Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

The spirochete Leptospira is the agent of leptospirosis, a zoonosis that primarily burdens tropical regions of the world.  Moist conditions promote the survival of Leptospira in soil and fresh water. Although Leptospira could survive out in wet environments if they had to, they thrive in the kidneys of rats and other maintenance hosts, where they form dense masses lining the inner surface of the kidney tubules.  The spirochetes spill into the urine that forms in the tubules, which drain into the bladder.  Animals colonized by their "preferred" serovar (immune type) shed Leptospira throughout their lives without ever showing signs of illness.  The tainted urine ends up contaminating soil and water with infectious Leptospira.

Humans aren't regarded as long-term carriers of Leptospira.  Rather, they are deemed "accidental" (incidental) hosts who may suffer serious complications of acute disease, including kidney failure and lung hemorrhage.  Humans get infected when they come into contact with contaminated water or soil or following direct exposure to infectious animal urine or tissue.  Leptospira enters through cuts in the skin or mucous membranes.  From there the motile spirochete spreads via the bloodstream and invades internal organs, including the kidneys, where they remain for the duration of the disease.  Patients typically stop releasing Leptospira into their urine after they recover from the illness, presumably because the spirochetes have been eliminated from their kidneys.  However, there have been a few reports of Leptospira excreted in urine months or even years following recovery from leptospirosis.  The truth is that no one has ever done a systematic study to determine how common the chronic carrier state is in humans.

A team of investigators from the United States and Peru set out to find long-term carriers of Leptospira.  Their study is described in the February issue of PLoS Neglected Tropical Diseases.  The authors examined the inhabitants of a rural Amazon village of Padrecocha near the city of Iquitos, Peru, where leptospirosis is endemic. The tropical climate is ideal for the survival of Leptospira in the moist environment favored by the spirochete.  Indeed, in an earlier study the authors detected infectious strains of Leptospira in the streams and wells serving the village.  Cattle, pigs, dogs, and rats, all potential carriers, freely roam the area.

Ganoza and colleagues wanted to determine what percentage of the villagers were chronic carriers of Leptospira.  They first identified villagers who were not recently infected with Leptospira.  Out of the 314 healthy villagers enrolled in the study, 102 (32.5%) had no clinical or serological evidence of recent infection; they did not recall experiencing a fever during the previous year (fever is a typical symptom of leptospirosis), and they tested negative for newly-acquired Leptospira infection by IgM ELISA.

The investigators next identified those whose kidney were colonized by Leptospira among the 102 who were not newly infected.  Since Leptospira living in the kidney tubules are shed into urine, they screened urine samples by nested PCR using primers targeting the 16S rRNA gene of Leptospira.  To exclude false-positive signals, the investigators screened the PCR-generated DNA (amplicon) by dot blot analysis with a Leptospira 16S rRNA probe.  Many false positive signals occurred because their Leptospira PCR primers also hybridized to the 16S rRNA gene from Atopobium vaginae, a bacterium recently found to be associated with vaginosis. 

When urine from the 102 "long-term" healthy individuals was screened, Leptospira DNA was found in 6 (5.9%).  Sequencing of the 16S rRNA gene revealed that the carriers were colonized with L. interrogans, L. fainei, and L. licerasiae.  So it turns out that the asymptomatic carrier state is not as rare as initially believed.  More than 1 in 20 individuals who had been healthy for at least a year were colonized with Leptospira in their kidneys.

The investigators found seven additional individuals colonized with Leptospira by screening urine from the other 212 individuals in the study.  Overall the percentage of shedders of Leptospira among all healthy individuals, irrespective of when they were infected, was 4.1% (13/314).  The concentration of Leptospira in the urine of shedders, as measured by quantitative PCR, was low, in the 10²-10⁴/ml range.  In contrast, rats may shed up to 10⁸ spirochetes/ml!

