Clinicopathologic and atypical features of naturally occurring leptospirosis in dogs: 51 cases (2000–2010)

Lindsay E. Tangeman Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Meryl P. Littman Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Abstract

Objective—To determine clinicopathologic features, percentage of atypical abnormalities, antibody titers against Leptospira serogroups, and importance of convalescent titers in dogs with leptospirosis.

Design—Retrospective case series.

Animals—51 dogs with leptospirosis.

Procedures—Criteria for inclusion were at least 1 positive microscopic agglutination test (MAT) result (titer ≥ 1:1,600 in vaccinated dogs, titer ≥ 1:800 in nonvaccinated dogs, or ≥ 4-fold increase in convalescent titer), a complete medical record (including leptospirosis vaccination date, reason for initial evaluation, and CBC, serum biochemical analysis, and urinalysis results), and clinical signs or laboratory findings consistent with leptospirosis.

Results—Initial clinical signs, temporal distribution, and signalment were similar to previous reports. Convalescent MAT titers were necessary for diagnosis in 45% of cases. Atypical abnormalities included radiographic evidence of pulmonary disease in 10 of 23 dogs and hepatic involvement alone in 7 of 51 dogs. Other abnormalities included proteinuria in 34 of 51 dogs, thrombocytopenia in 26 of 51, coagulopathy in 7 of 24 dogs, hypoalbuminemia in 14 of 51 dogs, and glucosuria in 9 of 51 dogs. Significant associations were found between antibodies against serogroup Grippotyphosa and renal involvement and serogroup Icterohaemorrhagiae and hepatic involvement.

Conclusions and Clinical Relevance—Increased awareness of atypical abnormalities may decrease misdiagnosis of leptospirosis in dogs. Results of concurrent infectious disease testing should be interpreted with caution; misdiagnosis of leptospirosis could pose a public health risk. Convalescent titers were necessary to identify infection when acute testing results were negative. Further research is needed to determine the true associations between antibodies against identified serogroups and clinical features.

Abstract

Objective—To determine clinicopathologic features, percentage of atypical abnormalities, antibody titers against Leptospira serogroups, and importance of convalescent titers in dogs with leptospirosis.

Design—Retrospective case series.

Animals—51 dogs with leptospirosis.

Procedures—Criteria for inclusion were at least 1 positive microscopic agglutination test (MAT) result (titer ≥ 1:1,600 in vaccinated dogs, titer ≥ 1:800 in nonvaccinated dogs, or ≥ 4-fold increase in convalescent titer), a complete medical record (including leptospirosis vaccination date, reason for initial evaluation, and CBC, serum biochemical analysis, and urinalysis results), and clinical signs or laboratory findings consistent with leptospirosis.

Results—Initial clinical signs, temporal distribution, and signalment were similar to previous reports. Convalescent MAT titers were necessary for diagnosis in 45% of cases. Atypical abnormalities included radiographic evidence of pulmonary disease in 10 of 23 dogs and hepatic involvement alone in 7 of 51 dogs. Other abnormalities included proteinuria in 34 of 51 dogs, thrombocytopenia in 26 of 51, coagulopathy in 7 of 24 dogs, hypoalbuminemia in 14 of 51 dogs, and glucosuria in 9 of 51 dogs. Significant associations were found between antibodies against serogroup Grippotyphosa and renal involvement and serogroup Icterohaemorrhagiae and hepatic involvement.

Conclusions and Clinical Relevance—Increased awareness of atypical abnormalities may decrease misdiagnosis of leptospirosis in dogs. Results of concurrent infectious disease testing should be interpreted with caution; misdiagnosis of leptospirosis could pose a public health risk. Convalescent titers were necessary to identify infection when acute testing results were negative. Further research is needed to determine the true associations between antibodies against identified serogroups and clinical features.

Leptospirosis is a zoonotic disease of worldwide distribution.1,2 Leptospires are spirochete bacteria classified by serovar. There are > 250 known pathogenic serovars, and immunity is believed to be serogroup specific.3–6 A recently developed canine combined subunit vaccine offers immunization against serogroups Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona.

