Brucellosis, as one of the most ancient and widespread zoonotic diseases which affects humans as well as the animals, is caused by a Gram-negative, facultative intracellular bacterium of the genus Brucella [1]. Many morphological variations were reported among the types of Brucella genus, which attributed to their virulence and presence of Smooth (S) or Rough (L) Lipopolysaccharide (LPS) in their outer membranes [2]. The presence of SLPS protects the organism initially from the responding of immunity and enhancing their abilities for surviving within hosts [3]. Within macrophages and monocytes, the species of SLPS were capable for preventing the ill cells from presenting of antigens (Ag) by T-helpers and enable the pathogen for hindering apoptosis through the ill cells [4]. 

In several developed countries, brucellosis has been eradicated through test and slaughter, vaccination and restriction of animal movements; while in other parts of the world such as the Mediterranean region, the Middle East, Western Asia and parts of Africa and Latin America, the prevalence of human and animal brucellosis is high [5]. However, the countries with the highest disease incidence rates are located within Asia but with the major gaps exist in prevalence data [6]. Many studies found that the organisms have the capability to transmit from animals to animals by different methods such as inhalation of infected aerosols with the bacterium, in particular in crowded and dry conditions, as well as by ingestion of food and grass contaminate by the organism. Direct contacting between the bacterium and skin injuries or mucous membrane in contaminated environments and from animals shed the organism venereally or through the infections of neonate were also reported. In adults, infections in naïvely pregnant is mostly resulted in abortions or given weak births. The abortive fetuses, fetal fluid and placentas are heavy load with the organism that contaminates the environments. Infected livestock have the ability for shedding the organism through their discharge and dairy products, additional contaminate the environments and poses an infection threaten for suckling and other animals [6-8].       

Worldwide, diagnosis of brucellosis cannot depend on a single test or symptoms of the disease; hence, utilization of more one diagnostic method is necessary to obtain a reliable detection of infection [8]. Although, direct isolation and detection of bacterium by culture consider more accurate in diagnosis, this method remains difficult and having several dangerous to workers [9]. Also, isolation cannot apply practically to testing many animals or for prevention and controlling of infection [10, 11]. Different serological assays were developed recently to detect specific anti-Brucella spp. antibodies in both human and animal populations such as Rose-Bengal test (RBT), standard-tube agglutination test (STAT) and Enzyme-linked immunosorbent assay (ELISA) [12-14]. In field, the application of one more diagnostic assay can provide additional advantages in detection of infection. Hence, the current work designed to investigate the prevalence of camel brucellosis in Wasit province (Iraq) using RBT, STAT and ELISA with estimation relationship between the positive findings obtained by these diagnostic assays and some animal risk factors; age and sex [15].

Ethical Approval

The license of present study was obtained by the Scientific Committee of the College of Science in the University of Wasit (Wasit, Iraq).

Samples Collection

Totally, 200 camels of different ages and sexes at many regions in Wasit province were subjected for this study during January to June (2022). Each animal was subjected for draining 10 ml of jugular venous blood using free-anticoagulant vacationer tubes. In laboratory, the tubes of blood sample were centrifuged (3000 rpm/15 minutes) and the obtained sera were kept frozen in 1 ml Eppendorf tubes.

Serological Assays

Following the manufacturer instructions of RBT and STAT (Bioveta, Czech) as well as ELISA (Sunlong Biotech, China), the sera were prepared, tested and read.

Statistical Analysis

GraphPad Prism (version 6.0.1) Software was used to analysis of obtained results by the t-test and One-Way ANOVA. Differences between values were considered significant if p<0.05 (15).

The findings of totally 200 serum samples tested by RBT, STAT and ELISA were 2.5%, 6.5% and 18.5% positive camels, respectively (Table 1).

Relationship between the obtained results showed that 2.5% were positives by both ELISA and RBT, 16% positives by ELISA only; while, no positives by RBT only (0%). However, 81.5% of samples were negative by both assays (Table 2).