The study unearthed another surprise.  All 13 individuals who were shedding Leptospira at the time of the study (including the 6 chronic carriers) were women.  The proportion of women with Leptospira DNA in their urine (13/13, 100%) was significantly higher than the proportion of women in the group lacking detectable DNA in their urine (199/301, 66%, p = 0.011).  This result raises the possibility that women are more likely to become persistent carriers than men.  However, the authors pointed out that men were underrepresented in the study sample.  Less than one third of the villagers enrolled in the study were men.  Most of the other men were away at work when the authors were recruiting people for the study.  Agricultural occupations, which bring workers into contact with environmental sources of Leptospira, are well-known risk factors for infection by the spirochete in endemic areas.  Hence, male shedders of Leptospira may have been inadvertently excluded from the study.

Another surprising result was that sera from all six chronic carriers failed to agglutinate Leptospira by MAT (microscopic agglutination test), a standard serological test used to check for Leptospira infection whether it occurred recently or years ago.  The authors mentioned that this was entirely consistent with old studies failing to detect agglutinating antibodies in the sera of some maintenance host animals excreting Leptospira. However, another possibility is that the "chronic human carriers" may have actually acquired asymptomatic infections very recently.  They could have been enrolled in the study before the anti-Leptospira IgM and agglutinating antibodies had enough time the accumulate to the cut-off values selected for the IgM ELISA and MAT, respectively.  Although the authors discounted the possibility of newly acquired asymptomatic infections accounting for the seronegativity of the shedders, they recommended a longitudinal study to clarify the issue.

The authors posed several questions raised by their study:

• Does persistent Leptospira infection of human kidneys have any subtle effect on their function? If so, is antibiotic treatment warranted?
• Are some strains of Leptospira more likely than others to persistently infect the kidneys of humans?  .
• Can persistent human shedders be a source of transmission of Leptospira to other humans (and animals)?

Future studies will need to include urine cultures to demonstrate that Leptospira shed by human carriers are alive.

In conclusion, this is an important study that challenges the simplistic notion that humans are incidental hosts of Leptospira.  The reality appears to be more complicated.

Featured paper

Ganoza, C.A., Matthias, M.A., Saito, M., Cespedes, M., Gotuzzo, E., & Vinetz, J.M. (2010). Asymptomatic renal colonization of humans in the Peruvian Amazon by Leptospira. PLoS Neglected Tropical Diseases, 4 (2) DOI: 10.1371/journal.pntd.0000612

Related paper

Ganoza, C.A., Matthias, M.A., Collins-Richards, D., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E., & Vinetz, J.M. (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine, 3 (8) DOI: 10.1371/journal.pmed.0030308

A fresh approach towards a Lyme disease vaccine: targeting the tick

We tend to focus on the pathogen when thinking about how the immune system responds to tick-borne infections.  Ticks also provoke an immune response, even those that don't harbor any infectious agent.  Animals that are repeatedly bitten by ticks will eventually develop immunity to the tick.  There's even a commercial tick vaccine called TickGARD, which protects cattle against infestation by the tick Boophilus microplus.  TickGARD is formulated with a protein located in the midgut of B. microplus.

Animals that are immune to ticks also resist infection by some tick-borne pathogens, including Borrelia burgdorferi (Nazario et al., 1998).  Vaccines designed from a single tick component may also protect host animals from infectious agents.  A tick vaccine formulated with the tick cement protein 64TRP protected mice from being killed by the tick-borne encephalitis virus introduced by its vector, Ixodes ricinus (Labuda et al., 2006).

These earlier studies prompted Erol Fikrig's group at Yale to devise a vaccine that targets an Ixodes tick protein required by B. burgdorferi for a successful infection.  They focused on Salp15, a protein found in tick saliva.  Salp15 binds to the B. burgdorferi surface protein OspC as the spirochete passes through the salivary gland on its way into the skin of the victim.  Salp15 is one of the many bioactive salivary proteins that dampen the immune system to allow the tick to remain attached for several days so that it could complete its blood meal.  B. burgdorferi exploits Salp15 to fend off the immune response in the early stages of infection.  Since Salp15 coats B. burgdorferi, a vaccine targeting Salp15 could make B. burgdorferi vulnerable to killing by the immune system.  Indeed, Salp15 antiserum was able to enhance phagocytosis of Salp15-coated B. burgdorferi by mouse macrophages in vitro.

