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Corresponding author: Christos K. Koutinas, Companion Animal Clinic, Veterinary Faculty, Aristotle University of Thessaloniki (AUTh), Nikeas 6 street, Thessaloniki 54453, Greece; e-mail: email@example.com.
Background: Canine leishmaniasis (CanL) is a common cause of epistaxis in dogs residing in endemic areas. The pathogenesis of CanL-associated epistaxis has not been fully explored because of the limited number of cases reported so far.
Hypothesis: Epistaxis in CanL could be attributed to more than 1 pathomechanism such as hemostatic dysfunction, biochemical abnormalities, chronic rhinitis, and coinfections occurring in various combinations.
Animals: Fifty-one dogs with natural CanL.
Methods: The allocation of 51 dogs in this cross-sectional study was based on the presence (n = 24) or absence (n = 27) of epistaxis. The potential associations among epistaxis and concurrent infections (Ehrlichia canis, Bartonella spp., and Aspergillus spp.), biochemical and hemostatic abnormalities, and nasal histopathology were investigated.
Results: Hypergammaglobulinemia (P= .044), increased serum viscosity (P= .038), decreased platelet aggregation response to collagen (P= .042), and nasal mucosa ulceration (P= .039) were more common in the dogs with epistaxis than in those without epistaxis. The other significant differences between the 2 groups involved total serum protein (P= .029) and γ-globulin (P= .013) concentrations, which were higher, and the percentage platelet aggregation to collagen, which was lower (P= .012) in the epistaxis dogs.
Clinical Importance: CanL-associated epistaxis appears to be the result of multiple and variable pathogenetic factors such as thrombocytopathy, hyperglobulinemia-induced serum hyperviscosity, and nasal mucosa ulceration.
Canine leishmaniasis (CanL, Leishmania infantum) is perhaps the most common protozoan canine disease in the Mediterranean countries, with infection rates as high as 63%.1 Five to 15% of CanL dogs demonstrate epistaxis. Interestingly, CanL accounted for 48% of cases in a recent retrospective study of 61 dogs with epistaxis residing in Northern Greece.2–5
The pathogenesis of epistaxis in CanL has not been fully elucidated, most likely because of the limited number of natural CanL cases investigated so far. To our knowledge, only 1 study has addressed the pathogenesis of epistaxis in CanL, in which the hemostatic profile and arterial blood pressure were evaluated in 19 dogs with natural CanL (6 with epistaxis), whereas nasal mucosa histopathology was investigated in 10 dogs (3 with epistaxis).6 In that study, impaired platelet function and ulcerative rhinitis were implicated as triggering factors of epistaxis. However, the limited number of dogs subjected to nasal histopathology did not allow correlation between the severity and histologic type of rhinitis and the development of epistaxis. Furthermore, the hypothetical role of comorbid factors such as infectious agents (eg, Ehrlichia canis, Bartonella spp., Aspergillus spp.), renal or liver disease, and serum hyperviscosity was not taken into account.
The purpose of the present study was to investigate the possibility of correlations among concurrent infectious diseases, biochemical and hemostatic abnormalities or nasal histopathology, and the appearance of epistaxis in natural CanL cases in an attempt to shed more light into its pathogenesis.
Materials and Methods
Fifty-one dogs admitted to the Companion Animal Clinic (CAC) from September 2002 through July 2005 entered the study. Dogs were eligible to enter the study if the diagnosis of CanL was confirmed, if they had not received antileishmanial or other medications for at least 60 days before admission, and if informed consent was obtained from the owners. Diagnosis of CanL was established by (a) the compatibility of clinical signs or conditions (data not shown), (b) the direct observation of L. infantum amastigotes in Giemsa-stained smears made from lymph node (44/51) or bone marrow (BM) (50/51) aspirates, and (c) seropositivity.a Dogs were allocated into group A (n = 24), which included those exhibiting at least 1 episode of epistaxis within 1 week before admission (4/24), upon admission (12/24), or during their hospitalization (8/24), and group B (n = 27), dogs that had never shown historical or clinical evidence of epistaxis episodes.
