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.