Despite the promising in vitro results, Salp15 as a vaccine was only partially protective in animal studies.  55-60% of mice actively immunized with Salp15 or passively immunized with Salp15 antiserum ended up infected with B. burgdorferi following challenge with infected Ixodes scapularis ticks.  Moreover, ticks were able to feed normally on mice immunized with Salp15, indicating that the animals did not acquire tick immunity.

Where Salp15 shined was in improving the efficacy of another Lyme vaccine.  The OspA vaccine requires several doses to achieve maximum protection against B. burgdorferi.  When Salp15 was combined with OspA, a single dose of the mixture spared 70% of mice from infection, whereas only 10-20% of mice immunized with a single dose of OspA or OspA plus Salp25D (an irrelevant tick salivary protein) were protected.  Thus future Lyme disease vaccines that target a component of the spirochete could also include Salp15 to enhance their protective capacity.

Scientists are undoubtedly examining other tick proteins as potential vaccines against ticks and the pathogens they transmit.

Featured paper

Dai, J., Wang, P., Adusumilli, S., Booth, C.J., Narasimhan, S., Anguita, J., and Fikrig, E. (November 19, 2009).  Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent.  Cell Host & Microbe 6:482-492.  DOI: 10.1016/j.chom.2009.10.006

Other references

Labuda, M., Trimnell, A.R., Licková, M., Kazimírová, M.,Davies, G.M., Lissina, O., Hails, R.S., and Nuttall, P.A. (April 2006).  An antivector vaccine protects against a lethal vector-borne pathogen.  PLoS Pathogens 2(4):e27.  DOI: 10.1371/journal.ppat.0020027

Nazario, S., Das, S., De Silva, A.M., Deponte, K., Marcantonio, N., Anderson, J.F., Fish, D., Fikrig, E., and Kantor, F.S. (June 1998).  Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity.  American Journal of Tropical Medicine and Hygiene 58(6):780-785.  Link

Tigecycline fails to eradicate persisting Borrelia burgdorferi

Antibiotics are usually successful in treating Lyme disease, especially if administered early.  The problem is that some patients continue to experience symptoms even after completing the recommended treatment regimen.  Although the current IDSA guidelines assert that the lingering symptoms are not due to persisting Borrelia burgdorferi,  the mouse model of Lyme disease clearly demonstrates the survival of live (albeit disabled) spirochetes following treatment with ceftriaxone, one of the antibiotics used to treat disseminated Lyme disease. As I wrote in an earlier post, the key question that must be answered is whether the lingering spirochetes are responsible for the persisting symptoms.  If so, a more potent antibiotic that could eliminate all of the spirochetes (or enough of them to allow the immune system to quickly mop up the rest) would be desired.

The newer antibiotic tigecycline was recently approved for treating skin and intra-abdominal infections caused by complex mixtures of bacteria.  Tigecycline is a tetracycline antibiotic, which blocks translation of mRNA into proteins by sticking tightly to the 30S ribosome subunit of bacteria.  In turns out that tigecycline exhibits greater antimicrobial activity than ceftriaxone (and doxycycline, another Lyme antibiotic) against B. burgdorferi, at least in the test tube.

