Detection Of Brucellosis Research Articles

Penggunaan Imunostik sebagai Uji Serologi untuk Deteksi Brucella abortus pada Sapi (APPLICATION IMMUNOSTICK ASSAY FOR SEROLOGICAL TEST BRUCELLA ABORTUS IN BOVINE)

Serological test is one of diagnostic method to detect pathogenicity brucella. Several methods are being improving such as Rose Bengal Test (RBT), Complement Fixation Test (CFT) dan Enzyme-linked Immunosorbent Assay (ELISA). Immunostick has an accuracy equivalent to ELISA and is easy to apply in the field so it is possible to be applied as a rapid test for brucellosis detection. The study aim was to know sensitivity and specificity of immunostick that were used to detecte antibody Brucella abortus using commercial antigens of B. abortus Strain 19 (S19) and B. abortus Strain 99 (S99). The test have compared with ELISA. The tests were conducted in two stages, namely (i) immunostick ability to detect antibodies in seropositive and seronegative serum, and (ii) the immunostick result were compared with ELISA result in serum grup that were be know and unknown status. A total of 250 serums were examined and result indicated that immunostick can be detect B. abortus antibodies in cattle serum with sensitivity 100%. Immunostick specifity were 45,45% for B. abortus S99(1) antigen; 78,79% for B. abortus S99(2) antigen and 51,52% for B. abortus S19 antigen. When the test compared with ELISA, the sensitivity 82,86% and the spesifity were 52,31% for B. abortus S99(1) antigen; 93,54% and 79,71 for B. abortus S99(2) antigen and 82,86% and 58,46% for B. abortus S19 antigen.

Comparison of rose bengal plate test, serum agglutination test, and indirect enzyme-linked immunosorbent assay in brucellosis detection for human and goat samples

Comparison of rose bengal plate test, serum agglutination test, and indirect enzyme-linked immunosorbent assay in brucellosis detection for human and goat samples

DNA Amplification Technique for Detection of Bovine Brucellosis

Brucellosis is one of cattle diseases which causes a very significant economic loss and categorized as zoonotic disease. Early detection of Brucellosis in livestock is very important to prevent the spread of disease to livestock and humans. The success of Brucellosis control depends on rapid, sensitive and specific detection methods. The aim of this paper is to review several methods of Brucellosis detection in cattle. Currently, the detection of Brucellosis in Indonesia is using serological and isolation methods. The latter method is the gold standard of Brucellosis diagnosis, however, its sensitivity is low. Therefore, molecular techniques with DNA amplification have been developed and applied in many countries both in livestock and humans because they are more sensitive, specific and rapid in detecting Brucella sp in blood, milk and semen samples. Various DNA amplification methods for detection of Brucellosis that have been developed including polymerase chain reaction (PCR), finger printing and loop-mediated isothermal amplificatiom (LAMP). Both PCR and LAMP are more sensitive and specific in detecting Brucella sp than conventional techniques. PCR technique has advantages in detecting Brucella sp species to serotype and biovar levels. In addition, PCR reagents are cheaper and easier to obtain than LAMP eventhough, LAMP procedure is simpler and faster.

Comparative evaluation of conventional serological tests and in-House recombinant OMP31 (rOMP31)-Based elisa for the detection of caprine brucellosis

Sero-detection of brucella antibodies was done in 300 serum samples randomly collected from aborted goats by the classical serological tests like rose bengal plate test (RBPT), serum tube agglutination test (SAT) and rOMP31-based indirect-Enzyme-Linked Immunosorbent Assay (i-ELISA) and dot-ELISA. The seroprevalence for brucellosis was 22.66, 11.33, 24.33 and 17.33% with RBPT, SAT, i-ELISA and dot-ELISA, respectively. The sensitivity and specificity of different tests varied from 56.25 to 87.50% and from 75.00 to 90.00%, respectively. It was found that rOmp31-based ELISA is the most suitable test with 87.5% sensitivity and 90.0% specificity for detection of brucellosis in goats.

Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella

Abstract Background: It is of great importance to quickly and accurately detect Brucella abortus and Brucella melitensis from clinical and non-clinical samples because of their high prevalence and high risk in causing brucellosis, a life-threating infectious disease affecting both humans and animals. Methods: The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared. Results: The mean range [cycles threshold±standard deviation (CT±SD)] for the amplified samples was 15.39–25.15 by real-time PCR. The melting peak range (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak height. Conclusions: The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing Brucella quickly and precisely. Quick recognition of Brucella species could decrease its prevalence among humans and animals and mitigate economic loss.

A pilot program for clinical Q fever surveillance as a first step for a standardized differential diagnosis of abortions: Organizational lessons applied to goats farms

Multiple challenges have to be faced both for animal and public health in the fields of infectious abortion surveillance especially when zoonotic diseases are concerned: ensuring early detection of brucellosis, improving outbreak surveillance as well as animal health management. Thus, a global approach to diagnosis of abortive infectious diseases was discussed within the framework of the French platform for animal health surveillance by a collaborative multi-stakeholder group. Q fever appeared as a relevant target to initiate this new approach in order to test the different steps, from the implementation in the farms to the analysis of the results. Therefore, a pilot study was set up in 2012, for three years, in ten départements (administrative unit of 5500Km2 on average) covering 1493 goat farms. It focused on abortive episodes defined as at least three abortions over seven days or less. A farm-level diagnosis approach, i.e. the interpretation of combinations of results from several animals was used. Therefore, the decision-making was based on the results of two qPCR applied on vaginal swabs (individual or pooled analysis) and, if necessary, on further serological analyses, taking account of the vaccination status of herds. The National Reference Laboratory had a key role as it was involved in validation and harmonization of the analytical process. A total of 114 abortive episodes from 105 goat farms were investigated. Among them, 16.7% were considered due to Q fever (related herds considered as “clinically affected” according to EFSA recommendations: 18.1 %). Between the eight départements where goat farms had notified series of abortions, the proportion of abortive episodes attributed to Q fever varied greatly from 0% to 36.4%. In addition to reinforcing knowledge about Q fever, this study enabled obtaining useful field feedbacks on feasibility and acceptability of such surveillance programs before extension to other abortive infectious agents and to the whole territory.

Diagnosis of Cattle Brucellosis by PCR and Serological Methods: Comparison of Diagnostic Tests

Brucellosis is one of the most widespread zoonotic diseases globally. This chronic, contagious disease mainly transmitted from cattle, sheep, goats, pigs and camels through direct contact with blood, placenta, fetuses or uterine secretions or through consumption of contaminated raw animal products such as unpasteurized milk or cheese. Brucellosis is endemic in Iran. The aim of this study was to compare the molecular and serological tests for detection of brucellosis. In a cross-sectional study, the blood specimens were collected from 92 unvaccinated cattle during the time period September 2014 to July, 2015.. The serum samples were aliquoted and stored at −20°C until tested. The brucellosis diagnosis was established by indirect ELISA (iELISA), Rose Bengal, 2ME, Wright and polymerase chain reaction (PCR) method. In this investigation frequency of brucellosis by using Wright, 2ME, Rose Bengal, and iELISA methods were 42.3% (n= 39), 50% (n= 71), 78.2% (n=72), 75% (n=69), 86.2% (n=76), respectively. The specificity and sensitivity of iELISA was higher than the other tests. The 72 positive Rose Bengal samples in PCR were shown to be positive by both genes and 20 negative Rose Bengal samples were shown negative by both samples. Prevention of brucellosis mainly involves education, food quality and personal hygiene. Efforts should be made by responsible authorities. The results of this study showed specificity and sensitivity of indirect ELISA (iELISA) test is more than other methods. So, brucellosis diagnosis using of this test is recommended.