To generate our reference intervals for specific hemostatic tests such as buccal mucosal bleeding time (BMBT), prothrombin time (PT), partial thromboplastin time (PTT), and platelet aggregation, a group of clinically healthy dogs (n = 25) was recruited (19 males and 6 females; 21 purebreds and 4 crossbreds; age range, 1–5 years, median, 1.5 years). These dogs were seronegative for L. infantum and E. canisb and were admitted for routine vaccination (n = 18) or kept in CAC for teaching purposes (n = 7). Nasal biopsies also were taken from the latter 7 dogs to enable blind evaluation of nasal histopathology.
Physical examination was followed by CBC, serum biochemistry and urinalysis. In dogs with platelet counts >80,000/μL (n = 47), BMBT was assessed.7,c Prothrombin time and PTT were measured with an in-office analyzer.8,d To measure thrombin time (TT), fibrinogen, and von Willebrand factor (vWf) concentration, citrated plasma was packed in dry ice and transported to the Clinic of Small Animals, Hannover School of Veterinary Medicine, Hannover, Germany; handling conditions and instrumentation were in accordance with the laboratory's standards.9,e,f,g Fibrin(ogen) degradation products (FDPs)h were measured in 7 dogs with laboratory evidence of impaired secondary hemostasis. Platelet aggregation was evaluated with a turbidometric aggregometeri within 4 hours after collection. Platelet-rich plasma was prepared by centrifugation of citrate-anticoagulated blood and adjusted to 200,000 platelets/μL.10 One patient and 1 control sample (healthy dog) were evaluated simultaneously. Adenosine diphosphate (ADP)j (5 mM/mL) and collagenk (50 μg/mL) were used as aggregation agonists. Each sample was analyzed twice and the mean aggregation percentage was calculated for each agonist.
Serum protein electrophoresis (SPE) was performed on cellulose acetate membrane.l Serum viscosity relative to water was calculated using a micro-Ostwald viscosimeterm at 21.5–22.5 °C, and its value was recorded as the mean of 2 measurements per sample.11
E. canis seroreactivity was investigated in all 51 dogs, whereas radial immunodiffusion was applied for the detection of anti-Aspergillus spp. antibodies only in group A dogs.12 Polymerase chain reaction (PCR) amplification of E. canis 16S rRNA gene and of the 16S–23S rRNA intergenic spacer (ITS) of Bartonella genus was performed on BM aspirates.13,14
Serial arterial blood pressure measurements were taken with an ultrasonic Doppler flow detector.n Open-mouth radiographic examination of nasal cavities was carried out under general anesthesia in 17/24 group A and 15/27 group B dogs, and followed by rhinoscopy with a rigid 2.4 mm rhinoscopeo; 3 pinch biopsies were obtained from each nasal cavity (1 each from the frontal nasal cavity, nasal conchae, and ethmoidal conchae, or as dictated by the lesions). In the remaining dogs (8 group A and 12 group B), in which euthanasia had been elected by their owners, postmortem nasal biopsies were procured. All biopsies were processed according to standard procedure and stained with hematoxylin–eosin. Histopathologic evaluation was performed in a blinded fashion. The pattern of lesions was recorded and the severity of these changes was scored with a 0–3 subjective scale.
The homogeneity between group A and B dogs in terms of breed, sex, and age distribution was examined by Pearson's χ2 or Student's t-tests. Reference values for BMBT, PT, PTT, and platelet aggregation responses (for both ADP and collagen) were generated from the normal dogs as mean ± 1.96 standard deviation (SD) intervals.15 The comparison of hematology, serum biochemistry, coagulation, SPE, serum viscosity, and arterial blood pressure values between groups A and B was done by Student's t-tests. Subsequently, these variables were dichotomized (normal or abnormal) and the resulting frequencies, the seropositivity rate and the frequency of positive PCR for E. canis, were compared between A and B groups with Pearson's χ2 or Fischer's exact tests. The Mann-Whitney U-test was employed to compare the median scores of histologic lesions and the Pearson's χ2 test to compare the frequencies of histopathologic patterns between group A and B dogs. Finally, the strength of the linear associations between serum viscosity and γ-globulin concentrations as well as BMBT and creatinine concentration was estimated with Pearson correlation coefficients. SPSS for Windows 15.0p software was employed for the statistical analysis, and null hypotheses were rejected when P values were < .05.
