First seroprevalence and molecular identification report of Brucella canis among dogs in Greater Cairo region and Damietta Governorate of Egypt

Background and Aim: Given the rise in stray and imported dogs in Egypt over the past 5 years, it is surprising that no report of Brucella canis infection in dogs or humans has been documented in Egypt’s published papers. This study aimed to detect the presence of antibodies against the rough (B. canis) and smooth Brucellae among dogs in Egypt and to characterize the Brucella species circulating in dogs. Materials and Methods: Blood samples (n = 449) were collected from owned and stray dogs in the Greater Cairo region (n = 309) and Damietta governorate (n = 140). The apparent, true, and total seroprevalence of canine brucellosis caused by B. canis infection were calculated using the 2-mercaptoethanol tube agglutination test (2-ME TAT) and rapid slide agglutination test (RSAT). We used the rose Bengal test (RBT) and the buffered acidified plate antigen test (BAPAT) to check the serum samples from dogs for the presence of antibodies against smooth Brucellae. Three polymerase chain reaction (PCR) assays - Bruce-ladder PCR, B. canis species-specific PCR (BcSS-PCR), and Abortus Melitensis Ovis Suis (AMOS)-PCR - were used to determine the Brucella species in the buffy coats of the serologically positive dogs. Results: The overall apparent and true prevalence of B. canis infection in dogs were estimated to be 3.8% and 13.2%. The estimated true prevalence in stray dogs (15%) was higher than in owned dogs (12.5%). The BAPAT and the RBT using smooth antigens revealed that 11 (2.4%) and 9 (2%) were positive. Bruce-ladder PCR targeting eryC, ABC, and Polysaccharide deacetylase genes was able to identify B. canis in nine out of 17 buffy coat samples. AMOS-PCR identified the eight undetermined Brucella species by Bruce-ladder PCR as Brucella abortus (n = 4) and Brucella melitensis (n = 4). To exclude the presence of Brucella suis, a one-step species-specific BcSS-PCR was performed and specifically amplified all B. canis DNA (n = 9) the same as did the Bruce-ladder PCR. Conclusion: To the best of our knowledge, this is the first report of B. canis detection in dogs in Egypt. Molecular identification of B. abortus and B. melitensis in the Egyptian canines highlights the role of stray dogs in brucellosis remerging in Brucellosis-free dairy farms. Brucella canis infection can be diagnosed specifically with the one-step BcSS-PCR. The obtained results set-an-alarm to the veterinary authorities to launch plans to control this disease in dogs.


Introduction
The genus Brucella comprises 12 species, six of them are classical species, including; Brucella abortus, Brucella melitensis, Brucella suis, Brucella ovis, Brucella canis, and Brucella neotomae [1,2], and six recently isolated species, namely; Brucella ceti, Brucella pinnipedialis, Brucella microti, Brucella inopinata, Brucella papionis, and Brucella vulpis [3,4]. Among all of the Brucella members, four species are most commonly involved in causing the disease in humans, namely; B. melitensis, B. suis, B. abortus, and B. canis. Brucella canis is a highly infectious pathogen that infects primarily dogs, leading to reproductive disorders and abortion. Despite the increasing international movement of dogs, there might be a growing risk of B. canis as a member of the genus Brucella. The Brucella organism causing canine brucellosis was first isolated by Carmichael in 1966 in the USA from aborted beagles and was termed B. canis [5]. Although B. canis is a rough organism, it is still pathogenic to canines and humans. The first report on B. canis infection in humans was in 1968 by the national Centers for Disease Control and Prevention of the US Public Health Service, by a laboratory technician who handled viable organisms. Brucella canis infection in female dogs is often linked to reproductive problems leading to infertility, abortion, and endometritis, even though many instances are clinically mild. However, a wide range of non-reproductive conditions, such as discospondylitis, endophthalmitis, and chronic uveitis, can also manifest [6]. Along with non-specific clinical symptoms such as fatigue, decreased appetite, and weight loss, lymphadenitis is another common condition in both male and female dogs [7]. Dogs infected with B. canis displayed prostate gland inflammation, epididymitis, orchitis, and scrotal edema [6]. The B. canis transmission between male and female dogs during mating periods is proven to disseminate organisms in seminal fluid leading to venereal transmission [8]. Besides, B. canis could be shed in the infected dog's urine, blood, and saliva. Brucella canis infection in dogs occurs most commonly from ingestion of vaginal discharges contaminated with huge numbers of organisms after an abortion [8]. Once these canines are infected, the infection is either self-eliminated within 2-3 years or develops into a lifelong infection [7]. The antibiotics used to treat B. canis infections are mostly ineffective [7].
Human infections with B. canis are relatively mild compared to those caused by other Brucella species [9]. However, all clinical features of Brucellosis in humans due to infection with B. canis are similar to those induced by other pathogenic Brucella species. The disease is transmitted to humans through direct and indirect transmission and is considered a classical debilitating zoonosis causing undulant fever [10]. The rapid slide agglutination test (RSAT) and the 2-mercaptoethanol tube agglutination test (2-ME TAT) are the serological methods most frequently used to diagnose B. canis in dogs [1]. These serological tests have limitations of low sensitivity and variable specificity [11]. Bacteriological examination of the causative agent through blood culture is usually applied to isolate B. canis. However, bacteremia is frequently intermittent or absent in chronic infections. Due to these shortcomings, different molecular techniques have been developed for the rapid and direct detection of B. canis as real-time polymerase chain reaction (PCR), Bruce ladder multiplex PCR, and B. canis species-specific (BcSS) PCR [12][13][14]. The world population of dogs is estimated at around 900 million [15]. The estimated stray dogs population in Egypt is about 15 million [16]. Dogs that are held by owners are registered and identified by collars or micro-chips, while unowned dogs include stray or guarding animal-farms dogs are not identified or registered or controlled. Brucella melitensis bv3 and, less frequently, B. abortus bv 1 are the predominant Brucella strains circulating in livestock in Egypt, according to a number of studies used to determine the prevalence of various Brucella strains in domestic farm animals [17,18]. Only two studies accidentally isolated B. abortus from two stray female dogs roaming on infected dairy cattle farms [19,20]. To the best of our knowledge, B. canis has not been reported in dogs or humans in Egypt in any published report.
This study aimed to detect the evidence of B. canis antibodies in dogs' sera in two areas for the first time in Egypt and to molecularly identify the Brucella DNA from the blood buffy coat of infected canines.

