Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.
Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.
This paper reviews the literature on leptospirosis in Malaysia from its first description in 1928 until the present day. Most of the early reports were on investigations of leptospirosis in wildlife and man and up-to-date, thirty-seven leptospiral serovars from thirteen serogroups have been bacteriologically identified. The thirteen serogroups are: Australis, Autumnalis Bataviae, Canicola, Celledoni, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Pomona, Pyrogenes, Sejroe and Tarassovi. Rats have been ascribed as the principal maintenance host of leptospires in Malaysia. However, serovars from the Pomona, Pyrogenes and Sejroe serogroups have yet to be isolated from rats. It is considered that the majority of leptospirosis cases in man were due to association of man with an environment where rats were plentiful. Recent investigations on domestic animals disclosed a high prevalence of infection in cattle and pigs and they were suspected as being the maintenance host for serovar hardjo and pomona respectively. There is ample scope for research in leptospirosis, particularly in the epidemiology and control of the disease in domestic animals. The strategy to control the infection in domestic animals and man in Malaysia is bound to be different from that of the temperate countries, basically due to the presence of a large number of leptospiral serovars in wildlife, further confounded by geographical and financial constraints.
A leptospiral isolate from a bovine kidney was found to be antigenically different from all previously recognized serovars of Leptospira interrogans based on the cross-agglutinin absorption test. The new serovar belongs to the Sejroe serogroup, and the name Leptospira interrogans serovar unipertama is proposed for it, with strain K2-1 as the reference strain.
A cross-sectional serological survey of domestic animals in West Malaysia revealed that 25.5% of the animals examined had agglutinating antibodies to one or more antigens belonging to Leptospira interrogans. Significant prevalence of infection was observed in cattle (40.5%), buffaloes (31%) and pigs (16%). The Sejroe serogroup was shown to be the principal one involved in cattle and buffaloes, and to a lesser extent the Tarassovi and Pomona serogroups. Evidence of infection in domestic animals by strains bearing the other seven antigens appeared insignificant and was indicative of sporadic infection. A majority of the large (semi-intensive) cattle and buffalo farms demonstrated a high prevalence of leptospiral infection. In both species of domestic animals mentioned above, the prevalence of infection was significantly higher (P = 0.01) in the semi-intensive farms than in the smallholdings. Amongst cattle, the droughtmasters had the highest prevalence whilst the Kedah-Kelantan (an indigenous breed) had the lowest prevalence of leptospiral infection. In general, the temperate breeds of cattle had a significantly (P = 0.01) higher prevalence of infection than local breeds. Leptospiral infection in goats and sheep was shown to be sporadic, and the Pomona serogroup was the principal leptospiral serogroup involved in these small ruminants. The prevalence of infection in pigs was observed to decline during the study period, and it is suspected that pigs in West Malaysia are the maintenance host for serovar pomona whilst cattle are the maintenance host for serovar hardjo. Overall, it appears that domestic animals in Malaysia will play a bigger role in the epidemiology of leptospiral infection with the advent of sophisticated farming.
This paper reviews the literature on human leptospirosis in Malaysia from its first description in 1925 until the present day. Fletcher diagnosed the first case of human leptospirosis in Malaysia in 1925. Following Fletcher, many investigations on human leptospirosis in Malaysia disclosed a high prevalence of infection. These investigations indicated that the disease was endemic in the country. Examination of 1993 suspected human cases of leptospirosis by Tan indicated 28 % of the cases were positive. In a recent survey, 2190 serum samples from patients with different clinical manifestations in the country disclosed 12.6% were positive for antibodies to leptospires. The risk to leptospiral infection with respect to occupation, location, sex, race and age groups was demonstrated. Both civilians and military personnel were affected. Thirty-seven serovars from thirteen serogroups have been identified in the country. Recent studies on animal leptospirosis showed that the disease was highly endemic in the animal population. It is considered that the majority of leptospirosis cases in humans were due to association of man with animals and disease-infected environment.