The study population included 36 (70.6%) purebreds representing 15 different breeds, and 15 (29.4%) crossbreds. Thirty-two (62.7%) of the animals were intact males and 19 (37.3%) were intact females, ranging in age from 6 months to 11 years (median, 4 years). No differences could be found between group A and B dogs regarding breed, sex, and age distribution.
The hematologic and biochemical abnormalities of 51 dogs are presented in Table 1. Thrombocytopenia was documented in 6/24 group A (median platelet number, 202,000/μL; SD, 85,775/μL) and in 13/27 (median platelet number, 192,814/μL; SD: 89,640/μL) group B dogs. No difference was found between the 2 groups regarding the frequency of thrombocytopenia and mean platelet count. Hyperproteinemia, hyperglobulinemia, and hypoalbuminemia were the most common biochemical abnormalities. Total protein concentration was significantly higher (P= .029) in group A (mean, 9.49 g/dL; SD, 1.78) than in group B (mean, 8.37 g/dL; SD, 1.78). Based on SPE-generated protein fractions, hypergammaglobulinemia (reference value, <1.6 g/dL) was noted more frequently (P= .044) in group A (22/24) compared with group B (17/27) dogs. Also, the mean concentration of γ-globulins was higher (P= .013) in the former group (3.12 g/dL; SD, 1.47 versus 2.16; SD, 1.13). Seven group A dogs demonstrated a narrow globulin spike in the γ-globulin region indicative of monoclonal or oligoclonal gammopathy.
Table 1. Frequency of hematologic, biochemical, and urinalysis abnormalities in dogs with symptomatic leishmaniasis (Leishmania infantum) with (group A) and without epistaxis episodes (group B).
Hemostatic abnormalities of 51 dogs are presented in Table 2. The frequency of impaired platelet response to collagen-induced aggregation was higher (P= .042) and the mean percentage of platelet aggregation to the same agonist was lower in group A compared with group B (group A, 19.6%; SD, 15.5; group B, 35.4%; SD, 24.0; P= .012) dogs. Bleeding time (group A, 215 seconds; SD, 124.5 seconds; group B, 210 seconds; SD: 93.2 seconds) was positively correlated with serum creatinine concentration (P= .003).
Table 2. Frequency of hemostatic abnormalities in dogs with symptomatic leishmaniasis (Leishmania infantum) with (group A) and without epistaxis episodes (group B).
Group A (n = 24)
Group B (n = 27)
Higher frequency in group A than in group B dogs (P= .042).
(↑), prolonged time or increased concentration; (↓), decreased concentration or aggregation; PTT, partial thomboplastin time; FDPs, fibrin(ogen) degradation products; vWf, von Willebrand factor; ADP, adenosine diphosphate.
Serum viscosity was increased (normal value: < 2) in 26/51 dogs, and increased serum viscosity was significantly more common (P= .038) in group A (16/24) than in group B (10/27) dogs, although mean values (2.28; SD, 0.44 and 2.03; SD, 0.51 for groups A and B, respectively) did not differ. γ-Globulin concentration was positively correlated with serum viscosity (P < .001).
E. canis seropositivity was found in 8/24 group A and 15/27 group B dogs, whereas PCR amplification of the E. canis 16S rRNA gene was positive in 3/24 group A and 5/27 group B seropositive dogs; no statistical significance was found. Bartonella spp. 16S–23S rRNA ITS was PCR-amplified in 1 dog from each group. Only 1 group A dog was seropositive for Aspergillus spp., but with no radiographic, rhinoscopic, or histopathologic evidence of nasal infection.
Systemic hypertension (systolic arterial pressure ≥ 180 mmHg)16 was documented in 3/23 group A and 2/27 group B dogs, but no difference was found in terms of its frequency and mean value (group A mean: 155.6; SD, 20.2; group B mean: 144.4; SD, 23.7) between groups. One of 3 group A and both group B dogs with systemic hypertension had proteinuria and renal failure, as defined by serum creatinine concentration ≥1.4 mg/dL and urine protein-to-creatinine ratio of 0.5.17
Nasal radiography was invariably unremarkable. Rhinoscopic and postmortem examination of the nasal mucosa revealed mild to moderate mucopurulent discharge in 9/24 group A and 8/27 group B dogs. Mucosal petechiation and echymoses were observed in 9/24 and 7/27 group A and B dogs, respectively, whereas the corresponding figures for erosions or ulcers of variable size and depth were 4/24 and 2/27.
