Case report
A 4-year-old Labrador Retriever, male, born in Spain and living in the Apulia Region (southern Italy) was presented to the referring clinician with a recurrence of dermatitis at flat thighs, abdomen and testicles. The dog was treated previously with cortisone and fluid therapy with no positive outcomes. Clinical diagnosis showed palpable abdomen, internal organs evaluable and a productive cough. The dog was regularly vaccinated and treated against ectoparasites.
Chest x-ray examination revealed not altered structures. Abdominal ultrasound revealed chronic cystitis with thickening of the bladder wall with irregular margins. A serum chemical profile and complete blood count (CBC) revealed eosinophilia, whereas other parameters were within normal limits.
A macroscopic examination of urine revealed haematuria, while a microscopic examination of urinary sediment revealed the presence of several C. plica eggs. The dog was treated with febendazole at a dose rate of 50 mg/kg/day for 10 days and showed a rapid recovery, with resolution of all clinical signs.
Flotation solutions choice for FLOTAC and Mini-FLOTAC and urine preservation
To determine the optimum flotation solution for FLOTAC and Mini-FLOTAC and the possibility to preserve urine, two FSs: sodium chloride (FS2), specific gravity (s.g. 1200) and zinc sulphate (FS7) (s.g. 1350) were compared using either fresh urine and urine fixed with 5% formalin (because could be a good alternative method of preservation of urine from collection until analysis).
For this aim one hundred and twenty ml of urine were collected from the C. plica infected dog (see Case report paragraph).
The urine was divided in two aliquots (Aliquot 1 and Aliquot 2) of 60 ml each: Aliquot 1 was analyzed as fresh, Aliquot 2 was fixed with 3 ml of a 37% formaldehyde solution and analyzed after 7 days. Each of two aliquots was carefully homogenized and 6 tubes were filled with 10 ml of urine, to have 3 replicates for each of the two FSs used.
The tubes were centrifuged at 240 g for 5 minutes and the supernatant poured off and discarded. The tube with the remaining pellet was then filled with each FS until 11 ml level mark and slowly agitated.
The suspension was transferred into the two chambers of a FLOTAC apparatus using a disposable pipette. The apparatus was centrifuged at 120 g for 5 min, and translated to separate the floating eggs from the urinary debris. All eggs visible through the ruled window of the reading disc were counted under a microscope at 100× total magnification.
Comparison of FLOTAC, Mini-FLOTAC and urine sedimentation techniques
The following three techniques were compared for the diagnosis of C. plica in dog urine: the FLOTAC basic technique[12, 17], the Mini-FLOTAC technique[18] and the urine sedimentation technique[25].
Fresh urine and FS2 (for FLOTAC and Mini-FLOTAC) were used based on the results obtained from the previous experiment (see Flotation solutions choice for FLOTAC and Mini-FLOTAC and urine preservation paragraph).
Two aliquots (Aliquot 3 and Aliquot 4) of 200 ml each of urine were collected from the same dog naturally infected by C. plica. Each aliquot of urine was accurately homogenized and divided in 18 tubes each filled with 10 ml of urine, to have 6 replicates for each of the three diagnostic method. The tubes were randomly assigned to the three techniques.
For the urine sedimentation technique, the tubes were left to room temperature for 1 h, the supernatant was poured off[25] and the tubes were centrifuged at 2000 g for 2 minutes. After the supernatant was discarded, the pellet has been examined on a glass slide.
For the FLOTAC and Mini-FLOTAC techniques, the tubes were centrifuged at 240 g for 5 minutes, the supernatant was poured off and 6 ml of FS2 were added to tubes to fill the FLOTAC chambers, whereas 2 ml of FS2 were added to tubes to fill the Mini-FLOTAC chambers (for both techniques the entire pellet was examined).
For each diagnostic technique, C. plica eggs were counted for all replicates using a light microscope at 10× total magnification. The analytic sensitivity of all the three techniques was 1 egg per 10 ml of urine.
Statistical analysis
The arithmetic mean eggs per 10 ml of urine, standard deviation (SD), and coefficient of variation (CV) were calculated for the two FSs, preservation method and diagnostic technique. Differences between FSs were analyzed using one-way ANOVA with post hoc Fisher’s least significant difference (LSD). All statistical analyses were carried out using STATA version 10.0 (Stata Corp.; Texas, USA). In addition, a likelihood ratio test of the equality of the CV of normally distributed populations was performed using software developed by the Statistical Services at the Forest Products Laboratory (USA; http://www1.fpl.fs.fed.us/covtestk.html).
Consent
Written informed consent was obtained from the owner of dog to publish data and information in this report.