Barthold and colleagues tested tigecycline to see if it could eradicate B. burgdorferi from persistently infected mice.  Groups of mice infected for 4 months with B. burgdorferi were treated with ceftriaxone (10 mice), a low dose of tigecycline (7 mice), or a high dose of tigecycline (9 mice).  A control group was sham treated with saline.  Three months after treatment was completed, the mice were examined to see if the spirochetes were still in the tissues.  As you might expect, B. burgdorferi DNA was detected by PCR at high levels in multiple tissues in all 10 mice that were administered saline, and the spirochetes were successfully cultured from the tissues.  In the ceftriaxone group, as shown in an earlier study by Barthold's lab, low levels of B. burgdorferi DNA were detected in leg joints from all 10 mice.  Although B. burgdorferi could not be cultured from the ceftriaxone-treated mice, ticks that fed on the mice were able to transmit the spirochetes to immunodeficient (SCID) mice, where B. burgdorferi was detected by PCR at the end of the experiment.  Since transmission requires active penetration of B. burgdorferi through several tissue barriers, the spirochetes that remained following antibiotic treatment must have been alive, although they could not be cultured.  Moreover, several B. burgdorferi mRNA transcripts were detected in some of the ceftriaxone-treated mice, another hint that the spirochetes remained viable (mRNA, unlike DNA, is extremely labile and would quickly degrade in dead bacteria).

How well did tigecycline work?  Despite its heightened potency against B. burgdorferi in test tube experiments and its much longer half-life in mice, tigecycline didn't work any better than ceftriaxone in eliminating the spirochetes, even at the higher dose.

The studies performed by Barthold's group raises several questions:

• Would prolonging antibiotic treatment eventually eliminate the spirochetes?  Curiously, tigecycline was administered to the mice for only 10 days.
• Do the spirochetes that remain following antibiotic treatment cause disease?  So far, the answer appears to be, "No."  Unlike the saline-treated mice, the antibiotic-treated mice did not exhibit any signs of disease.  Necropsies failed to reveal a inflammatory response against the spirochetes remaining in the tissues.
• Do the spirochetes that survive antibiotic treatment give rise to disease later?  The earlier study by Barthold's group showed that although viable, the spirochetes were slowly diminishing in number in the tissues of mice that were treated with antibiotics.  If the mice were followed for a longer period of time, would the disabled spirochetes eventually disappear or revive to elicit a relapse of disease?
• Is the mouse model even relevant to human Lyme disease?  Borrelia burgdorferi has evolved to persist in the mouse, its natural host, and may act differently in humans.  Obviously the experiments presented here can't be performed on humans, but an animal model that is more relevant to human Lyme disease may be more appropriate for addressing the issues raised by Barthold's work.

Featured paper

Barthold, S.W., Hodzic, E., Imai, D.M., Feng, S., Yang, X., and Luft, B.J. (February 2010).  Ineffectiveness of tigecycline against persistent Borrelia burgdorferi.  Antimicrobial Agents and Chemotherapy 54(2):643-651.  DOI: 10.1128/AAC.00788-09

Related paper

Hodzic, E., Feng, S., Holden, K., Freet, K.J., and Barthold, S.W. (May 2008).  Persistence of Borrelia burgdorferi following antibiotic treatment in mice.  Antimicrobial Agents and Chemotherapy 52(5):1728-1736.  DOI: 10.1128/AAC.01050-07

Did spirochetes kill off the Indians in Massachusetts before the Mayflower landed?

The coast of present-day Massachusetts was inhabited by several Native American tribes in the early 17th century.  Fishermen, traders, and explorers from the Old World encountered the Indians during their occasional travel through the area.  However by the time the Mayflower landed in Plymouth in 1620 to establish a colony, a mysterious epidemic had ravaged coastal New England, killing up to 90% of the indigenous population during the years 1616 through 1619.  Experts have yet to agree on the cause of the epidemic.  Smallpox, plague, and yellow fever, all highly lethal diseases, have been blamed.

Native American tribes of southeastern Massachusetts, approx. 1620 (Figure 1 from Marr and Cathey)

An article in the new issue of Emerging Infectious Diseases offers leptospirosis, caused by Leptospira spirochetes, as another possible agent of the 1616-1619 epidemic.  This is based not on any new information but on an examination of the lifestyle of the Native Americans of early 17th century New England.

