1. BRUCELLOSIS - AN UNKNOWN AND UNDERDIAGNOSED INFECTION IN BULGARIA

Brucellosis is still the most common zoonosis worldwide. Despite this, it is unknown and underdiagnosed infection in non-endemic areas. For some parts of the world it became a re-emerging infection. After several decades brucellosis re-emerged in Bulgaria. In 2005 an outbreak of imported human cases was detected and soon after, two autochthonous outbreaks occurred (2006 and 2015) with a total of 161 persons diagnosed.

genus Brucella and so far, there is no evidence for its pathogenicity for humans. Furthermore, another two strains (BO1 and BO2), similar to B. ovis were isolated from human clinical samples. Based on phenotypic and molecular analyses they were considered as a novel species, named B. inopinata (14,15). The last described species, B. vulpis sp. nov., was isolated from Austrian red foxes and carries the Brucella specific IS711 and bcsp31, but 5% of its genome was acquired from different soil bacteria (16,17). Brucellae are small Gram-negative coccobacilli or short rods, which are facultative intracellular parasites. They are non-motile, non-spore forming, without true capsules, pili and natural plasmids. Although brucellae are aerobes, some types require additionally CO 2 , especially for primary isolation. All members are fastidious and need rich peptone media supplemented with blood and/or serum. The lipopolysaccharide (LPS) of the cell wall is the immunodominant antigen. It cross-reacts with other Gram-negative bacteria such as Yersinia enterocolitica O9, Escherichia coli O157, E. hermannii, Salmonella enterica O:30, Vibrio cholerae O1 and Stenotrophomonas maltophilia (18). The protein antigens (outer membrane and cytoplasmic) have a protective effect against brucella by stimulating the cellular and humoral immune response in infected individuals. They are common for both -smooth and rough types and do not cross-react with other Gram-negative bacteria (19).

SOURCES AND ROUTES OF INFECTION
The main source of infection for humans are domestic animals. Their significance is determined by the most common host species (  (27), breast feeding (28), or possible sexual transition (29). In endemic areas the infection rates are the same in children and adults and most often the disease occurred after consumption of contaminated food. In non-endemic areas, such as Bulgaria, mainly adults are infected (occupational or imported cases) (20, 30).

PATHOGENESIS
Brucellae tend to invade and survive in the host. They manage to avoid intracellular killing in phagocytic cells and multiply in macrophages. Through the circulatory system they reach the regional lymph nodes and afterwards -various organs, mainly the reticuloendothelial system. These processes determine the diverse clinical manifestations of the disease (31). The intracellular survival of bacteria is facilitated by virulence factors such as LPS components, superoxide dismutase and some outer membrane proteins (19, 32). Protection of the macroorganism is carried out by anti-LPS antibodies and T cell-mediated activation of macrophages by the protein antigens (19). The antibody production by B lymphocytes is of small importance for the immune protection, but has a great diagnostic value.

CLINICAL PRESENTATION
Brucellosis has a variety of nonspecific symptoms and can mimic other infectious and noninfectious diseases. The clinical presentation depends mainly on the stage of the disease. The early symptoms include: fever, sweats, malaise, anorexia, headache, joint and muscle pains, depression. The so called "undulant fever" could appear. Physical abnormalities are generally few and include lymphadenopathy (10-20%), splenomegaly, and/or hepatomegaly (20-30%). Because of the untypical presentation of the disease, it often remains unrecognised or misdiagnosed, especially in non-endemic areas (33, 34). Chronic brucellosis (duration more than 12 months) is presented with recurrent relapses of the above-mentioned symptoms or focal infections. Antibiotic therapy in this phase is less effective. The most common complications (92%) are musculoskeletal: sacroiliitis, peripheral arthritis, spondylitis, bursitis, incl. in Bulgarian patients (34), followed by affection of reproductive, nervous and cardiovascular systems, and more rarely lung, kidney, eye and skin manifestations (33 . Several regiments are recommended: doxycycline 6 weeks + rifampicin 6 weeks or doxycycline 6 weeks + streptomycin for 2-3 weeks (gentamicin 1 week). The use of ciprofloxacin and co-trimoxazole is optional (esp. pregnant women and children younger than 8 years), but without any priority over the above-mentioned antimicrobial agents. Therapeutic failure or relapses are generally not caused by resistance, but by premature discontinuation of the treatment. Patients with relapse are usually retreated with the same antimicrobial agents, but often the result is not satisfactory. Recurrence of brucellosis may occur from a persistent focus of infection that requires additional treatment, for example surgical drainage. Due to serious side effects human vaccines are not applicable. Decrease of the incidence is achieved through control of the infection in animals and the contamination of dairy and meat products. Because of the low infectious dose in aerosols (10 to 10,000 cells depending on the species) B. melitensis, B. abortus, and B. suis are listed as category B potential bioterrorism agents (45, 46).

