Lyme disease   Lyme disease  PDF    (129  KB)

 

Lyme disease is a vector (Ixodes ricinus) transmitted bacterial infection. The spirochaete pathogen was identified in 1982 and is today called Borrelia burgdorferi. The genus Borrelia includes several species, of which the groups B.recurrens and B.duttonii (human relapsing fever), B.anserina (agents of avian spirochaetamia) and B.burgdorferisensu latu (causative agent of Lymedisease) are classified as pathogenic.

Borrelia are transmitted by vectors (ticks andlice), and with the exception of B.recurrens and B. dutonii they all have a reservoir in wild animals.The Borrelia spirochaete have all contractileaxial filaments, located under a multi-layered outer shell that gives the spirochaete their typicalspiral form and motility. The group of B. burgdorferisensu latu (B.b.s.l.) includes several species, such as Borrelia burgdorferi strictu sensu, B. afzelii and B. garinii.

At present the species B. burgdorferi sensu strictu (B. burgdorferi ss), B. afzelii and B. garinii are discussed as the agents of Lyme disease, although other species such as B. lusitaniae and B. valaisiana are now a suspected pathogen, since its detection in the human CSF.
         

 Distribution
 

The geographical distribution of the pathogenic Borrelia species is very different. For example, in North America the distribution of B. burgdorferi ss is close to 100%, while in Europe this pathogen exists only to 10%. Nearly 90% of European-Borrelia species are B.afzelii and B. garinii.
        

Reservoir
 

The pathogen reservoirs are forest mammals and birds that get infected with Borrelia from tick larvae in the first blood meal. With a further blood meal, the nymphs will already transmit Borrelia. Depending on the area up to 75% of adult ticks are infected with Borrelia, while a single tick can be infected with several Borrelia species.
 

Tick infection
 

If a tick gets infected with Borrelia with its blood meal, the agents first migrate in the ticks gut, where they start to express a specific surface pattern the surface protein A (Outer surface protein A = Osp A). With this surface protein, the Borrelia is in a position to settle permanently inthe mid gut. If there is now an attachment of  the tick to the warm skin of the host and blood flows in the tick gut, the Borrelia begins to change their surface structure. Temperature rises and pH changes and no more Osp A is expressed.

Instead, another surface protein is expressed: the Osp C. This surface protein allows the bacteria to penetrate the intestinal wall of the tick and migrate via haemolymph to the saliva glands. The period from the beginning of this change is about 24 - 48 hours with rare cases reported to have a shorter migration time. With the saliva Borrelia are transmitted into the host and begin to spread.
   

Infection of the host
 

After the invasion of Borrelia the host reacts witha non-specific immune response (immigration of neutrophils, macrophages, plasma cells). This usually gets suppressed by anti-phlogistic substances (prostaglandins) from the tick saliva. Consequently, there is an active migration of the bacteria through the host tissue. Granulocytes get attracted, degranulate and thus a massive inflammatory reaction is triggered. Borrelia carrya glycosaminogly can receptor that prefers to bind to tissue with a high proportion of collagenfibres such as in joints, heart and pericardium, brain and meninges. After the first innate, nonspecific immune response the host organism starts with the production of specific antibodies, first immunoglobulin M and later immunoglobulinG. In experimentally infected dogs IgG antibodieswere already present 4-6 weeks after infectionand showed persistence for more than 1 year.

Despite the specific immune response by thehost, Borrelia survive the immune response ofthe host to a great extent due to continuouschanges in their surface proteins. Especially thesurface protein VlsE and the Osp C have a largevariability of its DNA sequences. This results in afailure of the host to eliminate the pathogen.Although the VlsE protein consists in part ofvariable DNA sequences, constant areas alsoinduce antibodies that bind to early shares. Dueto constant variation though a proportion of antibodiesis produced without corresponding sites on the surface molecules of many bacteria. Thus, despite antibodies production a further spread of Borrelia through the organism is possible.

  

Clinical signs of Lyme Disease in the dog
 

The clinical signs of a Borrelia infection can varysignificantly. Only 5% of dogs infected are reportedto develop clinical signs. In experimentallyinfected dogs, however, nearly 75% of the animals show clinical symptoms. In contrast to humans erythema migrans is rarely seen in dogs. In the early phase of infection fever up to 40.5 °C, inappetence, and fatigue is seen for one to two days. This is followed by an asymptomatic phase of several weeks (or several months) duration. After this time the animals often show lameness for the first time lasting only a few days and often disappearing without any treatment. At this stage most patients are presented in a veterinary practice for the first time. The severity of lameness varies from severe to mild and can be intermittently. The lameness may occur mutually.
 
