Mol ecular bi ology aspects of Leptos pira

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In R FLP anal ys is , the DNA s ampl e is broken into pi eces b y rest ri ction enz ym es and t he res ulting restrict ion fragm ents are separat ed according to thei r l engt hs b y el ectrophoresi s. However, in P FGE is a t echnique us ed for the separation of large DNA molecul es b y appl ying an el ectri cal fi eld that periodi cal l y changes di recti on t o a gel m atrix. The P FGE method is comm onl y consi dered a gold st andard in epidemiol ogi cal studi es of pat hogeni c organism s.

Genom e sequencing of L. int err ogans serovar Lai and C openhegeni have been comp l et ed which can facilit at e underst anding of molecul ar mechanism s of l ept ospiral pathogenesis and als o to help to identi f y novel vaccine and di agnosti c m arker candi dat es. The leptos pi ral genome consists of two ci rcular chromosom es of approxim at el y 5,000 kb in tot al size (Baril et al ., 1990; Zuerner, 1991; Boursaux -Eude et al ., 1998; and Picardeau et al., 2008). The genome is larger compared with t he genom es of ot her s pirochetes such as Treponema spp and Borr elia spp whi ch indi cat es t he abili t y of L ept ospi ra spp to li ve wit hin divers e environm ents (Fras er and Fras er et al ., 1997) . The genom e compris es of a 4,400-kb chromosom e and a sm all er 350 -kb chromosom e ( Zuerner, 1991).

Leptos pires cont ain t wo s ets of 16S and 23S rR NA genes but onl y one 5S rRNA gene and th e rR NA genes are widel y s paced ( Fukunaga et al ., 1989; Baril et al ., 1992).

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The stud y of l ept ospi ral geneti c s is hampered b y the lack of a trans form ation s ys tem ( Zuerner et al., 1993; Kal ambaheti et al., 1999 ).

Li ttl e is known about genetic exchange am ong L ept ospi ra, alt hough lat eral t rans fer has been suggest ed (P ena -Moct ezum a, 1999). Recentl y, a shuttl e vector was devel oped usi ng the t emperate bact eriophage LE1 from L. biflexa (S ai nt Girons et al., 2000). This mi ght provide a bett er understandi ng of Leptospira at the mol ecul ar level . Another limitat ion to the geneti c anal ys is of these bact eri a i s the l ack of access or y geneti c repli cons (li near or circular pl asmi ds ). Therefore, much of the research on the mol ecul ar bi ology of L eptospir a has us ed m ol ecular c loni ng and het erologous express ion of genes i n suitable bact eri al hosts .

A number of l ept ospiral genes have been cloned and anal yz ed, includi ng several for amino aci d s ynthesis, rRNA, ribosom al protei ns, R NA pol ym erase, heat s hock prot eins , out er mem brane prot ei ns, fl agell ar prot ei ns and lipopol ys accharide ( LPS) s ynthesis ( Dohert et al., 1989;

Fukunaga, and Mi fuchi 1989; Di ng and Yel ton, 1993; Shang et al., 1996;

Li n et al ., 1999 and 1997; Pena -Moctez uma et al., 1999; Zuerner et al., 2000; R enesto et al ., 2000; Bulach et al ., 2000 ).

The us e of PCR t echnique i n epidemi ol ogi cal studies provi des a novel basi s for t yping lept ospi res using primers from species or st rain -speci fic sequences (Gravekamp et al ., 1993; Murgi a et al., 1997).

19 1.4 Epidemiology

Leptos pirosis has a worldwide dist ributi on due to a wide range of anim al reservoirs (Faine et al., 1999). Fi gure 1.4 shows t he sources and c ycl es of l eptospiral i nfecti on. The pat hogenic lept ospi res s hed i n t he uri ne of the carri er anim als contami nate the environ ment and cause hum an and anim al infection s throughout the world. Rats are the m ain reservoi r host but other wild and domes tic anim al s are also known to perform t his functi on. Reservoir host s can cross -i nfect each other via urine or congenit all y, and s exual transmissi on i s known to occur i n m an y species.

Hum ans are acci dent al host and are not capabl e of becoming carri ers .

Leptos pires are natural l y aquati c organis ms and are found i n fres h wat er, dam p soil , veget ation, and mud. The y s urvive i n thes e envi ronm ents for months or even years, t hus al lowing re -i nfection among t he animal hosts . Floodi ng aft er heav y rai nfall m a y spread the organism because, as water saturat es t he soil , l ept ospi res pass di rectl y ont o surface wat ers . The organi sms di e when dr y, or in aci d condi tions (pH <7.0). The sources of non -occupational l eptospi rosis i n t emperat e clim at es are m ai nl y rodents and dogs and are s pread through l ei sure activiti es and t ravel (Fai ne, 1999). Leptospi rosi s is consi dered an occupati onal hazard in farm ers and vet erinari ans as well as dai r y, slaughterhous e and s ewer workers, miners, rice and sugarcane fi eld workers, and mil itar y pers onnel .

