Protein-losing enteropathy and PLN are chronic and often fatal diseases in SCWTs. Diagnosis typically is made when dogs are at a mean age of 4 to 6 years old.1 In the United States, the prevalence is estimated to be as high as 10% and median survival time reportedly is only months after the disease is diagnosed.1 Several clinicopathologic markers can be used to help identify dogs with this syndrome, including decreases in serum albumin and globulin concentrations; an increase in UP:C; an increase in fecal concentration of α-proteinase inhibitor; microalbuminuria; and, at later stages of PLN, increases in SUN and creatinine concentrations.1–3 The pathogenetic mechanisms of PLE and PLN in SCWTs are unknown, but results for examination of renal biopsy specimens from SCWTs with PLN are consistent with a sclerosing immune-complex disease of the glomerulus.1 An immune-mediated process may also be implicated in the pathogenesis of PLE in SCWTs because food allergies play an important role in the natural progression of disease.2,4
Circulating autoantibodies (ie, pANCAs) are associated with diet-responsive diseases in dogs of various breeds that have chronic enteropathies.5 The detection of pANCAs has also been used for decades as a reliable diagnostic test for subtypes of inflammatory bowel disease and autoimmune glomerulonephritis in humans.6,7 Titers for pANCAs are determined by use of an indirect immunofluorescence assay against granulocytes, whereby a characteristic pattern of perinuclear staining can be detected in pANCA-positive samples.7 Sensitivity and specificity of pANCAs for diagnosing inflammatory bowel disease in dogs with chronic enteropathies are 55% and 95%, respectively.8
Therefore, we hypothesized that pANCAs play a role in the development of PLE and PLN diseases in SCWTs. The purpose of the study reported here was to determine whether pANCAs are associated with clinicopathologic markers of disease in SCWTs and to evaluate their usefulness as a possible early marker of PLE and PLN.
Materials and Methods
Animals—Two groups of dogs were included in the study. One group comprised 21 SCWTs (13 purebred SCWTs and 8 SCWT-crossbred dogs) housed in a research colony at North Carolina State University. The control group comprised 7 mixed-breed dogs with X-linked hereditary nephropathy that were housed in a research colony at Texas A&M University and 12 healthy client-owned SCWTs that were ≥ 9 years old. Dogs at each of the university facilities were cared for in accordance with established principles.9 Care and use of dogs at the university facilities were reviewed and approved by the respective animal care and use committee at each university. The portion of the study involving client-owned dogs was approved by the Ethics Committee of the Royal Veterinary College, and owners signed a consent form allowing the determination of pANCA in serum samples obtained from their dogs.
Research Colony SCWTs
Serum samples from 13 purebred SCWTs and 8 SCWT-Beagle crossbred dogs from a research colony were retrospectively tested for pANCAs. The purebred dogs in this colony were the result of breeding 2 founder SCWTs, both of which developed PLE and PLN. One affected male SCWT and a clinically normal female Beagle were mated to yield the 8 crossbred dogs. Since birth, all dogs had been observed daily and clinicopathologic examinations had been performed every 3 months. Serum samples for the study were obtained from all dogs beginning when dogs were 6 months old and, subsequently, every 6 months until the time of the study reported here (n = 9 dogs) or until they were euthanatized because of progression of disease (12). Mean ± SD age of the 12 dogs at the time of euthanasia was 6.93 ± 2.5 years (median, 6.5 years; range, 2.25 to 9.9 years). Eighteen of 21 dogs had convincing evidence of clinical disease of PLE, PLN, or both, with the onset of disease defined as persistent hypoalbuminemia (< 2.4 g/dL) or proteinuria (UP:C > 1.0) during a period of at least 1 year. At each time point for sample collections, serum concentrations of albumin, globulin, and creatinine; SUN concentrations; concentration of α-proteinase inhibitor in the feces; and UP:C were determined and correlated with pANCA status.
Control Dogs
Serologic pANCA status was determined in 7 mixed-breed dogs with X-linked hereditary nephropathy (5 homozygous males and 2 heterozygous females) from a research colony. Mean ± SD age of these dogs was 1.5 ± 1.8 years (median, 0.6 years; range, 0.4 to 5.1 years). Hereditary nephropathy had been diagnosed in each of these dogs by use of direct genetic testing for the causative COL4A5 mutation that is responsible for the development of X-linked hereditary nephropathy.10,11 At the time serum samples were obtained, all 7 dogs had an increase in UP:C (mean ± SD, 12.3 ± 5.5; reference range, < 0.5), 5 dogs had an increase in serum creatinine concentration (0.9 to 9.4 mg/dL; reference range, 0.5 to 1.5 mg/dL), and 5 dogs had a low serum albumin concentration (1.6 to 2.9 g/dL; reference range, 2.4 to 3.6 g/dL). In addition, serum samples were obtained from 12 healthy SCWTs that were ≥ 9 years old and that did not have any evidence of protein-losing disease. Mean ± SD age of these dogs was 10.3 ± 1.4 years (range, 9 to 13 years). Serum samples were evaluated for evidence of pANCAs. Collection of blood samples from these dogs was performed as part of a clinical health check at the Royal Veterinary College in London. Medical history of each dog was obtained and revealed no evidence of food allergy or other diseases. Results of physical examination were unremarkable, and results of a CBC, serum biochemical analysis (including measurement of total protein and albumin concentrations), and urinalysis of a midstream sample were within the respective reference ranges.
