BACKGROUND: Brucellosis or Malta fever is one of the most prevalent zoonotic diseases considered as a health and economic concern.OBJECTIVES: The current study aimed to employ several methods to detect Brucella in blood and milk samples of saanen goat and use a safe and definitive method to diagnose this disease.METHODS: In this study, 122 blood samples and 122 milk samples were collected from saanen goats. After culture and serological-based isolation methods (RBPT, Wright, 2ME, and Ring test), DNA was extracted from all the blood and milk samples. PCR was carried out using B4 and B5 primers on all the extracted DNAs in order to detect the B. abortus and B. melitensis; PCR was carried out with Br.a and Br.m primers.RESULTS: The results of all the blood samples were negative, but bacterial growth was observed in three milk samples, which was detected in biotyping, biovar 1 melitenensis. The PCR results for detection of Brucella spp. of nine blood samples and nine milk samples were positive. Using mPCR primers, B. melitensis were identified through all the nine milk and blood samples.CONCLUSIONS: Herein, we found that better bacterial diagnostic system and choosing an appropriate technique for rapid detection, such as PCR and Real Time PCR, in addition to popular awareness and other functions of national veterinary medicine institute could control the diseases and decrease their incidence successfully.

BACKGROUND: Brucellosis is one of the most dangerous worldwide infectious zoonotic diseases that are common between ruminants and human. Consumption of infected milk and by-products is the major transmission source to human. In Iran, sheep compared to cow, has a higher rate of contamination with brucellosis. Therefore, early detection and precision could be a starting point for any efficient program to control the disease in human and animals. For brucellosis monitoring, milk ring test (MRT) is recommended but the test is not reliable in sheep herds. Perhaps a more realistic outcome could be achieved by changing the antigen used in MRT. OBJECTIVES: Comparison of two Brucella abortus and Brucella melitensis antigens in MRT for detection of Brucella antibodies in milk, as well as monitoring contamination of  ewe’s milk in Dezful region by detection of B. abortus and B. melitensis genes using PCR. METHODS: In this research, 220 milk samples from 16 different herds were collected from Dezful region’s nomadic at Khuzestan province. As the first step, MRT by two antigens, B. abortus and B. melitensis, were conducted on the samples. Next, the samples were subjected to detect Brucella genes using PCR technique. RESULTS: Results showed that 47 (21/3 %) out of 220 cases were positive by MRT test, in terms of both antigens of B. abortus and B. melitensis. In PCR, out of 220 samples, only 9 (4%) samples were positive for specific genes of B. melitensis which were MRT positive as well. CONCLUSIONS: A significant difference between B. abortus and B. melitensis antigens was not observed in MRT. Although the nature and basis of PCR and MRT methods for the diagnosis of brucellosis is different but a significant difference between the results obtained by PCR and MRT showed that MRT even by changing of antigens is still not authentic. Considering that various methods of identification have their limitations, it is recommended that in ewe’s milk samples, in addition to using a serological method as screening, PCR and culture methods should be used for definitive diagnosis.

OBJECTIVE To evaluate a hypervariable octameric oligonucleotide fingerprints (HOOF-Prints) assay for identification of and discrimination between wild-type and vaccine strains of Brucella melitensis. SAMPLE Brucella melitensis vaccine strain M5 and wild-type strain M43. PROCEDURES 8 pairs of primers (alterable, octameric nucleotides) were designed on the basis of a biological analysis of 8 flanking sequences in the DNA of B melitensis. The HOOF-Prints technique was used to identify wild-type and vaccine strains of B melitensis. Phylogenetic analysis of short, polymorphic fragments of DNA from B melitensis strains M5 and M43 was performed. RESULTS Variable-number tandem repeat DNA segments of B melitensis vaccine strain M5 and wild-type strain M43 were successfully amplified by means of PCR assay. All target gene fragments ranged in size from 100 to 300 bp. Separate phylogenetic analysis of each Brucella strain revealed considerable differences between the vaccine and wild-type strains. CONCLUSIONS AND CLINICAL RELEVANCE The results of this study suggested the HOOF-Prints assay may be useful for discriminating vaccine strains of B melitensis from wild-type strains. This ability could allow discrimination between animals that are seropositive because of vaccination against B melitensis and those that are seropositive because of B melitensis infection and could decrease the likelihood of importing Brucella-infected animals.

