Abdul Rani Bahaman

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia

A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

BackgroundCell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.ResultsThe proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.ConclusionsThe model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

Modeling the potential distribution of sun bear in Krau wildlife reserve, Malaysia

Abstract Information and data on the distribution of wildlife species become important when strategizing the management and conservation of the species. Species distribution models have increasingly been used as a predictive tool in wildlife conservation planning and management. This research assessed the distribution of the sun bear and identified the relevant distribution descriptive variables in the Krau Wildlife Reserve, Pahang, Malaysia using Ecological Niche Factor Analysis (ENFA). Marginality and specialization factors showed that the sun bear habitat is very specific compared to the entire study area and its niche breadth was slightly narrow. Among all the habitat suitability algorithms available in Biomapper software package, median with the extreme optimum algorithm was found to have the best predictive capabilities by means of a continuous Boyce index. Results showed that sun bear can rarely be found in high altitudes, prefers lowland Dipterocarp, and avoids Mountain Ericaceous and Mixed Hill Dipterocarp forests. It also prefers to live nearby rivers, tends to avoid villages and prefers regions with fine silty-clay and loamy textures soils.

Retrospective Study of Leptospirosis in Malaysia

Abstract Leptospirosis is a bacterial disease transmitted to humans and animals by direct or indirect contact with urine or body fluids from infected animals especially rodents. Infection can be associated with wide clinical spectrum varying from asymptomatic to severe multi-organ syndrome with life-threatening consequences. We conducted a review of published studies on incidences, case reports, sero-epidemiological surveys from year 2000 to 2015 using different electronic data bases. Our study revealed that majority of the studies were conducted in Peninsular Malaysia and predominantly among high-risk human groups. Most of the studies on domestic animals were conducted in the 1980s; hence, the current status of leptospirosis among domestic animal population remains largely unknown. There tend to be a sharp rise in incidence rate among human population in the year 2014 which was attributed to flooding and heavy rainfall experienced as well as recreational activities. Several gaps in epidemiological knowledge were also disclosed.

Production and Characterization of a Polyclonal Antibody of Anti-rLipL21-IgG against Leptospira for Early Detection of Acute Leptospirosis

Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

Major epidemiological factors associated with leptospirosis in Malaysia

INTRODUCTION Leptospirosis is a zoonotic disease caused by a diverse pathogenic leptospira species and serovars. The disease is transmitted directly following contact with infected urine and other body fluids or indirectly after contact with water or soil contaminated with infected urine. OBJECTIVES While a wide range of domestic and wild animals are known to be reservoirs of the disease, occupation, international travel and recreation are beginning to assume a center stage in the transmission of the disease. The objective of this study is to review available literatures to determine the extent to which these aforementioned risk factors aid the transmission, increase incidence and outbreak of leptospirosis in Malaysia. STUDY DESIGN The review was conducted based on prevalence, incidence, and outbreak cases of leptospirosis among human and susceptible animals predisposed to several of the risk factors identified in Malaysia. METHODS Literature searchers and reviews were conducted based on articles published in citation index journals, Malaysian ministry of health reports, periodicals as well as reliable newspapers articles and online media platforms. In each case, the newspapers and online media reports were supported by press briefings by officials of the ministry of health and other agencies responsible. RESULTS The disease is endemic in Malaysia, and this was attributed to the large number of reservoir animals, suitable humid and moist environment for proliferation as well as abundant forest resources. Over 30 different serovars have been detected in Malaysia in different domestic and wild animal species. This, in addition to the frequency of flooding which has increased in recent years, and has helped increase the risk of human exposure. Occupation, recreation, flooding and rodent population were all identified as an important source and cause of the disease within the study population. CONCLUSION There is an urgent need for the government and other stakeholders to intensify efforts to control the spread of the disease, especially as it greatly affect human health and the tourism industry which is an important component of the Malaysian economy. The risk of infection can be minimized by creating awareness on the source and mode of transmission of the disease, including the use of protective clothing and avoiding swimming in contaminated waters. Moreover, improved diagnostics can also help reduce the suffering and mortalities that follow infection after exposure to infection source.

Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti‐HBs) using a surface plasmon resonance (SPR) chip‐based approach with Pichia pastoris‐derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X‐100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098–0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme‐linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross‐reactivity with other antibodies tested. The ability of SPR chip‐based assay and ELISA to detect anti‐HBs in human serum was comparable, indicating that the SPR chip‐based assay with its multiple screening capacity has greater advantage over ELISA.

Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay

BackgroundBrucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis.MethodsIn this study, omp25, omp28 and omp31 of B. melitensis were cloned and expressed using prokaryotic pET-32 Ek/LIC system and their respective rOMPs were combined as one coating antigen to develop rOMPs I-ELISA. Three groups of BALB/c mice were used to elicit antibody response. Group 1, infected with B. melitensis strain 0331 field strain; group 2, injected with B. melitensis Rev.1 vaccine strain and group 3, infected with Yersinia enterocolitica O:9. Antibody responses in three groups of mice were investigated using Rose Bengal plate test (RBPT) and rOMPs I-ELISA.ResultsThe production of rOMP25, rOMP28 and rOMP31 of B. melitensis were achieved and Western immunoblotting analysis demonstrated their reactivity. The RBPT was unable to differentiate the vaccinated mice (group 2) and mice infected with Y. enterocolitica O:9 (group 3) and categorized them wrongly as positive for brucellosis. In contrast, the rOMPs I-ELISA was able to differentiate the mice infected with B. melitensis strain 0331 (group 1) from both of group 2 and group 3, and recorded 100% sensitivity and 100% specificity. ConclusionsThe results of this study suggested that rOMPs of B. melitensis has potential diagnostic ability to differentiate the FPSR in serological diagnosis of brucellosis.

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

A study of the Mut+ phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L−1 Dry Cell Weight (DCW) and 26.07 mg L−1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with MutS expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut+ phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut+ phenotype. The results from this study demonstrated that expression of HBsAg with Mut+AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut+ phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.

Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection

Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars.