The use of skin delayed-type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: a review

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed-type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross-react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero-diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross-react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross-reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold > or =2 mm or > or =1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase > or =2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test-and-slaughter, an increase of > or =1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.     

A review of camel brucellosis

We reviewed the literature on camel brucellosis. The seroprevalence of brucellosis in camels appears to follow two distinct patterns: a low (2-5%) prevalence in nomadic or extensively kept camels and a high (8-15%) prevalence in camels kept intensively or semi-intensively. The infection is caused by different biotypes of Brucella abortus and Brucella melitensis. Many gaps exist in the literature on the epidemiology of camel brucellosis. There is no clear policy in any of the camel-keeping countries regarding the control of brucellosis in camels. We suggest whole-herd vaccination in low-prevalence countries and test-and-slaughter followed by vaccination in high-prevalence countries.     

The use of skin delayed‐type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: A review

Summary

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed‐type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross‐react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero‐diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross‐react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross‐reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold ≥2 mm or ≥1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase ≥2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test‐and‐slaughter, an increase of ≥1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.     

Risk factors associated with camel brucellosis in Jordan

During the period between February, 2004 and December, 2006, a cross-sectional study was performed to investigate some epidemiological aspects related to camel brucellosis in Jordan. Four hundred twelve camel sera from 37 herds were randomly collected and analyzed using Rose Bengal plate test and complement fixation test. A structured pre-tested questionnaire was administered to collect information on camel herd health and management. A multivariable logistic regression model was constructed to investigate risk factors associated with seropositivity to Brucella antigens. Moreover, the incidence of Brucella-specific abortion was investigated in 7 camel herds located in different locations in Southern Jordan. The true prevalence of Brucella-seropositive in camels was 12.1%. Thirteen (35.1%) herds had at least one positive camel. The seroprevalence of brucellosis in camels was significantly higher in the southern part of Jordan than that in central or northern Jordan. The multivariable logistic regression model on both individual and herd levels revealed large herds and contact with small ruminants as risk factors for Brucella seropositivity. On the other hand, using disinfectants was identified as a protective factor (OR = 0.8; 95% CI: 0.1, 0.9) only on the camel herd level. The incidence of Brucella-caused abortion was 1.9%. Brucella melitensis biotype 3 was isolated from 4 aborted camel fetuses.     

The efficacy of the skin delayed-type hypersensitivity using a Brucellin prepared from a mucoid strain of Brucella abortus to detect brucellosis

Eight-hundred-and-ninety-six cattle belonging to herds officially designated Brucella-free, and 190 cattle belonging to infected herds were tested with the skin delayed-type hypersensitivity (SDTH) test, using brucellin (273) prepared from a mucoid strain of Brucella abortus. An increase in skinfold thickness > or = 2 mm was considered a positive SDTH test. The serum agglutination test, complement fixation test and bacteriological examination were used to confirm SDTH test results. Results show that 6 of the 896 uninfected cattle tested positive in the SDTH test, indicating a 99.3% specificity. Of the 44 cattle that tested serologically or bacteriologically positive, 33 tested positive in the SDTH test, indicating a 75% sensitivity. The value of the SDTH test was demonstrated by its ability to detect infection earlier than serological tests, and by confirming infection in cattle with ambiguous serological test results. An increase in skinfold thickness > or = 1 mm in cattle in suspected herds should not be ignored, as it may indicate specific sensitization. We recommend the use of the SDTH test in combination with serological tests for early diagnosis of brucellosis in cattle.     

