Serological and molecular techniques for the diagnosis of Brucellosis

Science & Technology Development Journal, 22(4):400-408 Open Access Full Text Article Review 1College of Life Science, Northwest University, Xi’an, Shaanxi, P.R China 710069 2Department of Microbiology, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan Amir Ullah 3College of life science, Northwest University, Xi’an, Shaanxi, China 4College of biotechnology, Tianjin University of Science and Technology, Tianjin 5Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China 6Department of Microbiology, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan Correspondence Mujeeb ur Rahman, College of Life Science, Northwest University, Xi'an, Shaanxi, P.R China 710069 Email: mujeeb@stumail.nwu.edu.cn Serological andmolecular techniques for the diagnosis of Brucellosis Mujeeb ur Rahman1,*, Amir Ullah2, Haroon 3, MuhammadBilal4, FazalMehmood Khan5, MuhammadNaveed6 Use your smartphone to scan this QR code and download this article ABSTRACT Brucellosis is known as undulant fever or Malta fever, caused by the genus Brucella. It is the most common human zoonosis. The disease is worldwide distributed and causes significant economic losses. In animals, it causes abortion, reduction inmilk production, and infertility. While brucellosis in humans is a debilitating disease with various clinical manifestations that may lead to death in some cases. Control of disease in animals needs proper diagnosis, permanent monitoring of brucellosis- free herds, and removal of infected animals. The current review will discuss the serological and molecular techniques daily used for the determination of brucellosis in animals and humans. Key words: Brucellosis, Serological, Molecular, Diagnosis, Tests INTRODUCTION Human Brucellosis is a significant zoonosis with a worldwide geographical distribution. The causative agents of brucellosis belong to the genus Brucella. The traditional human’s disease generally caused by B. melitensis, B. abortus, and B. suis. Brucellosis mostly transmitted to humans through direct contact with infected animal secretions, placentas, or aborted fe- tuses and by the consumption of unpasteurized milk and milk products. In cattle, brucellosis causes re- duce fertility, stillbirth, late birth, and reduced milk production resulting in significant economic losses. While in humans, its clinical manifestations are non- specific such as undulant fever, insomnia, malaise, nervousness, repression, and sexual impotence. Bru- cellosis in humans is also known for various organ involvement, causing meningitis, encephalitis, endo- carditis, orchitis, arthritis, and prostatitis. Addition- ally, in pregnant women, brucellosis causes sponta- neous abortions1. It is challenging to diagnose brucellosis because signs and symptoms are almost similar to other infections; the causative agent usually grows very slowly in blood culture, and also the serodiagnosis is complicated 2. Brucellosis can be diagnosed by using several serolog- ical tests using Brucella antibodies, but the gold stan- dard remains isolation and identification of the bac- terium. Cultural observations of Brucella are time consumable, non-sensitive, and hazardous to lab staff. Various attempts were made to diagnose brucellosis for more than one century. Brucella diagnosed by us- ing a combination of tests to avoid false-negative re- sults3. Therefore, this study aims to review diagnostic tech- niques used for the isolation, screening, epidemiolog- ical surveillance, and confirmatory for brucellosis in humans and livestock. DIRECT SMEARMICROSCOPIC EXAMINATION The microorganism can be identified by microscopic examination of stained smear from secretions, fetuses, and exudates like vaginal discharges, placenta, using modified Ziehl-Neelsen (ZN) staining. This can pro- vide a predictive diagnosis of brucellosis, especially with serological support. Brucellae are not a true acid- fast bacillus but show resistant to decolorization by week acids. They seem like short rods or coccobacilli, mostly arranged singly but occasionally in pairs or small groups. They appear as coccobacilli or short rods, usually arranged individually but sometimes in pairs or small groups. Organisms such as Chlamydia abortus and Coxiella burnetii can resemble Brucella. The diagnoses of Brucella can sometimes be mislead- ing by Coxiella burnetti, Chlamydophila abortus, and Chlamydia psittaci because these bacterial strains are superficially similar to Brucella strains4. To identify and isolate B. melitensis accurately it is best to used vaginal swab andmilk samples of goats and sheep and culture these samples on culture media Farrell,s selec- tive media5. CULTURAL ISOLATIONOF BRUCELLA ORGANISM Brucella may be isolated from the placenta, fetus, vaginal swab, colostrum, milk, semen, the secretion Cite this article: ur Rahman M, Ullah A, H, Bilal M, Mehmood Khan F, Naveed M. Serological and molecular techniques for the diagnosis of Brucellosis. Sci. Tech. Dev. J.; 22(4):400-408. 