DIAGNOSIS
Brucellosis is a disease with wide range of symptoms and multiple clinical forms. Diagnosis is difficult and complex. Confirmed diagnosis is based on laboratory data interpreted along with the clinical and epidemiological ones.

LABORATORY EXAMINATION
Culturing is performed mainly on blood samples. Other more rarely used clinical materials are bone marrow, cerebrospinal fluid (CSF), pleural and synovial fluids, urine and tissue or abscess materials. It is extremely important to avoid contamination of the samples, because of the prolonged time for cultivation. Serum or less commonly CSF, are used for serological testing. PCR is performed mainly on whole blood or serum. Clinical samples should be handled very carefully, although human tissues do not contain high numbers of brucellae (47). Serological testing should be performed without using special precautions, other than personal protective equipment (PPE). In a case of positive culture, strict precautions are required due to the dangerous numbers of organisms presented. Subculturing and other manipulations with living brucellae must be performed by using practices and procedures required for Biosafety level 3. Direct detection of brucellae. It could be performed both with direct immunofluorescent microscopy (DIFM) and molecular methods. DIFM could be used for examination of clinical samples, as well as for identification of bacterial isolates. It is also a valuable method for preliminary diagnosis in case of suspected bioterrorism. PCR tests for direct detection of Brucella DNA use inactivated samples, which is a great advantage considering the low infectious dose. However, the specificity and sensitivity of PCR vary between laboratories due to the lack of standardisation regarding the sample type and processing, the target genes, the visualisation of products and others (48). Blood and serum are most commonly tested with PCR, but various other clinical materials also could be used (49). Some authors propose serum as a more suitable for PCR, because of the lower quantity of inhibitors in it, as well as its easier processing (50). A number of genus specific primers targeting bscp31, omp-2, 16S rRNA, IS711, and other genetic elements are used ( Table 2).
Species-specific primers are applied rarely, mainly for epidemiological and scientific purposes, since in general the particular type of Brucella is irrelevant for the therapy (48). Real-time PCR techniques for detection of Brucella spp. are also developed (51, 52, 53, 54). These methods have a high sensitivity (<10 cells/reaction) and specificity (99-100%), due to the usage of more than one marker for detection of Brucella DNA in clinical specimens. In recent years a number of multiplex methods based on real-time PCR are designed (55).
Cultivation. Isolation of the causative agent remains "gold standard" for confirmed diagnosis. Blood culture is leading in the bacteriological examination for brucellosis, but some data indicates that the amount of bacteriais low (1.3 -1000 CFU/ml), even in the acute phase of the disease (56). Bone marrow is the most suitable material in sub-acute brucellosis, after a negative blood culture and/or previous antibiotic treatment. Other samples are tested rarely. Due to the possibility for contamination of the blood cultures during their prolonged cultivation, as well as the high risk for the laboratory personnel, biphasic culture media are recommended for isolation of Brucella spp. (57). The bottles are incubated at 35-37 o C, in humid atmosphere, with 5-10% CO2 (mainly for B. abortus). They are inspected daily until visible growth on the agar phase is observed. Brucella colonies appear not earlier than the fourth day, but the majority of positives occur between the 7th and 21st day. Therefore, conventional culture methods require 21 days to 6 weeks. Before discharging the bottle, a blind subculture is performed (57, 58). Lysis-centrifugation method is faster (2-4 days), but increases the risk for laboratory accidents, as well as for contamination of the blood culture (59). The automated systems for continuous monitoring of samples, such as BACTEC and BacT/Alert greatly increase the sensitivity of the method and reduce the time for detection of brucellae (57, 60). For other materials like bone marrow, CSF, synovial fluid, and various tissue homogenates biphasic media, as well as blood and chocolate agar could be used. Because Brucella spp. are fastidious, enriched media such as Trypticase soy agar, Heart infusion agar, Brucella agar, and Columbia agar should be used. Addition of blood in different concentrations and 5-10% horse serum enables the growth of the demanding species. The plates are incubated at the above-mentioned conditions for 10 days. For materials with small concentration of bacteria (urine, milk), enrichment could be achieved via inoculation of Identification. Suspected colonies are subcultured on blood or chocolate agar. Brucella spp. are small Gram-negative coccobacilli that form non-haemolytic colonies on blood agar, and do not grow on MacConkey agar. The latter is useful for differentiation from other small Gram-negative coccobacilli that could be isolated from the same clinical materials (Table 3).
Brucellae are slow-growing and therefore visible growth of subcultures could be seen not earlier than 48 hours. Colonies are small, smooth, raised, transparent, with regular edge and shiny surface. Nonsmooth variants occur, especially after longer subcultivation. Only B. ovis and B. canis have stable nonsmooth (R) form. The presumptive identification on genus level is based on morphological, biochemical and serological criteria. Brucellae are oxidase, catalase and urease positive, and show positive slide agglutination reaction with specific B. abortus and B. melitensis antisera (19). The use of commercial identification systems is not reliable, if Brucella spp. is not included in the database. As mentioned above, identification to genus level is sufficient for the etiological treatment. PCR can be used for screening after therapy when Brucella DNA is not detectable if the treatment is successful (62, 63). In the cases of relapse PCR tests could become positive again, which is helpful for evaluation of the  Typing is performed in highly specialised and well equipped laboratories. Phenotypic methods include: sensitivity to dyes (growth in the presence of methionine and basic fuchsin); speed of urea hydrolysis; production of H2S; phage sensitivity (Tb, Wb); reaction with monospecific (A and M) sera; determination of the S/R morphology. Based on these phenotypic characteristics B. suis, B. abortus and B. melitensis are divided into biovars (

