Trans- species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt

Aims: This study aimed to identify the


| INTRODUCTION
Brucellosis is a common anthropozoonosis caused by members of the genus Brucella and 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, 11 species of Brucella have been described, of which Brucella melitensis is the most serious and virulent to humans (OIE, 2018). Furtheremore, B. melitensis is widely spread among ruminant population especially in Mediterranean region, Latin America and Central and Western Asia (Benkirane, 2006). 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).
Seropreavlence of brucellosis in different regions of Egypt was investigated using rose bengal plate agglutination test as screening test followed by the complement fixation test as a confirmatory test. In Kafr El-Sheikh and Giza governorates, the seroprevalence of brucellosis in sheep flocks was estimated as 41.3% and 11%, respectively (Abdel-Hamid et al., 2017;Hegazy et al., 2011). Brucella melitensis bv3 is the most common and predominant strain isolated from different animal species from almost all Egyptian governorates Abdel-Hamid et al., 2020). In Egypt, large ruminants are usually reared either as single species or mixed with small ruminants and equines. There are also different rearing systems, 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 could play a major role in the spread of B. melitensis, as they pass across different Egyptian governorates, searching for pasture, and come in to contact with other ruminants . 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 in Brucella sp., which show >98% similarity in their nucleotide sequences (Corbel & Brinley-Morgan, 1984;Halling et al., 2005). Genus-specific PCR techniques targeting 16S rRNA or genes coding for Brucella membrane proteins have previously been used (OIE, 2018). 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 sp. 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 used 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 (repeat size ≤8 bp) loci termed Panels 1 and 2, respectively; the latter is subdivided into Panels 2A and 2B (Kattar et al., 2008;Vergnaud & Pourcel, 2006). 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:// micro besge notyp ing.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 & 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 investigated the genetic diversity and strains' relatedness of B. melitensis in Kafr El-Sheikh Governorate, Egypt under various husbandry systems. Also, it highlights the epidemiology of brucellosis, potential causes of brucellosis control failure and the genetic relatedness of the local Egyptian Brucella genotypes with their peers, based on the MLVA-16 metadata, worldwide.

HEGAZY ET AL.
Committees and the international laws and regulations concerning ethical considerations in research.

| Study area
Kafr El-Sheikh Governorate is about 134 km north of Cairo, the fourth largest city in the Nile Delta of Lower Egypt, and it consists of 10 districts and 206 villages with a population exceeding three million according to the Central Agency for Public Mobilization and Statistics. It is an agricultural governorate with a high density of livestock and human population. Sheep are raised either in small numbers kept in the household by farmers or in village flocks managed by shepherds (Aidaros, 2005). One shepherd would often keep sheep from many different owners; as a result, animals from different households are part of the same flock for grazing and breeding during most of the year. Some village flocks are sometimes combined to make a large flock managed by more than one shepherd. The flocks are reared in a free-range system, where animals graze freely all over the country as there is no restrictions for animal movement in Egypt (Aidaros, 2005). These flocks could have other ruminant species as cattle, buffaloes or goats. On the other hand, cattle and buffaloes are being raised as household animals, mobile herds just like sheep or in farms. Kafr El-Sheikh Governorate has large animal markets in which livestock owners and traders from different parts of Egypat purchase and sell their animals.