400 History  Received: 2019-08-31  Accepted: 2019-11-21  Published: 2019-12-31 DOI : 10.32508/stdj.v22i4.1709 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Science & Technology Development Journal, 22(4):400-408 of nonlactating udders, the testis and the sites of clin- ical localization such as hygroma fluids or infected joints. While the microscopy samples include various lymph nodes, spleen, the pregnant or premature post parturient uterus, the udder, and male reproductive organs6. At the research site, mostly culturing tests are used to diagnose brucellosis. Culturing of Bru- cella from blood is useful in the case of bacteremia, which does not always exist but culturing milk gives a positive response to show the presence of Brucella. Samples of liver, udder, lymph nodes, spleen, and other organs used for culturing the purpose of brucel- losis. Phenotypic characters including CO2 require- ment, phage typing, and biochemical tests, are of great deals while using culture techniques for the identifica- tion of Brucella organisms and other problems in cul- turing are time-consuming, trained interne and ap- plications of bio-safety7. To culture brucella, broth or agar can prepare from powder media. Solid me- dia, including tryptose agar, trypticase soy agar, and dextrose agar are used to identify and isolate Brucella at the primary level. However, species like B. Hovis and B. canis can be cultured in media by the addi- tion of 5-10 % sterile bovine or equine serum to it8. The optimum pH for the growth of Brucella is 6.6-7.4, whereas, optimum growth temperature ranges from 36 to 38 C9. Growth of other microbes and contam- inants can be prevented using selective media such as Kuzdas and Morse and Farrell,s Morse 5,10. Farrell’s medium has some drawbacks because some of Brucella strains such as B. melitensis, B. ovis, and B. abortus cannot show healthy growth. Therefore, Thayer- Martin medium is slightly modified and then used in combination with Farrell’s medium to get bet- ter growth of these Brucella species 2. MOLECULARMETHODS The molecular procedure often based on PCR am- plification is dominantly used for identification and typing to reduce the problem and hurdles of micro- biological testing11. DNA isolation is an initial and essential step of PCR as its feature has a consider- able impact on method sensitivity 12. Initially, for bacterial determination, PCR has been developed 13. Also, now, these processes are applied for the iden- tification of brucellosis in humans and animals’ clini- cal samples. The use of a single pair of primer act to the bacterial DNA sequence, such as 16 S-23s RNA operon, 15711 or BCSP31 genes with PCR is a reli- able technique for the detection of brucellosis14. Us- ing a mixture of some primer’s pairs for magnifica- tion of BCSP31, OMP2B,OMP31 genes, encoding the external membrane proteins. It is easy to detect the four Brucella species: B. melitensis, B. suis, B. abor- tus and B. canis. The mixture of seven PCR reac- tions is another to allocate favoritism between bru- cella six species. PCR techniques used for the detec- tion of some Brucella abortus biovars, which differen- tiated between S19 and RB51 strain of B. abortus and allowed for vaccination against pathogenic strain15. Multiplex PCR To boost the affective prevention and of brucellosis, a quick and precise method is required. Several stud- ies have developed a PCR based assay for the differ- entiation of Brucella species. It has been revealed that the two multiplex PCR, called AMOS (B. abor- tus, B. melitensis, B. ovis and B. suis) and Bruce-ladder PCR assay can discriminate most of Brucella species such as marine mammal and vaccine strain B. abor- tus RB51, B. abortus S19 and B. melitensis 16. It al- lowed identification evidence of the four of Brucella species (B. abortus, B. melitensis, B. ovis and B. suis) and was titled AMOS PCR for the main correspon- dence of species name. AMOS PCR cannot detect the similar species single biovar but identify just the pair biovars of each of the same species, sooner af- ter this technique has been promoted to differentiate more biovar and recognize brucella S 19 RB51 vaccine strain16,17. Moreover, as the PCR system convey high contamination risk and needs equipment for visual- ization, it is less favorable for routine diagnosis pur- pose. So real-time PCR systems have been established that are quicker and less prone to contamination and thus use more clinically. Real-time PCR The real-time PCR method is highly specific, sensi- tive, reproducible and quicker than the conventional PCR. The quantitative real-time (qRT) PCR permits both identification and quantification of the PCR product in real-time, but it is synthesized18. It has also been possible to differentiate the species and even at the biovar level through real-time PCR. This tech- nique