Detection of brucellosis through active surveillance, Armenia, 2014

ObjectiveIn the spring of 2014, people from vulnerable households in allmarzes of Armenia were examined with the aim of active surveillance.IntroductionBrucellosis is a serious disease caused by bacteria of the Brucellagenus. It principally affects ruminants but may be transmitted tohumans. Registration of cases in cattle farms causes considerableeconomic losses and creates favorable conditions for mass infectionamong humans. In Armenia the expansion of animal industries andurbanization are the main reasons for occurrence and developmentof brucellosis.MethodsBlood was sampled from people on farms reported as havinginfected animals. Blood samples were tested by the Wright-Huddleston method. The standard case definition of brucellosis wasused for diagnosis. A questionnaire-based interview was carried outamong the population to identify the form of contact with animals andto analyze epidemiological links. During the investigation provisionswere followed in governmental decree RA 19.01.2006 N480-Nand brucellosis prevention, epidemiology, diagnosis, treatment,preventive measures.ResultsA total of 11160 people from 1054 households were enrolled in thestudy, of which 3625 (32.5%) underwent a laboratory examination.Nearly 6% (641) refused to be tested. Over 6% of those tested (226)were positive for antibodies to Brucellae. Of these, 129 (3.5%) hadchronic brucellosis. Those testing positive for brucellosis were treatedappropriately. These included 203 (90%) adults and 23 (10%) below14 years old; 147 (65%) were male and 79 (35%) were female.Of those diagnosed with brucellosis, working in animal husbandryaccounted for 46.6% (106), while those who harvested milk accountedfor 37.6% (85) and those using raw milk made up 15.4% (35).ConclusionsCases were most frequently reported among people 20-55 years ofage; the highest percentage of positives were among 41-45 year oldmales who had contact with infected animals. The main risk factor foracquiring brucellosis is animal husbandry.

Detection of Brucellosis in Sika Deer ( Cervus nippon ) through Loop-mediated Isothermal Amplification (LAMP).

Brucellosis (Brucella bovis) in sika deer ( Cervus nippon ) can cause enormous losses to stag breeding, especially in areas in which stag breeding has become an important industry. It also poses a threat to humans because it is a zoonotic disease. Use of the loop-mediated isothermal amplification (LAMP) assay has been poorly described in the diagnosis of brucellosis in deer. We developed a LAMP assay targeting the omp25 gene sequence to detect brucellosis in sika deer. The reaction can be completed in 60 min at 63 C and, with a detection limit of 17 pg, it was more sensitive than conventional PCR, with its detection limit of 1.7 ng. No cross-reactivity was observed with four bacteria: Escherichia coli , Salmonella enterica subsp. enterica, Clostridium pasteurianum , and Pseudomonas aeruginosa . We used 263 samples of blood to evaluate the reaction. The percentage of agreement between LAMP and PCR reached 91%; relative specificity reached 87%, and relative sensitivity reached 100%. The results indicate LAMP can be a simple and rapid diagnostic tool for detecting brucellosis in sika deer, particularly in the field, where it is essential to control brucellosis in deer with a rapid and accurate diagnosis for removal of positive animals.

Sero-Prevalence and Molecular Detection of Brucellosis among Febrile Patients in West Darfur State, Sudan

Brucellosis is a worldwide zoonosis with a high degree of morbidity in humans. It was formerly known as Mediterranean fever, Malta fever or undulant fever. The aim of this study to determine the prevalence of brucellosis among febrile negative malaria patients consume raw milk in West Darfur State, Sudan. In this cross sectional study, one hundred and half blood samples were collected from febrile patients and examined by Rose Bengal (RBPT), ELISA, SAT and PCR methods. The results showed 55 samples were positive by RBPT method, 67 samples were positive by SAT, 72 samples were positive by ELISA and 110 sample positive by nested PCR. Deficiency of awareness of brucellosis with prevailing routine habit of consumption raw milk and close contact with infected animals can function as mean of infection to human beings, molecular methods and could be a useful tool for the detection of Brucella spp.