pANCA assay—The pANCA status of each dog was determined by use of indirect immunofluorescence.8 Blood samples were collected into serum tubes, and serum was harvested and subsequently frozen at −20°C until analysis. Briefly, slides of canine granulocytes were incubated for 1 hour with serum (1:10 dilution) obtained from the dogs. Cells were washed, a secondary fluorescein isothiocyanate–labeled antibodya was added, and cells were allowed to incubate for 1 hour in a humid chamber at 22°C. After a final wash, slides were dried and mounted with fluorescence mounting medium. Slides were separately evaluated by use of fluorescence microscopy by 2 authors (KA and BL) who were unaware of the source of the sample. Sera from dogs that resulted in a specific pattern of perinuclear staining in granulocytes were considered to have positive results for pANCA, whereas no staining or an atypical staining pattern of granulocytes (including evidence of intranuclear staining) was considered a negative result for pANCAs.
Statistical analysis—The Fisher exact test was used to determine associations between pANCA status and each clinicopathologic variable. Data for age were tested for a normal distribution, and a paired t test was used to determine differences between mean age of the first positive pANCA result and mean age of the onset of clinicopathologic abnormalities. Significance was set at values of P < 0.05. All analyses were performed with commercially available statistical software.b
Results
Research colony SCWTs—A total of 259 samples were evaluated for pANCA status, which represented a mean of 12 samples/dog (range, 3 to 16 samples/dog). Of the 13 purebred SCWTs, 6 had positive results for pANCAs at each measurement point during their lives. Of these 6 dogs, 5 developed definitive evidence of disease (1 dog with PLE, 3 dogs with PLN, and 1 dog with both diseases). One purebred SCWT had negative results for pANCAs throughout its life but developed definitive evidence of disease (euthanatized because of progressive PLN at 5 years of age). The remaining 6 SCWTs had positive results for at least 2 samples during their lives (mean, 80% of tests had positive results; range, 25% [2 positive results for 8 samples] to 90% [9 positive results for 10 samples]). All 6 of these SCWTs developed definitive evidence of disease (2 dogs with PLN, 2 dogs with PLE, and 2 dogs with PLE and PLN).
None of the 8 SCWT-Beagle crossbred dogs had only positive or negative results for all tests conducted throughout their lives. However, all 8 of these dogs had positive results for pANCAs at least 4 times (mean, 70% of tests had positive results; range, 25% [4 positive results for 16 samples] to 88% [14 positive results for 16 samples]). Six of these 8 dogs developed definitive evidence of disease (1 dog with PLE, 3 dogs with PLN, and 2 dogs with PLE and PLN).
When considering all dogs with positive and negative results for pANCAs throughout their lives, 11 of 18 (61%) dogs had negative results for pANCAs for samples obtained early during their lives, but they seroconverted before developing evidence of diseases. These dogs continued to have positive results for pANCAs once PLE or PLN (or both) was diagnosed.
Control dogs—None of the 7 dogs with X-linked hereditary nephropathy or 12 healthy SCWTs > 9 years old had positive results for pANCAs.
Correlation of pANCA status with clinicopathologic variables—We did not detect a significant association between a positive pANCA status and a decrease in serum globulin concentration (P = 0.5), increase in SUN concentration (P = 0.2), increase in serum creatinine concentration (P = 0.5), increase in UP:C (P = 0.5), or increase in fecal concentration of α-proteinase inhibitor (P = 0.2). However, a positive pANCA status was significantly (P < 0.001) associated with hypoalbuminemia (< 2.4 g/dL) at the time serum samples were obtained.
The first positive result for pANCA testing was 2.4 years before the onset of hypoalbuminemia (n = 18 dogs with confirmed disease). Mean ± SD age of dogs at the time of the first positive result for pANCA testing was 1.69 ± 1.2 years, whereas mean age of dogs at the time of onset of disease (ie, hypoalbuminemia) was 4.19 ± 1.6 years. These values differed significantly (P < 0.001). Mean age of dogs at the time of euthanasia (12/18 dogs with confirmed disease were euthanatized) was 6.93 ± 2.5 years. This value differed significantly (P = 0.001), compared with the value for age of dogs at the time of onset of disease (mean age at time of euthanasia for the 12 dogs, 6.93 ± 2.5 years; mean age at onset of disease for the 18 dogs, 4.19 ± 1.6 years; Figure 1).
Box-and-whisker plots representing age at first positive pANCA test result, age at onset of clinical disease (defined as persistent hypoalbuminemia [< 2.4 g/dL]), and age at euthanasia (n = 12 dogs) for 18 dogs affected with PLE, PLN, or both. The horizontal line in each box represents the median; the top and bottom of each box represent the 75th and 25th percentiles, respectively; and the whiskers represent the 95th and 5th percentiles. Mean ± SD age at first pANCA test result was 1.69 ± 1.2 years, mean age at onset of clinical disease was 4.19 ± 1.6 years, and mean age at euthanasia was 6.93 ± 2.5 years.
