How a new species of Lyme disease bacteria was discovered

A new agent of the tick-borne illness known as Lyme disease has emerged in the upper Midwest.  The bacterium is genetically related to Borrelia burgdorferi, until now believed to be the only cause of Lyme disease in the United States.  The name proposed for the bacterium is Borrelia mayonii because the work was conducted at the Mayo Clinic.  B. mayonii has not been detected in patients outside of the Midwest (so far).  The findings are described in The Lancet Infectious Diseases.

The new species was discovered at the Mayo Clinic during routine testing of specimens (blood, cerebral spinal fluid, and joint fluid) received from all regions of the U.S.  Over 100,000 specimens collected from 2003 through 2014 were tested for Lyme disease bacteria by real-time PCR . The PCR probes were designed to detect the oppA1 gene from Borrelia species belonging to the Lyme disease group, known in the scientific literature as "B. burgdorferi sensu lato."  The Lyme disease group comprises 18 species that fall into the same genetic cluster within the genus Borrelia.  They include species known or suspected to cause Lyme disease (B. burgdorferi, B. garinii, B. afzelii, B. spielmanii, B. valaisiana, B bissettii, B. bavariensis, and B. lusitaniae) and another ten species that do not cause illness.  The PCR probes do not react with DNA from species belonging to the other cluster of Borrelia, the relapsing fever group.

The key to the discovery of the new species was the melting temperature analysis routinely programmed onto the end of real-time PCR runs.  The oppA1 PCR products amplified from B. burgdorferi strains have melting temperatures of 63.6 through 64.9°C.  For other Lyme disease species, the melting temperature ranges from 52.3°C (B. valaisiana) to 59.2°C (B. californiensis).  Therefore, the melting temperature of the oppA1 PCR product was used to distinguish B. burgdorferi from other Lyme disease Borrelia.

Over 9,000 specimens were collected from the states of Minnesota, Wisconsin, and North Dakota from January 2012 through September 2014.  102 were PCR positive, and most of the PCR products had the melting temperature profile of B. burgdorferi.  However, six had melting temperatures ranging from 60.4°C to 61.2°C, too low to be B. burgdorferi but too high to be any other member of the Lyme disease group.  The novel spirochetes were cultured from the blood of two of the patients.  The DNA sequence of several "housekeeping" genes of the new isolates differed enough from those of other Borrelia species to signify that a new Borrelia species has been found.  The investigators named the new spirochete Borrelia mayonii.  No specimen collected from other regions of the U.S. exhibited the atypical melting temperatures, and neither did any collected earlier than 2012 from the Midwest.  These findings led the authors to conclude that B. mayonii has recently emerged in the upper Midwest and that the six patients are the first known cases of Lyme disease to be caused by the new species.

The investigators also collected Ixodes scapularis ticks in Wisconsin.  PCR and melting temperature analysis showed that 19 of 658 ticks (2.9%) were positive for B. mayonii, 195 (29.6%) positive for B. burgdorferi, and two positive for both.

One striking feature of B. mayonii infections is the large number of spirochetes circulating within the patients.  The densities ranged from 420,000 to 6,400,000 bacterial cells per milliliter, at least a hundred times higher than observed in the blood of patients with B. burgdorferi infections.  The numbers were high enough that spirochetes could be seen in blood collected from one of the patients.

Fig. 1b from Pritt et al., 2016

The six patients had many of the typical Lyme disease symptoms:  headache, neck pain, muscle aches, joint pain, and fatigue.  Although mild fever is also common in Lyme disease, two of the six patients had severe fevers with temperature readings approaching 40°C (104°F).  Four had nausea or were vomiting, which are also uncommon Lyme disease symptoms.  Two patients were hospitalized because of the severity of their illness.  Lyme disease may be missed in those infected with B. mayonii because of the unusual symptoms.

The standard two-tier antibody test, which uses B. burgdorferi antigens to detect reactive antibody, may help with the diagnosis.  Blood specimens from five of the six patients were tested.  Four patients either tested positive or, if negative initially, tested positive with blood drawn weeks later.  The one patient who tested negative had blood drawn only on the first day of illness, so it's likely that the antibody response hadn't kicked in fully.  The test appears to help with the diagnosis of Lyme disease caused by B. mayonii, but the number of patients tested was too small to draw firm conclusions.

The authors conclude:

In view of the differing clinical manifestations for patients infected with the novel B burgdorferi sensu lato genospecies, it is likely that Lyme borreliosis is not being considered—and therefore not diagnosed—in some patients with this infection. The clinical range of illness must be better defined in additional patients to ensure that physicians can recognise the infection and distinguish it from other tick-borne infections. Many tick-borne pathogens have global distribution, therefore studies are needed to establish the geographic distribution of human beings and ticks infected with the novel B. burgdorferi sensu lato genopecies. Finally, clinicians should be aware of the potential role of oppA1 PCR for diagnosing infection with this novel pathogen.

Reference

Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL, & Petersen JM (2016). Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. The Lancet. Infectious diseases. PMID: 26856777