BRUCELLOSIS AROUND THE WORLD
Brucellosis is one of the most common zoonosis in the world (74). It remains a major problem in the Middle East, especially in Syria (above 100% 000 per year). Turkey has annual incidence of 8-50% 000 (20, 75). In the rest of Asia, the incidence is still high in Mongolia (50-100% 000 ), but the rate of brucellosis in some former Soviet republics (Kyrgyzstan, Kazakhstan, Tajikistan, Azerbaijan) significantly increases and new foci of the disease appear. The incidence in Australia, Canada, and the USA is low (<2 cases per million). According to data from the Centre for Disease Control and Prevention about 80 new cases are diagnosed annually in the USA (76). Mexico is the main source for importation of human brucellosis in USA. In Latin America, the incidence is generally low, with the exception of Mexico and Peru (10-50 cases per million) and to a lesser extent for Argentina(2-10 cases per million) (20). The Mediterranean basin is one of the major endemic regions for brucellosis (77, 18). In the European Mediterranean countries, the incidence has been reduced significantly through overall control of animal brucellosis. Ovine and carpine brucellosis, caused by B. mellitensis occurred naturally in Bulgaria in the past, but the country has been considered as free since 1941 (19). In the following more than filthy years few epizootics due to importation of infected animals were registered (83, 84).
Bovine brucellosis in Bulgaria was first reported by Acad. Stefan Angelov in 1924. He described 188 cases in cows for a period of ten years (1924)(1925)(1926)(1927)(1928)(1929)(1930)(1931)(1932)(1933) In 2006, after a period of more than 40 years during which Bulgaria was brucellosis-free, the first autochthonous cases were detected by the NRL HMRI. This was the start of an outbreak, caused by B.melitensis, which occurred after illegal import of goats from Greece. By the end of 2008 several regions were affected (81). In four of them (Smolian, Haskovo, Yambol, and Stara Zagora) an epidemiological link between human and animal cases was established. The highest incidence of the disease was registered in 2007 when 58 new cases were detected (0,74% 000 ). The majority of them occurred in Haskovo region. All infected persons have had contact with Brucella-positive animals and/or consumed dairy products from their milk. The epidemiological investigation showed that sale of infected animals without certificates led to the spread of the disease in 11 villages of three districts. By the end of 2008 a total of 88 autochthonous human cases were registered. All Brucella-positive animals (496 goats, 117 sheep and 7 cattle) were destroyed. The stringent measures undertaken by the medical and veterinary authorities led to a significant decrease in human case numbers during 2009-2014 with 0 to 4 reports per year without any epidemiological link.
In July 2015 a patient was diagnosed with brucellosis. He was resident of Kyustendil district and without any connection with the regions affected in 2006-2008. By the middle of August an outbreak focus was found with 31 newly registered cases (92). B. melitensis was identified as the causative agent. A total of 36 patients were diagnosed until October 2015 (93). Based on data obtained during the investigation, breeding of animals and consumption of unpasteurised milk and homemade soft cheese appeared as the main risk factors for transmission of the disease. All infected animals were destroyed (94).
The investigation conducted by different governmental authorities pointed out that importation of infected animals from a neighbouring country, where the disease is endemic, was the reason for the occurrence of both recent brucellosis outbreaks in Bulgaria. As stated previously, free movement of goods and of people between EU Member States is a fundamental policy of the Community but has some negative epizootological and epidemiological effects, one of which is the re-emergence of brucellosis caused by B. melitensis in Bulgaria (81).