Brucellosis and Q-fever seroprevalences of nomadic pastoralists and their livestock in Chad

As a part of a research-and-action partnership between public health and veterinary medicine, the relationships between the seroprevalences of brucellosis and Q-fever in humans and livestock were evaluated in three nomadic communities of Chad (Fulani cattle breeders, and Arab camel and cattle breeders). Nomad camps were visited between April 1999 and April 2000. A total of 860 human and 1637 animal sera were tested for antibodies against Brucella spp., and 368 human and 613 animal sera for Coxiella burnetii. The same indirect ELISA was used for livestock and human sera, and the test characteristics for its use on human sera were evaluated. Twenty-eight people were seropositive for brucellosis (seroprevalence 3.8%). Brucella seroprevalence was higher in cattle (7%) than other livestock, and brucellosis seropositivity was a significant factor for abortion in cattle (OR=2.8). No correlation was found between human brucellosis serostatus and camp proportions of seropositive animals.

Q-fever-seropositive blood samples were taken from 11 Arab camel and 4 Arab cattle breeders (seroprevalence 1%). Being a camel breeder was associated with Q-fever seropositivity in humans (OR=9). Camels had the highest Q-fever seroprevalence (80%) among livestock species.

Although there was high-risk human behaviour for the acquisition of brucellosis and Q-fever from livestock through raw-milk consumption (98%) and contact with placentas of livestock (62%), we concluded that seroprevalences in humans were relatively low (likely due to limited active foci in livestock).     

A practical method for the production of Brucella skin test antigen

Skin test antigens for the diagnosis of brucellosis were produced from the rough Brucella abortus strain 45/20. The production of two products termed Brucellin B and Brucellin W are described. The method described for the production of Brucellin W is recommended as an improved practical method of Brucellin production. The Brucellin products described were equal in sensitivity to that of a commercially available product, Brucellergen, which is used in New Zealand. Brucellin B was extensively tested in non-infected cattle and its specificity was equal to that of Brucellergen. Recommendations for the standardisation of skin test reagents for the diagnosis of brucellosis are made. Intradermal testing for brucellosis in cattle should be used only for the identification of infected herds and not as an individual animal test.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Differentiation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: the use of specific lymphocyte transformation and brucellin skin tests

The lymphocyte transformation test (using an in vitro whole-blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non-exposed cows. Lymphocytes from Brucella-inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia-infected and non-exposed cattle. Four of the five cows infected with Yersinia enterocolitica type 09 and all four control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

牛羊布鲁菌病（brucellosis）的诊断方法

布鲁菌病是一类由各型布鲁菌感染而引起的重要的人畜共患病之一。被感染的动物最常见的临床表现为流产、胎盘炎、附睾炎、睾丸炎等。人感染布鲁茵病后,多呈慢性经过,可损害人体多种器官。虽然病原学鉴定是一种常规有效的诊断布鲁茵病的方法之一,但由于其对操作人员安全以及实验室级别的要求非常严格,加之从患病动物体内分离病原体的过程既耗时又具有危险性,在布鲁茵病临床诊断实践中推广应用困难。布鲁菌病血清学诊断方法显示出较高的特异性和敏感性．但难以鉴别布鲁菌各型野毒株以及疫苗株感染。鉴于上述问题．深入开发和研制更高特异性、敏感性和稳定性的新型布鲁茵病诊断方法在兽医临床实践中势在必行。主要从细菌学、血清学和分子生物学等方面对布鲁茵病诊断方法研究情况进行了综述,以期为相关研究提供参考。     

The ELISA for the examination of hare sera for anti-Brucella antibodies

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.     

Prevalence of Brucellosis in Horse North-East of Iran

Brucella preferentially infects cattle, swine, sheep, and goats. However, some epidemiological surveys have been carried out to investigate nonruminants, such as horses. Horse brucellosis has been found in clinical cases, but there are few epidemiologic patterns. Between May 2008 and April 2009, a total of 120 horses were screened for brucella infections in Mashhad, Iran, by the rose bengal test and the tube agglutination test. Sera from three horses were found positive by rose bengal test and tube agglutination test, and therefore the prevalence rate was 2.5%. In horses, the highest individual seroprevalence was in an animal kept close under the intensive system, with other animals such as cattle, sheep, and goats. The zoonotic aspects of brucellosis from the horse must, therefore, be considered because the disease is important from a public health standpoint. The present study documents the first serological evidence of Brucella spp. infection in horses in Iran.     

