DIAGNOSIS

Diagnosis of LB is difficult even in humans, and labora­tory confirmation of a borrelial infection is needed for all manifestations of Lyme borreliosis, with the exception of typical skin lesions.

Samples from Animals

Cultivation is the ‘gold standard’ of diagnosis of B. burgdor­feri. In culture, the LBS are microaerophilic and slow- growing. They grow best in liquid media at 30—35°C and have complex nutritional requirements. The medium in common use is a modified BSK (Barbour, Stoenner and Kelly). However, as the numbers of viable bacteria in tissues and blood may be very low and the organism is very fastidi­ous, negative results do not exclude infection. The sensitivity is very variable, but may be satisfactory for samples from skin, for example ear biopsies or needle aspiration from skin in areas where ticks collect. The sensitivity is very low for fluids such as blood, joint fluid or cerebrospinal fluid (CSF).

Genus- and species-specific polymerase chain reaction (PCR) methods can be used to detect borrelial DNA of both viable and non-viable organisms and is now a stand­ard approach for direct detection of infection in ticks, hosts and clinical specimens. Given that the number of spirochaetes in infected tissues or body fluids is generally very low, appropriate, controlled procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Quantification of the borrelial burden in host tissues can be determined by either competitive PCR or real-time quantitative PCR(2⁶).

In both cultivation and PCR it is important to realize that mixed infections occur, and that different strains have different tissue tropism. Negative results may not be regarded as conclusive, and positive isolation of one species in a sample may not exclude the presence of others.

Serology is often used on human samples, usually as a two-step procedure, starting with enzyme-linked immu­nosorbent assay (ELISA) followed by an immunoblot of positive specimens. These methods have many challenges, owing due to the antigenic complexity of European LBS and their variable expression of antigens. In wild animals, results of serologic tests not validated for the host species should be interpreted with caution. Serology is most appropriate when used to evaluate exposure at the popula­tion level. The seroprevalence should be expected to be low in non- competent hosts, as sensitivity of antibody detection in early, localized disease in humans is only between 20 and 50%(¹7).

In human cases of neuroborreliosis, an index based on the ratio between borrelia-specific antibodies in CSF and serum relative to the ratio between total amount of antibod­ies in CSF and serum, is used as a diagnostic criterion.

Xenodiagnosis is the method of choice for evaluating host infectivity to feeding ticks. The fact that a non-fed, non-infected tick larva or nymph acquires borrelial infec­tion when feeding on a given host species and still has viable borrelia in its midgut in the next developmental stage is regarded as a proof of this host’s reservoir compe­tence. However, this method is labour-intensive and requires long- term handling and restraint of animals. A more practical method for assessment of reservoir capacity may be to compare the prevalence of infection among larvae that have fed on the host with that of questing larvae in the same area.

Detection in Ticks

I n addition to cultivation and PCR, viable and motile Borrelia may be visualized directly by using dark-field microscopy on suspension of the midgut of ticks.

Identification of Host Blood Meal Source

Different techniques have recently been developed for identification of host blood meal source in ticks. Com­bined with isolation of borrelial DNA from the same ticks, this provides an interesting tool for analysis of transmis­sion dynamics and host associations.