Clinical features of leptospirosis may vary by infecting serovar, geographic location, and host immune response.4,6,7 The classic disease is characterized by renal disease or concurrent renal and hepatic disease.8,9 Recently, leptospirosis has been associated with pulmonary hemorrhage, coagulopathies, electrolyte disturbances, and vasculitis in humans and dogs.10–13 Links between infecting serogroup and clinical features have been proposed,14 but a clear correlation has not yet been determined.4

Clinicopathologic changes are nonspecific but may reflect renal disease, hepatic disease, or both. A CBC may reveal thrombocytopenia.6 Urinalysis may reveal isosthenuria or hyposthenuria, with variable glucosuria or proteinuria.4 Recently, proteinuria has been increasingly recognized.8,12 Prolonged PT and PTT have been reported in 6% to 50% of canine cases.7,8,11 In Europe, evidence of pulmonary disease on thoracic radiographs in as many as 70% of canine cases has been reported and this finding is associated with a higher mortality rate.11

The MAT is presently considered the gold standard for diagnosis.4,6,8 The canine MAT detects antibodies against 5 to 7 serogroups including Autumnalis, Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona.4 Limitations of the MAT include possible false-negative results in the first weeks of illness, paradoxical reactions early in the course of disease, and lack of clear cutoff values.4,15 Typically, cross-reactions occur within the first 6 weeks of infection,15,16 and serogroups Autumnalis and Bratislava appear to be major cross-reactors. The American College of Veterinary Internal Medicine Leptospirosis Consensus Statement recommends that both acute and convalescent titers be measured, with convalescent titers measured 2 to 4 weeks after the acute titers and at least 7 to 14 days between successive titers.4 That statement and human literature indicate that a ≥ 4-fold increase in titer supports a diagnosis of recent infection.4,15,17 In previous reports, the diagnosis was routinely accepted on the basis of a titer ≥ 1:1,600 in vaccinated dogs and ≥ 1:800 in nonvaccinated dogs.4,15,18–20

Historically the serogroup with the highest MAT titer has been deemed the infectious serogroup.3,8,14 Recent reports3,15 in human and veterinary literature have questioned the accuracy of this technique. Presently, human MAT titers are obtained ≥ 2 weeks and as many as 4 to 6 weeks after the acute titer, to decrease the chance of paradoxical reactions.17,21–23 At this time, MAT titers are not believed to accurately predict the infectious serogroup.4,6,15,24

The objectives of the study reported here were to retrospectively review the clinicopathologic features of dogs naturally infected with leptospirosis, identify antibodies against Leptospira serogroups on the basis of acute and convalescent MAT titers, determine the percentage of atypical features, and evaluate the importance of convalescent titers. We hypothesized that many leptospirosis cases are misdiagnosed if a single acute titer is used and that atypical features may be more common than suspected.

Materials and Methods

Medical records of dogs with suspected leptospirosis at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania between January 2000 and January 2010 were reviewed. Criteria for inclusion were at least 1 MAT titer, a complete medical record (including most recent date of leptospirosis vaccination, reason for initial evaluation, CBC, serum biochemical analysis, and urinalysis results), and clinical signs or laboratory findings consistent with leptospirosis. Additional diagnostic test results that were reviewed but not required for inclusion were a convalescent MAT titer, coagulation profile, bacteriologic culture of urine, UPC, 3-view thoracic radiography, abdominal ultrasonography, and infectious disease testing, including tests for Dirofilaria immitis antigen; antibodies against Borrelia burgdorferi, Ehrlichia canis, or Ehrlichia chaffeensis and Anaplasma phagocytophilum or Anaplasma platysa; and antibodies against Rickettsia rickettsii,b Bartonella vinsonii or Bartonella henselae,c and Babesia canis.c All diagnostic tests were performed during the time of illness.

The MAT was performed in compliance with International Leptospirosis Society standards.d Results included titers against the serogroups Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona (51/51 dogs). Additionally, 12 of 51 dogs had results for serogroup Autumnalis and 41 of 51 had results for serogroup Bratislava. In accordance with previous studies and the American College of Veterinary Internal Medicine Leptospirosis Consensus Statement, titers were considered consistent with infection if ≥ 1:1,600 in vaccinated animals or if ≥ 1:800 in nonvaccinated animals or if a ≥ 4-fold increase was detected in a convalescent titer.