Differentation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: The use of specific lymphocyte transformation and brucellin skin tests

Summary

The lymphocyte transformation test (using an in vitro whole‐blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non‐exposed cows. Lymphocytes from Brucella‐inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia‐infected and non‐exposed cattle. Four of thefive cowsinfected with Yersinia enterocolitica type 09 and allfour control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

Evaluation of surveillance strategies for bovine brucellosis in Japan using a simulation model

Bovine brucellosis is caused by Brucella abortus and induces abortions in female cattle, with other cattle at risk of infection from the aborted fetus or contaminated placenta. In Japan, the number of cases has dramatically reduced due to national surveillance and eradication strategies. Bovine brucellosis is now believed to be eradicated in Japan. Here, we examine the surveillance strategies currently in place for early detection of infected cattle in the event of a future reintroduction of the disease. We compared current serological surveillance for the dairy population with bulk-milk surveillance and abortion surveillance, and used time to detection as the main criterion of surveillance efficacy. A stochastic individual-based model (IBM) was developed to simulate disease transmission within and between farms. Using outputs from the transmission model, a comparison of surveillance strategies was simulated. For evaluation of the robustness of the parameter values used in the transmission model, a sensitivity analysis was conducted. For the purpose of evaluating the direct costs of each surveillance strategy, the annual number of samples to be tested and the annual number of farms to be visited were estimated. Our results indicated that current serological surveillance with 60-month test intervals is not effective enough for rapid detection of a brucellosis outbreak. Bulk-milk surveillance appeared the most effective method based on the early detection of infected cows and a reduced number of samples required. The time to detection for abortion surveillance was greater than that of bulk-milk surveillance but varied widely depending on the reported ratio of abortions. Results from the surveillance model were consistent when alternative scenarios were applied to the transmission model. Although our model cannot exactly replicate an actual brucellosis outbreak, or the results of surveillance, our results may help decision-makers to choose the most effective surveillance strategy.     

Serology as an aid to diagnosis: uses and abuses

The importance of correct interpretation of serological test results, common sources of error and problems associated with tests are discussed. In bovine brucellosis, a disease which is ideally suited to serological diagnosis, foetal contact with infection may cause the calf to be a serologically negative carrier. The immune tolerant animal resulting from foetal contact with virus is a major problem in the serological detection of border disease. In Johnes' disease and to a lesser extent in Brucella ovis and leptospiral infections, problems associated with sensitivity and specificity of the tests are stressed. Serovar specificity, cumbersome test procedures and negative tests in the incubation period cause difficulty in the interpretation of serological test results for leptospirosis. The importance of clinical examination, herd histories and alternative diagnostic procedures is important in all diseases. Wherever possible, flocks or herds should be maintained in specific disease-free state. Selection of stock from accredited herds or flocks is the most certain method of buying non-infected animals.     

Evaluation of cattle for experimental infection with and transmission of Brucella suis biovar 4

OBJECTIVE: To determine whether cattle can become persistently infected with Brucella suis biovar 4, whether the organism can be transmitted vertically or horizontally, and whether tests for bovine brucellosis are diagnostic. DESIGN: Observational study. ANIMALS: 24 pregnant cows and their calves and 6 bulls. PROCEDURE: Cows and bulls were housed separately in groups of 6 with each group consisting of 3 cattle experimentally infected with B suis biovar 4 and 3 na?ve animals. Cattle were observed for clinical signs daily; blood samples were collected weekly. Clotted blood from each sample was submitted for bacterial culture. Serum was tested with an indirect ELISA and the standard tube agglutination test (STAT), buffered plate agglutination test, brucellosis card test (BCT), and complemen't fixation test (CFT). Tissues collected at necropsy were submitted for bacterial culture and histologic examination. RESULTS: All 15 inoculated cattle seroconverted on 2 or more serologic tests, and bacteria were isolated from 4 inoculated cows at necropsy. There was no bacteriologic evidence of vertical or horizontal transmission, and none of the cattle developed clinical abnormalities or gross or histologic lesions. Results of the indirect ELISA were positive for all inoculated cattle. The other tests gave variable results; the CFT, STAT, and BCT yielded negative results for at least 1 of the 4 cattle from which the organism was isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle-to-cattle transmission of B suis biovar 4 is unlikely. Serologic tests for bovine brucellosis should be used cautiously when attempting to identify cattle with rangiferine brucellosis, as they do not discriminate between the 2 diseases and vary in their ability to detect exposed cattle.     

