Antigen presentation in the bloodstream: How invariant NKT cells are activated by Lyme disease spirochetes

The spirochete Borrelia burgdorferi is the tick-borne agent of Lyme disease, which affects the joints, nervous system, and heart.  After being deposited into the skin by an infected tick, the spirochete must enter the bloodstream so that it can circulate in the blood to gain access to its target organs.

The host doesn't sit idly as B. burgdorferi establishes an infection.  Invariant natural killer (iNKT) cells are one of the tools deployed by the immune system in its battle against the Lyme spirochetes.  Scientists know this because B. burgdorferi-infected mice lacking iNKT cells ended up with more spirochetes in their tissues and greater joint swelling than mice with a complete immune system.²

iNKT cells are an odd type of T cell.  Like other T cells, iNKT cells have a T cell receptor (TCR), yet they also express protein markers used to identify natural killer (NK) cells.  What makes the iNKT cell invariant is its TCR α chain, which comes only in the version dubbed Vα14 in mice and Vα24 in humans.  Even the β chain of the TCR of iNKT cells is restricted to three types in mice and just one in humans.  The lack of variation is unusual because the α and β TCR chains of conventional αβ T cells come in many forms in each individual, resulting in millions of varieties of TCRs.  This enables conventional αβ T cells to recognize a wide range of microbial peptide antigens when displayed by an MHC molecule on the surface of an antigen-presenting cell (see figure below).  In contrast, the TCRs of iNKT cells recognize a limited set of glycolipids displayed by the antigen-presenting cell's CD1d molecule, which structurally resembles MHC.  So far these glycolipids have been found only in Sphingomonas and B. burgdorferi.

Antigen recognition by T cells.  The "X" represents variable T cell receptor chains.
Figure 1 from ref. 3.

The structures of the B. burgdorferi glycolipids recognized by iNKT cells are shown below.  BbGL-IIc is recognized by mouse iNKT cells, and BbGL-IIf reacts with human iNKT cells.⁴

Structures of B. burgdorferi glycolipid antigens recognized by iNKT cells.  Figure 3d from ref. 3.

iNKT cells are activated when their TCR binds to BbGL-II complexed with CD1d.⁴  The activated iNKT cells secrete cytokines that elicit the appropriate immune response against the spirochetes.  How these cytokines promote killing of B. burgdorferi remains unknown.

To view the process of iNKT cell activation, scientists have recently obtained video footage of the early stages of the immune response to Borrelia burgdorferi circulating in the bloodstream of mice.¹  The study by Lee et al., which appeared in the April issue of Nature Immunology, complements two earlier studies that revealed how the Lyme disease spirochete escapes from the bloodstream of mice to invade the surrounding tissues.^5,6

The investigators employed fluorescence video microscopy to watch the immune cells in action following injection of an engineered B. burgdorferi strain expressing green fluorescent protein (GFP) into the bloodstream.  Although the spleen is better known for filtering bloodstream pathogens, the liver was selected for observation because iNKT cells make up 30% of the T cells in the liver.  In contrast, iNKT cells represent only 2.5% of T cells in the spleen.  Moreover, mice missing their spleen were able to limit B. burgdorferi infection as well as mice having a spleen, suggesting that the spleen is not critical in fighting bloodstream B. burgdorferi.

iNKT cells reside in the liver's sinusoids, which are the specialized capillaries that carry blood through the liver.  Similar to what other investigators have observed, the authors saw iNKT cells creeping along the inner surface of the liver sinusoids in healthy mice (see video below).

iNKT cells crawling within the liver sinusoids of a mouse genetically altered to express green fluorescent protein (GFP) in iNKT cells.  The iNKT cells glow bright green.  The elapsed time is shown at the top right.  Video 2 from ref. 1.

The investigators wanted to figure out which of the antigen-presenting cells found in the liver presented borrelial glycolipid to iNKT cells.  The answer?  After the spirochetes were injected into the bloodstream, they were quickly captured by Kupffer cells, the specialized blood-filtering macrophages that also reside in the liver sinusoids (see figure below).  Unlike iNKT cells, Kupffer cell remained stationary.

Capture of fluorescent B. burgdorferi (thin green bodies) by Kupffer cells (arrowhead).  Kupffer cells are stained red.  B. burgdorferi that avoided capture can be seen bound to the endothelium, trying to escape from the bloodstream into the liver tissue (arrow).  Figure 2e from ref. 1.