Citation: American Journal of Veterinary Research 69, 10; 10.2460/ajvr.69.10.1301
Sensitivity and specificity for positive result for pANCA testing to predict PLE or PLN in SCWTs—Calculated sensitivity and specificity for use of pANCA testing to predict disease in the tested dogs (18 affected dogs vs 12 healthy SCWTs > 9 years old) were 0.95 (95% CI, 0.72 to 1.00) and 0.8 (95% CI, 0.51 to 0.95), respectively. Positive predictive value for the test was 0.86 (95% CI, 0.63 to 0.96), and negative predictive value was 0.92 (95% CI, 0.62 to 1.00). The calculated overall accuracy was 0.86 (95% CI, 0.72 to 0.96).
Discussion
The study reported here provides evidence that pANCAs may be associated with PLE and PLN in SCWTs and that pANCAs may be useful for early detection of these diseases in SCWTs. The majority (17/18) of dogs in this study that developed definitive evidence of disease had positive results for pANCAs at least twice during their lives, but none of the 19 control dogs had positive results when tested for pANCAs. Seven of the control dogs originated from a research colony of dogs with confirmed X-linked hereditary nephropathy, and they carry a genetic mutation in basement membrane (type IV) collagen that leads to progressive glomerular disease.10,11 It is also true that X-linked hereditary nephropathy serves as a method for studying Alport syndrome, which has not been associated with ANCAs in humans.10 In theory, these dogs should be ideal control animals because they represent dogs with a hereditary glomerular disease but have a completely different pathogenesis and cause than those postulated in ANCA-associated diseases. The other control dogs were older SCWTs that never developed evidence of PLE or PLN and that were therefore unlikely to develop protein-losing disease.1
Results from the study reported here predicted good sensitivity and specificity (95% and 80%, respectively) for pANCA testing to detect protein-losing diseases in SCWTs; however, larger prospective studies in the population of SCWTs in which investigators evaluate many dogs with and without disease will be necessary to confirm these data. In humans with ANCA-associated diseases, pANCAs or ANCAs have been used to diagnose small-vessel vasculitides with high accuracy. Sensitivity and specificity of pANCAs and ANCAs used to detect Wegener's granulomatosis or microscopic polyangiitis are as high as 70% to 80% and 98%,12 respectively, which is similar to the predicted accuracy for our data reported here.
Typically, dogs in our study had the first positive results for pANCAs 1 to 2 years before onset of hypoalbuminemia. This may prove helpful in identifying and monitoring dogs that will develop disease later in life and may increase opportunities for early treatment, such as feeding a hypoallergenic diet to young dogs.3 Additionally, early detection of dogs that will eventually develop PLN or PLE may allow breeders to gradually decrease the incidence of these diseases by removing animals predisposed to these diseases from the breeding pool.
Rather than only being markers, ANCAs are proposed to be implicated in the pathogenesis of small-vessel vasculitides in humans. Mean age of onset of disease in humans with ANCA-positive vasculitides is 65 to 74 years of age, and the appearance of pANCAs or ANCAs usually precedes the onset of disease.13 This fact concurs with findings in the SCWTs of our study because most dogs had negative results for pANCAs early in life but seroconverted before onset of disease and then continued to have positive results once a diagnosis of PLE or PLN (or both) was established. In addition, an increase in seropositivity of pANCAs can precede the onset of clinical relapses in humans with ANCA-associated diseases and is proposed to assist in noninvasive monitoring of such patients.14 On the other hand, in a prospective study5 in which investigators evaluated pANCA status in dogs with food-responsive inflammatory bowel disease, pANCA titers did not correlate with clinical activity of disease.
Interestingly, a positive test result for pANCAs was significantly associated with a decrease in albumin concentration in the study reported here but was not significantly associated with other indicators of disease, such as serum concentrations of globulin and creatinine, SUN concentration, UP:C, and fecal concentration of α-proteinase inhibitor. This may imply that more advanced disease or concurrent disease, the hallmark of which is a decrease in serum albumin concentration, is associated with a positive pANCA test result. Again, this finding is in agreement with results in humans because patients with positive test results for pANCAs are likely to develop disease later in life.14
Analysis of results of the study reported here suggested that pANCAs may be associated with PLE and PLN in SCWTs. Larger studies in which investigators evaluate the pathogenesis of protein-losing diseases in SCWTs will be necessary to elucidate the role of pANCAs in the development of these diseases.
Abbreviations
| ANCA | Anti-neutrophilic cytoplasmic autoantibody |
| CI | Confidence interval |
| pANCA | Perinuclear anti-neutrophilic cytoplasmic autoantibody |
| PLE | Protein-losing enteropathy |
| PLN | Protein-losing nephropathy |
| SCWT | Soft Coated Wheaten Terrier |
| UP:C | Urinary protein-to-creatinine ratio |
Sheep anti-canine IgG:FITC, Serotec AA132, Kidlington, Oxford, England.
NCSS statistical software, version 2005, NCSS, Kaysville, Utah.
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