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ADVANCES IN LEPTOSPIRA AND LEPTOSPIROSIS RESEARCH STARTING FROM EARLY SIXTIES TO THE NEW MILLENNIUM
Arabinda Palit

Courtesy : Festschrift - Dr. S. Ramachandran

Introduction
Since the return of Columbus’s voyage in fifteenth century from the so-called “New World”, the attention of microbiologists and that of the western civilization turned to the spirochaetes. It must have been a devastating experience amongst Columbus’s voyagers to America after some of them had an intimate contact with ladies in the ‘New World”. Since then syphilis spread very rapidly in Europe, which was a virgin country for the Treponema pallidum infection. Interestingly, “Treponematosis” has been found in the skeletal remains of pre-Colombian Indians (1). The disease was so devastating in Europe that the contemporary poet Girolamo Fracastoro mentioned about it in a poem he wrote in 1530, this was twelve years after the London College of Physicians was founded in 1518 and 196 years before the first British medical faculty was founded at the Edinburgh University in 1726 (2).

In the past 40 years, scientific knowledge has advanced substantially on the family Spirochaetaceae but so far there is no vaccine against syphilis. This is primarily because it is still not possible to grow Treponema pallidum at a reasonable cost in sufficient cell mass in any artificial culture medium. However, the vaccinology in the case of animal spirochaetes (Leptospiraeceae) has advanced considerably due to the advent of the artificial growth medium developed by Ellinghausen and McCullough (3) and Johnson and Harris which is also known as the EMJH medium (4).

Classification and nomenclature
The order Spirochaetales is comprised of helical, motile organisms normally 6-12 m long and 0.I m in thickness. The order Spirochaetales is divided into two families, Spirochaetaceae and Leptospiraeceae. The pathogenic genera in the former family are Treponema causing syphilis and Borelliae causing “Lyme disease” in humans. In the Leptospiraeceae family, the pathogenic genera are Leptospira interrogans and Leptospira borgpetersenii. These two genera cause a wide range of infections in humans (Weil’s disease) and animals (Leptospirosis). Both families mentioned above have other non-pathogenic genera.

The principal morphological difference so far known between the two families is that in Leptospiraeceae the terminal ends of the organism are hooked like the end of a coat hanger whereas the same is not the case with Spirochaetaceae family.

For the purpose of brevity, I shall confine myself to the Leptospiraeceae family only.

The classification of Leptospiraeceae is based on serology, which is done by the agglutinin absorption technique, the concept of which was first recognized by Borg-Petersen in 1944 and subsequently adopted by Wolff and Broom (5,6). For details on classification, one may refer to Kemty and Dikken (7).

The basic taxon in this classification system is the serovar within which there are serotypes and strains, The serologically related serovars were placed in a group designated as a serogroup. This was initiated by Borg-Petersen (5), The term serogroup is not exactly defined but for practical purposes it is an unavoidable component of the current serological classification system. As it stands, there are 46 known serovars divided into 20 serogroups (7). An updated list of serovars of the L. interrogans species is also available (7). However, this classification system is not really complete in all respects as it is becoming apparent with the advent of more specific techniques like monoclonal antibodies and restriction endonuclease analysis known as REA (8,9). Further fine distinguishing properties are coming to light and are helping to develop “genotypes” within the same strain classified serologically. To cite an example, in the case of Serovar hardjo in the serogroup Sejroe, there are two genotypes taking part in animal infection, Hardjoprajitno and Hardjobovis (10,11,12,13). This latter aspect is further discussed below in the vaccine section. The WHO taxonomic subcommittee on Leptospira is constantly keeping vigilance on the development of new techniques that are being applied in this area and the classification of Leptospira is being continually updated.

Morphological structures and related protective antigens
Leptospira is a helical organism with hooked ends; this could be easily visualized under a dark field microscope with an appropriately prepared, unstained fresh material e.g. infected urine, blood, etc. Electron microscopic studies have revealed that it has an outer envelope or sheath, below this lies the protoplasmic cylinder (PC). At either end of the PC is attached one axial filament, which is synonymous to the flagella of the Gram-negative organisms (14,15,16,17).