Nasal histopathology results are summarized in Table 3. According to the predominating cellular infiltrate (Fig 1a–d), lymphoplasmacytic rhinitis appeared to be the most common histolopathologic pattern (24/50), followed by mixed cell (8/50), granulomatous (6/50), and neutrophilic (4/50) rhinitis. In 4/24 group A and 3/26 group B dogs, a dual histopathologic pattern was found in different biopsies (Table 3). In addition, epithelial ulceration was observed in 6/24 group A and 1/26 group B dogs. Neutrophilic vasculitis was found only in 1 group B dog. No significant difference was found regarding the frequency of the overall nasal pathology or the mean lesional score between the 2 groups, apart from epithelial ulceration in group A dogs (P= .039). In the biopsies of 4 group A and 5 group B dogs, the nasal mucosa appeared normal. L. infantum-laden macrophages either in lesional or in normal mucosa were observed in 3/24 and 5/26 of group A and B dogs, respectively.
Table 3. Frequency of histopathologic lesions and their total scoring (mean ± SD) in the nasal mucosa in dogs with symptomatic leishmaniasis (Leishmania infantum) with (group A) and without epistaxis episodes (group B).
Group A (n = 24)
Group B (n = 26)
Concurrent discrete histopathological patterns were found in 4 group A (2 lymphoplasmacytic/neutrophilic, 1 with mixed-cell/neutrophilic, and 1 with mixed-cell/granulomatous rhinitis) and 3 group B (1 with lymphoplasmacytic/granulomatous, 1 with mixed-cell/granulomatous, and 1 with mixed-cell/lymphoplasmacytic rhinitis) dogs.
b Bacterial colonies, eosinophilic infiltration, fibrosis.
c Overlap with other histopathological patterns.
d Lesional score for each animal was the mean of all specimens screened.
This study was designed to determine the underlying pathomechanism of epistaxis occurring in natural CanL. To that end, we investigated the potential involvement of multiple clinical and laboratory abnormalities and the possible role of concomitant infections. Dogs with CanL and secondary renal or liver disease or E. canis coinfection were not excluded from the study, because their confounding effect was included in the scope of the study. Epistaxis, on the other hand, has never been reported in the experimental disease.18–21
Results of the present study indicate that no single abnormality invariably leads to the development of epistaxis in CanL, but suggest a multifactorial mechanism combining impaired platelet function, increased serum viscosity attributed to hyperglobulinemia, and nasal mucosa ulceration.
Hemostatic abnormalities have been investigated in several clinical and experimental studies of Leishmania-infected dogs with6 or without19,22–26 epistaxis. Thrombocytopenia in these animals was mostly of insufficient severity to trigger spontaneous bleeding, but thrombocytopathy reflected by sluggish platelet aggregation or prolonged BMBT has been reported as the most consistent hemostatic abnormality.6,19,23,24 The present study provides ample documentation of a substantial reduction in platelet aggregation, in terms of both frequency and percentage of platelet response to collagen, in Leishmania-infected dogs exhibiting epistaxis. Interestingly, collagen has been shown to be more sensitive than ADP in demonstrating impaired platelet aggregation in CanL.24–26 This abnormality would be unlikely to exclusively account for epistaxis, because it is an in vitro indicator of thrombocytopathy and its association with clinical bleeding has been inconsistent.19,24–26 Nevertheless, it might play a synergistic role in the induction of epistaxis in this disease. Not all dogs with impaired collagen or ADP aggregation demonstrated prolonged BMBT, and the latter was no more common or more severely prolonged in dogs with epistaxis, in contrast to what has been reported before.6 Bleeding time is a less sensitive in vivo test of platelet function, the prolongation of which usually indicates a platelet aggregation or adhesion defect, especially in canine and human uremic patients.10,23,27,28 Secondary and tertiary hemostatic defects do not seem to play an important role in the development of CanL-associated epistaxis, although FDPs were increased in all 7 dogs that had been tested.6
To our knowledge, this is the 1st report of increased serum viscosity in CanL. Hyperviscosity was positively correlated with hypergammaglobulinemia and documented in a significantly higher proportion of dogs with epistaxis (69.6%) compared with those without epistaxis (40%). CanL is an example of nonneoplastic disease that frequently leads to dysproteinemia and possibly to hyperviscosity syndrome.29–33 In theory, the latter may result in bleeding tendency, attributed mainly to thrombocytopathy resulting from decreased platelet adhesiveness owing to the coating effect exercised by the globulins, thus impairing the binding between vWf and the GPIb-IX platelet membrane receptor and rarely to direct vascular damage.32,34–36 However, no convincing evidence of other hyperviscosity-associated clinical signs (eg, neurologic, ophthalmologic) was documented in any of our dogs. Moreover, mean relative viscosity did not differ between the 2 groups (2.28 and 2.03 in groups A and B, respectively), and clinical signs attributable to hyperviscosity syndrome are expected with values > 4–5.11,34 Therefore, increased serum viscosity, with hyperglobulinemia-induced thrombocytopathy as its main pathogenetic component, appears to play primarily a contributory role in the pathogenesis of CanL-associated epistaxis.