can be used for the quick diagnosis of chronic serologically positive brucellosis and for acute brucel- losis when blood and serum samples of recognized clinical presentations are examined 19. These assays are developed for targeting 16 S-23 S internal tran- scribed spacer region (ITC) and the genes coding omp25 and omp31, bcsp31, and IS711 20. For the detection of bacteria at the genus level, the bcsp31 gene target can be suggested. Species-specific recog- nition verifying the initial diagnosis by second gene target such as IS711 21 (Table 1). Many multiplex 401 Science & Technology Development Journal, 22(4):400-408 real-time PCR methods are developed for the im- mediate identification ofMycobacterium tuberculosis complex (MTC) and Brucella species. These meth- ods amplify the IS711, bcsp31 and omp genes for the identification of Brucellas species and target the IS6110, senX3-regX3 and cfp31 genes for the recog- nition of the MTC22. Sanjuan-jimenez et al. revealed three molecular targets of MTC (senX3-regX3, cfp31, IS6110) and three molecular targets (bcs31, IS711, omp2a) of Brucella for their instantaneous identifi- cation by a multiplex real-time PCR 23–25. However, the sensitivity and specificity of PCR for Brucella dif- fer between laboratories, and hence, standardization is needed. Serological diagnosis Several serodiagnosis methods are found for the determination of brucellosis 26. However, some of the tests are satisfactory sensitive and specific like indirect enzyme-linked immunosorbent assay (i- ELISA), competitive enzyme-linked immunosorbent assay (c- ELISA), Milk ring test, complement fixa- tion test (CFT) and the fluorescence polarization as- say (FPA)26,27. In each and every epidemiological sit- uation, no single serological test is sufficient, all of which have limitations, particularly when it comes to screening individual animals Fluorescence polariza- tion assay (FPA), Complement fixation test (CFT) and ELISA are considered more suitable for international trade than serum tube agglutination test (SAT). The buffered Brucella antigen tests (BBATs), i.e., the Rose Bengal Test (RBT) and the buffered plate agglutina- tion test (BPAT), as well as the ELISA and the FPA, are sufficient screening tests for brucellosis control at the national or local level28. If necessary, positive re- actions can be retested using an appropriate confirma- tory strategy. Agglutination test Serological diagnosis of brucellosis first completed through an agglutination test29. The primary agglutination antibodies IgM and IgG2 detected through these tests similar to serum agglutination test (SAT)30. Due to cross-reaction by IgM antibodies created in the competition ofB. abortus sequences and other closely to Brucella species, therefore, its sensi- tivity is good, and specificity is low31. This test was rejected for international trading. Antiglobulin (Coombs) test The direct Coombs test is also known as the direct antiglobulin test (DAT) was the first time discovered by Coombs, Mourant, and Race in 1945 and is still an essential assay for the diagnosis of autoimmune hemolytic anemia (AIHA.TheDAT can identify com- plement (C3) and RBC-bound IgG that opsonizes RBCs32. The serum agglutination test gives negative or suspected results, so a Coombs test used for con- firmation of results. Due to the advantage of this test to detect incomplete antibodies of IgG types that com- bine with cellular antigens, this test is used for the epi- demiological study but does not increase agglutina- tion reaction (Table 1). To save time, this test modi- fied to a microtiter plate set up. The limitation of this test it is not suggested for the diagnosis of vaccinated animals11. The 2-mercaptoethanol test The 2-MET are two forms that use either 2- mercaptoethanol33 or dithiothreitol34. Dithiothre- itol has recommended, because of the toxicity of 2- mercaptoethanol. The disulfide of IgM is being con- densed to themanometric molecule and unable to ag- glutination essentially calculate IgG unable to agglu- tinate. However, IgG can also be decreased in the procedure, providing false-negative results (Table 1). Though in general, reduction of IgM increases speci- ficity35. The test not suggested for the global trade due to not eradication vaccinal antibodies. The 2-MET is, however, used prominently for national control and eradication programs36. Buffered plate agglutination test (BPAT) The BAPT test was developed to detect Brucella spp antibody. BPAT is an easy cheap and uniform agglu- tination test. It utilized antigen at pH of 3.65, which is prepared from B. abortus S119.3 whole cells dyed with crystal violet and brilliant green colors. The test is responsible for false-positive results because of the prozoning effect and vaccinal antibodies 36. Due to the reduction of non-specific test reactions, this test is very beneficial. It has directed for IgG testing37. Brucellin allergic skin test (BAST) The skin test is an allergic test that measures Brucella spp’s unique cellular immune response. Brucellin al- lergic skin test (BAST) based on a delayed-type hyper- sensitivity reaction with a maximum sensitivity at 72 hours post-inoculation. This delayed type of hyper- sensitivity reaction is measured at the site of inocula- tion by the increase in skin thickness. The test is spe- cific to complement serological tests for the diagno- sis of bovine brucellosis, and thus decrease the figure of false-positive reactions significantly by distinguish- ing brucellosis from other cross-reacting organisms27. 