The axial filament at each end runs only half way through the protoplasmic cylinder. The presence of more than one axial filament at each terminal of the PC has also been reported by Chang and Faine (17). It has further been suggested that the axial filament is protein in nature (16,17) similar to that of the flagellar material of Gram-negative organisms. The protective efficacy of purified axial filament has not yet been elucidated, although methods for purification have been worked out by more than one group of workers (16,17).

Anderson and Johnson in 1968 made a significant observation (18). They noted that specific antibodies in the presence of the complement could disrupt the outer envelope of both pathogenic and non-pathogenic species and the protoplasmic cylinder could be disrupted by lysozyme. The electron microscopic studies of the purified outer envelope has revealed that it is mostly triple layered but 5-7 layers are also not rare (18,19). The serological, immunogenic, and some of the chemical properties of the purified outer envelope have also been revealed by Anderson and Johnson (18) and Palit et al. (19,22).

Chang and McComb (20) found that the nonpathogenic Biflexa patoc1 strain contained the erythrocyte sensitizing substance (ESS), which was genus specific in the haemagglutination test. Subsequently, Palit et al. in 1974 demonstrated that the ESS resides in the purified outer envelope preparation of the organism (19). The protective efficacy of the purified outer envelope from serovar canicola, Hond Utrecht IV strain has been demonstrated to be protective in hamster against homologous virulent challenge by Auran, Johnson and Ritzi (21). From the same organism Palit et al. worked out the chemical nature of the purified outer envelope containing polysaccharide, lipid, protein, amino sugars and at least 16 identifiable amino acids (22).

To our knowledge there is no published work on the protective efficacy of the purified flagellar preparation of pathogenic leptospira; the insufficient yield of purified axial filament may be a limiting factor.

Extra-cellular protective antigens
Our unpublished findings suggest that the cell-free extra-cellular material of appropriately grown virulent Leptospira serovar pomona is protective against homologous virulent challenge in appropriately immunized hamster. However, this will need further experimental work to find out the lasting immunity of such protective antigen(s) in both laboratory animal, hamster and target species. It is therefore imperative that some of the protective antigens are present in the outer envelope of pathogenic Leptospira and possibly in the cell-free extra-cellular growth material of virulent serovar pomona. No such work has been done with purified axial filament and protoplasmic cylinder of the pathogenic Leptospira. Under the circumstances it is logical to use killed, whole culture vaccine with appropriate adjuvant to immunize the livestock.

Vaccine development
Leptospirosis is a zoonotic disease. It is viewed by many human vaccine protagonists that mass vaccination of livestock will minimize the incidence of human leptospirosis in occupational groups of humans, particularly those who are involved in animal industries. However, the spread of the disease will remain open through the contaminated urine of carrier animals like rats, wild animals, etc. Unless the demand for the human vaccine is high, no commercial manufacturer is willing to invest for the development of human vaccine. It is imperative that for the manufacture of human vaccine, one must develop an appropriate protein or serum free medium which will support growth and elaborate all the protective antigens at least to the same extent as is the case with the EMJH medium containing bovine serum albumin fraction. Once these challenges are met, then only one can think of developing a safe human vaccine against various serovars of pathogenic Leptospira.

However, some countries like Japan and China are using killed, whole culture cells, which are grown in animal serum medium for vaccinating rice field workers and some other occupational groups. This could be considered as somewhat desperate measure as such human vaccines have considerable post-vaccination local and systemic reaction with varying degrees of severity.

Animal vaccines
The development of animal vaccines depends on the particular need occasioned by the prevalence of pathogenic serovars in a particular country. As it stands, in Australia and New Zealand animal vaccination is conducted with serovars pomona and hardjo (Hardjobovis) for cattle and serovars pomona and tarassovi for domestic pigs (23,24).

In New Zealand the need to include serovar copenhageni (icterohaemorrhagiae) along with serovars hardjo and pomona has also been addressed and a trivalent vaccine is available for use in cattle (25). In U.K. and Ireland the incidence of serovar hardjo infection has prompted the use of the monocomponent hardjo vaccine. A highly protective vaccine has been developed by the author and is being used.