A study was carried out to report the phylogenetic analysis of Brucella abortus and Brucella melitensisby using molecular techniques from samples submitted to the Regional Veterinary Laboratory, Bukit Tengah.In this study, identification and genetic characterization of Brucella isolated samples using molecular analysis based on IS711 sequence between localisolates and foreign countries accesses in GenBank was done successfully. A total of 31 samples were isolated for Brucella species and then were amplified byPCR, directly sequenced and compared genetically to published sequences which were obtained from GenBank. The most common Brucella species that was found in both bovine (76.5%) and caprine (85.7%) through diagnostic samples in Regional Veterinary Laboratory, Bukit Tengah, was Brucella melitensis. PCR and sequencing were confirmed positive with 76.5% for Brucella melitensis, 23.5% for Brucella abortus and 23.5% for mixed infectionfrom the total of 17 bovine samples. In caprine, the detection of Brucella melintesis and Brucella abortus showed 85.7% and 21.4% respectively meanwhile total mixedinfection showed 21.4%. These clustering between local isolates of Brucella melitensis were phylogenetically related to other Asian countries such as Singapore,Yemen and Saudi Arabia. The Neighbour Joining Analysis clustered the Brucella abortus local isolates for both bovine and caprine were most closely related to India,Iran, Italy and USA. Interestingly, all the isolates within Malaysia have a close relationship (>95%) with the low level of genetic diversity. When local isolates arecompared to GenBank data, it gives an indication on the possible sources of these infections. Eventually, it will improve the import and export policies to controlbrucellosis in Malaysia.

Brucella melitensis is the Brucella species most frequently associated with brucellosis in humans. It is also the causative agent of the disease in goats and other ruminants. Although significant aspects of the pathogenesis of infection by this intracellular pathogen have been clarified, several events during invasion of host cells remain to be elucidated. In this study, infections of human macrophages from the THP-1 monocyte cell line were conducted with B. melitensis Bm133 wild-type strain and a strain of Salmonella serovar Enteritidis as a control. A multiplicity of infection of 100 was used in trials focused on defining the relative expression of syntaxin 4 (STX4), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, in the early events of phagocytosis (at 15, 30, 45, and 60 min). Immunoblot assays were also done to visualize expression of the protein in cells infected with either bacterial strain. The expression of STX4 was not significantly different in cells infected with B. melitensis strain Bm133 compared to that observed in cells infected with S. Enteritidis. When the expression of STX4 mRNA was inhibited with short or small interfering, or silencing, RNA in the THP-1 cells, the survival of B. melitensis was significantly reduced at time 0, when gentamicin treatment of cultures was begun (after 1 h of phagocytosis), and also at 2 h and 12 h after infection.

Results of a double agar gel immunodiffusion (Ouchterlony) test that contained a polysaccharide (poly-B) antigen of Brucella melitensis strain B115 were compared with those of 5 other serotests. To determine the sensitivity and specificity of the immunodiffusion, standard tube, 2-mercaptoethanol, Rivanol, card, and complement fixation tests, sera obtained from 1,328 vaccinated, infected and seronegative cattle, 56 of which had been examined bacteriologically, were used to evaluate the humoral response to Brucella sp. The poly-B antigen confirmed infection in 87.5% of the 56 cattle from which Brucella abortus biotype 1 had been isolated, and in 96.6% (205/212) of a group of cattle suspected to be infected on the basis of results of conventional serotests. Likewise, sera from 4 groups of vaccinated cattle did not react with poly-B antigen, whereas they did not react in conventional tests. The poly-B antigen was more specific in detecting infected cattle even in a group of vaccinated adults. A useful strategy to identify infected cattle might be screening, using a combination of the Rivanol and card tests together with the agar-gel immunodiffusion test containing poly-B antigen.

Forty-nine fresh goat’s milk samples produced by local farmers and sold in market for public consumption as well as raw goat milk in Johor, Malaysia were analysed for total plate count(TPC) , E. coli, Coliform, Brucella melitensis, Brucella abortus,Coxiella burnetii as well as aflatoxin M1 (AFM1) content, as measures for food safety. The mean counts per ml for TPC were 4.90 x 105, 6.50 x 105, 1.60 x 105 and 1.48 x 106 for pasteurised, unpasteurised and unknown (status of pasteurisation) milk sold in the market as well as the raw milk from milkcollection center (MCC), respectively. Among pasteurised samples, only one had TPC count higher than the permitted level whereas the rest were all within the permitted level. The mean counts per ml for E. coli were <1.00 x 102 for pasteurised and unknown milkwhereas 1.67 x 101 for unpasteurised and 1.18 x 102 for raw milk. The mean counts per ml for coliform were 9.53 x 103, 9.76 x103, 1.20 x 102 and 1.16 x 104 for pasteurised, unpasteurised, unknown milk and raw milk, respectively. Overall, no significantdifferences on the bacterial counts in both pasteurised and unpasteurised milk. All milk samples were negative of B. melitensis and B. abortus, but one unknown sample fromthe market and two raw samples from MCC were positive of C. burnetii through the ELISA test. The unknown sample from the market showed the presence of C. burnetii when further analysed microscopically. Meanwhile, no sample exceeded the permitted level of AFM1 in milk.