402 Science & Technology Development Journal, 22(4):400-408 The test is more specific to RBPT and CFT in condi- tions of its specificity (exceeding 99%). The skin test is highly specific, but its weak sensitivity makes it a good herd test, but not an individual certification test. Thus, it is often suggested for use at the herd level as a positive test in unvaccinated animals38. Complement fixation tests The IgM isotypes incompletely damaged during the inactivation process, so the CFT test mostly detects the IgG isotypes antibody. After the IgM type, the antibodies IgG1 types usually appear. The SAT and CFT best performed the control and surveillance of the disease. The test indicates an association with the recovery of Brucella from artificial recovery or natu- rally infected animals. Although the test is rapid and precise, it does not permit differentiation between an- tibodies due to infection from vaccinal antibodies39 (Table 1). Other hurdles consist of a high figure of reagents and controls required to perform the test. Moreover, each time the assay is set up, a high number of titrations are necessary, and an explanation of the results is subjective due to variation in procedures40. Rarely, there is direct activation of complement by serum (anti-complementary activity) and the incapa- bility of the test to be agreeable for usewith hemolyzed serum samples. The laborious nature of this test and the need for highly- trained personnel and suitable laboratory facilities make the CFT less ideal for use in developing countries31. The complement fixation test may give false adverse reactions because the anti- bodies of IgG2 type obstruct the complement fixation. Despite all these problems, the complement fixation test is broadly used analysis because it is a most ac- ceptable and specific serological test for the diagnosis of brucellosis, so it is a suggested test for international trade41. MILK RING TEST (MRT) Fleischer developed amilk ring test (MRT) in 1937 42. Fleischer promoted adoption of the serum agglutina- tions test to identify the accurateness of antibodies against Brucella species in milk named the MRT. It is suggested as a screening test to check Brucellosis is bulk tank milk43. TheMilk ring test (MRT) is mainly an agglutination test done by cream or whole milk. Hematoxylin Brucella stained cells are added to milk and incubated to occur the reaction. Through the Fc portion of a fat molecule, the immunoglobulins present in the milk attached to fat globules 44. MRT detects the IgM and IgA immunoglobulins. This test may be useful for an individual animal or to pooled milk samples by using the maximum volume of milk, comparative to the pool size3. In the milk ring test, the abnormal milk caused a false adverse reaction due tomastitis, milk from the late lactation, and due to the presence of colostrum45. Due to the low concentra- tion of lacteal antibodies or lacking fat, clustering, fac- tors inmilkmay also cause a false-negative result. De- spite all these problems, the milk ring test is very suc- cessful, it is the method of choice in dairy herds, and it is a low-cost screening test as compared to other46. Primary Binding Assays Primary binding tests directlymeasure the interaction of antibodies and antigens while traditional serologi- cal tests, such as acidified agglutination tests or com- plementary fixation tests (CFTs), measure secondary phenomena such as agglutination or complementary activation. The first binding assay technique developed due to some limitations in conventional methods of Brucella diagnosis. This test can find the humoral antibodies to Brucella species very rapidly and accurately47. Due to a short time of exposure, the vaccine has low effi- ciency, so it eliminates very soon by the immune sys- tem, but when a natural antigen enters the host has long exposure and has high energy and not removed by the immune system48. Therefore, to defeat this problem, the fluorescent polarization assay (FPA) and a competitive enzyme-linked immunosorbent assay were developed (cELISA). These tests can differenti- ate vaccinated animals or animals affected by cross- reacting microorganisms like Escherichia coli O: 116 and O: 157, Salmonella Urbana O: 30, and Yersinia enterocolitica serotype nine from naturally- infected animals. Because of these capabilities, it is possible to decrease the amount of false-positive reactions49. Lateral Flow Assay (LFA) The simplified ELISA technique known as lateral flow assay (LFA) is used to detect antibodies of a specific antigen in samples of blood, serum, and milk. The method based on the attachment of antibodies speci- fied to immobilized antigen on a strip (cellulosemem- brane matrix) that is involved in detecting specific IgM and IgG antibodies in all stages of the diseases 3. The main advantage of this technique that it does not require any electrical equipment, but the only refrig- erator is used to store the test kits, and this technique is limited in the formation of visible bands because of many ingredients in reaction50. 