During the next several hours, the captured spirochetes were engulfed and broken up by the Kupffer cells so that BbGL-II could be loaded onto CD1d and displayed on the cell surface.  At 8 hours post injection, iNKT cells started to cluster and form stable contacts with Kupffer cells.  The iNKT cells were attracted to Kupffer cells churning out the chemokine CXCL9, a potent iNKT cell attractant.  The evidence for this was that injection of antibodies against the CXCL9 receptor, located on the iNKT cell surface, blocked clustering of iNKT cells.  Interaction of the Kupffer and iNKT cells was accompanied by increased blood and liver levels of the cytokine IFN-γ (interferon-gamma), a sign that the iNKT cells were being activated.

Left panel:  Liver 24 hours after injection of a GFP+ strain of B. burgdorferi into the bloodstream of a mouse.  Arrows indicate spirochetes (thin green bodies) that were not captured.  Kupffer cells are stained a red. The iNKT cells are the large bright green bodies.  The bright iNKT clusters overwhelm the faint red Kupffer cells, which are difficult to see.  Right panel:  To obtain a more convincing image showing contact between Kupffer cells and iNKT cells, a 3D reconstruction of the optical sections through the liver was performed.  Rotation of the image reveals interactions between Kupffer and iNKT cells.

Not all spirochetes were captured.  The investigators saw B. burgdorferi escaping from the sinusoids into the surrounding liver tissue even as other spirochetes were trapped by nearby Kupffer cells (see figures above).  Spirochetes circulating throughout the host probably escaped into other organs in the same manner.  Indeed, large amounts of  B. burgdorferi DNA were detected by PCR in several organs, including the liver, three days after the spirochetes were injected.  Although one doesn't usually think about the effects of Lyme disease on the liver, the authors pointed out that a mild hepatitis is common in Lyme disease patients.  In one prospective study, 40% of Lyme disease patients had at least one liver test abnormality.⁷

By now you may be wondering why the liver would devote such a high percentage of its T cells towards recognizing glycolipids that aren't found on most bacteria.  One answer is that microbes lacking the proper glycolipids may activate iNKT cells indirectly.³  For example, Salmonella typhimurium uses its LPS to coax antigen-presenting cells into making an endogenous glycolipid that gets presented to the iNKT cell by CD1d.⁸  It is also possible that glycolipids that are recognized by iNKT cells are present in other bacteria but are yet to be discovered.


Featured paper

1. Lee, W.Y., Moriarty, T.J., Wong, C.H.Y., Zhou, H., Strieter, R.M., van Rooijen, N., Chaconas, G., & Kubes, P. (2010). An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells Nature Immunology, 11 (4), 295-302 DOI: 10.1038/ni.1855

Other references

2.  Tupin, E., Benhnia, M.R., Kinjo, Y., Patsey, R., Lena, C.J., Haller, M.C., Caimano, M.J., Imamura, M., Wong, C., Crotty, S., Radolf, J.D., Sellati, T.J., and Kronenberg, M. (2008).  NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi.  Proc. Natl. Acad. Sci. USA 105(50):19863-19868.  DOI: 10.1073/pnas.0810519105

3.  Tupin, E., Kinjo, Y., and Kronenberg, M. (2007).  The unique role of natural killer T cells in the response to microorganisms.  Nature Reviews Microbiology 5(6):405-417.  DOI: 10.1038/nrmicro1657

4.  Kinjo, J., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M. R., Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C., and Kronenberg, M. (2006).  Nature Immunology 7(9):978-986.  DOI: 10.1038/ni1380

5.  Moriarty, T.J., Norman, M.U., Colarusso, P., Bankhead, T., Kubes, P., and Chaconas, G. (June 20, 2008).  Real-time high resolution 3D imaging of the Lyme disease spirochete adhering to and escaping from the vasculature of a living host.  PLoS Pathogens 4(6):e1000090.  DOI: 10.1371/journal.ppat.1000090

6. Norman, M.U., Moriarty, T.J., Dresser, A.R., Millen, B., Kubes, P., and Chaconas, G. (October 3, 2008). Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host.  PLoS Pathogens 4(10):e1000169.  DOI: 10.1371/journal.ppat.1000169

