbel et al., 2006). The infective dose is low (10-100 bacteria) and is easily transmitted to humans by ingestion, direct contact, and inhalation causing undulant fever and other severe health problems (Christopher et al., 2010). In ruminants, it is a common cause of contagious abortion, decrease in milk production, and infertility (McDermott et al., 2013). Until now, eleven species of Brucella  have been described, of which Brucella melitensis  is the most serious and virulent to humans (OIE, 2018). The serious nature of B. melitensis  is related to its widespread presence in ruminant populations, high pathogenicity in humans, difficulties in its control compared to other species of Brucella , and its high virulence (Cloeckaert et al., 2002). Moreover, the high mobility of small ruminants, which are the preference host of B. melitensis , facilitates its dissemination between farms and regions (McDermott et al., 2013).
In Egypt, the seroprevalence of brucellosis in sheep flocks was estimated to be 41.3%, and 11% in Kafrelsheikh and Giza governorates (Hegazy et al., 2011; Abdel-Hamid et al., 2017). B. melitensis  bv3 is the most common and predominant strain isolated from different animal species from almost all Egyptian governorates (Abdel-Hamid et al., 2016; Abdel-Hamid et al., 2020). In these contexts, large ruminants are reared either as single species or mixed with small ruminant and equines. In addition, different raising-systems exist, for instance, ruminants graze during the day and are then kept in pens at night, while other farmers rear their ruminants indoors or in mobile herds. The shelterless small ruminant mobile flocks play a major role in the spread of B. melitensis , as they pass across different Egyptian governorates and come in to contact with other ruminants whilst grazing (Hegazy et al., 2016). This animal husbandry method is common in the majority of Middle Eastern countries and has resulted in the contact of different species of ruminant for a long period of time.
Limited genetic polymorphisms exist on Brucella spp.  which show >98% similarity in their nucleotide sequences (Corbel and Morgan, 1984, Halling et al., 2005). Genus specific PCR techniques targeting 16S rRNA or genes coding for Brucella  membrane proteins have previously been used. Other molecular techniques such as the repeat of short nucleotide sequences or the variable number of tandem repeats (VNTR) have being used to differentiate Brucella  species and biotypes depending on the wide variation in the number of these repeats (Christopher et al., 2010).
Multiple locus VNTR (MLVA-16) has been efficiently employed in epidemiological studies to identify Brucella  strains targeting 16 loci and seeking their genetic associations (Maquart et al., 2009). The MLVA-16 includes eight minisatellite loci (repeat size of ≥9 bp) and eight microsatellite loci (repeat size ≤8 bp) loci termed panel 1 and panel 2 respectively, this latter subdivided into panels 2A and 2B (Vergnaud and Pourcel, 2006, Kattar et al., 2008). This is a powerful discriminatory tool in subtyping bacteria of high genomic homology regardless of their geographic origin such as Brucella  (García-Yoldi et al., 2007). However, further studies on large numbers of isolates from different countries are required to improve strain relatedness and to enhance the MLVA database “http://microbesgenotyping.i2bc.paris-saclay.fr”; a database that contains the VNTRs metadata of more than 5000 isolates (Kattar et al., 2008). The efficiency of MLVA to detect the genetic divergence between different isolates of Brucella  is judged by the Hunter-Gaston diversity index (HGDI) which includes the results of each marker of the panels separately as well as in combination (Hunter and Gaston, 1988).
The use of the MLVA technique could play an important role in explaining the geographic distribution of some genotypes, and the role of animal movement and animal trade in spreading the infection. The MLVA technique allows for the characterization of species and biovars of Brucella   and allows for new strains to be traced back to their original source (De Massis et al., 2019; Wareth et al., 2020). Furthermore, the technique could provide more information on the biology of Brucella  in different animal species, which is essential to undertake effective control measures against brucellosis.
Thus, this study will investigate the genetic diversity and strains relatedness of B.  melitensis  in Kafrelsheikh governorate, Egypt. The study will also investigate the epidemiology of brucellosis, the causes of control failure in the study areas, and the genetic relatedness of the local Egyptian Brucella  genotypes with their peers on the MLVA-16 worldwide database.