Development and Evaluation of a Rapid Dipstick Assay for Serodiagnosis of Bovine Brucellosis

Fast, cheap and sufficient serodiagnostic tools needs to be developed for the early detectionof brucellosis. Currently the tools cannot differentiate an active infection from vaccinated, norcan it differentiate other bacterial infections with lipopolysaccharides, especially Yersiniainfections. In this study, we purified recombinant outer membrane protein 10 and 28(rOmp10,rOmp28), and a dipstick assay(indirect or sandwich) was constructed with single(rOmp10 orrOmp28) and combined rOmps(rOmp10 and rOmp28) from Brucella(B.) abortus 544 to evaluatebovine Brucella positive serum collected during the beginning of the Korean outbreak from2006 to 2015. In application with single rOmp, rOmp10(70%; indirect, 92.11%; sandwichdipstick) and rOmp28(72.5%; indirect, 86.84%; sandwich dipstick) had comparable results. Inaddition, results indicated that dipstick with combined rOmps(rOmps10 and rOmp28) weresuperior in detecting positive serum samples, at 85% indirect and 100% sandwich dipstick. Surprisingly, the results were the same in detecting negative results at 97.78% for both singleand combined indirect dipsticks. The dipstick tools with rOmp10 and rOmp28 would be usefulfor a rapid screen method for bovine brucellosis.

Deteksi Spesies Brucella pada Kambing di Rumah Potong Hewan Jakarta

Brucellosis is a zoonosis and occupational diseases transmision. The diseases caused by bacterial and attack multiple species of animals. Common species that infects goats as the most pathogenic species (zoonotic) is Brucella melitensis; however, the species B. abortus could also infect goats. The study purposed to find out the brucellosis seropositive in goat in Jakarta slaughterhouse and to detect caused agent of brucellosis. Sampling was done through slaughtered goats that come from brucellosis endemic area. The samples were collected fromslaughtered mature female goats i.e serum, goat milk, vaginal swab, mamary gland, limphoglandula supramamary, limph, and uterus. The detection method was used i.e patological lession, serological, culture and PolymeraseChain Reaction (PCR) technique. The serological detection of brucellosis in goats was done parallelly between Rose Bengal Test (RBT), Complement Fixation Test (CFT) and Enzyme Linked Immunosorbent Assay (ELISA). The results of this study demonstrated that out of the 119 serum samples serologically tested, negative for RBT, one was positive for CFT and none were positive with ELISA. Patological observation in the Brucella predilection organs, there were 5 goat carcases showed pathological lession (vagina discharge, hemoragy at limphand limphoglandula, crumbly limph and there were pus in uterus). The serum samples that had reacted positively and the organs with pathological lesion were confirmed further with PCR, bacterial isolation and identification.The PCR test results and the culture of milk samples, vaginal swabs and organs did not reveal any Brucella spp bacteria (B. abortus, B. melitensis, B. ovis dan B. suis) and also vaccine strains of RB51. Based on these results, it was concluded that brucellosis in goats on Java Island was a 0.84% seropositive (confidence interval 95%; 0.00826 - 0.00854) (1/119), although the species of Brucella that had infected them remains unknown.

Bacteriological and molecular detection of brucellosis with special reference to the effect of disinfectants on isolated strains

Bacteriological and molecular detection of brucellosis with special reference to the effect of disinfectants on isolated strains

Diagnostic and Epidemiologic Study of Human Brucellosis at Al-Najaf province

Background: Maltese fever is one of the dangerous occupational diseases for dairy producers, processor and staff cattle slaughterhouse, veterinarians and laboratory personnel. Infection accompanies a wide range of clinical symptoms that require the use of effective diagnostic tests. The choice of the epidemiological diagnosis in the region and the decision of the infection depends on the size of the infestation., Clinical information provided by the patient must include the geographical area, work type Was traveling to rural areas or affected countries this disease Is a family member with the disease, treatment he takes the patient what is positive and negative impact on the patient. Germ Malta fever tend to tissue rich in blood vessels Aims of study: The study aims to evaluate the epidemiological aspects of the population according to gender and age. And the study of the property and the sensitivity of the two tests, the first is the use of a conventional test (Rose Bengal), and modern molecular technique (RT- PCR) and compare the two results. Methodology: Type of study is a diagnostic study includes 41 blood samples of patients with suspected Malta fever, in both gender diagnosed questionable, the ages were between 14-45 years. Patients were attending to the province of Al-Najaf Al-Ashraf of rural area surrounding the governorate and the center of the province, and attend private clinic on January to December 2015 in order to evaluate serum infection of Malta carried out using serological testing and technology of molecular test method Results: A total of 41 patients with suspected brucellosis had been included in this study. The most frequent age group was more than 35(29.3%) years. The percentage to an urban cases were (58.54%) and the percent of rural area. Brucellosis, variations during one year shows positive predictive value=50%, Negative predictive value=95.2% and Accuracy =73.1 % of diagnosis of serological compared to Real time, PCR proportion of 41 patients. High sensitivity (90.9 %) has detection of Brucellosis by Real Time PCR, and the Specificity=66.7%. Conclusion: Estimated incidence in most regions injury (rural areas) may be less compared to the younger population. Medical safety is very important in the laboratory. Organization of the CDC classifies Iraq in high incidence areas with Malta fever. Recommendations: Avoid unpasteurized hand milk products and fresh milk which is not boiled. Clinical laboratories must be under the terms of health safety when isolate the pathogen. And early treatment of the disease at the onset of clinical signs of the disease eliminates protect against chronic doubled.