7.  Horowitz, H.W.,  Dworkin, B., Forseter, G., Nadelman, R.B., Connolly, C., Luciano, B.B., Nowakowski, J., O'Brien, T.A., Calmann, M., Wormser, G.P. (June 1996).  Liver function in early Lyme disease.  Hepatology 23(6):1412-1417.  DOI: 10.1002/hep.510230617

8.  Mattner J., DeBord, K.L., Ismail, N., Goff, R.D., Cantu III, C., Zhou, D., Saint Mezard, P., Wang, V., Gao, Y., Yin, N, Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L, Savage, P.B., and Bendelac, A. (March 24, 2005).  Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections.  Nature 434(7032):525-529.  DOI: 10.1038/nature03408

Related posts

• Watch videos of the Lyme disease spirochete escaping from the bloodstream of live mice!
• The Lyme disease spirochete hijacks fibronectin to escape from the bloodstream

Congenital syphilis, upward trend (again) in the United States

Syphilis can be deadly if passed from an infected mother to her unborn child. The most recent CDC data show that 6.5% of U.S. infants with congenital syphilis (CS) in 2008 were stillborn or died within 30 days of birth.¹

Newborns with CS who are destined to live begin to show signs of disease within the first few weeks of life.  The main features of CS in early infancy include fever, skin lesions, enlarged liver and spleen, and a chronic runny nose ("snuffles"), which may be tinged with blood.  Bone lesions may lead to Parrot's pseudoparalysis, a condition so painful that the infant will refuse to move the affected extremities.  Ongoing damage to bony tissue may later lead to childhood deformities including saddle nose, sabre shins, and Hutchinson incisors (notched central incisors).  Other late signs of CS include inflammation of the cornea and sudden hearing loss.

The lesions and deformities associated with congenital syphilis are sparked by Treponema pallidum, a spirochete that can cross the placenta from the mother's bloodstream.  The probability of transmission to the fetus depends on how long the mother has been infected with T. pallidum.  The risk of transmission is lower in mothers at later stages of syphilis.  After crossing the placenta, the spirochete invades the fetal organs.  The continuing immune response to persistent T. pallidum infection causes the damage seen in CS.  Early treatment of the mother with penicillin, at least 30 days before delivery, is essential to stop the disease.

The rate of congenital syphilis in the United States has started to creep back up after plummeting over two decades.¹  The incidence of congenital syphilis has gone up from 8.2 cases per 100,000 live births in 2005 to 10.1 in 2008 with most of the increase having occurred in the South.  CS rates in infants born to black mothers have gone up from 26.6 in 2005 to 34.6 per 100,000 live births in 2008 and now account for half of all CS cases.  Since CS is transmitted from mothers with syphilis, CS rates have historically tracked the combined primary and secondary syphilis rate seen in women, which has also started to climb (see figure below).  What factors account for the increased incidence of syphilis?  In one Alabama county, increased syphilis rates in black women were linked to crack cocaine use and the exchange of sex for money or drugs.²  Although more studies are needed to determine whether the same factors are linked to syphilis throughout the South, it should be pointed out that the same factors were associated with the previous syphilis epidemic that peaked in the early 1990s, when there were several thousand yearly cases of CS as opposed to the several hundred seen today.³

Figure from CDC¹

Now that the upward trend in the CS rate has been recognized, public health authorities in partnership with community-based groups must allocate some of their scarce resources to reverse the trend.  With prenatal care and prompt treatment, congenital syphilis can be prevented.

1. Centers for Disease Control and Prevention (CDC) (April 16, 2010). Congenital syphilis - United States, 2003-2008. MMWR Morbidity and Mortality Weekly Report 59(14):413-417.  link

2. Centers for Disease Control and Prevention (CDC) (May 8, 2009).  Primary and secondary syphilis - Jefferson County, Alabama, 2002-2007.  MMWR Morbidity and Mortality Weekly Report 58(17):463-467.  link

3. Nakashima, A.K., Rolfs, R.T., Flock, M.L., Kilmarx, P., and Greenspan, J.R. (Jan-Feb 1996). Epidemiology of syphilis in the United States, 1941-1993.  Sexually Transmitted Diseases 23(1):16-23.  PMID: 8801638

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