| Sampling, animals and specimens
According to the national control programme for brucellosis in Egypt, the public health authority has to notify the general organization for veterinary services (GOVS) of any cases of human brucellosis. Hence, GOVS collects blood samples from all animals owned by the notified positive human cases to identify their infection status and cull the positive ones. In the current study, samples were collected from animals reared in various husbandry systems (farms, mobile flocks and households), across different districts of Kafr El-Sheikh governorate, Egypt (Table1). Farms 'A' and 'B' have cattle and buffalo, farms 'F' and 'G' have only cattle, mobile flock 'C' has only sheep, mobile flock 'D' has mixed breeds of (goats, cattle and buffaloes) and mobile flock 'E' has sheep, goats, cattle and buffaloes. The remaining samples were taken from individual household ruminants that could have had contact with other animals. The individual household animals were five ewes and one cow.
Samples were given a code such as G-Cow6 when derived from cow number 6 of farm 'G' or HH-Ewe83 for ewe number 83 from the household.
For the aim of Brucella isolation and identification, specimens were collected from seropositive animals in four farms (A, B, F and G), three mobile flocks (C, D and E) and six households. These animals were targeted upon the confirmed seropositivity, abortion at the third trimester and/or the notification of a brucellosis case among the humans in contact with these animals. Farm 'G' is a dairy farm with 119 cows that had no history of abortion, nor Brucella spp. infection declaration, no history of introducing animals from outside the farm and had no contact with small ruminants. In 2017, the farm received a replacement cow of unknown brucellosis status for the first time, and 2 months later, abortions had occurred in 20% of the herd.
Full data about total animal numbers, seropositive cases, localities, specimens concerning farms, mobile flocks and household animals under the field of this study were shown in Table 1. Samples for bacterial isolation and identification were collected from live seropositive animals and upon slaughtering the seropositive animals through the national control programme. Supra-mammary (n = 81), retropharyngeal lymph nodes (n = 9), testicles (n = 1), fetal membranes (n = 52), spleen (n = 79) and milk samples (n = 40) were collected from seropositive live and slaughtered animals.

| Bacterial strains isolation, phenotypic characterization and molecular typing
The phenotypic characterization of Brucella isolates was done at the genus level based on colony morphology, urease activity, oxidase and catalase production. Then, species determination was carried out by phage lysis using Tbilisi (Tb), Izatnagar (Iz), Weybridge (Wb) and rough-canis (R/C) phages. Agglutination with monospecific A, M and R antisera besides, CO 2 requirement, H 2 S production, growth on thionin and basic fuchsin (20 µg/ml in serum dextrose agar) were performed to identify Brucella at the biovar level. Full typing at these three levels was done according to Alton et al. (1988) and OIE (2018). DNA was extracted from bacterial culture harvested in phosphate-buffered saline with PH 7.2 and inactivated at 100℃ for 15 min using QIAamp® DNA Mini Kit (Qiagen). DNA concentrations were measured by NanoDrop™ 2000/2000c Spectrophotometers (Nanodrop Technologies). Molecular typing using AMOS-PCR described by Bricker and Halling (1994) and Bricker et al. (2003) was conducted under the following conditions: initial denaturation at 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 55℃ for 40 s and 72℃ for 45 s, with a final extension of 72℃ for 10 min. The extracted genomic DNA from the B. melitensis bv3 reference strain Ether (ATCC 23458) was used for the allele assignment control.  Dahouk et al., 2007;Le Fleche et al., 2006) were performed for B. melitensis bv3 isolates (n = 41). For the phylogeny, dendrograms were performed after uploading the VNTRs data and estimating the Brucella genotypes online through the MLVA bank for microbe genotyping (http://micro besge notyp ing.i2bc.paris -saclay. fr). Dendrograms seeking the genetic similarities among the 41 Brucella strains were based on the categorical coefficient with distance calculation and unweighted pair group method with arithmetic mean using BioNumerics version 7.6 (Applied Maths).

| Bacteriological examination and identification
A total of 41 Brucella isolates were obtained in this study. Phenotypically, all Brucella isolates (n = 41) proved to be B. melitensis bv3 by fitting the identification scheme (Alton et al., 1988;OIE, 2018). AMOS-PCR showed a B. melitensisspecific band of 731 bp. Thirty-one isolates were obtained from 28 sheep and three goats (the preferential host), and 10 isolates were obtained from nine cattle and one buffalo (nonpreferential hosts) as shown in Figure 1. No Brucella isolates were identified from farms 'A' and 'B' regardless of the seropositive cases.

genotypes with previously reported genotypes outside the study area
Genetic similarities of different genotypes from different districts were identified (Figure 1) in Egypt are shown in the dendrogram, Figure 2. Genotypes 61-Egy-Bm3-Sharq and 18-Egy-Bm3-Kshkh isolated from Sharqia and Kafr El-Sheikh governorates (Sayour et al., 2020) are 100% identical with our genotypes M17_K.E and M11_K.E, respectively. Out of the 118 Brucella strains, 115 were grouped with the 41 B. melitensis strains of this study into one cluster with a similarity coefficient of approximately 96%, Figure 2.