Ethical approval
The Animal Health Research Institute's Research Ethics Committee for Experimental and Clinical Studies approved the study protocol, which adhered to the guidelines of the World Health Organization and the Helsinki Declaration.

Study period and location
The study was conducted from June 2019 to January 2022. Blood samples were collected from randomly selected adult dogs (n = 449) in two different regions in Egypt. The first one was the Greater Cairo region (owned dogs) which includes the following; all cities in Cairo Governorate as well as Giza, 6 th of October city, Sheikh Zayed City in the Giza Governorate, Shubra El Kheima, and Obour in the Qalyubia Governorate with the aid of personalized dog owners. The second one was the Damietta Governorate (stray dogs) from Damietta city, Faraskur city, and Kafr Saad. Serum samples were separated from the blood, divided into aliquots, and preserved at -80°C until being examined. The reason why these areas were selected is that the greater Cairo Province is the largest metropolitan area in Egypt, the largest urban area in Africa, the Middle East, and the 6 th largest metropolitan area in the world, with a total population estimated at 20,901,000; area: 1,709 km 2 ; density: 10,400/km 2 [21]. While Damietta governorate is famous for having dairy farms where stray dogs cross the fences of these farms.

Study design and sampling
The sample size was estimated using the online WinEpi tool [22]. An estimated sample size of 449 blood samples with and without sodium citrate was collected from owned dogs in the Greater Cairo region (n = 309) and from stray dogs located in Damietta governorate (n = 140). These samples were tested for the detection of Brucella antibodies (RSAT and 2-ME TAT) and Brucella DNA by three PCR assays for the detection of smooth and rough Brucellae circulating in the blood of dogs in the study area.

Estimation of seroprevalence
Using the online WinEpi tool [22], we estimated the minimum sample size to be 385 dogs. This was calculated based on the population size of 15 million dogs, a confidence level of 95%, an accepted type 1 error of 5%, and a minimum expected prevalence of Available at www.veterinaryworld.org/Vol.16/January-2023/28.pdf 50% (First report of B. canis prevalence estimation in Egypt). The estimated sample size was adjusted to be 449 blood samples.
The apparent prevalence (AP) of canine brucellosis caused by B. canis infection was calculated by dividing the total number of seropositive dogs identified by both RSAT and 2-ME TAT by the total number of tested dogs. We calculated the true prevalence of canine brucellosis caused by B. canis using the formula TP = (AP+CSp-1)/(CSe+CSp-1) [23,24] using the combined sensitivity (CSe) and combined specificity (CSp) of RSAT and 2-ME TAT. Based on the reported sensitivities and specificities by Keid et al. [25], the CSe of both RSAT and 2-ME TAT was calculated [22] to be in series CSe of 28.1% and CSp of 99.9%.

