Brucellosis re-emergence after a decade of quiescence in Palestine, 2015-2017: A seroprevalence and molecular characterization study.

Brucellosis is an endemic disease in many developing countries and ranked by the World Health Organization among the top seven "neglected zoonoses". Although a Palestinian brucellosis control program was launched in 1998, the disease re-emerged after 2012. Interestingly, a similar re-emerging pattern was reported in the neighbouring Israeli regions. The aim of this work was to characterize the re-emerging strains and delineate their genetic relatedness. During 2015-2017, blood samples from 1324 suspected human cases were analyzed using two serological tests. Seropositive samples were cultured, and their DNAs were analyzed by different genetic markers to determine the involved Brucella species and rule out any possible involvement of the Rev.1 vaccine strain. The rpoB gene was sequenced from nine isolates to screen for rifampicin resistance mutations. Multi locus VNTR analysis (MLVA-16) was used for genotyping the isolates. The molecular analysis showed that all isolates were Brucella melitensis strains unrelated to the Rev.1 vaccine. The rpoB gene sequences showed four single nucleotide variations (SNVs) not associated with rifampicin resistance. MLVA-16 analysis clustered the isolates into 22 unique genotypes that belonged to the East Mediterranean lineage. Altogether, our findings show that the re-emergence of brucellosis was due to B. melitensis strains of local origin, the Palestinian and Israeli control programs' weaknesses could be a major factor behind the re-emergence of the disease. However, other socioeconomic and environmental factors must be investigated. Moreover, strengthening brucellosis control programs and enhancing cooperation between all stakeholders is essential to ensure long-term program outcomes to fight brucellosis.

Improving sensitivity of single tube nested PCR to detect fastidious microorganisms.

Single Tube Nested PCR (ST-nPCR) is of value to clinical laboratories with limited settings for the detection of fastidious microorganisms. The detection sensitivity of ST-nPCR is dependent on ensuring minimal leftovers of outer primers during the second round of the reaction. In this work, we investigated various approaches to optimize the performance of outer primers, including decreasing outer primer concentrations; using antisense oligonucleotides to block outer primers; using chemically modified inner primers; and using Q5 Taq polymerase that lacks 5'-3' exonuclease and strand displacement capabilities. These solutions were tested on C. abortus and C. psittaci, which are both fastidious intracellular bacteria that are difficult to diagnose. The best obtained result was by using Q5 Taq polymerase. A detection limit with a range between 0.1 and 1 ag was achieved, which corresponds to a range between 0.2 and 2 copies of the plasmid positive control. This level of sensitivity is comparable or even better than the sensitivity achieved by TaqMan probe based real-time PCR assays. The assay was validated using 70 veterinary clinical samples from small ruminant abortions and 10% of these samples gave positive results. In conclusion, sensitivity of ST-nPCR to detect fastidious microorganisms can be improved by using Taq polymerases that lacks 5'-3' exonuclease. The proposed assay is affordable and applicable to a wide range of fastidious pathogens and can be suitable for laboratories with limited settings.