Structure and Morphology

Leptos pires are ti ght l y coil ed spi rochet es , us ual l y 0.1 µ m b y 6 µm to 0.1 µm b y 20 µm, but cultures ma y occasionall y contai n much longer cell s (Fai ne et al., 1999). The cell s have pointed end s which are usuall y bent into a distincti ve hook shape (Fi gure 1.1). A pai r of axial fil am ents l yi ng in the peripl asmi c s pace and around whi ch t he bact eri um i s 'coil ed' act s as endofl agell a enabl ing it to move b y turning and wri ggli ng. Le ptos pires are gram negative but t oo t hin t o be visible under t he ordinar y micros cope. Dark -fi eld mi croscop y is most oft en us ed for lept ospi res obs ervat ion . All l eptospires look ali ke with onl y minor differences ; therefore, m orphol ogy does not help t o diffe renti at e bet ween pat hogenic and saproph yt i c l ept ospi res or bet ween the vari ous pat hogeni c lept ospi res. Leptospires have a t ypical double m em brane struct ure in comm on with other spirochetes, in which the c yt oplasmi c m embrane and peptidogl ycan cell wal l are clo sel y ass oci at ed and are overlaid b y an out er m em brane (Haake 2000). Leptos piral li popol ys accharide has a com positi on simi lar to that of other gram -negative bacteri a but has lower

5

endotoxin activi t y (Pri ya et al ., 2008). The most out er l a yer of lept ospi r es cons ist of out er envelope (OE) which is compos ed of prot ein, lipid and lip opol ys accharide ( LPS) m oieti es .Various anti gens,not abl y thos e associ at ed wit h the LPS are locat ed in the out er envel op OE regi on (Haake, 1991).

6

Figu re 1. 1 S canni ng el ectron micrograph of Leptos pira spp. s howi ng t he corks crew appearance of t he ba ct eri um. (www.cherokeeanim alclinic.com)

7 1.3.2 Taxonomy and Classification

The o rder Spirochaet ales has three fam ilies nam el y Brach yspi raceae, Spirospi raceae and Leptos piraceae. Wit hin L eptospi rac eae t here are onl y three genera i.e. L eptonema, Turneria and Lept ospi ra. P rior to 1989, the genus L ept ospi ra was di vided i nto t wo s peci es bas ed on anti genic cl assi fi cation nam el y pat hogenic L ept os pira int er rogans and saproph yti c Leptospir a bifl exa (Faine, 1 982). Both L. int err ogans and L . bifl exa are further divided into numerous serovars that is the basi c tax on us ed to cl assi f y t hese bact eri a and is defined b y aggl utinati on aft er cross -abs orption wit h hom ologous anti gen s (Dikken et al ., 1978; Faine, 1984;

Kmet y, 1993; Gust avo, M., & M athi eu , P., 2009 ). Over 60 serovars of L. bifl exa have been recorded (Fai ne, 1984). Serovars having ant i geni c similariti es are formed i nto serogroups, and t here are over 230 pat hogeni c L. int err ogans serovars whi ch are di vided i nto 25 serogroups (Tabl e 1.1 ). Different strains with s mall anti geni c di fferences can sometim es be found within cert ain serovars. Identifi cation and cl assi fi cation of L eptospira species is important becaus e s ome species have hos t preferences.

During recen t years the t axonom y of l ept ospi res has undergone a st ate of transiti on from an anti geni c to a genetic cl assi fi cation. The geneti c cl assi fi cation is bas ed on DNA -DNA hybri dizat ion which has reveal ed considerable heterogeneit y i n pathogeni c speci es. Thi s me thod has been the basis of the division of leptospires into genom ospeci es ( Yasuda et

8

al., 1987; R amadass et al., 1990; R amadas s et al., 1992; Perol at et al. , 1998; Brenner et al., 1999; R om ero et al., 2006 ). Based on the genet i c cl assi fi cation as determined b y DNA-DNA h ybridi zation, there are currentl y 20 speci es of Leptos pira. Thes e s peci es can be furt her di vided into pat hogenic, non -pathogeni c and opportunisti c/ possibl y pat hogeni c.