Urine was collected in a sterile fashion, and bacteriologic culture was performed in-house in 44 of 51 cases. Any urine sample that yielded bacterial growth was subsequently excluded from urinalysis and UPC determination. The CBC and serum biochemical tests were performed in-house, and blood smears were reviewed by a board-certified clinical pathologist when abnormalities were present. In all cases, 3-view thoracic radiography was performed and radiographs were reviewed by a board-certified radiologist. Thoracic radiographs were obtained at the time of initial evaluation as part of diagnostic testing. Abdominal ultrasonography was performed by a board-certified radiologist.

Leptospirosis cases were classified via organ involvement on the basis of serum biochemical profile and urinalysis results as renal only, hepatic only, or concurrent renal-hepatic. Renal-only involvement was defined by increased (compared with reference range) BUN concentration, increased creatinine concentration, or both, with or without glucosuria, but without increases in ALT, AST, ALP, or GGT activities or total bilirubin concentration. Hepatic-only involvement was defined by an increase in 3 of 5 liver-related variables (ALT, AST, ALP, and GGT activities and total bilirubin concentration), without an increase in BUN or creatinine concentration and without glucosuria. Concurrent renal-hepatic involvement was defined by an increase in concentration of BUN, creatinine, or both as well as 3 of the 5 liver values, with or without the presence of glucosuria, or by an increase in 3 of 5 of the liver values with concurrent glucosuria. Urine specific gravity results were not used in determining organ involvement because time of sample collection was not always stated and may have been influenced by IV fluid therapy.

Only dogs with their highest antibody titer against a single serogroup (Grippotyphosa, n = 20; Icterohemorrhagiae, 13; Pomona, 8) were included in the statistical analysis for association between serogroup with the highest antibody titer and type of organ involvement. When both an acute and convalescent MAT titer was performed, the serogroup identified by the convalescent titer was used.

Statistical analysis—Because of the low number of dogs in the study, associations were determined by use of the Fisher exact test. All tests were 2 tailed, and a value of P < 0.05 was considered significant.

Results

Of 138 dogs with suspected leptospirosis, 87 were excluded. Eighty-four were excluded because of inconsistent MAT findings (titer < 1:1,600 in vaccinated dogs or < 1:800 in nonvaccinated dogs), 1 dog was excluded for lack of a complete medical record, and 2 dogs were excluded for lack of a CBC, serum biochemical profile, or both. All 84 dogs excluded for inconsistent MAT findings had only an acute titer measured.

Fifty-one dogs met the criteria for inclusion. Mean age of the dogs was 6.5 years, (range, 0.33 to 18.33 years). Eighteen (35%) were spayed females, 3 (6%) were sexually intact females, 13 (26%) were castrated males, and 17 (33%) were sexually intact males. Affected breeds included mixed (n = 9), Golden Retriever (4), Cocker Spaniel (3), English Springer Spaniel (3), German Shepherd Dog (3), Rottweiler (3), Labrador Retriever (2), Pug (2), Siberian Husky (2), West Highland White Terrier (2), and 1 each of Airedale Terrier, Australian Shepherd Dog, Beagle, Bichon Frise, Boxer, Cairn Terrier, Chihuahua, Dalmatian, Doberman Pinscher, Jack Russell Terrier, Maltese, Mastiff, Miniature Pinscher, Pembroke Welsh Corgi, Pomeranian, Shar-Pei, Staffordshire Terrier, and Toy Poodle.

For 28 (55%) dogs, leptospirosis was diagnosed by use of a single MAT titer in a serum sample obtained within 72 hours after initial evaluation. Twenty-three (45%) dogs required a convalescent MAT (7 to 21 days after the acute titer was measured) for diagnosis. When available, the serogroup identified via the convalescent titer was used. Diagnosis was most common in the fall months with 24 of 51 (47%) cases occurring between September and December. The MAT titers revealed antibodies against serogroups Grippotyphosa (n = 20 dogs), Icterohaemorrhagiae (13), Pomona (8), Autumnalis (2), and Hardjo (1), with equal titers against Grippotyphosa and Icterohaemorrhagiae (3), Grippotyphosa and Pomona (2), Bratislava and Icterohaemorrhagiae (1), and Grippotyphosa and Bratislava (1). No dogs had positive results for Canicola or Bratislava alone. The range of antibody titers was 1:800 to 1:102,400, with a median titer of 1:1,600. The highest reported antibody titer of 1:102,400 was against serogroup Grippotyphosa. The lowest reported antibody titer of 1:800 was against multiple serogroups. Thirty-nine dogs had never been vaccinated against leptospirosis, 12 were vaccinated in their lifetimes, and 9 were vaccinated within a year prior to diagnosis. Of the 9 dogs vaccinated within a year of diagnosis, 3 had convalescent titers consistent with infection and none were vaccinated within 3 months of their positive MAT result. Microscopic agglutination test titers of dogs vaccinated within a year of diagnosis ranged from 1:1,600 to 1:51,200.