Association between STAT and ELISA reported that 5.5% were positives for both ELISA and STAT, 1% positive for STAT only and 13% positives for ELISA only and 80.5% (161/200) were negatives for both assays (Table 3).

The findings dealt with the distribution of positive results among sex and age factor were variable significantly (Table 4). For sex, positive findings were reported in males by STAT (2.44%) and ELISA (12.2%) but not by RB (0%). In females, there were 3.15%, 7.55% and 13.22% (32/159) positive camels by RBT, STAT and ELISA, respectively. Regarding age, positive results of camels aged 1-4 years were 1.35%, 4.05% and 10.81%; whereas in those aged ³ 4 years, there were 3.18%, 7.94% and 23.02% by RBT, STAT and ELISA, respectively (Table 4).

Many countries have tried to eradicate brucellosis from livestock animals, but the disease has been additional spread with several cases in humans occurring each year where the disease has not been eradicated from animals [16]. Worldwide, brucellosis has a public health hazard as it interferes with the trading of animal products and interferes with the free grazing of animals [17]. Our findings reported that the prevalence of brucellosis in camels using different diagnostic assays was variable significantly. The RBT-Brucella antibody prevalence in Iraq alone has been estimated at 3.03% [18] and 6.73% [19], while the global Brucella spp. prevalence was variable ranging 0-51% [19-21]. Several studies have performed to evaluate of disease in camels and for explaining differences in results between different serological methods [23,24].

Table 1: Total Finding for Application of Three Serological Diagnostic Techniques

Significance *(p<0.05)

Table 2: Relationship Between the Findings of ELISA and RBT

Table 3: Relationship Between the Findings of ELISA and STAT

Table (4): Distribution of Positive Results Among Sex and Age Factors

Significance * (p<0.05)

However, the method for choosing a reference test represented a challenging task. For brucellosis, isolation of the bacterium is the gold test, but it requires long incubation times and often leads to incubation failure [25,26]. Developing of other diagnostic methods such as RBT as fast, easy and cheap tool with no risk of infection for workers had contributing effectively in screening of infection in humans as well as animals; however, this technique has several disadvantages such as the high probability of side effects presence of inhibitory antibodies and low sensitivity and specificity in healthy and diseased individuals, especially in endemic areas [28,29]. Regarding STAT that considers as the first advanced serological test based on vaccination for the diagnosis of brucellosis with a respective 92–100% and 90-99% for sensitivity and specificity when compared to RBT [30]. Although, this test can distinguish vaccine antibody responses from true infections, this can lead to negative false results due to incompatibilities and, in some cases, cross-reactivity with other infections [31]. Therefore, brucellosis identification should support by highly sensitive and specific method to decreasing the persistent area of lesions. In comparison to many serological assays, ELISA demonstrated greater specificity values in particular in endemically stable areas [32,33], with its ability to distinguish between acute and chronic stages of disease [34]. The ELISA kit served in this study uses the LPS of Brucella spp. as an antigen to detect specific antibodies in sera of chronic brucellosis [35].

Regarding sex, RBT and STAT revealed a higher prevalence of antibodies to Brucella in female camels compared to male camels, while ELISA showed no significant sex differences. This might be due to exposing of females to more physical stress tan males which caused by milk production and pregnancy in addition to erythritol exiatance in only females [36,37]. Other researchers have obtained insignificant variation in between males and females [38,39]. All age groups can be affected by brucellosis, but young animals are more resistant to brucellosis than adults and incidence be increased with age in older animals. This can be explained by the fact that immunity to various diseases can decline with age due to a variety of factors, including stressors and repeated exposure to infections [40,41].

The prevalence of camel brucellosis was higher than expected in particular with the ELISA assay that revealed higher reliability than the traditional usually applied assay, RB. Hence, authors recommended to using of ELISA in screening schemes of brucellosis in camels and even in other domestic animals. Furthermore, studies in other areas in Iraq are necessary to confirm the real prevalence of camel brucellosis. As well as, the role of camels in transmission of the disease to other domestic animals or even humans should be aimed in other carried studies.

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