F I G U R E 2
Comparative relatedness between the multilocus variable-number tandem-repeat analysis (MLVA-16) data of our genotypes with those previously reported in Egypt 8 |
All genotypes and strains identified in this study belonged mainly to the Western Mediterranean clonal lineage and were less likely related to the America's clonal lineage ( Figure  S1 and Figure 3). The highest similarity percentages were with neighbourhood Mediterranean and European countries. M12_K.E genotype is of 97% similarity with genotypes 2018Vergnaud#0133 and 2007AlDahouk#013, which were isolated from humans in France (1978) and Tunisia (1992), respectively. M3_K.E genotype is 99% similar to the French genotypes 2018Vergnaud#0531 and 2018Vergnaud#0555, which were isolated in 1983 from cattle ( Figure S1). M14_K.E genotype is 99% similar to genotypes 2018Vergnaud#0381 and 2018Vergnaud#0408 isolated from humans in Belgium (1982), which, in turn, have a similarity of 98.5% with M4_K.E genotype ( Figure S1).

| DISCUSSION
Brucellosis is a worldwide re-emerging transboundary anthropozoonosis associated with huge economic losses and public health problems (McDermott et al., 2013). Brucella melitensis is endemic in ruminants in Middle Eastern countries, and previous studies have shown that current national brucellosis control programmes are not effective in eradicating or reducing disease prevalence in either animals or humans Eltholth et al., 2017;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 F I G U R E 3 Similarity coefficient-based minimum spanning tree of 19 Brucella melitensis biovar 3 genotypes of this study with that worldwide E. Mediterranean W. Mediterranean Egypt Americas | 9 HEGAZY ET AL. 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 programme of brucellosis includes the voluntary annual vaccination of heifers with B. abortus S19 and kids and lambs with B. melitensis Rev 1 vaccine, whereas in some dairy farms, adult naive cows are vaccinated with a commercial B. abortus RB51 vaccine (Refai, 2002). The vaccination programme for both small and large ruminants is limited and does not cover all animal population (Eltholth et al., 2017). This may be due to the unsustainability of vaccination programmes 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 noninfected 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 prevalence of B. melitensis among small and large ruminants in Egypt.
Our results indicated that B. melitensis could be circulated and spread among cattle population, cow to cow transmission, in the absence of sheep and goats (preferential host). These findings are in agreement with previous observations (Godfroid, 2017;Wareth et al., 2020) 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 cannot 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 transmitting 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 long-term, widespread prevelance of the disease in Egypt. These results are matching with those reported by (El-Sayed and Awad 2018). Also, the result could be from a small mutation that occurs in the Brucella sp. genome .
Interestingly, in this study, we found more than one genotype from isolates obtained from the same animal, and this was also found by (Mathew et al., 2015) for B. abortus. Detection of both B. 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 transspecies transmission. Another reason for this finding may be the lack of cross protection between genotypes. This may reflect the absence of biosafety and poor hygeinic 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. melitensis genotypes 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 other governorates. Also, 100% genetic similarity was recorded between two Brucella genotypes (61-Egy-Bm3-Sharq and 18-Egy-Bm3-Kshkh) recovered from Sharqia and Kafr El-Sheikh 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. melitensis across different geographical regions of Egypt. This may be attributed to the lack of control of animal movement 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 suggest 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). In general, the importation of live animals is also a risk factor for the diversity of brucella genotypes (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.
In conclusion, this study showed a wide diversity of B. melitensis bv3 genotypes among different ruminant species in Egypt and the risk of uncontrolled local and international animal movement. It also indicates that this is a potentially major cause of failure for national control measures for brucellosis. Our novel findings on the biology and epidemiology of Brucella spp. are important for reviewing the current strategies for the control of brucellosis in Egypt and other countries with similar production systems. It will also support the selection of the proper vaccinal strain originating from the predominant genotype.