Clinical signs that precipitated initial evaluation included inappetance or anorexia (n = 29), lethargy (22), vomiting (21), polyuria and polydipsia (12), diarrhea (6), weight loss (4), anuria (2), difficulty breathing (1), and ataxia (1). In addition, 18 dogs were referred because of acute renal failure, 4 were referred for hemodialysis, and 3 were referred because of hepatic disease.

Complete blood count and serum biochemical abnormalities included thrombocytopenia, azotemia, high serum liver enzyme activities and total bilirubin concentration, hyperphosphatemia, hypoalbuminemia, and hypokalemia. Urinalysis abnormalities included isosthenuria, hyposthenuria, proteinuria, and glucosuria as determined via urine dipstick testing and calculation of the UPC. Proteinuria was identified in 34 of 51 (66%) dogs (Table 1). Urine protein-to-creatinine concentration ratio was abnormal in 14 of 19 dogs. Four dogs had positive results of bacteriologic culture of urine (all with Escherichia coli); these samples were excluded from urinalysis and evaluation of proteinuria, glucosuria, and UPC.

Table 1—

Clinicopathologic variables in 51 dogs with leptospirosis.

VariableMedian (range)Proportion (%) of dogs with abnormal valuesReference range
BUN (mg/dL)68 (6–213)38/51 (75)5–30
Creatinine (mg/dL)3.8 (0.6–15.2)36/51 (71)0.7–1.8
ALT (U/L)91.5 (10–2,460)26/51 (51)16–91
AST (U/L)62 (15–1,766)24/51 (47)23–65
ALP (U/L)191.5 (23–2,445)30/51 (59)20–155
GGT (U/L)16 (3–136)12/51 (24)7–24
Total bilirubin (mg/dL)0.43 (0–44.2)19/51 (37)0.1–0.5
Phosphorus (mg/dL)6.9 (3–19.6)28/51 (55)2.8–6.1
Albumin (g/dL)2.8 (1.6–5.4)14/51 (27)2.5–3.7
Potassium (mmol/L)4.2 (2.6–5.8)18/51 (35)4.0–5.2
Urine protein (dipstick)2+ (trace to 3+)34/51 (66)< 2+ if USG < 1.012, or trace if USG < 1.008
Urine glucose (dipstick)Trace (trace to 3+)9/51 (18)Negative
UPC1.85 (0.15–7.34)14/19 (73)< 0.5
USG1.012 (1.005–1.040)24/51 (47)1.013–1.045
PT (s)8.4 (6.1–13.2)7/24 (29)6.8–10.2
PTT (s)13.5 (10.4–17.9)6/24 (25)10.7–16.4
Platelet count (× 103/μL)168.5 (2.5–647)26/51 (51)177–398