Mastitis in lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia.

Quarter milk samples (n = 543) from 152 traditionally managed lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia were examined to determine the prevalence of camel mastitis and identify its bacterial causes. Out of 152 camels examined, 19 (12.5%) were diagnosed as clinical mastitis cases based on clinical signs and bacteriological examinations. Of the 257 California Mastitis Test (CMT) positive quarter milk samples 162 (63.0%) yielded pathogenic bacteria. A positive correlation was observed between CMT positive results and presence of major pathogens in camel milk samples. The main mastitis pathogens isolated were Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, S. dysgalactiae, and other species of streptococci, Pasteurella haemolytica and E. coli. Results of the present study suggest that mastitis in Afar camels is prevalent, Gram-positive cocci are the major isolates from camel milk samples and the CMT can be used as a screening test for the detection of mastitis in camels.     

Comparison of conventional and non-conventional techniques for the diagnosis of bovine brucellosis in Sudan

The objective of the present study was to detect brucellosis in suspected dairy cattle in Khartoum State, Sudan using the conventional serological tests and tests done on milk in comparison to a PCR-based technique. Milk and blood samples collected simultaneously from suspected brucellosis cows (n = 147) in 12 different dairy farms around Khartoum State were used in the study. Overall, 54 (36.7%) of the total milk samples were positive according to the milk ring test (MRT), while 29 (19.7%) of the serum samples were positive according to the Rose Bengal test (RBT); microscopy on modified Ziehl-Neelsen-stained slides detected 13.6% of the cases, and recovery of Brucella species on both Brucella medium and tryptic soya agar was 7.5%. Thirty-three (22.4%) samples were found positive on PCR-amplified IS711 which were then taken as positive brucellosis cases. The differences of RBT and PCR-IS711 from MRT were highly significant (P < 0.05). MRT detected more cases of bovine brucellosis compared to RBT, PCR, microscopy, and culture. MRT is recommended as a noninvasive test compared to RBT, and it is less expensive compared to PCR and culture.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

Evaluation of a nanobody phage display library constructed from a Brucella-immunised camel

Brucella are invasive gram-negative bacteria that multiply and survive within eukaryotic cells causing brucellosis. Syrian (and Middle East) health and economy sectors are still affected by this disease causing a serious national problem that needs to be solved. Here, a strategy was developed to introduce a new generation of binders, known as Nanobodies (Nbs) in our combat against Brucella. These Nbs, recombinant single-domain variable fragments derived from camelid heavy-chain antibodies are very stable and highly soluble, making them a useful tool in numerous biotechnological and medical applications. In this work and without having access to purified antigens (Ags), a camel was immunised successfully with heat-killed Brucella melitensis strain Riv1 as demonstrated by the high titer of Ag-specific heavy-chain antibodies in the serum. Lymphocytes of the immunised camel were isolated and their Nb genes were cloned in a relatively large library of 10(8) individual transformants, of which 81% contained an insert with the proper size of a Nb gene. Phage display expression of the Nbs from this library and pannings on the Brucella lysate resulted in a clear enrichment of three distinct Nb-displaying phages (phage-Nbs), referred to as NbBruc01, 02 and 03, with specificity for Brucella. Producing these binders in a pure, soluble form, as well as identifying their specific targets, which are likely to be immunodominant Ags in Brucella, is expected to open wide perspectives for following the vaccination, diagnosis and treatment of brucellosis.