In addition to arthritis symptoms also a cardiac, anervous and other organ involvement is reported.A serious complication is the development of glomerulonephritis with subsequent kidney failure due to deposition of antibody antigen complexes in the basal membranes. These animals show lethargy, emaciation, vomiting and they develop peripheral oedema due to the loss of albumin. Fatalities are reported.  Azetonaemia, microalbuminuria and proteinuria are the dominating findings.
 
Glomerulonephritis as a complication is mainlyobserved in the Golden and Labrador Retrieverand the Bernese Mountain dogs.
 
 
lab result

      

  
Potentials and limits of the diagnosis
 

The practitioner has several methods at hand to diagnose a Borrelia infection. One of the first detection methods was the immunofluorescence assay (IFAT), in which serum was applied to antigen-covered slides. Then, a fluorescent secondary antibody was added and the formation of antigenantibody complexes in its intensity was assessed using a fluorescence microscope.
 
Since the specificity of the ELISA developed subsequently is higher this method is preferred nowadays for testing dog sera. In the ELISA amicrotitre plate is coated with Borrelia antigen and serum is added. An enzyme labelled secondary antibody as conjugate is pipetted into the wells and the test is incubated. Added substrateresults in a colour change according to theamount of specific antibodies bound. Since the abacterial lysate is used as antigen, the ELISA allows only a statement concerning the totalamount of specific antibodies, i.e. a precise differentiation of  the antibodies detected (vaccination/ infection) is not possible.
  
Our data reveal a seroprevalence of roughly 4%for Ig M and 29% for Ig G antibodies in 2007 when we look at nearly 12,700 blood samples.
 
Borrelia serology results 2007

 
We have to state though, that since these animalswere selected for testing in the practices thepercentage does not represent the overall prevalencewithin the country. It does represent percentageswe can expect to be positive whenexamining animals with clnical signs suspect of borreliosis.

The golden standard in Borrelia diagnostic is the two-stage diagnosis using ELISA and Western Blot. In a Western blot different antigens of the bacterial organism are separated by size in an electric field and than transferred to a nylon ornitrocellulose membrane. In the subsequent incubation of the membrane with the test serum antibodies bind to several different locations around the membrane. A secondary antibody reveals the binding sites. The assessment of Western blots is determined by the specificity of the various bands; e.g. ideally reactions specific for vaccination early phase or late phase infectioncan be differentiated.Whenever ELISA and Western Blot are combined the practitioner has a combination of tests with high sensitivity and high specificity likewise at hand. An analysis of nearly 5000 Borrelia blotsin the years 2006 to 2008 at LABOKLIN showed positive results for the specific infection VlsE band in 5% of the samples, p100 was seen in 17% ofhte cases, and OsPC in 13%. OpsA being specific for vaccination was seen in approximately 38% of the samples.


 
Borelia Blot dog


Another option for diagnosing Borrelia pathogensis the detection by use of PCR (polymerase chainreaction). In this case primer sequences multiplyBorrelia DNA. Different markers can be used tomake the reaction visible.The value of a PCR is, however, limited by theselection of suitable sample and by the concentrationof pathogens. As part of a chronic infectionand although in many locations the pathogenis suspected, the concentration of DNA may bevery low and therefore the PCR may show anegative result. While a positive PCR is proof ofan infection, a negative PCR never rules it out.In 2007 15% of the ticks tested in our lab 15%were positive for Borrelia using a PCR (n=247).Multiple infections with other pathogens(Babesia, Ehrlichia, TBD) were not detectable.
 
Borrelia -PCR  in ticks 2007

   
Culture theoretically is an alternative for demonstratethe pathogen in suspect samples. Since this procedure demands special media (Barbour-Stoenner-Kelly medium) and is very time consuming with difficult preanalytic procedures (theduration may take up to 6 weeks and the samples must be taken absolutely sterile to prevent contamination with bacteria and fungi that will inhibit growth of Borrelia) PCR is method of first choice.
 
Dark field microscopy is another alternative way to detection of the pathogen. Major disadvantage of  this method is a low sensitivity, therefore it is consequently succeeded by PCR.

Sample material may be tissue samples or synovial for PCR as well as for culture.
        

Therapy
 

Drug of choice is Doxycyclin p.o. 10 mg/kg bid for30 days. In case of adverse reactions Amoxicillinp.o. 20 mg/kg bid for 30 days is recommended.
       

Therapy control
 

Serology for therapy control should, if at all, only carried out with long inervals between the samples.Antibodies to Borrelia persist long, thus serology cannot really help in differentiating between successfully and not successfully treated animals.
        

 

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