Serological tests
The following serological tests were used to check the serum samples from the dogs for the presence of antibodies specific to the rough and smooth Brucellae: The RSAT, the 2-ME TAT, rose Bengal test (RBT), and the buffered acidified plate antigen test (BAPAT). DNA of Brucella canis and other smooth Brucellae were identified in buffy coat samples separated from whole blood collected from seropositive dogs by three types of conventional PCRs (AMOS-PCR, Bruce-ladder PCR, and BcSS-PCR).
Brucella canis rough antigen that was used in the RSAT and 2-ME TAT was prepared from heat-killed cells of B. canis reference strain RM666 suspended in a formalized phosphate-buffered saline solution. Brucella canis 2-ME TAT antigen was obtained from the National Veterinary Services Laboratory (NVSL) Ames IA 50010, USA. Both RSAT and 2-ME TAT were carried out in this study to detect antibodies against B. canis, according to the technique adopted by Alton et al. [1].
RBT and BAPAT originating from smooth Brucella abortus antigens were performed in this study to test dog blood samples for the detection of smooth Brucella antibodies according to the techniques described by Alton et al. [1] and World Organization for Animal Health (WOAH) [2].

Polymerase chain reaction assays
A. DNA Purification from Buffy Coat Samples with the Higher Purity™ Blood Genomic DNA Extraction Kit (Canvax Biotech, Spain).
Buffy coats fractions were separated after centrifuging 5 mL of citrated whole blood at 2500 × g for 10 minutes at room temperature (15°C-25°C), then transferred into a 15 mL tube containing 1500 μL of S1 buffer. This process is followed by gently mixing and centrifugation at 2000× g for 5 min. The supernatant was removed and the pellet was treated with proteinase K (250 μL) and S2 buffer (5 mL), incubated for 1 h at 55°C in a water bath, and then allowed to cool at 15°C-25°C. The S3 buffer was then added, followed by centrifugation. The supernatant was then gently mixed and transferred to a fresh 15 mL tube containing 5 mL isopropanol. The process involved centrifuging the sample, removing the supernatant using a pipette, and drying the pellet. The tube was centrifuged at 2000 × g for one minute after the pellet had been washed with 5 mL of 70% ethanol. In addition, we dried the pellet after removing the supernatant with a pipette. We added 500 μL of Buffer EB to the pellet, mixed it for 5 s at medium speed in the vortex, and then allowed it to incubate for 1 h. The eluted DNA was stored at −20°C.

Oligonucleotide primer
Primers supplied by Eurofins Scientific (USA) and the cyclic conditions are listed in Table-1 [26].
A. Fifty μL reaction containing 25 μL of Emerald-Amp Max PCR Master Mix (Takara, Japan), 1 μL of each primer at a concentration of 20 pmol (for a total of 6 μL of used primers), 14 μL of water, and 5 μL of DNA template was used to utilize the primers. Thermal cycler 2720 from Applied Biosystems (USA) was used to perform the reaction.
• C.1. AMOS-PCR [27] A 25 μL reaction containing 12.5 μL of Emerald-Amp Max PCR Master Mix (Takara, Japan), 1 μL of each primer at a concentration of 20 pmol (for a total of 3 μL of used primers), 4.5 μL of water, and 5 μL of DNA template were used to test the primers. Thermal cycler 2720 from Applied Biosystems was used to carry out the reaction.
D. Analysis of the PCR Products of Bruce-ladder PCR, AMOS-PCR, and BcSS-PCR.
The products of PCR were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1× TBE buffer at 15°C-25°C using gradients of 5V/cm. For gel analysis, 40 μL of the Bruceladder PCR products were loaded into each gel slot. Whereas after the BcSS-PCR and the AMOS-PCR, each gel slot was loaded with 15 μL of the DNA products. The Gene-ruler 100 bp DNA ladder (Fermentas, Thermo, Germany) was used to determine the fragment sizes. The gel was photographed by the gel documentation system (Alpha Innotech, Biometra), and the data were analyzed through computer software.