A cross-sectional serological survey of dairy cattle in Taranaki in 1979-80 indicated that 62% (551/891) of the animals had evidence of Leptospira interrogans serovar hardjo infection as disclosed by the microscopic agglutination test. Titres to Leptospira interrogans serovar pomona were demonstrated in only 4% (23/591) of the animals examined. The high prevalence of hardjo infection is suggestive of an endemic infection whilst the low level to pomona is indicative of sporadic infection. In a detailed examination of 10 herds, 9 revealed high (55%-91%) prevalence of serological reactions to hardjo and the herd profiles of titres, indicated that the animals had become infected at one to two years of age. A field strain of hardjo from cattle as well as the usual laboratory strain (hardjoprajitno) was incorporated in the test but there were no significant differences between the results given by the two antigens.
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.
A total of 157 stool samples were examined for Group A rotaviruses in diarrheic children admitted to 8 different major hospitals in Malaysia. The overall incidence rate in this study was 19.7% (31 of 157) with a variation of 9.5% to 39.1% in different locations. Majority of the infections detected were in those under 2 years of age and there were fewer admissions in the older age group. The stool samples were initially screened for rotavirus Group A by latex agglutination method and followed by RNA electrophoresis. The size and the characteristics wheel-shaped morphology of the viral preparations when examined by electron-microscopy further confirmed the presence of rotaviruses in the positive stool samples. Analysis of the RNA pattern showed that majority of the isolates, 51.6% (16 of 31) were Type IIC ('long' with comigration of RNA segments 7 and 8), 35.5% (11 of 31) with Type IIG ('long' with comigration of segments 7, 8, 9), 9.7% (3 of 31) with Type IG ('short' with comigration of RNA segments 7, 8, 9) and 3.2% (1 of 31) of mixed or atypical pattern. It appeared that over a 12 year interval, only one new or unusual rotavirus electropherotype was found. This is the first comprehensive report on the electropherotypes of rotaviruses covering eight different geographical locations in Malaysia and the data obtained is useful for understanding the geographic distribution and types of rotaviruses transmitting in Malaysia.
A cross-sectional bacteriological survey of cattle in West Malaysia revealed 14.4% (32/222) had leptospiral infection. Isolates were obtained from all except one herd with prevalence of infection in herds ranging from 0-44.8%. A small number of buffalo urine samples were examined and all of them were found to be negative. A leptospiral isolate obtained from a bovine kidney proved to be a new serovar of Leptospira interrogans and the name unipertama was assigned to it. Six other leptospiral serovars were isolated, namely canicola, australis, javanica, ballum, pomona and hardjo. All six serovars were isolated for the first time in cattle in Malaysia. Cattle in Malaysia appear to be the maintenance host for serovar hardjo. The presence of the other serovars in cattle was probably due to contact with the maintenance hosts, pigs for serovar pomona and rodents for the other three serovars. It appears that the epidemiology of leptospiral infection in cattle in Malaysia is similar to that reported overseas.
A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.
Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected National Service Training Centres (NSTC) in Kelantan and Terengganu. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) medium, incubated at room temperature for 1 month and examined under the dark-field microscope. Positive growth of the leptospiral isolates were then confirmed with 8-Azaguanine Test, Polymerase Chain Reaction (PCR) assay and Microscopic Agglutination Test (MAT). Fifteen cultures (10.34%) exhibited positive growths which were seen under dark field microscope whilst only 20% (3/15) were confirmed as pathogenic species. based on 8-Azaguanine Test and PCR. Serological identification of the isolates with MAT showed that hebdomadis was the dominant serovar in Terengganu. Pathogenic leptospires can be detected in Malaysian environment and this has the potential to cause an outbreak. Therefore, precautionary steps against leptospirosis should be taken by camp authorities to ensure the safety of trainees.
