Sunday, March 29, 2009

Leptospirosis outbreak among strawberry harvesters in Germany

ResearchBlogging.orgHumans can become infected with the spirochete Leptospira if they have wounds that contact contaminated soil or water. Leptospira live in the kidneys of reservoir animals and are released in urine into the environment. The majority of leptospirosis cases occurs in tropical locales, where the warm, moist conditions promote the survival of Leptospira in the environment. Flooding following heavy rainfall can cause large epidemics of leptospirosis in these regions of the world. A research article published in the March 15th issue of Clinical Infectious Diseases by Desai and colleagues describes an unexpected outbreak of leptospirosis among strawberry harvesters in Germany, a country not known for tropical weather. The July 2007 outbreak, while small by the standards of those that occur in tropical countries, was Germany's largest since the 1960s. The authors searched for factors that may have caused the epidemic.

The outbreak occurred among laborers who worked on a strawberry farm near Düren. The strawberry harvesters were seasonal workers from Romania, Slovakia, and Poland. Among the 153 who were employed on the farm during the outbreak, 24 were stricken with leptospirosis, and 13 were hospitalized. Fortunately, no one died. Most who became sick had antibodies against the Leptospira serogroup Grippotyphosa in their blood.

In September 2007, the authors conducted a retrospective cohort study to identify risk factors for acquiring leptospirosis. The strawberry harvesters were questioned about possible sources of exposure to Leptospira before the outbreak, including the presence of wounds, contact with rodents, and consumption of unwashed strawberries. Logistic regression of the data revealed two statistically significant risk factors. First, the odds of the harvesters acquiring leptospirosis increased with the number of days worked with unprotected hand lesions (OR, 1.1; 95% CI, 1.04-1.1). Leptospira probably entered the lesions as the workers reached into the water-logged soil. "Accidental contact with rodents" was listed as the other significant risk factor (OR, 4.8; 95% CI, 1.5-15.9), although the nature of the contact was not described by the authors. Of course recall bias may have skewed the results of the retrospective study. However, there is nothing unusual about these risk factors; contact of skin wounds with moist soil laden with Leptospira and exposure to infected rodents are typical means of acquiring leptospirosis.

The common vole was the main suspect in contaminating the strawberry field with Leptospira. More than 10 mouse holes per square meter were found in the field and surrounding areas when the site was examined in September 2007. Kidneys of voles captured in and near the field were infested with the spirochete. Leptospira was successfully cultured from the kidneys and were identified as members of the serogroup Grippotyphosa, the same serogroup that the antibodies in the patients' blood were targeting.

The strawberry field outbreak is reminiscent of the large epidemics of leptospirosis that sickened thousands of German agriculture workers from the 1920s through the 1960s, with the Leptospira serogroup Grippotyphosa carried by voles and hamsters responsible for many cases. In contrast, most German cases of leptospirosis of late are related to activities outside of work. Recreational activities involving water and exposure at home to infected pets and contaminated soil while gardening are common risk factors. Icterohaemorrhagiae, with rats as the reservoir host, is now the most prevalent serogroup in Germany. So why did a 1960s-style leptospirosis outbreak occur in 2007? The authors propose that the unusual weather in the months preceding the outbreak was a critical factor.

Europe experienced its warmest autumn in 2006 since recordings began, and the warm weather continued through the winter into 2007. In Germany, heavy rainfall accompanied the high temperatures in the months preceding the outbreak. May and June 2007 were Germany's wettest months since 1901. The tropical conditions may have augmented the amount of food available to the voles. In addition, the vole population, which naturally fluctuates every 3-5 years, may have already been at the peak of its cycle in 2007. A population explosion of infected voles may have increased the density of Leptospira residing in the strawberry field. Because the wet, warm conditions were favorable to survival of Leptospira, it is easy to imagine how an outbreak would have occurred among workers who spent days reaching into the contaminated soil with unprotected hands.

In conclusion, the authors present leptospirosis as a potential model for an infectious disease affected by global warming. Only time will tell whether the leptospirosis outbreak caused by the confluence of warm weather and heavy rainfall in a normally temperate region was a statistical anomaly or a sign of things to come.

