BIOLOGY: The Spirochaete: Classification

Spirochaetes were first identified by morphological characters and motility using dark-field microscopy. The first Borrelia species microscopically observed was Borrelia recurrentis by Otto Obermeier in 1868. Later, the analysis of 16S ribosomal gene sequences confirmed the order Spirochaetales as one of the bacterial phyla. This Order is divided into five families, Borreliaceae, Breznakiellaceae, Sphaerochaetaceae, Spirochaetaceae, Treponemaceae, and one not validly published family “Termitinemataceae”. For further information on the taxonomy of (not only veterinary and medically important) spirochaetal microorganisms, please see the List of Prokaryotic Names with Standing in Nomenclature (LPSN, https://lpsn.dsmz.de/ check under “browse by rank” the class Spirochaetia). The family Borreliaceae comprises the genera Borrelia and Cristispira.

Genomic analyses of bacterial species have benefitted from new sequencing technologies developed since the turn of the century. This has had a massive influence on bacterial taxonomy and with this on bacterial nomenclature. When bacterial genera are being divided, it leads to name changes and that in turn may introduce challenges to diagnosis of bacterial infections by medical professionals. The genus Borrelia was no exception and in 2014 a paper was published to divide the genus into relapsing-fever Borrelia and the Lyme borreliosis group of species (i.e. B. burgdorferi sensu lato) which was then termed “Borreliella”. This division of the genus was not widely supported and a publication re-instated the name “Borrelia” for all members of the genus. Since this did not allow the designation of new names, in some databases (such as GenBank) members of the B. burgdorferi sensu lato species complex are still named “Borreliella” although in other databases (such as LPSN), Borrelia is given as the correct name for species within the B. burgdorferi sensu lato complex. The Borrelia MLST database hosted at the University of Oxford (https://pubmlst.org/borrelia) also uses Borrelia for all members of the species complex.

BIOLOGY: The Genus Borrelia
The genus Borrelia is divided into species that form clusters in phylogenetic trees such as the relapsing-fever species, reptile-associated species and species of the B. burgdorferi sensu lato complex.

Historically, relapsing-fever Borrelia species were classified according to their arthropod vector, but the taxonomy now relies on genetic criteria. Approximately 20 named relapsing-fever species are transmitted by soft ticks (genera Ornithodoros or Argas), B. miyamotoi by hard ticks (the same Ixodes species as for B. burgdorferi sensu lato) or by the human body louse as it is the case for Borrelia recurrentis . Borrelia species that have been reported to cause relapsing fever in humans include B. duttonii, B. crocidurae, B. hispanica, Ca. B. kalaharica in Africa, B. persica in Asia, B. hermsii and to a lesser extent B. turicatae and B. parkeri in Western North America and B. miyamotoi in North America and Eurasia. Borrelia anserina, the type species of the genus, has a world-wide distribution, and can cause avian borreliosis in fowl. Borrelia theileri is another species that has a world-wide distribution, it fell historically into the relapsing-fever group but is vectored by the hard-tick Rhipicephalus. It can cause bovine spirochaetosis.

 Reptile-associated species are transmitted by hard ticks and the pathogenic potential (medical or veterinary) of these species is unknown.