Rats infected with Leptospira may have stowed away in the ships that sailed from Europe to the New World.  Because Leptospira lives in the kidney tubules of chronic carriers, infected rats released into the New World would have contaminated their surroundings every time they urinated.  Since Leptospira can survive in moist soil and fresh water, indigenous rodents and other animals could have become chronically infected with Leptospira, further spreading the spirochete throughout the region. The Indian lifestyle provided plenty of opportunities for exposure to Leptospira through skin abrasions and swallowing of contaminated water or food.  Their high-risk activities included the following:

• walking around barefooted
• storing food accessible to rodents
• swimming and bathing in streams and ponds
• working on moist soil to raise and harvest crops

Leptospira has little effect on the health of carrier animals yet can cause humans to fall ill.  Many escape with what may be confused with a mild case of the flu, but some end up suffering with life-threatening symptoms.  Eyewitnesses of the 1616-1619 epidemic reported that victims were afflicted with skin lesions, severe headaches, yellowing of the skin (likely jaundice), and bloody nose (possibly from lung hemorrhage), which are all symptoms of the severe form of leptospirosis.  Even today leptospirosis can be deadly with reported fatality rates of greater than 50% among those with severe lung hemorrhaging.

While the authors should be commended for even considering a disease of a spirochete that is often ignored (at least by those in the developed world), I don't think Leptospira is what killed off the Indians. One strong argument against leptospirosis being the cause of the 1616-1619 epidemic is that Leptospira is not hardy enough to survive the cold winters that Mother Nature inflicts upon New England.  Since the fatalities continued through the winter, leptospirosis is unlikely to be the culprit.

Whatever the cause, the epidemic may have been a pivotal event that facilitated English colonization of coastal Massachusetts since the surviving Indians lacked the capacity to resist the newcomers.

Featured paper

Marr, J.S., & Cathey, J.T. (2010). New Hypothesis for Cause of Epidemic among Native Americans, New England, 1616–1619 Emerging Infectious Diseases, 16 (2), 281-286 DOI: 10.3201/eid1602.090276

The Lyme disease spirochete has flagella but doesn't use them to penetrate the gut of the feeding tick

The Lyme disease agent Borrelia burgdorferi possesses flagella, which are the thin motility structures owned by many members of the bacteria world.  Flagella propel bacteria towards their destination by spinning (read this post to see how flagella function in Borrelia).  It has been assumed B. burgdorferi spin their flagella whenever they need to move from one location to another.  A recent paper in The Journal of Clinical Investigation has demonstrated otherwise, at least for B. burgdorferi in the midgut of a feeding Ixodes (blacklegged) tick.

Borrelia burgdorferi spends much of its life cycle lying dormant in the midgut of Ixodes ticks.  The spirochetes lightly pepper the inner surface of the midgut cell lining, with a few spirochetes also hiding between cells.  None live at the base of the cells at the basement membrane surrounding the midgut.  The spirochetes wake up and multiply only when the tick attaches to an animal or human and imbibes blood.  A few days into the blood meal, some spirochetes eventually breech the basement membrane and enter the hemocoel, the fluid-filled space between the tick organs where they must avoid the phagocytes patrolling the area.  From there the spirochetes invade the salivary glands, which can then release B. burgdorferi-tainted saliva into the skin of the victim.  After completing its satisfying meal of blood, the tick detaches from the skin of the victim, who may end up suffering from Lyme disease.

Dunham-Ems and colleagues wanted to follow the spirochetes in the midgut as ticks took their meal of blood.  They engineered a strain of B. burgdorferi expressing green fluorescent protein so that they could watch the spirochetes in the gut by fluorescence microscopy.  They allowed ticks with the green B. burgdorferi strain in their midguts to feed on laboratory mice.  24, 48, and 72 hours after the ticks were placed on the mice, the investigators removed the midguts and examined the organ by fluorescence microscopy to see what the spirochetes were doing.  Surprisingly, they never saw motile spirochetes in the midgut even though the spirochetes eventually found their way at 72 hours into the hemocoel, where they were highly motile.