One hundred and sixty eight rats were trapped from the National Service Training Centres (NSTC) in Kelantan and Terengganu from October 2008 to May 2009. Microscopic agglutination test (MAT) was performed to detect the presence of agglutinating antibodies to Leptospira among the rats caught. All the MAT positive rats were identified as Rattus tiomanicus. In Kelantan, 17.3 % (14/81) of the rats had leptospiral antibodies to serovars Icterohaemorrhagiae (12.3%), Canicola (2.5%), Ballum (1.2%), and Pyrogenes (1.2%). In Terengganu, 18.4% (16/87) of the rats had antibodies to serovars Icterohaemorrhagiae (15%), Canicola (1.1%), Pyrogenes (1.1%) and Hebdomadis (1.1%). This study indicated that Leptospira serovars were prevalent in the rat population in the study areas and could be a source of infection to humans. Therefore, control of the rat population in all NSTC is critical to prevent outbreaks of leptospirosis amongst the NSTC trainees.
Pseudorabies (Aujeszky's disease) is an economically significant disease of swine known to cause central nervous disorders, respiratory disease, reproductive failure and mortality in infected pigs. In attempts to eradicate the disease from becoming endemic, early detection is important to prevent further economic losses and to allow for detection and removal of infected pigs in domestic herds. Thus, a rapid and sensitive technique is necessary for the detection of the virus. For rapid and simple examination, an immuno - chromatographic lateral - flow assay system based on immunologic recognition of specific pseudorabies virus antigen was developed by utilising, as signal generator, colloidal gold conjugated to secondary antibody to detect primary or sample antibody in the sera of pseudorabies infected animals. The pseudorabies virus used as a capture antigen in the test strip was first cultivated in VERO cell culture and then purified by sucrose gradient separation to produce the viral protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted well with the hyperimmune serum (HIS). The antibody detection system is basically composed of colloidal gold - labelled antibodies fixed on a conjugate pad, and the complementary pseudorabies antigen immobilised onto a nitrocellulose membrane forming capture zone. If the target antibody is present in a specimen, the colloidal gold-labelled antibody will form a complex with the antibody sample. Subsequently, the formed complex will migrate to the capture zone and is then bound to the solid phase via antigen - antibody interaction. As a result, a signal marker is generated by the accumulation of colloidal gold for detection confirmation. The results obtained demonstrated that the optimum combination of pseudorabies antigen needed as the capture reagent and gold conjugate as secondary antibody recognition marker was at a concentration of 0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid - based test strip towards pseudorabies antibodies was high with a detection limit of 1 to 10,000 - dilution factor. The specificity of the assay was 100% with no cross - reaction being observed with other sera or antibodies. Accurate reading time needed for confirmation of the assay can be completed in 5 min with a whole blood sample of 25 microl. The colloidal gold - labelled antibody is stable at room temperature for 6 months or more (data not shown). Findings from this study indicated that the solid - based test strip assay system provided high sensitivity and specificity for the detection of pseudorabies at low levels of antibody concentration. The assay was rapid, simple, cheap, and does not require any sophisticated equipment. Thus, the solid based test strip will be a useful serological screening technique or for rapid diagnosis of an infectious disease in target populations of animals characterised by heterogeneous antibody responses.
Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis.
Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.
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.
ELISA and immunoblotting techniques were used to examine the humoral immune response to Pasteurella multocida, in bovine sera from Indonesia and Malaysia. Elevated levels of antibody to a crude lipopolysaccharide preparation were found in vaccinated animals. In addition to the response to lipopolysaccharide, antibodies from the vaccinated cattle strongly labelled five to six of the 40 protein bands in this organism.
The effect of enclosure design on diurnal activity and stereotypic behaviour was assessed in 17 adult Malayan Sun bears (Helarctos malayanus), kept either in barren indoor enclosures or relatively enriched outdoor enclosures. Locomotion was the most frequent activity observed in the indoor bears, followed by resting. In contrast, conspecifics housed outdoors spent most of the time resting. Eleven forms of stereotypic behaviours were recorded in the bears, with pacing being the most common. The frequency and repertoire of stereotypies were significantly higher in the indoor bears irrespective of enclosure size. Novel forms of locomotor (forward-reverse pacing) and oral (allo-sucking) stereotypies were recorded. Oral stereotypies were predominant in the bears housed indoors, while patrolling was confined to the outdoor bears. Enclosure complexity significantly influences activity budget and occurrence of stereotypic behaviours, highlighting the importance of appropriate enclosure design and enrichment for the welfare of captive bears.