Brucellosis almost always causes fever, which may be associated with night sweats and weight loss [3-6]. Common treatment includes doxycycline and aminoglycosides, although complicated cases, such as brucellosis combined with endocarditis, neurobrucellosis, may require stronger medicatinos administered for a longer duration and challenging treatment process [7-9]. Other than direct contact with infected animals, the disease is also foodborne; food habits are very difficult to change, in eastern of Turkey also cheese made with unboiled milk, which will assure many future cases of foodborne disease [10,11]. Brucella melitensis, Brucella abortus and Brucella suis are the three species generally associated with human disease, in our region B. melitensis common cause of the brucellosis. Rare cases of human infection with Brucella canis have been reported, while human cases of Brucella ovis and Brucella neotomae infection have not been reported. Little is known about the capacity of the new Brucella species to cause infection [10]. One possible laboratory acquired infection with a marine mammal isolate has been reported [12] and one specific sequence type (ST27) has been associated with three human infections in Peru and New Zealand [10,12]. Interestingly, the patients had had no contact with marine mammals; however contact with raw fish was a common feature of the three cases of patients. B. melitensis bv2 found in catfish in Egypt, suggesting that fish may constitute a novel source of infection [10,13]. There is some other types of brucella; Brucella canis, Brucella suis, Brucella ovis, Brucella ceti, Brucella pinnipedialis, Brucella cetaceae, Brucella neotomae [14]. In endemic areas like our region Brucella melitensis is the most common type [15].

The aim of this study was to investigate the seroprevalence of brucellosis titters in 5 years patients with using the rose Bengal agglutination test and coombs brucella test. Coombs brucella test is a microaglutination test as sandwich ELISA model. The sensitivity and specifity are found to be 94-95% and 98-99%. The aglutinin detect IgG, IgM and IgA. Test is effective in detecting the agglutinating and non-agglutinating immunglobulins in acute and chronic brucellosis caused by B. abortus, B. melitensis and B. suis.

MATERIALS AND METHOD

A total of 761 brucellosis cases admitted to our clinic, the Department of Infectious Diseases and Clinical Microbiology of Kafkas University Hospital, over a 4 year period from 2014 to 2017, were included in the study. A retrospective study was undertaken and patient files were investigated for their gender, age, rose Bengal positivity and brucella coombs titters, as well as clinical outcomes. Firstly rose Bengal plate test, the procedure described by Foster et al. [15] was followed with little modification by Unver et al. [16]. Brucella Rose Bengal Plate Test antigen was prepared from Brucella abortus S99 strain, standardized with Brucella antiserum and stained with Rose-Bengal. The antigen was left at room temperature for 15 min before use and shaken well. On a clean plate, 0.05 ml milk serum was dropped. Then, 0.05 ml Rose Bengal Lam Test antigen was added to this. The antigen and milk serum were mixed and spread over an area with diameter 1.5 cm. The plate was left in the air for 2 min by turning by hand. Formation of clusters of coarse particles was assessed as positive while homogeneous appearance was assessed as negative. Reactions were categorized as 0, +, ++, +++, according to [17] where: 0=means no agglutination, + = barely perceptible agglutination (using magnifying glass), ++ = fine agglutination, some clearing and +++ = clumping, definite clearing. Those samples identified with no agglutination (0) were regarded as negative, while those with +, ++ and +++ were regarded as positive. This test was performed following the procedure described by Unver et al. [16] mixing 1.30 μL serum mixed with equal volume of antigen. The plates were shaken for 4 min and any agglutination that appeared within this time was recorded as a positive reaction. It can detect B. abortus, B. melitensis and Brucella suis. In chronic infections there is 30% false negatives can be observed.

After the agglutination Brucella coombs test were performed. The procedure of test: two wells are used for each serum and well for positive control and well 1 for negative control are used for each run.

·        5 μl of serum is diluted with 195 μl saline solution in a test tube (1:40 titer). Mix well while diluting the serum sample.

·        2 wells for each serum sample and 2 wells for positive-negative control are emplaced to the microplate frame and 50 μl MCBT diluent is added to the each well.

·        The diluted serum sample is added to first well, which 50 μl of (1:80 titer). It is mixed well by pipetting 3-4 times than 50 μl of 1:80 diluted sample is added to the other well (1:160 titer), 50 μl of diluted sample is discarded from last well.

·        5 μl of positive and negative control solution is added to the control wells.

·        50 μl of antigen is added to all wells (First well 1:160 titer, second well 1:320 titer).

·        The plate is incubated in the special locked box with a piece of wet cotton for 24 h.

·        The results are evaluated after the incubation period. Solutions are in order: 1/40, 1/80, 1/320, 1/640, 1/1280, 1/2560, 1/5120.

RESULTS

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