Clinical application of three methods for the detection of brucellosis

Objective To detect serum in high risk population, three methods were used and their advantages and disadvantages was compared. Methods In accordance with the surveillance standard for brucellosis (GB 16885-1997) and diagnostic criteria (WS269-2007) in the prescribed method, rose-bengal plate agglutination test (RBPT), standard-tube agglutination test (SAT) and enzyme linked immunoassay assay (ELISA) were used to detect and analysis of its diagnostic significance in the high risk population of sheep farm of Guoyang county in Anhui province. Results The positive rates of RBPT, SAT and ELISA were 19.1%, 12.1% and 16.3% in 257 blood samples, respectively.Compared to SAT, the sensitivity, specificity, accuracy and the area under the ROC curve of RBPT were 88.5%, 91.8%, 91.4%, 0.81, respectively, which of ELISA were 93.9%, 95.1%, 94.9%, 0.88. Conclusion The sensitivity, specificity, accuracy and the area under the ROC curve of ELISA were higher than those of other methods.Proper method, early surveillance and effective technology can help to control the occurrence and epidemic of in the actual test work promotion. Key words: Brucellosis; Rose-bengal plate agglutination test; Standard-tube agglutination test; ELISA method; Detection

Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP.

Rapid and accurate detection of Brucella abortus and Brucella melitensis from clinical samples is so important because antibiotic treatment has major side effects. This study reveals a new method in detection of clinical samples of brucellosis using real-time PCR and high-resolution melt (HRM) curve analysis. 160 brucellosis suspicious samples with more than 1/80 serum antibody titers were collected and the results were compared with the RFLP method. In order to amplify the sequences for HRM analysis, vdcc, int-hyp and glk and for RFLP, omp2a and omp2b with PstI and Hinf1 restriction endonuclease were used. At last, the accuracy and specificity of the two methods were compared with each other. Out of these 160 samples, multiplex real time PCR showed 108 positive samples (67.5%), including 56% B. melitensis and 44% B. abortus; whereas in PCR-RFLP 52 out of 160 samples were positive, where recognition of two species were accordant with HRM analysis, separation was based on the size of the amplified fragment. Using the designed primers and performing the assay, we confirmed this method to be much faster and have lower cost with more than 99% accuracy compared to methods such as RFLP. The present study showed that this technique, which scans gene segments and creates an analysis pattern for detection of clinical samples, is useful and more dominant compared with PCR-RFLP. Thus, this method can be used for brucellosis detection, and clinical and epidemiological research.

Detection of animal brucellosis by cultural method

Brucellosis, an infectious disease of many vertebrate species, is an important re-emerging zoonosis with a worldwide distribution. The present research was aimed for detection of brucellosis in animals by isolation and identification of Brucella species isolates by conventional method and PCR. Clinical specimens (61) which included vaginal swabs (48), foetal membranes (08), semen samples (03) and abomasal contents of aborted foetuses (02) were subjected for isolation on Brucella Agar Medium (BAM), wherein 15 Brucella spp. isolates were recovered with isolation rate of 24.59%. All 15 conventionally identified Brucella abortus species isolates were further confirmed by 2 different PCR assays using genus specific BCSP31 and species specific IS711 (AB and BM) primers. The BCSP31 (223 bp) PCR assay was simple, effective and sensitive in detection of Brucella isolates at genus level, whereas, IS711/AB PCR was useful to detect isolates at species level. In IS711/AB PCR, abortus specific amplification product of 498 bp was produced in all 15 isolates confirming their identity as B. abortus, whereas none of isolate showed B. melitensis specific amplification in IS711/BM PCR.

Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis.

BackgroundBrucellosis is the most common bacterial zoonosis, and serological tests are routinely used in brucellosis control and eradication programs. In order to improve the accuracy of serological diagnostic method used in bovine brucellosis detection, this study developed an improved competitive ELISA with higher specificity and good sensitivity.ResultsThis study prepared 12 monoclonal antibodies against smooth Brucella lipopolysaccharide. One monoclonal antibody 3 F9, presented C epitope specificity, was used to develop a competitive ELISA for the serological detection of bovine brucellosis. The competitive ELISA, a commercial competitive ELISA kit, the rose-bengal plate agglutination test, and a microplate agglutination test were all used in the detection of 6 hyperimmune antisera against other commonly cross-reacted bacterial pathogens and 110 clinical bovine serum samples. The results of the test comparisons indicated that the competitive ELISA had higher specificity than the commercial competitive ELISA kit and RBT, and comparable sensitivity with the commercial ELISA kit.ConclusionsThis study provided a valuable detection tool with high specificity and good sensitivity, which prevent the wrong-culling of bovines in the eradication campaigns of bovine brucellosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0436-3) contains supplementary material, which is available to authorized users.

Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members.

BackgroundBrucellosis is considered as endemic zoonotic disease in the country of Georgia. However, the burden of the disease on a household level is not known. Therefore, this study sought to determine the benefits of active surveillance coupled to serological screening for the early detection of brucellosis among close contacts of brucellosis cases.MethodsWe used an active surveillance approach to estimate the rate of seropositivity among household family members and neighboring community members of brucellosis index cases. All participants were screened using the serum tube agglutination test (SAT). Blood cultures were performed, obtained isolates were identified by a bacteriological algorithm, and confirmed as Brucella spp. using real-time PCR. Further confirmation of Brucella species was done using the AMOS PCR assay.ResultsA total of 141 participants enrolled. Of these, 27 were brucellosis index cases, 86 were household family members, and 28 were neighboring community members. The serological evidence of brucellosis in the household member group was 7% and the rate at the household level was 21%. No screened community members were Brucella seropositive. Majority of brucellosis cases were caused by B. melitensis; only one index case was linked to B. abortus.ConclusionWe found evidence of brucellosis infection among household family members of brucellosis index cases. B. melitensis was the most common species obtained. Findings of this active surveillance study highlight the importance of screening household family members of brucellosis cases and of the use of culture methods to identify Brucella species in the country of Georgia.

Seroprevalence and comparison of different serological tests for brucellosis detection in small ruminants.

Aim:The aim was to study the seroprevalence and efficacy of the different serological tests used for detection of antibody against Brucella species in small ruminants of Banaskantha district of North-Gujarat.Materials and Methods:Total 1000 serum samples comprising of 485 from sheep and 515 from goat tested for detection of antibodies against the Brucella species by three different serological tests viz., Rose bengal plate test (RBPT), Standard tube agglutination test (STAT), and Indirect Enzyme-linked immunosorbent assay (I-ELISA).Results:The seroprevalence of brucellosis in small ruminants was 11.30%, 11.10%, and 8.80% by RBPT, STAT, and I-ELISA, respectively. The seroprevalence of brucellosis was found to be higher in sheep than goats. The sensitivity of RBPT was found slight more than STAT, but the specificity of both tests was same. In this study, the overall agreement of RBPT and STAT with I-ELISA was found 92.50% and 92.30% in small ruminants, respectively.Conclusion:I-ELISA was a better serological test as compared to RBPT and STAT in the sense of sensitivity, specificity, and rapidity and it could be advocated for screening of brucellosis in sheep and goats.