If the spirochetes in the midgut remained nonmotile during tick feeding, how did they reach the basement membrane? The few spirochetes that initially populated the midgut multiplied exponentially and formed growing networks of spirochetes on the cell surfaces as the tick drank blood from the mice.  By 72 hours the networks eventually coalesced, encasing many gut cells in spirochetes (see the figures below).  Spirochetes at the base of the encased cells were poised to penetrate the basement membrane and invade the hemocoel.  All of this happened without B. burgdorferi ever spinning its flagella.  Only when they broke through into the hemocoel did the flagella start spinning.

Figure 4F-H from Dunham-Ems 2009.  Confocal fluorescence microscopy of a midgut from a nymph that fed on a mouse for 72 hours.  Panel F shows a network of spirochetes (green) attached to the inner surface of the midgut.  An optical section taken 24-26 µm into the lining of the midgut (panel G) reveals aggregates of spirochetes surrounding the cells. Panel H shows that some spirochetes have made it to the basement membrane, which is found 50 µm below the surface.  The midgut cell membrane is stained in red.  Scale bars = 25 µm.  Some of the gut cells are extremely large because they are differentiating as part of the digestion process.

Figure 5 A and B from Dunham-Ems 2009.  Silver stain of sections from ticks that fed for 48 hours (panel A) and 72 hours (panel B).  The edges of the epithelial cells are easier to see than in the previous figure.  Arrows point to aggregates of spirochetes (hairy bodies).  At 72 hours at least one cell is encased in spirochetes.  Scale bars = 25 µm.  Some of the cells are extremely large because they are differentiating as part of the normal digestion process of the tick (dc, differentiated cells; uc, undifferentiated cells). 

The investigators also found that something in the tick midgut inhibited the motility of B. burgdorferi.  They placed a bit of minced midgut from a tick that had been feeding on a mouse for 72 hours at the edge of a gelatin matrix containing motile fluorescent B. burgdorferi.  (Because of their helical shape, spirochetes love to move about in viscous substances such as gelatin.)  Most of the spirochetes near the tissue ceased moving and remained motionless throughout the 15 minute viewing period.  In contrast, the spirochetes continued moving when mouse blood was placed at the edge of the gelatin matrix.

Why does B. burgdorferi employ a nonmotile mode of penetration of the cell lining of the tick midgut?  Is there some advantage for the spirochete to avoid using their flagella?  As blood is known to be a powerful chemoattractant for B. burgdorferi, the authors offered the following explanation:

These results, although counterintuitive at first blush, make sense; if blood in the midgut acted as a chemoattractant, spirochetes would never disseminate during feeding.

Hence the "inhibitor" of motility released by the tick gut serves as a signal to the spirochete to not spin their flagella.

To me, this explanation isn't satisfying.  It would seem simple for B. burgdorferi to have evolved a regulatory scheme that would allow the spirochete to temporarily uncouple blood chemotaxis from flagellar motility so that they could bore through the gut lining in minutes rather than days. There must be a reason why B. burgdorferi chooses to take its time to penetrate the gut lining.

Perhaps B. burgdorferi delays its journey to the salivary glands to allow the feeding tick to properly prepare the skin, which is an inhospitible environment for both tick and spirochete.  As the tick feeds, it releases a brew of anti-immune factors into the skin to protect itself from attack by the immune system.  Early arrival of B. burgdorferi to the salivary gland would release the spirochetes into the skin before the anti-immune factors have taken full effect, potentially allowing the host immune system to eliminate the spirochetes before they could establish an infection.

Reference

Dunham-Ems, S.M., Caimano, M.J., Pal, U., Wolgemuth, C.W., Eggers, C.H., Balic, A., & Radolf, J.D. (2009). Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks. Journal of Clinical Investigation. 119(12):3652-3665. DOI: 10.1172/JCI39401