Additional diagnostic tests included coagulation profiles, thoracic radiography, abdominal ultrasonography, and testing for concurrent infectious diseases. Dogs undergoing hemodialysis (n = 4) were excluded from coagulation profile review because predialysis coagulation profiles were not available. Coagulation profiles were available for 24 dogs. Abnormalities included high PT, PTT, and fibrin degradation product concentrations. Both PT and PTT were concurrently increased in 4 of 24 (17%) dogs (Table 1). Thoracic radiographs were abnormal in 10 of 23 (43%) dogs. Abnormalities included an interstitial pattern (n = 4), pleural effusion (3), alveolar pattern (2), linear pulmonary vascular pattern (1), and sternal lymphadenopathy (1). Abdominal ultrasonograms were available for 38 of 51 dogs. Abnormalities included a hyperechoic renal cortex (n = 16), sediment in the gallbladder (16), bilateral renomegaly (12), mild to moderate abdominal effusion (9), bilateral pyelectasia (8), heterogeneous hepatic parenchyma (7), decreased renal corticomedullary junction distinction (7), hypoechoic hepatic parenchyma (5), thickened gastric wall (4), hepatomegaly (3), bilaterally irregular renal surface (3), mesenteric lymphadenopathy (3), splenomegaly (2), hypoechoic pancreatic parenchyma (2), hyperechoic hepatic parenchyma (2), and 1 each for hyperechoic splenic parenchyma, splenic nodule, thickened small intestinal wall, and renal calculi (unilateral). Abdominal ultrasonography results were within normal limits in 4 of 51 dogs. Infectious disease test results were positive for D immitis antigen in 0 of 21 dogs, and antibodies were detected against B burgdorferi in 8 of 25 (32%) dogs, R rickettsii in 1 of 18 dogs (weak positive result), A phagocytophilum or A platys in 0 of 9 dogs, B canis in 0 of 1 dog, B henselae in 0 of 1 dog, B vinsonii in 0 of 4 dogs, and E canis or E chaffeensis in 0 of 25 dogs.

Classification by organ involvement for type of infection revealed renal-only involvement in 26 of 51 (51%) dogs, concurrent renal-hepatic involvement in 17 of 51 (33%) dogs, hepatic-only involvement in 7 of 51 (14%) dogs, and neither in 1 of 51 (2%) dogs. Serogroups were evaluated for the highest antibody titer, organ involvement, and evidence of pulmonary changes. Dogs with antibody titers in multiple serogroups were not considered in statistical analysis. Positive associations were found between antibodies against serogroups Grippotyphosa and renal-only infection (P = 0.02) and between antibodies against Icterohemorrhagiae and hepatic-only infection (P = 0.01). No significant associations were found between antibodies against other serogroups and organ involvement. No significant association was found between antibodies against specific serogroup and pulmonary involvement. However, 3 of 4 dogs with necropsy findings consistent with pulmonary involvement had their highest antibody titer against serogroup Icterohemorrhagiae. It is important to note that associations were based on the convalescent titers when available (20/51 dogs) and a single MAT titer in 24 of 51 dogs.

Necropsy results were available for 5 dogs. Necropsy results were consistent with leptospirosis and included the presence of spirochete organisms detected via the Warthin-Starry stain, pulmonary fibrosis, vasculitis and thromboemboli, renal tubular necrosis, and hepatocellular necrosis.

Discussion

Results of this study were largely in agreement with previous studies4–7,9,20,25,26 regarding temporal patterns, signalment, and clinical signs. Previously recognized findings, including proteinuria, thrombocytopenia, coagulopathy, hypoalbuminemia, and glucosuria, were identified. Other previously recognized abnormalities such as uveitis, cardiac manifestations, and intussusceptions were not identified. The study also identified atypical features, including the single clinical sign of respiratory distress in 1 dog, referral because of hepatic disease, radiographic evidence of pulmonary disease in 43% of dogs, and hepatic-only infection in 14% of dogs. Results of this study also stressed the importance of use of convalescent titers in accurately diagnosing leptospirosis because 45% of cases would have been misdiagnosed without a convalescent titer. Associations between antibodies against specific serogroups and clinical features were identified, but these findings should be interpreted with caution because MAT titers cannot accurately predict the infecting serogroup.4,6,15,24

The most common clinical signs in this study were consistent with those described in the American College of Veterinary Internal Medicine Consensus Statement.4 However, atypical clinical signs and reasons for initial evaluation, such as respiratory difficulty and referral because of hepatic disease, were also observed. A study11 from Europe reported respiratory tract signs in as many as 62% of dogs at the time of diagnosis. Although this was not observed as commonly in the present study (difficulty breathing was reported in 2% of dogs), a large portion of dogs had thoracic radiographic abnormalities (43%). The discrepancy between studies may be a result of location (Europe vs North America) or may be a reflection of client-reported complaints (the present study) versus veterinarian-reported abnormalities. Additionally, respiratory tract signs usually appear between days 4 and 6 of disease11; it is possible we evaluated radiographs in a more acute setting (thoracic radiographs were obtained at initial evaluation as part of the diagnostic protocol) before radiographic changes were evident. Finally, it is possible that many dogs evaluated because of respiratory tract disease alone were not tested for leptospirosis and consequently not included in the study. Dogs with primarily respiratory tract disease may receive a misdiagnosis if not tested for leptospirosis.