Although the need for the serovar hardjo vaccine is an important one in the USA, the prevalence of serovars pomona, icterohaemorrhagiae, canicola and grippotyphosa also warrant their inclusion in a vaccine (26).

It is unanimously accepted by most microbiologists that the principal consideration in development of a highly protective vaccine depends on several factors:-

The development and use of an appropriate growth medium and the procurement of appropriate strains of the organisms, preferably a local isolate from virulent infection.

The preservation of all important protective antigens as a pre-requisite during and after the manufacturing process of the vaccine.

Finally, the testing of every batch of the finished product to satisfy the appropriate compendium requirements e.g. BP, US CFR tests, etc. preferably by an independent authority.

Collectively, all the above-mentioned measures mostly lead to the use of a highly protective vaccine. To this effect various groups have done enormous amount of work and some of it is being discussed below.

Seed culture for vaccine(s)
The following concepts and approach are always highly productive in any vaccine development project:

Wherever possible use local isolates.
Ensure that the isolate is a recent one from preferably the target species for which the vaccine is being developed.
Authenticate the vaccine strain by a WHO reference laboratory on Leptospira.
Stock multi-dose seed culture in a substantial number in liquid nitrogen (27).

This will serve as the source of uniform seed lots for the manufacture of vaccine. Ensure the purity of the culture by appropriate serotyping and by the REA analysis (8,9) prior to the storage in liquid nitrogen. One can store the culture for number of years in liquid nitrogen without any change in the innate properties of the seed when it is subsequently resuscitated in an appropriate medium.

Growth for vaccine manufacture
It is also well known that some of the serovars of Leptospira are difficult to cultivate and in that respect the serovar hardjo is a notorious one. Leptospires require fatty acid for growth but the presence of some of the free fatty acids is detrimental to its growth (3,4).

The albumin-fatty acid broth is the most useful basic growth medium but appropriate growth trials are necessary to choose the right bovine serum albumin faction V and the supply of appropriate fatty acid source e.g. Tween (80 or 40) preparations.

The use of appropriate vitamins also help in the growth since one must achieve sufficient cell density in the final growth to be able to formulate vaccine containing in excess of 108 organisms per ml. of the final vaccine (23).

It is economical to grow the organism in 350 to 450 liters volume in a portable stainless steel tank, which will result in a vaccine batch of 1500 to 2000 liters after formalin killing, and in situ adjuvanting with aluminium hydroxide adjuvant. The final cell concentration of the vaccine may depend on the strain used in manufacture of the vaccine; it may range from 5 x l06 to 108 per ml. of the finished product.

In vaccine manufacturing process, to attain a high cell yield in the order of 109 per ml. is entirely possible by monitoring the pH, fatty acid supply and the level of toxic free-fatty acid, etc.

Monocomponent serovar hardjo vaccine for cattle
Having given a general outline on the approach to the leptospiral vaccine development, it may be prudent to develop the thoughts on a particular serotype vaccine like serovar hardjo which is a difficult one to grow and has worldwide need for a vaccine. The incidence of serovar hardjo is worldwide and currently it is a concern in USA, Australia, New Zealand, U.K., and Ireland as well as many other countries around the world.

As mentioned before, serovar hardjo, Hardjobovis and Hardjoprajitno genotypes are differentiable by the DNA analysis (11). However, the virulent challenge results have proven that there is 100 per cent cross protectivity between them. Therefore, vaccine manufactured from either of these two strains will have the same protective effect.

In the United Kingdom both variants of serovar hardjo are present whereas in Australia and New Zealand only Hardjobovis has been found (11,12,13). These findings prompted the need for development of the monocomponent serovar hardjo vaccine.

In the next few pages I shall outline on leptospiral vaccines development by a R and D team headed by me at the Commonwealth Serum laboratories, Melbourne (now known as “CSL Limited”).

During 1990, we started developing a monocomponent L. borgpetersenii serovar hardjo killed vaccine for cattle and sheep. It is also known as “Spirovac*”. It is a formalin killed, aluminium hydroxide adjuvanted vaccine. The vaccine was tested in cattle by virulent challenge, the details are outlined below.