Results and Discussion
Diagnosis of canine brucellosis is primarily based on serological examinations. However, infected dogs with B. canis usually give negative results by conventional serological tests based on smooth B. abortus antigen. This is because rough antibodies to B. canis do not cross-react with those induced by smooth species. For that, using a rough antigen obtained from B. canis is required to assess the immunoglobulin induced by B. canis.
The results of the RSAT and the 2-ME TAT performed on serum samples (n = 449) of the owned and stray dogs in the Greater Cairo province and Damietta governorate were 6.2% (28/449) and 3.8% (17/449) as presented in Table-3. Dogs were interpreted as positive when they gave positive reactions in both RSAT and 2-ME TAT (titers ≥ 1/150). These tests detect antibodies against surface antigens of Brucella spp., particularly antibodies against rough lipopolysaccharide (LPS). RSAT is a screening highly sensitive test that may give false positive results due to cross-reaction with other bacteria such as Pseudomonas, Bordetella bronchiseptica, Salmonella, Yersinia enterocolitica, and Escherichia coli [11,28]. In addition, the test can be negative early in the first 3-8 weeks post-infection [29]. The 2-ME TAT is more specific than RSAT as the 2-mercaptoethanol (2ME) acts as a disulfide bond reducing agent of the immunoglobulin (Ig)M, the class of immunoglobulin responsible for the cross-reaction with Gram-negative bacteria through splitting the disulfide bonds and depolymerizes the IgM pentamer. Therefore, any remaining agglutinating activity after 2ME treatment is due to 2ME-resistant IgG, which is the long-lasting specific class of immunoglobulins in B. canis infection [1].
The 2-ME TAT is considered a semi-quantitative test that is employed to confirm the results given by RSAT [30]. Serological diagnosis of canine brucellosis due to infection by B. canis is challenging. A combination of different serological tests is required for proper diagnosis. To avoid the false positive results, the buffy coats separated from the whole blood of the same seropositive dogs for B. canis by both RSAT and 2-ME TAT were further confirmed by molecular tools.
The calculated apparent prevalence of B. canis infection in owned dogs in Greater Cairo province was 11/309 (3.6%), while in stray dogs obtained from Damietta governorate was 6/140 (4.3%). The estimated true prevalence (Table-4) in both owned and stray dogs after including the errors represented in the combined sensitivities and specificities of RSAT and 2-ME TAT in the formula was 12.5% in owned dogs and 15% in stray dogs. The overall apparent and true prevalence were estimated to be 3.8% and 13.2%. Our results match the results of Daly et al. [31], who reported an overall apparent B. canis seroprevalence of 6.8% and adjusted the estimated true prevalence of 29.4% when they performed in-clinic screening of 3898 dogs over more than 4 years in two South Dakota Indian reservations. The results are also in line with Whitten et al. [32], who estimated the apparent seroprevalence of B. canis antibodies in dogs entering a Minnesota humane society during 1 year (2016-2017) to be 3.1% (22/943) and 3.5% (8/943) among stray and owner-surrendered dogs by using RSAT.  It is noteworthy to mention that the Egyptian government stopped random killing of stray dogs. Based on this fact, the number of stray dogs increased over the past 5 years. The true prevalence of canine brucellosis due to B. canis infection in stray dogs was found to be higher than in owned dogs in this study due to the absence of mating control in stray dogs compared to owned dogs [33]. Stray dogs are considered one of the main reasons for B. canis spreading in some European countries [34]. To the best of our knowledge, this is the first study to document B. canis in the Egyptian canine population.
On the other hand, the application of the BAPAT and the RBT on 449 canine sera revealed that 11 (2.4%) and 9 (2%) were positive. The BAPAT detected two positive samples more than RBT. Enhancing sensitivity was attributed to the final packed cell volume in the case of BAPA was that of 3%, whereas that of RBT was (4%), and the final pH following the addition of serum in BAPAT was (4.02), and (3.8) in RBT [1]. The main factors that cause the BAPAT to be a little more sensitive than the RBT are the reduced final packed cell volume of BAPA compared to RBT and BAPA's slightly lower final acidic pH [1]. The presence of smooth LPS detected by serological tests based on smooth B. abortus antigen of the BAPAT and RBT indicates infection of these dogs with Brucella species other than B. canis. However, in a previous study [35], using conventional serological tests targeting smooth Brucella antibodies, 6.48% of dogs were positive to smooth Brucellae. This relatively high positive percentage of smooth Brucella antibodies in dogs in such a study was because most dogs were located in and around the infected dairy farms during the outbreak.