The presence of thrombocytopenia was similar in the present study (26/51 [51%]), compared with previous reports4,11,13,14,25,26 (58%). Platelet counts ranged widely in the present study (2.5 × 103 platelets/μL to 647 × 103 platelets/μL; mean, 213 × 103 platelets/μL), making multiple pathogeneses possible. Although most platelet counts were only mildly decreased (possibly because of consumption, vasculitis, or both), a small portion was substantially decreased (suggestive of destruction, immune-mediated disease, or decreased production). Serum biochemical findings were largely in accordance with previous reports. Azotemia was found in 71% to 75% of cases (previous reports, 57% to 90%).8,11,14,26 The percentage of dogs with increased liver enzyme activities or analyte concentrations differed per variable, but overall an increase in at least 3 of 5 values was detected in 24 of 51 dogs. Hypoalbuminemia was identified in 14 of 51 dogs. Hypoalbuminemia may be secondary to decreased production associated with hepatic failure, renal loss, anorexia, or acute-phase proteins. Gastrointestinal tract loss cannot be ruled out because many dogs had gastrointestinal tract signs, although globulin and cholesterol concentrations were largely within reference ranges, making this less likely. Additionally, these dogs did not have evidence of liver dysfunction and their disease onset was acute, making hepatic failure less likely. Given the large proportion of dogs with proteinuria, hypoalbuminemia was likely attributable to renal loss27; however, given the retrospective nature of the study, other causes could not be definitively ruled out. It is also possible that IV fluid therapy affected protein concentrations. Hypokalemia was detected in 18 of 51 dogs. Many of these dogs were receiving IV fluid therapy, making it difficult to determine whether this resulted from dilution versus disease. Leptospira endotoxins affect Na-K-ATPase in nephrons, which may contribute to hypokalemia.28,29 Leptospira infection is reported to induce hypokalemic renal failure in humans caused by impaired tubular sodium re-absorption, but it is presently unknown whether this occurs in dogs,4,28 and further investigation is needed.

Urinalysis revealed evidence of isosthenuria, hyposthenuria, proteinuria, and glucosuria in many dogs. Although the effects of IV fluid therapy on USG were unknown in this study, Leptospira infection is known to impair renal concentrating ability.4 Proteinuria was detected in a large portion of dogs (34/51) and could be attributable to tubular or glomerular damage. Leptospirosis causes acute interstitial nephritis and tubular dysfunction and is suspected to cause mesangial proliferative glomerulonephritis.4,8,29 Consequently, proteinuria should be anticipated. The wide range of UPC results in the present study suggested both glomerular and tubular components.27 Assuming these cases reflected only leptospirosis infection, clinicians should be cautious in interpreting UPC results because the high magnitude of proteinuria may make differentiation from other diseases (eg, borreliosis-associated nephritis) even more difficult. Glucosuria was detected in 9 of 51 dogs, providing evidence of renal tubular disease.

Although previous studies4,13 have detected prolonged PT or PTT in 6% to 50% of dogs, little emphasis has been placed on coagulopathy as a clinical feature of leptospirosis. Coagulation profiles in the present study confirmed coagulopathies in up to 7 of 24 cases. Although there was no evidence of disseminated intravascular coagulation in this population, not all dogs had PT or PTT results. Results from this study reinforce the importance of monitoring for coagulopathies in affected dogs.