403 Science & Technology Development Journal, 22(4):400-408 Table 1: Comparisons of different diagnostic techniques Techniques Advantage Disadvantage Serum agglutination test Safe, inexpensive, and appropriate for primary screening Cross-reactivity with other mi- croorganisms, false-negative results in the early stages of infection, and prozone phenomenon ELISA Highly sensitive and specific, rapid, simple, and capable of distinguishing between acute and chronic stages Cross-reactivity Conventional culture Gold standard and specificity Time consuming, insensitive or low sensitive, and posing a risk for laboratory staff Sensitive for relapsing and chronic brucellosis Labor-intensive and time consum- ing Coombs antiglobulin agglutination test Lateral flow assay Easy, rapid, sensitive, and specific Expensive and possibility of cross- reactivity Sensitive and specificComplement fixation test 2-Mercaptoethanol A confirmatory test that allows selective quantifi- cation of IgG anti-Brucella Toxicity of mercaptoethanol, the possibility of IgG degradation by the 2-ME, which may lead to false negative results Fluorescence polarization immunoassay Highly sensitive and specific, and capable of distinguishing between acute and chronic stages Costly, need of trained laboratory technicians, and expensive equipment Rose Bengal plate agglutination test Cross-reactivity with the antibodies of other microorganisms, false-negative results in the early stages of infection, and prozone phe- nomenon PCR Rapid and accurate; can be performed on blood, serum, CSF, and other clinical samples; can yield positive results as early as 10 days after inoculation Expensive equipment, genus spe- cific Brucladder has low detection limit, and works only on pure cultures Real-time PCR Highly sensitive, specific, and rapid; can be per- formed on blood, serum, CSF and other clinical samples Expensive equipment 404 Science & Technology Development Journal, 22(4):400-408 Figure 1: Milk ring test result. Fluorescence Polarization Assay (FPA) Fluorescence Polarization Assay (FPA) is a homoge- neous immunoassay. Homogenous immunoassays are single-step assays that do not require repeated washing steps to remove unbound reactants as with conventional primary binding assays.This technique works on the principle of excitation of fluorescent molecules using polarized light to emit it, the emis- sion of light in the solution is inversely proportional to the rotation speed of the molecules. This speed is associated with the viscosity of the solution, tem- perature and gas constant, and molecular volume51. In the serology of brucellosis, a component of O- polysaccharide (OPS) of smaller molecular weight is labeled with fluorescein isothiocyanate to use as an antigen. In different samples of serum, milk, and blood if antibodies are present, they are rotated at a reduced rate because of presences of antibodies 52. Competitive Immunoassays This technique applied by usingmonoclonal antibody having a high affinity to antigen as compared to a cross-reacting antibody. This technique is mainly used because of its high specificity and involved in the detection of antibody isotypes (IgM, IgG1, IgG2, and IgA). The limitation related to this technique is less sensitive than direct immunoassay53. Rose Bengal Plate Test (RBPT) This test is mostly used to diagnose brucellosis in sheep, goats, and buffalo, and it was the first time used by Morgan for Brucella-infected animals. It is an in- ternationally recommended test for screening of Bru- cella detection in animals. The result obtained in a short time, but the limitation of this test is the sen- sitivity and specificity of RBPT antigen because of its cross-reactivity with other bacterial species such as E. coli O157, Vibrio cholera, and some Salmonella spp. The RBPT is spot agglutination technique, which we also called card test or buffered brucella antigen test27. In this test suspension of B. abortus, smooth cells are retained with Rose Bengal dye using a buffer of Ph 3.65. Low Ph is used to increase the sensitivity of test53. The test can also be used to show the pres- ence of IgM, IgG1, and IgG2 antibodies at neutral PH. This test may result in false-negative results, but it