Although the definitive confirmation of infection through the isolation of the causative agent is the gold standard for diagnosis [2], this procedure has low sensitivity, takes a long time, poses a biological hazard, especially for lab workers, and necessitates a BSL3 lab facility. Therefore, using molecular tools to detect causative agents increased the possibility of rapid and accurate detection of Brucella species.
We used buffy coats in this study instead of whole blood samples for DNA extraction as buffy coat yields more DNA than an equivalent volume of whole blood [36]. In addition, using the buffy coat in the BcSS PCR assay resulted in approximately 100 times higher sensitivity for B. canis direct detection if compared with whole blood [13]. Besides, whole blood contains PCR inhibitors such as heparin, hemoglobin that affects the DNA polymerase activity and decreases the amplification efficiency, IgG that binds to single-stranded genomic DNA, and the presence of antimicrobial drug residues [37].
Bruce-ladder is primarily designated to identify and differentiate all standard Brucella species, including B. abortus, B. melitensis, B. suis, B. canis, B. ovis,  and B. neotomae as well as Brucellae in marine mammals and the S19, RB51, and Rev.1 vaccine strains using a total of eight pairs of primers in a single step multiplex PCR [26]. To fit the purpose of the application of Bruce-ladder in identifying and differentiating expected Brucellae that may infect canines, three pairs of primers were selected to be used in the current study.
All positive canine blood samples for B. canis to rough antibodies (n = 17), and all those positive to smooth Brucella antibodies (n = 9) were subjected to DNA extraction from buffy coat and PCR analysis. Out of the 26 buffy coat samples subjected to DNA extraction, all reacting canine blood samples for B. canis to rough antibodies (n = 17), as well as those reacting to smooth Brucella antibodies (n = 9) were subjected to DNA extraction from buffy coat and prepared for PCR analysis. All 26 samples yielded DNA from the buffy coat fraction of the whole blood. However, nine samples gave negative results by Bruce-ladder PCR. This finding may be attributed to false positive results that may be given by serological test to the other Gram-negative bacteria sharing B. canis in antigenicity [6].
To detect different Brucella species DNA in canine blood, we investigated the Erythritol Catabolism gene (eryC) existing in all Brucella species except B. abortus S19 vaccine strain due to the deletion of the 702 bp in BMEI10427-BMEI10428 in this vaccinal strain. Since B. abortus S19 is not used or recommended for dog vaccination, the presence of the eryC gene in canine blood refers to any other species or biovars belonging to the genus Brucella. The eryC gene was also detected in B. abortus reference strain 544, B. melitensis reference strain 16M, and B. canis reference strain RM 666 (Figure-1). Out of 26 positive serum samples for rough and smooth Brucella antibodies, the DNA extracted from 17 buffy coat samples were amplified by Bruce-ladder and gave an amplicon size of 587 bp.
Using specific primers (BR0953f and BR0953r), it was attempted to recognize the ABC gene, which is responsible for the transporter binding protein, in B. canis DNA extracted from the buffy coats of canine blood samples. This resulted in an amplicon size of 272 bp of nine samples and only in the positive control of B. canis reference strain RM 666.
Polysaccharide deacetylase present in all Brucella species, including all members of B. abortus and B. melitensis in an amplicon size of 794 bp, which does not exist in B. canis. The absence of the 794-bp fragment distinguished B. canis from other species [26] and confirmed the presence of B. canis DNA in the buffy coat samples. Using the Bruce-ladder PCR targeting the Polysaccharide deacetylase gene to amplify Brucella DNA in the given samples (n = 19), Bruceladder PCR gave a specific DNA band (794 bp) in eight DNA samples (S8, S9, S16, S17, S18, S19, S21, and S22) as shown in Figure-1. The results of the Bruce-ladder PCR suggest that there are additional, as yet unidentified Brucella species (n = 8) other than B. canis. In order to identify these eight Brucella species, duplex AMOS-PCR was employed.
Application of Duplex AMOS-PCR [8] on eight samples targeting insertion sequence IS711, using B. abortus and B. melitensis specific primers revealed that four samples' numbers (S4, S6, S9, and S10) were identified as B. abortus with an amplicon size of 498 bp. The other four samples, namely; S3, S5, S7, and S8, showed a specific band at an amplicon size of 731 bp, indicating B. melitensis species (Figure-2).
Given into consideration that dogs' blood samples were collected from an area far away from pig farms and that those dogs have no history of contact with swine. Since the 272-bp DNA fragment amplified by the Bruce-ladder multiplex PCR is not restricted to B. canis. Furthermore, a 272-bp DNA fragment is presented in both B. suis and B. neotomae [26]. We tried BcSS-PCR assay, a one-step PCR assay (Figure-3), for the detection of B. canis samples positive by the Bruce-ladder multiplex PCR to make sure that there was no space for doubt. Kang et al. [14] created this sensitive assay, which specifically amplified all B. canis DNA extracted from the buffy coat of the whole-blood samples in a 300 bp band. The BCAN B0548-0549 area on B. canis chromosome II served as the basis for the creation of the specific PCR primer set employed in this experiment.