Since the 1980s interest in the agent of Lyme borreliosis (also called Lyme disease) has increased. When B. burgdorferi was originally described it was believed to be the only species responsible for Lyme borreliosis. However, in the last 25 years genetic investigations of a large number of strains clearly demonstrated that the genetic diversity of this species was greater than expected. To date, the B. burgdorferi sensu lato species complex can be divided into at least 20 species. Borrelia burgdorferi sensu stricto and B. bissettiae are present in Europe and in the USA but absent in Ixodes persulcatus in Eastern Europe and Asia;  B. afzelii, B. bavariensis (previously B. garinii ospA type 4), B. garinii, B. turdi in Eurasia; B. spielmanii, B. valaisiana and B. lusitaniae in Europe; B. japonica and B. tanukii are restricted to Japan, B. sinica and B. yangtzensis are present in China; and B. andersonii, B. americana, B. carolinensis, B. californiensis, B. kurtenbachii, B. maritima and B. mayonii in the USA. Borrelia species of the LB group found in South America include B. chilensis, B. ibitipoquensis and Candidatus B. paulista. PCR-based methods are used to identify these species and the most commonly used targets for DNA amplification, hybridisation and restriction polymorphism analysis were rRNA genes, and intergenic spacers, flaB and OspA genes. Since 2008 a Multilocus Sequence Typing (MLST) scheme is available that allows fine resolution between and within species and has been used for taxonomic discrimination. Intensification of whole genome sequencing has allowed the development of a core genome MLST (cgMLST) that permits even finer resolution of lineages within B. burgdorferi sensu lato. It has been suggested that Borrelia species which phylogenetically (16S rRNA/rrs sequences) cluster with relapsing fever could be responsible for Lyme-like diseases. Such borreliae, which are transmitted by hard ticks, have been identified in the USA (B. lonestari) and in I. persulcatus in Eastern Europe and Asia, I. ricinus in several European countries and I. scapularis and I. pacificus in the USA (B. miyamotoi).

Pathogenicity
Borrelia afzelii, B. bavariensis, B. burgdorferi sensu stricto, B. garinii, B. spielmanii and B. mayonii are assured human pathogenic and have been recurrently isolated from clinical cases of LB. There is sufficient evidence resulting from isolation from patients, PCR and serological data, that the division of B. burgdorferi sensu lato into genospecies has clinical relevance. Thus, B. burgdorferi sensu stricto is most often associated with arthritis, particularly in North America where it is the main cause of Lyme disease, B. bavariensis and B. garinii are associated with neurological symptoms and B. afzelii with the chronic skin condition, acrodermatitis chronica atrophicans (ACA). Interestingly, B. mayonii has been observed in the blood of infected persons with clinical episodes. Overlap between species in relation to clinical manifestations occurs and all cause the pathognomonic symptom erythema migrans (EM), though there is evidence in Europe that this early sign occurs more frequently in B. afzelii infections than in those caused by B. garinii. Borrelia valaisiana has not yet been isolated from any human sample, and thus, has been considered to be non-human pathogenic. Borrelia lusitaniae has been isolated from symptomatic humans in Portugal and is regarded as a human pathogen. Little information is available for B. bissettiae from the USA (a species mostly encountered in California) as no isolate belonging to this species has been obtained from a human patient in the USA. However, one case of LB was noted in Europe where B. bissettiae was isolated from CSF of a child without any travel history. In Asia a human case of LB was described and species identification points to B. yangtzensis  (although in the paper it was termed B. valaisiana).

The other genospecies have not been isolated from human cases of Lyme disease and are only known from isolates obtained from ticks or wild animals.

The complete sequence of B. burgdorferi (B31 strain) was published in 1997 and it is apparent that the spirochaete has few biosynthetic proteins and has no sequences for recognizable toxins. It is generally agreed that immunopathology plays a large part in the pathogenesis of B. burgdorferi sensu lato.

So far there has been little progress in identifying pathotypes within genospecies. However, small subsets of B. burgdorferi sensu stricto, characterised by particular variants of Outer Surface Protein C (OspC), have been reported to be particularly invasive in humans. However, genetic replacement of an “invasive” OspC-type against a non-invasive did not change the invasive behaviour of the recipient isolates, thus suggesting that other molecules are also involved in invasiveness of Borrelia. Nevertheless, the role of OspC in invasive behaviour is further emphasized by the observation that strains of spirochaetes lacking these proteins are never invasive.