Target animal vaccination and virulent challenge
A trial in target animal cattle was initiated during October 1993 to find out the 54 weeks post-vaccination efficacy of the “Spirovac” by virulent challenge.

Experimental design
Crossbred dairy-beef heifers between 8-10 months old and seronegative for serovar hardjo were used in this trial. A group of 9 animals were vaccinated with 2 x 2 ml. of the above mentioned vaccine, at 4-week intervals. A similar number of animals were kept as unvaccinated control in the same housing and pasture at the +CSL farm, Woodend, Victoria.

* “Spirovac” is a monocomponent serovar hardjo, formalin killed vaccine containing aluminum hydroxide as an adjuvant. It is manufactured and marketed worldwide by the +CSL Limited, 45-Poplar Road, Parkville, Victoria, Australia-3053.

All 18 animals were challenged 54 weeks after the first dose of the vaccine with a virulent strain of Hardjobovis, which was proven previously to be highly virulent for cattle. The stored seed lot was resuscitated from the liquid nitrogen storage for this purpose (23,27). The challenge route was intraperitoneal and the challenge dose was 9.l x l09 cells per animal where the total volume was adjusted to 3.5 ml. in EMJH medium for the convenience of animal inoculation.

The challenge criteria were based on post-challenge bacteriaemia followed by the colonization of challenge organisms in the kidneys. The latter condition was determined by periodic urine collection to look for excretion of organism through urine (Leptospiruria). The aseptically collected urine sample was appropriately processed for the challenge organism isolation and its serological identification. The processed urine was examined for the presence of leptospires under dark field microscope, culture medium inoculation and inoculation of young hamsters for the recovery of the excreted live organisms. All inoculated hamsters were sacrificed 15 days post-inoculation; kidneys were collected aseptically and appropriately cultured in the EMJH medium for the isolation and identification of the challenge strain. On the designated day of the urine collection, the processing of the samples and hamster inoculation were performed within a few hours of the collection of samples from cattle (23,24). The post-challenge observation period was 42-43 days. During this time, any animal which failed to show leptospiruria (kidney infection) was subjected to kidney culture for isolation or otherwise of the challenge organism. This was achieved without sacrificing the heifer but by performing a unilateral nephrectomy by appropriate surgical procedures. The details on the processing of the extracted kidney to isolate the organism are readily available as outlined by Palit et al. (23).

Trial results
The results of pre and post-vaccination serological response by the MA test (23) are presented in Table-1. Peak MA titers in vaccinated animals were observed 8 weeks post-vaccination and declined to its lowest value during weeks 40-54. The pre-challenge GM titers of the vaccinated group were 40. None of the unvaccinated control heifers showed any significant MA titer over the 54 weeks period, confirming that the herd remained free from the serovar hardjo infection during the entire pre-challenge experimental period.

On the day of virulent challenge, and 48 hours post-challenge, blood taken in sterile vacutainers containing lithium heparin were subjected to culture. All pre-challenge blood samples were negative over a period of 10 weeks of culture incubation period. The post-challenge samples of unvaccinated controls proved positive for the isolation of serovar hardjo. All vaccinated animals’ samples were negative. The post-challenge leptospiruria results demonstrate that there were no post-challenge kidney colonization by virulent organism in case of the vaccinated group but all unvaccinated heifers were positive. The 18 hamsters injected with the processed urine samples from the vaccinated animals, collected on 28-29 days post-challenge, were all culture negative but hamster injected with control animal’s urine was positive. These findings are consistent with the unilaterally nephrectomised kidney culture results of one excreting unvaccinated and 9 non-excreting vaccinated animal’s kidney culture results.

Table 1
Mean microscopic agglutination test titers of vaccinated and unvaccinated heifers

Weeks Post 1st Vaccination	Vaccinated	Unvaccinated
0 (1st Vaccination)	< 4	< 4
4 (2nd Vaccination)	219	-
6	3251	-
8	3792	-
16	878	-
24	110	< 4
30	59	-
40	55	< 4
50	35	< 4
54 Challenge	40	< 4

Table 2

Mean post-challenge microscopic agglutination test titers of vaccinated and unvaccinated heifers

Weeks Post Challenge	Vaccinated	Unvaccinated
1 (55) *	533	299
2 (56)	1755	2792
4 (58)	1755	5574

Chi-square = 704.45, P < 0.0001
* Figures in parenthesis are weeks post 1st vaccination.