also results in false-positive results due to the significant part to reactions with IgM in animals with the pre- vious vaccination. However, this test occurs actively right when the organisms are not vaccinated previ- ously, and the animal exposed to Brucella species. CONCLUSION The diagnosis of brucellosis in humans and livestock is not an easy task. The “gold standard” of Brucella identification is the recovery of the agent from the host, but it is time consuming and laborious method. Which can be done in highly equipped laboratories. For the diagnosis of brucellosis serological test has been developed more than a century ago, but still, a comprehensive test has not been established. The traditional serological procedure for the diagnostic of brucellosis is based on the recognition of antibodies, specific to surface LPS. Which is responsible for the low specificity of the test results. An alternative way to solve this problem is the identification of antibodies to Brucella specific proteins. It appears that there are 405 Science & Technology Development Journal, 22(4):400-408 Figure 2: Rose Bengal plate indicating agglutination. Right strong agglutination, moderate, noagglutina- tion. no sole immunodominant proteins, but to date, pro- teomic techniques permit analysis of whole Brucella proteome to determine a series of such proteins. The systemic biologymethodsmay not only effectively use in the diagnosis of brucellosis, but can also develop the understanding of fundamental biological processes in the Brucella infected body, including those leading to the large variability in the immune response. The molecular diagnosis method is the most commonly used for the diagnosis of disease. Because it is cost- effective, safe, and rapid as compared to bacteriolog- ical tests. PCR-base techniques for the identification of Brucella in biological samples are becoming an es- sential tool for the diagnosis of brucellosis at biovar and species levels. Although, PCR analysis of the sam- ple should be fully authenticated earlier, the daily use in laboratory testing for brucellosis. For the detection of Brucella DNA, the most promising method is real- time multiplex PCR. Also, the next-generation tech- niques can be used for organism diagnosis. Still, they are costly but becoming more accessible and popu- lar. Recently, for the recognition and genotyping of Brucella, the mass spectrometry approach was rec- ommended. This method provides reliable and fast identification of organisms at the species level, but it needed special sophisticated equipment, which is only available in big laboratories. All of the above meth- ods can be very accurate and sensitive, but they can’t be utilized in the field condition such as farms, where laboratory testing is available. Meanwhile, these are more suitable for the detection of humans Brucella, but not in livestock. Therefore, we believe that the development of a diag- nostic test for brucellosis is associated with an easy-to- use, quick test for initial diagnosis and high sensitivity and specific method for further laboratory testing. ABBREVIATIONS BBATs: buffered Brucella antigen tests BPAT: buffered plate agglutination test FPA: Fluorescence polarization assay i-ELISA: enzyme-linked immunosorbent assay ITC: transcribed spacer region LFA: lateral flow assay MRT: milk ring test MTC: Mycobacterium tuberculosis complex PCR: polymerase chain reaction qRT: quantitative real-time RBPT: Rose Bengal Plate Test RBT: Rose Bengal Test SAT: serum tube agglutination test ZN: Ziehl-Neelsen CONFLICT OF INTEREST The author shows no conflict of interest. AUTHORS’ CONTRIBUTIONS All the authors have equally contributed to this work. REFERENCES 1. Abdelbaset AE, Abushahba MF, Hamed MI, Rawy MS. Sero- diagnosis of brucellosis in sheep and humans in Assiut and El- Minyagovernorates, Egypt. International journal of veterinary science and medicine. 2018;6(S1):S63–S7. 2. Poester FP, Nielsen K, Samartino LE, Yu WL. Diagnosis of bru- cellosis. TheOpenVeterinary Science Journal. 2010;4(1). Avail- able from: 10.2174/1874318801004010046. 3. Nielsen K, Yu WL. Serological diagnosis of brucellosis. Prilozi. 2010;31(1):65–89. PMID: 20703184. 4. Marin G, Gamba RJ. A new measurement of acculturation for Hispanics: The Bidimensional Acculturation Scale for Hispan- ics (BAS). Hisp JBehavSci. 1996;18(3):297–316. Available from: 10.1177/07399863960183002. 5. Farrell ID. The development of a new selective medium for the isolation of Brucella abortus from contaminated sources. Res Vet Sci. 1974;16(3):280–6. PMID: 4369280. Available from: 10.1016/S0034-5288(18)33726-3. 406 Science & Technology Development Journal, 22(4):400-408 6. Acha PN, Szyfres B. Zoonoses and communicable diseases common to man and animals. vol. Volume 580. Pan Ameri- can Health Org; 2003. 7. Ismail AA. Knowledge, Attitudes and Practices Associated with Brucellosis in Small-holder Dairy Farms in Suburbs of Khartoum State, Sudan. EC Veterinary Science. 