E. canis infection, alone or in combination with CanL, frequently is implicated in canine epistaxis,5 but not in the present study, in which only a few dogs (3 group A and 5 group B) were truly infected, although seropositivity was much higher (8/24 and 15/27 group A and B dogs, respectively). Epistaxis in monocytic ehrlichiosis appears to be a clinical sign of a generalized bleeding tendency and has been mainly attributed to severe thrombocytopenia, usually as part of aplastic pancytopenia.5 None of these changes appeared in our study population. Similarly, coinfection with Bartonella spp. was a rare occurrence as defined by the results of BM PCR. Nevertheless, recent reports support the potential role of Bartonella species as a cause of epistaxis in dogs, alone or in conjunction with E. canis or other infectious agents.37,38
The role of systemic hypertension in the pathogenesis of canine epistaxis in general, and in CanL epistaxis in particular, remains an unresolved issue. Only a limited number of our dogs (5/51) were hypertensive and no significant differences were found between the 2 groups, as was also the case in a previous study.6 Epistaxis because of systemic hypertension has never been reported in the dog, and in humans this association still is controversial.5,39
Juttner et al6 reported that epithelial erosions and perivascular mixed-cell rhinitis were invariably seen in their CanL cases admitted either with or without epistaxis. The majority of our dogs also had inflammatory lesions of the nasal mucosa. Lymphoplasmacytic rhinitis was the predominant histologic pattern, followed by mixed-cell, granulomatous, and neutrophilic rhinitis. There was clear evidence of vasculitis only in 1 group B case, thus refuting the previous view that vasculitis is the most important underlying pathology of CanL epistaxis,40 but confirming again that CanL is a common cause of canine rhinitis especially in endemic areas of the disease. Mucosal ulceration, however, more frequently was seen in group A than in group B dogs, regardless of the underlying inflammatory pattern, thus raising suspicion of CanL in the majority of epistaxis cases. In some group B dogs, nasal mucosa hemorrhage (7/27) or ulceration was detected on rhinoscopy (2/27) and histopathology (1/26). Although none of these dogs had historical or clinical evidence of epistaxis, the possibility of minor nasal hemorrhage that went unnoticed in the past cannot be ruled out. These dogs may also exhibit future epistaxis episodes if they are not treated appropriately.
In conclusion, the results of this study indicate that a single pathogenetic mechanism cannot invariably be incriminated for epistaxis in natural CanL, but rather the interaction of various internal factors such as thrombocytopathy, hyperglobulinemia-induced serum hyperviscosity, and ulcerative or nonulcerative rhinitis most likely is responsible.
aSnap Leishmania, IDEXX, Westbrook, ME
bImmunoComb, Biogal-Galed, Kibbutz Galed, Israel
cSurgicutt Adult, International Technidyne Corp, Edison, NJ
nVet/Dopp Model 100, SDI Sensor Devices Inc, Waukesha, WI
oRhinoscope, Richard Wolf, Knittlingen, Germany
pSPSS for Windows 15.0, Chicago, IL
This project was financially implemented by the Companion Animal Clinic, Veterinary Faculty, Aristotle University of Thessaloniki, Thessaloniki, Greece. Dr P. Diniz was financially supported by IDEXX. All authors declare no conflict of interest. The authors express their gratitude to Dr H. Billinis and Dr A. Fytianou for their invaluable help in some of the laboratory testing, as well as to Dr S.K. Vakalopoulou for sharing her experience with us.