Featured paper

Desai, S., van Treeck, U., Lierz, M., Espelage, W., Zota, L., Sarbu, A., Czerwinski, M., Sadkowska‐Todys, M., Avdicová, M., Reetz, J., Luge, E., Guerra, B., Nöckler, K., & Jansen, A. (2009). Resurgence of Field Fever in a Temperate Country: An Epidemic of Leptospirosis among Seasonal Strawberry Harvesters in Germany in 2007 Clinical Infectious Diseases, 48 (6), 691-697 DOI: 10.1086/597036

Other references

Jansen, A., Schöneberg, I., Frank, C., Alpers, K., Schneider, T., and Stark, K. (2005). Leptospirosis in Germany, 1962-2003. Emerging Infectious Diseases 11(7):1048-1054.

Pappas, G., Papadimitriou, P., Siozopoulou, V., Christou, L., Akritidis, N. (2008). The globalization of leptospirosis: worldwide incidence trends. International Journal of Infectious Diseases 12(4):351-357.

Tuesday, March 10, 2009

Rhesus monkeys with Lyme spirochetes in the brain

ResearchBlogging.orgLyme disease has been mentioned in the news a lot lately. First it was a pet chimp mauling his neighbor last month in a Connecticut town. Many news accounts of the assault reported that Travis the chimp suffered from Lyme disease and had been given tea laced with Xanax to calm his erratic behavior prior to the attack. Investigators may never figure out what provoked Travis to attack Charla Nash. Was Lyme disease a factor? Maybe (although if I had to guess, it had more to do with a wild animal being kept as a pet, living where it didn't belong).

This past weekend a human reportedly suffering from long-term Lyme disease gunned down a pastor and injured several parishioners in an Illinois church. As with any topic having to do with chronic Lyme disease, experts disagree on whether Lyme disease can trigger aggression (but I'm pretty sure what the shooter's defense attorney believes). One prominent researcher even questions whether the alleged shooter truly had Lyme disease.

A blogger on doubts whether chimps could be stricken with Lyme disease. In reality, seeing a chimp with Lyme disease should not surprise us at all since another nonhuman primate, the rhesus monkey, has been used for years to study neuroborreliosis, the form of Lyme disease caused by infection of the nervous system. Neuroborreliosis is observed in up to 15% of untreated Lyme disease patients. I will present two studies in which scientists examined infection of the central nervous system of rhesus monkeys by the Lyme disease spirochete, Borrelia burgdorferi.

In a 2000 study by Cadavid and colleagues, the investigators found small numbers of spirochetes in the meninges and spinal cord nerve roots of rhesus monkeys four months following laboratory infection with B. burgdorferi by needle inoculation into the skin (see figure below). In humans, the inflammatory response to the spirochetes in the meninges and nerve roots can cause meningitis (headache, stiff neck, and sensitivity to light) and radiculoneuritis (shooting pains and abnormal skin sensations), respectively.

Figure 2b from Cadavid et al., 2000. A single B. burgdorferi cell in the anterior nerve root stained by immunohistochemistry with anti-B. burgdorferi antibodies.

The investigators found no convincing evidence for the presence of B. burgdorferi in the brain parenchyma of the infected rhesus monkeys. B. burgdorferi was not found in the brain by microscopy. On the other hand, B. burgdorferi DNA was detected in brain tissue by polymerase chain reaction (PCR). This result may suggest that the spirochete is present in the brain at low levels, although the authors believed that spirochetes in the meninges surrounding the brain was the real source of the DNA. It's also possible that the N40 B. burgdorferi strain used by the authors was incapable of invading the brain of rhesus monkeys and that another strain if injected into the skin would have found its way to the brain.

Two conditions associated with neuroborreliosis disrupt brain function in humans. The first is Lyme encephalopathy, which can cause fatigue and problems with concentration and memory. Brain activity is clearly affected in patients with Lyme encephalopathy, yet the underlying pathogenic mechanism remains an enigma. Many patients with this potentially disabling condition do not exhibit the classic signs of central nervous system inflammation such as the presence of white blood cells in their cerebral spinal fluid (CSF), production of antibodies against the infective agent in the CSF, or abnormal brain MRIs. Some neuroborreliosis experts have proposed that these cases of Lyme encephalopathy are a result of "toxic-metabolic" effects of infection elsewhere in the body (such as Lyme arthritis in the joints) spilling into the brain. Nevertheless, a low-level infection of the brain by B. burgdorferi cannot be ruled out. More obvious brain involvement is observed in neuroborreliosis patients with encephalitis, in whom brain lesions are detected by MRI, accompanied by white blood cells and production of anti-B. burgdorferi antibodies in the CSF. Symptoms include slight weakness or paralysis affecting one side of the body, spastic muscles, inability to feel sensations, and bladder dysfunction. In rare cases, patients may experience strokes or seizures. Patients with encephalitis are likely to have spirochetes in their brain, usually near blood vessels.