Pathogeni c Leptospi ra s peci es include: L. interrogans, L. ki rschneri, L.

santaros ai, L. w eilii, L. al exanderi, L. borgpet ers enii, L. als tonii and L . noguchii. Non pathogeni c Leptospira include: L. bifl exa, L. meyer i, L . kmet yi, L . vant hi eli i, L. t erpstrae, L. wolbachii, and L . yanagaw ae. Opport unist ic/ int erm edi at e Leptospira include: L. broomi, L . fai nei, L . inadai, L. licer asiae, and L. wolffii (Faine et al., 1999; Hawrami and Breuer., 1999; Nogva and Lill ehaug., 1999; Levett et al., 2001; Sl ack et al., 2006; Gustova & Mat hi eu., 2009 ).

Although anti geni c classi fi cation has been repl aced b y a genetic one, t he former is st ill widel y acc ept ed since s erovar i s the basis fo r taxonom y at the subspecies l evel (Elli s, 1991).

9

Table 1. 1 Serogroups and som e serovars of Leptos pira int err ogans sensu l at o (Levett, 2001).

Serogroup Serovar(s)

Icterohaemorrhagiae Icterohaemorrhagiae, Copenhageni, Lai, Zimbabwe

Hebdomadis Hebdomadis, Jules, Kremastos Autumnalis Autumnalis, Fortbragg, Bim,

weerasinghe

Pyrogenes Pyrogenes

Bataviae Bataviae

Grippotyphosa Grippotyphosa, Canalzonae, Ratnapura

Canicola Canicola

Australis Australis, Bratislava, Lora

Pomona Pomona

Javanica Javanica

Sejroe Sejroe, Saxkoebing, Hardjo

Panama Panama, Mangus

Cynopteri Cynopteri

Djasiman Djasiman

Sarmin Sarmin

Mini Mini, georgia

Tarassovi Tarassovi

Ballum Ballum, aroborea

Celledoni Celledoni

Louisiana Louisiana, Lanka

Ranarum Ranarum

Manhao Manhao

Shermani Shermani

Hurstbridge Hurstbridge

10 1.3.3 Growth and metabolic activiti es

All leptos pi res are chem oorganotrophs, growing i n aerobi c or microaerophili c environments and usi ng ox ygen as t he fi na l el ectron recept or. C yt ochrom e c, catalase, and oxidas e are present (Green et al., 1967 ). The onl y m ajor source of carbon and energy i s long -chain fatt y acids using bet a oxi dati on m etaboli c process, and t hese are ess ent ial t o tri gger growt h (Henneberr y, 1970). In addition, C O2 is also requi red for growth.

The growth curv e of lept ospi res foll ow t he s am e s tages as s een i n ot her bact eri a, except t hat the tim e scale i s longer. A t ypical generat ion tim e for pathogeni c l ept ospi res is about 6 -8 hours. However saproph yt ic lept ospi res grow fas ter i n about 2 -3 da ys as compared to t he pathogeni c ones that requi re about 4 -7 da ys . The y are aerobic, s o ox ygen is required and cultures benefit from gentl e aerati on or s haking. The yield from cult ures ma y be increased as much as one log b y aerat ion.

Saproph yti c leptos pires will grow at 11 -13ºC, al though optimu m devel opm ent occur s at 28 -30ºC (Johnson & Harris , 1967 ). The optimum growth of pathogeni c l ept ospi res is al so 28 -30ºC . H owever t he y will not grow at 13ºC . Other than di fferenti ating the two species b y growth at 13 ºC, the t wo species can be als o di fferent iat ed b y t he fact that , unl ike L.

interrogans, the saproph yti c L. bifl exa can grow in the presence of 8 - azaguanine and it s failure t o form spherical cells in 1M NaC l.

11

A prim ar y concern for cultivating s am pl es i s concent ration -i nduced l ys is where t he l ipas es produced b y the bacteri a begi n to outwei gh the abs orptive abilit y of the cul ture, and the form ati on of toxi c lipids . This can be ver y rapi d ei ther during the last port ion of the log curve or the stati onar y phas e, and can cause dama ge to the cult ures i f the y are not sub -cultured and di luted oft en enough (Adl er Adler & de la Pena Moct ezum a et al., 2010).