Samples and dogs
Dogs included in a previous study on the prevalence of B. burgdorferi in Bernese Mountain dogs were re-evaluated after 2.5 years (899 days), and after 3.0 years (1113 days) (median 2.7 years (992 days)) . This study focussed on including similar numbers of dogs of the following groups: Bernese Mountain dogs in which antibodies against B. burgdorferi were diagnosed, Bernese Mountain dogs in which no antibodies against B. burgdorferi were detected, control dogs (long haired large breed dogs but not Bernese Mountain dogs) in which antibodies against B. burgdorferi were diagnosed, and control dogs in which no antibodies against B. burgdorferi were detected. For the first study dogs were sampled between July 2002 and April 2003. There were 160 Bernese Mountain dogs and 62 control dogs. Dogs were re-evaluated between June and October 2005. The owners of 82 Bernese Mountain dogs and 62 control dogs evaluated in the previous study were contacted a second time in order to evaluate the response over time of clinical and laboratory parameters. For 29 Bernese Mountain dogs (35%) and 32 control dogs (52%) no second examination was possible because the dogs were either dead (25 Bernese Mountain dogs and 14 control dogs) or the owners could not be reached a second time, or were unwilling to participate again (4 Bernese Mountain dogs and 18 control dogs). The health status of the dogs was assessed using a questionnaire filled in by the owners. Answers relating to general health and lameness were compared between the first and the second examination to assess possible consequences of a B. burgdorferi infection. Owners were asked to judge if the general health of their dog was normal or abnormal, and if their dogs were lame or not at the time of the second evaluation. At both time-points routine laboratory tests of blood and urine samples were performed. For serologic testing samples were frozen at minus 80°Celsius.
Haematology and serum biochemistry
Laboratory tests included a complete blood count (CBC) and a serum biochemical analysis containing determination of bilirubin, glucose, urea, creatinine, total protein, albumin, cholesterol, sodium, potassium, chloride, calcium and phosphorus concentrations; as well as the activity of alkaline phosphatase, alanine transferase, aspartate transferase and amylase. Hematocrit, urea, creatinine, total protein and albumin values during the first and the second examination were compared to assess renal function and function of the filtration barrier. Dogs were deemed azotemic if creatinine was above 125 μmol/L and/or if urea was above 9.4 mmol/L.
Urinalysis consisted of a urine dip stick (Combur-Test, Roche Diagnostics GmbH, Mannheim Germany), microscopic examination of urine sediment, and determination of urine specific gravity. Results of urine protein measurements were considered only if fewer than 5 leukocytes per 400× field were counted in the urine sediment. The results of the protein measurement with the dip stick were recorded as negative or 1+ to 3+ positive. The urine protein-to-creatinine ratio (UPC) was measured on a Cobas-Integra analyzer (Roche, Rotkreuz, Switzerland). A difference in UPC between the first and the second examination was considered significant if it was 80% or more of the first value . Microalbuminuria was measured by a commercial rapid immunoassay for canine microalbuminuria (E.R.D.-Screen™-Test, HESKA®, Fribourg, Switzerland). Results were interpreted as negative, low-positive, medium-positive or high-positive according to the manufacturer's instructions. Urine specific gravity and results of the measurements of protein in urine were compared between the first and the second examination. If the dogs were azotemic, the azotemia was considered renal if the urine specific gravity was below 1.030.
Serologic testing of serum stored at minus 80°C was performed. Serologic tests of the samples of the first and the second evaluation were performed together. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against B. burgdorferi sensu lato was performed for all dogs according to established protocols as described previously [8, 10].
Briefly, a whole-cell sonicate of B. burgdorferi sensu stricto reference strain B31 (ATCC 35210), B. garinii N34, B. afzelii VS 461 and B. valaisiana VS 116 was used as antigen. Prior to serological testing serum samples were absorbed with a heterologous sorbant of washed formalin inactivated whole cells of Escherichia coli, Salmonella typhimurium, Brachispira hyodysenteriae, Bacillus subtilis and a total of ten serovars of Leptospira (L.) interrogans and L. borgepetersenii, respectively.
For each plate positive and negative control samples were applied. The cut-off was calculated from the serial measurement of the negative controls.
Western blot examinations to detect antibodies against B. burgdorferi were performed using a commercial test kit modified for dogs (Virion Ltd., Rüschlikon, Switzerland). Tests were performed according to the manufacturer's instructions and consisted of Western blot strips with defined partial antigens of B. burgdorferi sensu stricto and B. afzelii. Western blot results were interpreted according to criteria recommended for European species of B. burgdorferi sensu lato . Samples were called positive if bands at the level of the partial antigens p100, p58, OspC, p21 or wb18 were identified or if at least two bands at the level of the partial antigens p45, bmpa und wb30 were present. Bands at the level of the partial antigens OspB, OspA, OspD, wb22 und OspE were considered unspecific.
Dogs were considered to have antibodies against B. burgdorferi if both the ELISA and the Western blot were positive.
Data was recorded and analyzed using a commercial computer program (Statistical Package for the Social Sciences for Windows version 11, SPSS Inc, Chicago Illinois, USA). Data of the first and the second examination was compared using the Wilcoxon signed rank test for ordinal data and McNemar's change test for nominal data. Additionally, to compare age between dogs with and without poor general condition and between dogs with and without lameness the Mann-Whitney U test was used. For dogs re-evaluated after the first examination and those not re-evaluated the Mann-Whitney U test was also used for the comparison of hematocrit, urea, creatinine, protein, albumin, urine specific gravity and urine protein-to creatinine ratio. The Fisher's exact test was used to compare the occurrence of lameness between dogs re-evaluated a second time and those not re-evaluated. Differences were considered significant at P < 0.05. Bonferroni correction was applied in the comparison of haematology, serum chemistry and urine parameters.