We choose the one-step BcSS-PCR for the following reasons: (1) The detection limit of the BcSS-PCR is slightly lower than that of the 16S rRNA PCR and equal to the sensitivity of the BCSP31 PCR using primer pairs of B4/B5 [14]. (2) Using a well-designated PCR primer based on the BCAN B0548-0549 region in chromosome II of B. canis, it gives a 300-bp product that is not present in other Brucella species or genetically or serologically related bacteria [14]. (3) In addition, the PCR assay using JPF/JPR primer pairs of a gene coding for outer membrane protein (omp-2) that is employed to detect Brucella species demonstrated lower sensitivity than the BcSS-PCR [38].
In a previous study, four Brucella genes were evaluated by conventional PCR to identify B. canis DNA in canine blood, urine, and uterine discharges, including; the gene coding for the BCSP31 protein (primers B4 and B5), the ribosomal gene coding for the 16S-23S intergenic spacer region (primers ITS66 and ITS279), the gene coding for porins (primers JPF and JPRca), and the gene coding for the insertion sequence IS711 (primers O1 and O2) [39]. The gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples, but the 16S rRNA gene PCR assay gave false-positive results for the field strains of Ochrobactrum anthropi and Staphylococcus aureus [40,41] while the primers that amplify the insertion sequence IS711 were the most specific one compared with the other primers used in their study with the Sp: 99.66% (confidence interval 98.84-100) [39].
In Egypt, Brucella abortus was isolated from an apparently healthy Ballady (native) stray bitch roaming in an infected dairy cattle farm in Damietta governorate [20]. Nevertheless, while B. canis is the most common cause of canine brucellosis [42], occasional infections with B. melitensis, B. abortus, or B. suis may occur in dogs that have close contact with tissues or secretions of infected livestock animals, especially raw milk, aborted fetuses, and placentas [43]. Multiplex PCR Bruce-ladder assay was recommended by the WOAH as a quick and easy test for molecular identification and typing of more isolated Brucella species [2]. Buffy coat samples were used in this study as a source DNA that was amplified by multiplex PCR for detection and identification of B. canis was found practical, appropriate, and fit matrix for the purpose of B. canis diagnosis in dogs.
Brucella canis antibodies were detected in the two dogs' serum samples by RSAT and 2-ME TAT (S13 and S14). The DNA extracted from the buffy coat of these two serologically positive samples gave negative results to Bruce-ladder PCR (Figure-1). This may be explained by the fact that only a small number of B. canis cells, which produce little, undetectable DNA, might be present in the whole blood of these negative samples. This issue could be solved by pooling blood samples from the same suspect dog.
Although the obtained results exhibit evidence of the presence of B. canis among dogs in Egypt, the source of this Brucella species remains unknown. It might have originated and circulating in Egypt already for many decades or it might be of exotic origin and introduced to the country through crossing international borders with travelers with pet animal companions. Further studies should be conducted to isolate the organism and run WG sequencing to assess the phylogenetic relatedness of this strain with other international strains. Limitations of the present study are that the results refer to random samples dogs collected from dogs in Greater Cairo region and Damietta Governorate and do not reflect countrywide.

Conclusion
This article reports the presence of B. canis in Egypt for the first time and highlights this neglected zoonotic disease. For the first step of diagnosis, serological tests originating from rough B. canis antigen, particularly RSAT as a screening test and 2-ME TAT as a supplementary test, followed by PCR assays, was necessary for the accurate diagnosis. The alternative use of multiplex PCR Bruce-ladder assay using only three pairs of primers, followed by AMOS-PCR assay, has proved applicable for the identification of B. canis and other Brucella species in infected dogs. The one-step BcSS-PCR could be used as a single alternative, accurate, and valuable tool in the diagnosis of B. canis infection in the buffy coat samples. Detection of B. abortus and B. melitensis in canine blood revealed the role of stray dogs in remerging the disease in Brucellosis-free dairy farms in Egypt. The obtained results set-an-alarm to the veterinary authorities to launch plans to control this disease in dogs. One Health approach should be initiated for canine brucellosis interface with humans who come in contact with infected dogs.