In Table 2, post-challenge MA test results are presented. It demonstrates that the GM titer reached its peak on week 4 in unvaccinated controls and was 3.2 times higher than the peak titer of the vaccinated group; this suggests the presence of an active infection in the former group.

Conclusions
A 100 per cent protection of seronegative vaccinated animals recorded on 54 weeks post-vaccination virulent challenge demonstrates the efficacy of the monocomponent serovar-hardjo (“Spirovac’) vaccine. All unvaccinated controls succumbed to the challenge infection.

Vaccination and challenge trial with “Spirovac” at National Animal Disease Centre, Ames Iowa, USA
With the above mentioned “Spirovac” vaccine, Bolin et al. in 2001 (26) further confirmed the efficacy of the vaccine by challenging cattle via the conjunctival route. This finding further confirms the protective efficacy of the “Spirovac” developed by us in the CSL Limited.

Multivalent vaccines
Similarly, a highly efficacious bivalent serovar hardjo and pomona vaccine was developed at the CSL Limited to protect cattle (23). In this vaccine all Australian isolates were used. The respective seed cultures stored in liquid nitrogen were resuscitated separately in EMJH medium for vaccine manufacture (27). In case of the bivalent serovar hardjo and pomona vaccine for cattle, the hardjo component was tested by virulent challenge at 10, 16, 20, 24 and 54 weeks post-second vaccination (23). In this instance animals were vaccinated with the bivalent hardjo-pomona vaccine by administering 2 x 2 ml. doses of vaccines at 4-week intervals by the subcutaneous route.

The details on vaccine development, quality assurance testing to satisfy the British Pharmacopeia and the US code of Federal Regulation (US CFR) tests, the virulent challenge in cattle for serovar hardjo and in domestic pigs for serovar pomona are readily available (23). The virulent organism challenge criteria in target animals have been outlined earlier.

It is universally acceptable that any leptospiral vaccine which can prevent initial bacteriaemia and urinary excretion (leptospiruria) of virulent infection during the recommended protection period of the vaccine is a protective vaccine.

Calf hood vaccination in presence of colostrum derived maternal antibodies
Vaccination of young calves with bivalent hardjo-pomona vaccine in the presence of maternal antibodies at the age as young as 4 weeks had no untoward effect in eliciting protective antibodies. Vaccination of calves in the presence of maternal antibodies was found to reduce the serological response judged by the conventional MAT (Microscopic Agglutination test) titers but not the protective response of the vaccine (23,28).

Thus, the availability of highly protective animal leptospiral vaccines in various combinations is a reality. One may find several multivalent vaccines are readily available as commercial vaccines.

Summary
In the past 40 years of research on Spirochaetes, a substantial progress has been made. Research findings on the morphological structures, chemical composition and protective antigens as well as the development of the EMJH medium for the growth of Leptospira have helped substantially. The development of highly protective animal vaccines and their regular use have helped countries like New Zealand, Australia and to some extent Ireland. Vaccination of domestic animals to control the spread of the disease from animals to humans has made a substantial progress in the above mentioned countries, which employ a decent number of people in primary industries. It is now apparent that young animals in the presence of maternal antibodies could be vaccinated against leptospirosis without compromising on the development of protective post-vaccination immunity.

However, due to the lack of appropriate protein-free medium for the growth of T. pallidum, L. interrogans and L. borgpetersenii no suitable human vaccine is available.

Acknowledgement
I wish to thank my wife, Dr. Nandita Palit and my daughter Dr. Ishita Palit for their help during writing of this manuscript. Thanks are also due to Dr. (Mrs.) S. Ramachandran for being in regular contact with me while writing this manuscript.

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Authors Corresponding address:

Dr. Arabinda Palit
Managing Consultant,
179 Sterling Drive, Keilor East, Victoria, Australia 3033

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).