Lyme disease may also be associated with psychiatric illness, particularly depression. Rare cases of panic attacks, bipolar disorder, mania, obsessive-compulsive disorder, dementia, and violent outbursts have also been reported.

A recent study by Mario Philipp's group in Tulane, published recently in The American Journal of Pathology, described the potential effects of B. burgdorferi on brain cell activity of rhesus monkeys. Spirochetes injected into the skin with a needle or by tick bite are unable to make it into the brain of rhesus monkeys, as I explained above. Therefore, the investigators injected live B. burgdorferi spirochetes directly into the right side of the brains of rhesus monkeys. After two weeks, they found that an average of ~10% of oligodendrocytes at the injection sites had undergone death by apoptosis, as detected by fluorescent TUNEL staining (see figure below). In contrast, fewer than 2% of oligodendrocytes underwent apoptosis when sites in the left side of the brain of the same animals were injected with saline. Oligodendrocytes produce the myelin sheaths that surround the axons of neurons and promote transmission of electric impulses; one would expect the loss of oligodendrocytes to affect neuron function.

The in vivo results corroborated ex vivo experiments in which brain slices from rhesus monkeys were incubated with B. burgdorferi for up to eight hours. In the ex vivo experiments, apoptosis of both oligodendrocytes and neurons were detected by fluorescent TUNEL staining and with antibody against activated caspase 3, a more specific marker of apoptosis. The authors speculated that neurons undergoing apoptosis in vivo were removed by phagocytes and therefore went undetected in the brain injection experiment. The other major brain cell types, microglial cells and astrocytes, were spared from apoptotic death both ex vivo and in vivo.

Figure 4A from Ramesh et al., 2008. Oligodendrocytes are stained red with antibody to S-100. Nuclei of cells undergoing DNA fragmentation, a consequence of apoptosis, are TUNEL stained green. When the images are merged, the nuclei of oligodendrocytes undergoing apoptosis become yellow. B. burgdorferi, which are stained in blue, do not appear to be in physical contact with the oligodendrocytes.

Figure 4B from Ramesh et al., 2008. Percentage of oligodendrocytes undergoing apoptosis from 3 sites of spirochete (A-C) and saline (D-F) injections (click on image for larger view).

The Tulane group also detected myriad cytokines and chemokines such as IL-6, IL-1ß, and CXCL13 being produced in the B. burgdorferi-treated brain slices by immunofluorescence antibody staining and DNA microarray analysis. The authors concluded that B. burgdorferi invasion of the brain elicits production of a brew of inflammatory mediators that promote apoptotic death of oligodendrocytes and neurons. It may be true that brain invasion by B. burgdorferi in cases of human neuroborreliosis leads to production of these cytokines and chemokines, as IL-6, IL-1ß, and CXCL13 have been detected in the CSF of neuroborreliosis patients in earlier studies. However, the apoptotic cells observed by the Tulane group may be the unnatural consequence of small areas of the brain being exposed to extremely large numbers of spirochetes. Each site was injected with 5,000 spirochetes, a number far greater than the rare spirochetes observed by microscopy in the brains of neuroborreliosis patients. For the ex vivo experiments, a 2-mm section of brain was incubated with 20 million spirochetes, again a large burden of bacteria. More convincing would be finding apoptosis of oligodendrocytes and neurons in brain tissues in patients with Lyme encephalitis. Clearly more studies are needed to understand the events that really occur when B. burgdorferi invades the brain.


Ramesh, G., Borda, J., Dufour, J., Kaushal, D., Ramamoorthy, R., Lackner, A., & Philipp, M. (2008). Interaction of the Lyme Disease Spirochete Borrelia burgdorferi with Brain Parenchyma Elicits Inflammatory Mediators from Glial Cells as Well as Glial and Neuronal Apoptosis American Journal Of Pathology, 173 (5), 1415-1427 DOI: 10.2353/ajpath.2008.080483

Diego Cadavid, Tim O'Neill, Henry Schaefer, and Andrew R. Pachner (2000). Localization of Borrelia burgdorferi in the Nervous System and Other Organs in a Nonhuman Primate Model of Lyme Disease Laboratory Investigation, 80 (7), 1043-1054