Geographical distribution
The geographic distribution of species of the B. burgdorferi sensu lato complex is not uniform. It has been suggested that vector competence of Ixodes ticks for the different species plays a role in this. Of the nine species that have been recorded in the USA, only two (B. burgdorferi sensu stricto and B. mayonii) are human pathogenic. B. burgdorferi sensu stricto is responsible for the majority of Lyme borreliosis cases in North America. Cases of LB are not equally distributed in the USA, the majority is recorded in the Northeast, fewer cases are recorded in the Midwest or in California. Nine species are also known in Europe, five of which are human pathogenic (B. afzelii, B. bavariensis, B. burgdorferi sensu stricto, B. garinii and B. spielmanii). Borrelia afzelii is the most common species in Europe and it has been associated with the condition of acrodermatitis chronica atrophicans (ACA) in central Europe and in Scandinavia. Neurological symptoms (neuroborreliosis) are a common manifestation in Western Europe and B. garinii and B. bavariensis are most frequently associated with these cases. Whilst the bird-associated B. garinii is widely distributed, the rodent-associated B. bavariensis has a highly focal distribution although it has been found in many European countries including Denmark, The Netherlands, Germany, Austria, Slovenia, Slovakia.  Borrelia burgdorferi sensu stricto does not seem to dominate in any European region and appears to be absent in I. persulcatus in Russia and Asia.  Borrelia spielmanii has a focal distribution in Europe as it is associated with garden door mice as reservoir hosts. Borrelia valaisiana has been isolated from I. ricinus in Europe (Switzerland, the Netherlands, the UK, Germany, Scandinavia and Central Europe), and a single isolate from I. columnae in Asia. It seems to be the commonest genospecies (by PCR) in ticks in Ireland. Borrelia lusitaniae  has a focal distribution in many parts of Europe (France, Poland, Slovakia), but seems to be more common around the Mediterranean in Italy, Portugal, Algeria and Tunisia. Across the huge geographic range of I. persulcatus distribution from Eastern Europe to Asia, three Borrelia species are found, all of which are human pathogenic: B. afzelii, B. bavariensis and B. garinii. Other species found in Asia are associated with different vector ticks, i.e. B. japonica – I. ovatus, B. sinica – I. ovatus, B. tanukii – I. tanuki and B. yangtzensis – I. granulatus and their human pathogenic potential needs yet to be determined.

Reservoir hosts
Borrelia species can infect many hosts, whether they are reservoir competent or not. The definition of a reservoir host is that the Borrelia species can
i) infect the host,
ii) amplify in the host, and most importantly
iii) can be transferred from the host to a new tick (that is, maintaining the natural transmission cycle).
In early years of Borrelia research, there had been considerable speculation concerning possible differences in the ecology of the genospecies. In environmental studies investigating double infections in ticks, B. valaisiana was most often associated with B. garinii, and in other study both these species were shown to be transmitted from blackbirds (Turdus merula) to ticks. Later on, transmission experiments have shown that B. afzelii and B. bavariensis are associated with rodents, B. garinii and B. valaisiana with birds, and B. lusitaniae with lizards. Borrelia burgdorferi sensu stricto seems to occur frequently in both birds and rodents.
The finding that different genospecies have differential susceptibility to serum complement from particular host species in vitro has proved valuable in further indicating the reservoir host spectrum of each genospecies. However, some DNA studies have suggested that spirochaetes may circulate (perhaps by co-feeding) between ticks and supposedly incompetent hosts.
Additional data will be required before the pattern of host associations of all the different genospecies is confirmed.

Tick associations
Similar to reservoir host competence, vector competent tick species need not only acquire a gut infection with a Borrelia species but the Borrelia species must be able to
i) amplify in the tick,
ii) survive immune responses of the tick and invade the salivary glands,
iii) be transmitted to the next host.
There seems to be little association of the human pathogenic Borrelia species with tick species other than those imposed by the global distribution of spirochaetes and ticks. In Europe, a large range of Borrelia species have adapted to I. ricinus. The genospecies associated with the important human-biting tick in Eastern Europe and Asia, I. persulcatus, are B. afzelii, B. bavariensis and B. garinii. In the laboratory Ixodes persulcatus appears to be able to transmit B. burgdorferi sensu stricto but B. burgdorferi sensu stricto has not been recorded from this tick in the field and if so, only in areas where it is sympatric with I. ricinus. In contrast to the pathogenic genospecies, some of the non-pathogenic species are closely associated with a specific tick species and usually have a limited geographic distribution. For example, in Japan, B. japonica is strictly associated with I. ovatus, B. tanukii with I. tanuki, and B. turdi with I. turdus and I. frontalis in Asia and Europe. In the USA, B. andersonii is associated with I. dentatus.