2019;4:241– 50. 8. Atlas R, Snyder J. Reagents, stains, and media: bacteriology. Manual of Clinical Microbiology. American Society of Micro- biology; 2015. 9. Pacheco-Montealegre M, Patiño RE, Torres L, Jiménez S, Ro- driguez JL, Caro-QuinteroA. Highqualitydraft genomeofBru- cella abortus strain Col-B012, isolated from aHolstein cattle in Nariño Colombia, brings new insights into the diagnosis and the epidemiology of biovar 4 strains. PeerJ Preprints; 2017. 10. Kuzdas CD, Morse EV. A selective medium for the isola- tion of brucellae from contaminated materials. J Bacteriol. 1953;66(4):502–4. PMID: 13096514. Available from: 10.1128/ JB.66.4.502-504.1953. 11. Minda AG, Gezahegne MK. A review on diagnostic methods of brucellosis. J Vet Sci Technol. 2016;7(3). 12. Moussa I, Omnia M, Amin A, Selim S. Evaluation of the currently used polymerase chain reaction assays for molec- ular detection of Brucella species. Afr J Microbiol Res. 2011;5(12):1511–20. Available from: 10.5897/AJMR11.054. 13. Boschiroli ML, Ouahrani-Bettache S, Foulongne V, Michaux- Charachon S, Bourg G, Allardet-Servent A, et al. The Brucella suis virB operon is induced intracellularly in macrophages. Proc Natl Acad Sci USA. 2002;99(3):1544–9. PMID: 11830669. Available from: 10.1073/pnas.032514299. 14. Godfroid J, Nielsen K, SaegermanC. Diagnosis of brucellosis in livestock andwildlife. CroatMed J. 2010;51(4):296–305. PMID: 20718082. Available from: 10.3325/cmj.2010.51.296. 15. YuWL, Nielsen K. Review of detection of Brucella spp. by poly- merase chain reaction. CroatMed J. 2010;51(4):306–13. PMID: 20718083. Available from: 10.3325/cmj.2010.51.306. 16. Kang SI, Her M, Kim JW, Kim JY, Ko KY, Ha YM, et al. Advanced multiplex PCR assay for differentiation of Brucella species. Appl EnvironMicrobiol. 2011;77(18):6726–8. PMID: 21666028. Available from: 10.1128/AEM.00581-11. 17. Kattar MM, Zalloua PA, Araj GF, Samaha-Kfoury J, Shbaklo H, Kanj SS, et al. Development and evaluation of real- time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human bru- cellosis. Diagn Microbiol Infect Dis. 2007;59(1):23–32. PMID: 17532591. Available from: 10.1016/j.diagmicrobio.2007.04. 002. 18. Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt MS, Po- liak S, et al. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol. 2009;137(1-2):156–64. PMID: 19200666. Available from: 10.1016/j.vetmic.2008.12.023. 19. Dahouk SA, Nöckler K, Scholz HC, Pfeffer M, Neubauer H, Tomaso H. Evaluation of genus-specific and species-specific real-time PCR assays for the identification of Brucella spp. Clin Chem LabMed. 2007;45(11):1464–70. PMID: 17970716. Avail- able from: 10.1515/CCLM.2007.305. 20. Gee JE, De BK, Levett PN, Whitney AM, Novak RT, Popovic T. Use of 16S rRNA gene sequencing for rapid confirma- tory identification of Brucella isolates. J Clin Microbiol. 2004;42(8):3649–54. PMID: 15297511. Available from: 10. 1128/JCM.42.8.3649-3654.2004. 21. Huber B, Scholz HC, Lucero N, Busse HJ. Development of a PCR assay for typing and subtyping of Brucella species. Int J Med Microbiol. 2009;299(8):563–73. PMID: 19560966. Avail- able from: 10.1016/j.ijmm.2009.05.002. 22. Dahouk SA, Nöckler K, Tomaso H, Splettstoesser WD, Jungersen G, Riber U, et al. Seroprevalence of brucellosis, tularemia, and yersiniosis in wild boars (Sus scrofa) from north-eastern Germany. J Vet Med B Infect Dis Vet Public Health. 2005;52(10):444–55. PMID: 16364020. Available from: 10.1111/j.1439-0450.2005.00898.x. 23. Dahouk SA, Flèche PL, Nöckler K, Jacques I, Grayon M, Scholz HC, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods. 2007;69(1):137–45. PMID: 17261338. Available from: 10.1016/j.mimet.2006.12.015. 24. Dahouk SA, Sprague LD, Neubauer H. New developments in the diagnostic procedures for zoonotic brucellosis in humans. Rev Sci Tech. 2013;32(1):177–88. PMID: 23837375. Available from: 10.20506/rst.32.1.2204. 25. Özdemir M, Feyzioğlu B, Kurtoğlu MG, DoğanM, DağıHT, Yük- sekkaya, et al. A comparison of immuncapture agglutination and ELISA methods in serological diagnosis of brucellosis. Int J Med Sci. 2011;8(5):428–32. PMID: 21814476. Available from: 10.7150/ijms.8.428. 26. Ducrotoy MJ, Conde-Álvarez R, Blasco JM, Moriyón I. A re- view of the basis of the immunological diagnosis of ruminant brucellosis. Vet Immunol Immunopathol. 2016;171:81–102. PMID: 26964721. Available from: 10.1016/j.vetimm.2016.02. 002. 27. Manish K, Chand P, Rajesh C, Teena R, Sunil K. Brucel- losis: an updated review of the disease. Indian J Anim Sci. 2013;83(1):3–16. 28. Ali S, Ali Q, Neubauer H, Melzer F, Elschner M, Khan I, et al. Seroprevalence and risk factors associated with brucellosis as a professional hazard in Pakistan. Foodborne Pathog Dis. 2013;10(6):500–5. PMID: 23560424. Available from: 10.1089/ fpd.2012.1360. 29. Clavijo E, Díaz R, Anguita A, García A, PinedoA, Smits HL. Com- parison of a dipstick assay for detection of Brucella-specific immunoglobulin M antibodies with other tests for serodi- agnosis of human brucellosis. Clin Diagn Lab Immunol. 