A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis

Brucellosis is a highly zoonotic disease that affects animals and human beings.Brucella suisis the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, anN-formylperosamine O-polysaccharide–protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected withB. suisbiovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of “smooth” brucellosis in animals and humans.

The porcine brucellosis – evidence of the role of Yersinia enterocolitica O:9 in occurrence of false positive serological reactions

Forty four pigs with typical characteristics for false positive serological reactions (FPSR) were examined for the presence of Yersinia enterocolitica O:9. The positive reactions were observed in rose bengal test (RBT, N=23 sera), serum agglutination test (SAT, N=16), complement fixation test (CFT, N=9), indirect ELISA (i-ELISA, N=11) in first, and in RBT (N=14), SAT (N=8), CFT (N=7) and i-ELISA (N=18) in second examination, respectively. In bacteriological examination Y. enterocolitica was confirmed in 12 cases. Six of these isolates were identified with PCR as Y. enterocolitica O:9.

Value of Fluorescence Polarisation Assay in Comparison to Traditional Techniques in Diagnosis of Porcine Brucellosis

The aim of this study was the evaluation of fluorescence polarisation assay (FPA) in the diagnosis of porcine brucellosis in comparison with Rose Bengal test (RBT), serum agglutination test (SAT), complement fixation test (CFT), 2-mercaptoethanol test, and ELISA. Eight hundred seventeen sera from pigs, including 612 sera from healthy animals, seven sera from Brucella suis bv2 culture positive animals, and 198 sera classified as false positive, originated from confirmatory investigations, were used. All sera from healthy animals, negative in RBT, SAT, CFT, and ELISA were also negative in FPA. All sera positive in serological examination, originated from Brucella infected animals, were also positive in FPA. Among sera classified as false positive almost half of the samples tested (49.49%) reacted positively in FPA. The examinations confirmed the usefulness of FPA in diagnosis of porcine brucellosis, but the method, like the other tests, does not allow resolving the problem of discrimination cross-reacting from specific antibodies.

PUNCTURE OF BOAR’S EPIDIDYMIS FOR DIAGNOSING PORCINE BRUCELLOSIS

In Serbia, mostly along the Danube, Morava and Mlava rivers, in animals reared under extensive conditions a disease named “rednja” may be detected. In the settlements where abortion is diagnosed all the sows mate with one boar that is infected with brucellosis. The Veterinary Program designs the control of Brucellosis twice a year, but the boars raised in extensive system are not controled and they are not registered. The causative agent was only recently isolated, confirmed and typed as Brucella suis biotype 2 (7). The owners have not reported the cases of abortion because they had thought that the reason are mechanical injuries that occur during feeding and drinking. After the visit of a vet a suspicion of brucellosis is notified. The symptoms of this disease are nonspecific. Only after abortion and if the swelling of boar scrotum occurs Brucellosis can be suspected. Brucellosis can be diagnosed in pigs by clinical examination, isolation, identification and typing of pathogens, serological and biological examination. Blood sampling is usually done in the case of abortion for serology examination, and also the placenta or aborted foetus is used for determining the cause. In addition to the above mentioned, the most important samples for bacteriological examination are the mandibular, retropharyngeal and supramamary lymph nodes, boar semen, i.e. testis after castration. All these samples cannot be taken when the reports on abortion are untimely. This means, only blood samples for serological examination can be taken. However, the literature does not mention epididymal puncture as a possible sample that is important for bacteriological examination, in addition to blood sampling from live animals. When the boars are fixed for blood sampling, the puncture of the epididymis can also be carried out for isolation of Brucella suis. After the disinfection, a sterile needle is stitched about 1 cm in depth and about 1-2 ml of the content is aspirated. Occassional sanies does not interfere the testing. The material can be frozen and examined when there the conditions are favourable and the laboratory available. Epididymal puncture is recommended as a safe and reliable way of sampling the diseased animals for the isolation and identification of Brucella suis biotype 2. During the bacteriological examination it was found that other bacterial species did not affect the growth of Brucella suis.

Revised Genome Sequence of Brucella suis 1330

Brucella suis is a causative agent of porcine brucellosis. We report the resequencing of the original sample upon which the published sequence of Brucella suis 1330 is based and describe the differences between the published assembly and our assembly at 12 loci.