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Figu re 1. 4 Source and c ycl es of l ept ospi ral infection (Fai ne et al., 1999)

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Leptos pires ent er a ho st vi a port als s uch as dam aged s kin, certain m ucous membranes, the l ungs and conjuncti va m embranes. The y are not t hought to be capable of penet rating undam aged skin except where it has been expos ed to wat er for s ome ti mes and has s woll en signi ficantl y (www .leptospi rosi s.org/m edi cal ). The diseas e is not t ransmitted b y ingestion of food or b y i nhal ation of airborne particles (Faine et al., 1999). The s ource of human infect ion i s usuall y t hrough eit her di rect or indi rect contact wit h the uri ne of an i nfect ed anim al. The i nci dence is si gni fi cantl y hi gher in warm cl imat e countri es t han in t em perat e regions (Everard and Everard, 1993); this is m ai nl y due to the longer survival of lept ospi res i n t he warm, humi d environm ent .

In M al a ysi a l eptospi rosi s is endemi c ma i nl y due to the warm and humid clim at e. Rodents i n Mal a ys ia have been reported as the m ajor maint enance hos ts for vari ous l ept ospi ral serovars (Baham an& Ibrahim, 1988). The y s hed l ept ospi res in t heir urine, thus cont aminat ing t he environm ent. The inci dence of l eptospirosis has not be en well docum ent ed becaus e it has not been a noti fiabl e dis eas e until ver y recentl y. Foll owi ng Fl et cher, man y inves ti gat ions on hum an l ept ospi ros is in M al a ysi a dis clos ed a hi gh preval ence of the i nfection (Tan, 1970a;

Supram ani am, 1979; El Jalii, 2002). Seropreval ence for hum an infecti on as reported b y S upram ani am in 197 9 was bet ween 12% - 22% of t he popul ati on . In anot her stud y, preval ence of leptospi ral antibod y in norm al populati on was reported to be at 12% (Tan, 1970). Accord ing t o

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Tan (1964) rubber estat e workers were the most hi ghl y infected group foll owed b y w orkers handling sewage, drain s, town clea ning, forest r y and anti -m al ari a workers .

In another st ud y by Tan (1964) a tot al of 584 cases of p yrexi a of unknown ori gi n (P UO) were exam ined over a period of 4.5 years from different st at es i n Mal a ysia. The resul ts indicat ed that 29.6% of thos e cases were positi ve for l ept ospi ral infecti on which means that nearl y one out of t hree P UO cases i n M al a ysi a coul d possi bl y be due to lept ospi rosis.

Tan (1970a) carried out a stud y on 1993 suspected cas es of l ept ospi ros is in rural West Mala ysia and report ed 559(28%) were confirm ed positive.

The hi ghest number of cases occurred among Indi an m al es, aged 20 -40 years old. Tan et al (1986a ) exami ned 36 cas es of acut e renal failure admit ted to Kual a Lumpur General Hospit al during 1980 – 1983. She report ed that 16 (44.4%) of thos e cas es were due to l ept os pirosis. The infecting serovars were either s erovar C ell edoni or Pomona. Import ant serova rs is olated from hum an cases were C ani col a, Icterohemorrhagi ae, P yrogenes, Hebdom adis , and Autum nal is (Baham an & Ibrahim 1987).

Thus far 37 leptospiral s erovars from 13 s erogrou ps have been bact eri ologicall y identi fi ed in t his countr y i .e. Australi s, Autum nalis, Bat avi ae, C ani col a, C ell edoni, G ri ppot yphos a, Hepdom adis ,

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Ict erohaemorrhagi ae, J avani ca, Pomona, P yrogenes, Sej roe and T arassovi (Bahaman & Ibrahim , 1988).

Seasonal fl oodi ng and ot her recent drastic cli mat e changes have also contri but ed to the s pr ead of the disease, parti cul arl y those resulting with more s evere s ym ptoms. T he t wo fataliti es i n t he recent flooding in the stat e of J ohor , M alays ia were caused b y leptos pirosis (Badrul Hisham et al., 2009). In addition, the report of fat alit y among st ud ent s parti cipating i n nati onal servi ce was als o alarmi ng for M al ays ian healt h aut horiti es (www.re uters.com ).