Stability study of biological products especially living bacterial vaccines plays an important role for the determination of product changes in maintenance period, and ensures safety, efficacy and maintenance of biological properties of the vaccines. So, the objective of this study was to establish stability and keeping quality of the local Brucella melitensis Rev-1 vaccine using different types of stabilizers in lyophilization process. A long-term stability study was carried out for four batches of reduced-dose Brucella melitensis Rev-1 vaccine manufactured by veterinary serum and vaccine research institute using four different stabilizers. Stabilizers were: (A) sucrose and skimmed milk, (B and C) different concentrations of sucrose, sodium glutamate and gelatin, and (D) casein, sucrose and sodium glutamate. The quality control tests including colony forming unit, purity, dissociation and physicochemical tests on all batches until 12 months postproduction were performed. The obtained results indicated that in spite of collapse (shrinkage) of lyophilized cake in a number of bottles in batches prepared using stabilizer A, Brucella vaccine batches were stable and met the specification recommended by OIE 2012 for 12 months post-production in vaccine batches with stabilizers A and D.

Brucellosis, caused by Brucella melitensis, is a significantproblem for both public and animal health worldwide. The Rose Bengal plate test (RBPT) antigen from Brucella melitensis local isolates were developed in this study. The performance of the assay wasinvestigated using serum samples collected from goats. A total of 1063 serum samples obtained from goats were examined for thepresence of antibodies against Brucella by in-house RBPT (LRBPT), commercial RBPT (Veterinary Laboratory Agency – VLA, UK) and Compliment Fixation test (CFT). The sensitivity and specificity wascalculated using CFT as the gold standard. Out of 1063 goats sera analyzed 364 (34.24%), 335 (31.51%), and 373 (35.08%) were found to be positive by LRBPT, commercial RBPT and CFT, respectively. The sensitivity calculated for the LRBPT, was 90.1% compared to commercial RBPT 85.0%. However, the specificity of the LRBPT was lower (95.9%), than the commercial RBPT (97.4%). Furthermorethe LRBPT has better value of NPV (94.7%) than commercial RBPT NPV(92.3%). While the PPV, of the commercial RBPT is higher (94.6%) than LRBPT (92.3%) respectively. High sensitive and low cost LRBPT compared to cRBPT B. melitensis RBPT test was successfully developed in this present study. Therefore it was concluded that this diagnostic test kit can complement and replace the availablecommercial RBPT which is relatively more expensive and less sensitive in detection of brucellosis in goats. It could also be used for epidemiological surveillance of caprine brucellosis in Malaysia.

Brucellosis in goats is mainly caused by the bacterium Brucellamelitensis, which is one of the most important pathogenic species in the world. In Malaysia, the annual prevalence data of brucellosis was recorded in goats and the control strategy of the disease basedon test and cull of infected animals. This strategy has caused huge economic losses to farmers and government alike. Therefore, whole genome sequencing of B. melitensis local strain is essential forimproving the current vaccine. B. melitensis strain VRI 6530/11 wasobtained from veterinary research institute biobank, Ipoh. The strain was submitted for classical identification procedures and the total genomic DNA was extracted by using DNeasy blood and tissue kit(QIAGEN). The concentration and purity of DNA were determined by using agarose gel electrophoresis and spectrophotometer (DNA/RNA) assay respectively. The genome was sequenced by using IlluminaHiSeq platform with insert size ~200 bp. A total of 1.0 Gb data was generated from the sample. More than 95% of sequencing data was retained in the sample after quality filtering, this indicatethe sequencing reads are of high quality. Final assembly had 33 scaffolds with total size ~3.28 Mb, 44 contigs, GC content is 57.25%, N50 is 293,291. A total of 3,238 protein coding genes, 48 tRNAs and 3rRNAs were predicted and over 87% of the genes were functionally annotated. Genome sequencing of a local B. melitensis strain is the first of its kind in Malaysia and work from this study can contribute towards the development of a new effective vaccine for the control ofthe disease in the country.