2003;10(4):612–5. PMID: 12853393. 30. Praud A, Durán-Ferrer M, Fretin D, Jaÿ M, O’Connor M, Stournara A, et al. Evaluation of three competitive ELISAs and a fluorescence polarisation assay for the diagnosis of bovine brucellosis. Vet J. 2016;216:38–44. PMID: 27687924. Available from: 10.1016/j.tvjl.2016.06.014. 31. Segel GB, Lichtman MA. Direct antiglobulin (”) test-negative autoimmune hemolytic anemia: a review. Blood CellsMol Dis. 2014;52(4):152–60. PMID: 24411920. Available from: 10.1016/ j.bcmd.2013.12.003. 32. Rose JE, Roepke MH. PHYSICOCHEMICAL STUDIES ON POST- VACCINAL BRUCELLA AGGLUTININS IN BOVINE SERUM. Am J Vet Res. 1964;25:325–8. PMID: 14125895. 33. Klein GC, Behan KA. Determination of brucella immunoglob- ulin G agglutinating antibody titer with dithiothreitol. J Clin Microbiol. 1981;14(1):24–5. PMID: 7263851. Available from: 10.1128/JCM.14.1.24-25.1981. 34. Gupte S, Kaur T. Determination of brucella immunoglobulin G agglutinating antibody titer with dithiothreitol. Journal of clinical microbiology. 2015;14(1):24–5. 35. Nielsen K. Diagnosis of brucellosis by serology. Vet Micro- biol. 2002;90(1-4):447–59. PMID: 12414164. Available from: 10.1016/S0378-1135(02)00229-8. 36. de Glanville WA, Conde-Álvarez R, Moriyón I, Njeru J, Díaz R, Cook EA, et al. Poor performance of the rapid test for human brucellosis in health facilities in Kenya. PLoS Negl Trop Dis. 2017;11(4):e0005508. PMID: 28388625. Available from: 10. 1371/journal.pntd.0005508. 37. Bercovich Z, Güler L, Baysal T, Schreuder B, van Zijderveld F. Evaluation of the currently used diagnostic procedures for the detection of Brucella melitensis in sheep. Small Rumin Res. 1998;31(1):1–6. Available from: 10.1016/S0921-4488(98) 00111-4. 38. Zamri-Saad M, Kamarudin MI. Control of animal brucel- losis: the Malaysian experience. Asian Pac J Trop Med. 2016;9(12):1136–40. PMID: 27955740. Available from: 10. 1016/j.apjtm.2016.11.007. 39. Getachew T, Getachew G, Sintayehu G, Getenet M, Fasil A. Control of animal brucellosis: theMalaysian experience. Asian Pacific journal of tropicalmedicine. 2016;9(12):1136–40. Avail- able from: 10.1155/2016/8032753. 407 Science & Technology Development Journal, 22(4):400-408 40. Manual OT. Bayesian estimation of sensitivity and specificity of rose bengal, complement fixation, and indirect ELISA tests for the diagnosis of bovine brucellosis in Ethiopia. Veterinary Medicine International. 2009;2016. 41. Manual OT. Bovine brucellosis. Retrieved February 02, 2012 fromhttp. 2009. 42. Ali S, Akhter S, Neubauer H, Melzer F, Khan I, Ali Q, et al. Serological, cultural, and molecular evidence of Brucella in- fection in small ruminants in Pakistan. J Infect Dev Ctries. 2015;9(5):470–5. PMID: 25989166. Available from: 10.3855/ jidc.5110. 43. Morgan WJ. The serological diagnosis of bovine brucellosis. Vet Rec. 1967;80(21):612–20. PMID: 6067975. Available from: 10.1136/vr.80.21.612. 44. Spitsberg VL. Invited review: bovine milk fat glob- ule membrane as a potential nutraceutical. J Dairy Sci. 2005;88(7):2289–94. PMID: 15956291. Available from: 10. 3168/jds.S0022-0302(05)72906-4. 45. Al-Mariri A, Haj-Mahmoud N. Detection of Brucella abortus in bovine milk by polymerase chain reaction. Acta Vet Brno. 2010;79(2):277–80. Available from: 10.2754/avb201079020277. 46. Corbel MJ. Brucellosis in humans and animals. World Health Organization; 2006. 47. El-Eragi A, Salih MH, Alawad MF, Mohammed K. Evaluation of immunochromatographic assay for serodiagnosis of bovine brucellosis in Gezira State, Sudan. VetWorld. 2014;7(6):395–7. Available from: 10.14202/vetworld.2014.395-397. 48. Nielsen K, Cherwonogrodzky JW, Duncan JR, Bundle DR. Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Bru- cella abortus or vaccinated with strain 19. Am J Vet Res. 1989;50(1):5–9. PMID: 2465711. 49. Pedersen K, Bauer NE, Olsen S, Arenas-GamboaAM, Henry AC, Sibley TD, et al. Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas, USA. Zoonoses Public Health. 2017;64(8):647–54. PMID: 28391650. Available from: 10.1111/ zph.12359. 50. Chin CD, Linder V, Sia SK. Commercialization of microfluidic point-of-care diagnostic devices. LabChip. 2012;12(12):2118– 34. PMID: 22344520. Available from: 10.1039/c2lc21204h. 51. Jolley ME, Nasir MS. The use of fluorescence polarization as- says for thedetectionof infectiousdiseases. CombChemHigh Throughput Screen. 2003;6(3):235–44. PMID: 12678702. Avail- able from: 10.2174/138620703106298419. 52. O’Grady D, Byrne W, Kelleher P, O’Callaghan H, Kenny K, Heneghan T, et al. A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle. Vet J. 2014;199(3):370–5. PMID: 24507882. Available from: 10.1016/ j.tvjl.2014.01.008. 53. MacMillan AP, Greiser-Wilke I, Moennig V, Mathias LA. A competition enzyme immunoassay for brucellosis diagnosis. Dtsch Tierarztl Wochenschr. 1990;97(2):83–5. PMID: 2178906. 408