Concurrent infectious disease testing was performed in 25 of 51 dogs. Although positive test results for borreliosis were obtained in 8 of 25 (32%) dogs, no further confirmatory diagnostic tests were performed. Given the retrospective nature of the study, it was difficult to determine whether these dogs were truly infected. Interestingly, this study revealed a large percentage of dogs with proteinuria, hypoalbuminemia, and glucosuria, which could easily result in a misdiagnosis of borreliosis, nephritis, or protein-losing disease. Given that 8 of 25 dogs in this study had positive results for B burgdorferi, clinicians must be cautious in interpretation. However, positive test results for borreliosis may serve as a marker of exposure to wildlife and should increase clinician awareness of possible exposure to leptospires. Convalescent MAT titers with a ≥ 4-fold increase in titer are important for diagnosis of leptospirosis and argue against another diagnosis. Additional diagnostic findings, such as positive results of a Leptospira PCR assay, a low titer against or negative results of antibody testing for Lyme C6 antibody, or both, may indicate leptospirosis as the cause of proteinuria. Urine electrophoresis and evaluation of renal biopsy specimens may help differentiate glomerular from tubular proteinuria.8,29 A diagnosis of leptospirosis may carry a better prognosis, and a misdiagnosis could lead to substantial public health risk.

In the present study, the serogroups with the highest antibody titers were Grippotyphosa (20/51) and Icterohemorrhagiae (13/51). This large proportion of Icterohemorrhagiae cases may reflect the inner-city population of the study group and their potential exposure to rodent carriers. Microscopic agglutination test titers against serogroups Autumnalis and Bratislava were not available for all dogs, which may have affected results. Information regarding regional serovars may be useful in aiding diagnosis and determining vaccine strategy. However, annual vaccination failed to protect 9 dogs in the study. All of these dogs had clinical signs, and 3 had convalescent titers consistent with leptospirosis. Additionally, vaccinal MAT titers typically wane 3 to 4 months after vaccination.4,6 All dogs in the present study vaccinated within a year of diagnosis had a positive MAT result (titer ≥ 1:1,600) at least 4 months after vaccination, suggesting true infection. This information should be interpreted with caution because some dogs may have unusually high titers after vaccination.30 Additionally, some dogs may have titers indicative of a positive result without active infection.31 It is possible that the dogs with anti-Borrelia antibodies in the present study were truly infected with B burgdorferi and simply had high postvaccinal leptospirosis titers. However, 7 of 8 dogs with anti-Borrelia antibodies had either not received leptospirosis vaccination within the year or had never been vaccinated. The single dog with anti-Borrelia antibodies that had received a leptospirosis vaccination 9 months prior to evaluation had convalescent MAT titers confirming infection (1:200 to 1:1,600). These data suggest that leptospirosis may remain a differential diagnosis even for dogs with up-to-date vaccination histories.

Convalescent titers were necessary for diagnosis in 23 of 51 dogs, thereby reinforcing their importance. Eighty-four dogs were excluded from this study on the basis of an MAT titer inconsistent with leptospirosis, all of which had only an acute titer obtained. It is possible that, in these dogs, disease was misdiagnosed on the basis of the single titer and that, had convalescent titers been performed, more diagnoses of leptospirosis might have been made. Additionally, the serogroup with the highest acute MAT antibody titer was occasionally different from the serogroup with the highest convalescent MAT titer. Delayed seroconversion has been recognized in human medicine and can occur as many as 6 weeks after infection. If the same is true in dogs, perhaps veterinarians should consider determining convalescent titers even later (eg, 4 to 6 weeks after initial evaluation) than the current 2-week recommendation.4,15,16,18 More research is needed to determine the time frame in which dogs seroconvert.

Associations between antibodies against serogroups Grippotyphosa and renal involvement and Icterohemorrhagiae and hepatic involvement were found. No significant association was found between antibodies against a serogroup and the detection of pulmonary changes via radiography or necropsy. This was likely a result of low case numbers. However, 3 of 4 dogs with pulmonary changes consistent with leptospirosis at necropsy had their highest titer against serogroup Icterohemorrhagiae. More studies with larger case numbers are needed to investigate this association. Presently, MAT titers cannot accurately predict the infective serogroup, so further research and comparison between MAT titers and culture results is needed to determine the importance of these associations. Additionally, 24 of 51 associations were based on the acute MAT titer alone. Given that cross-reactions are possible in the first 6 weeks of infection, it is possible that associations made on the basis of single MAT titers may not be accurate.

Atypical clinical features including radiographic evidence of pulmonary disease and hepatic involvement alone were identified in the present study. It is presently believed that hepatic infection almost always occurs in conjunction with azotemia and that renal failure alone is the most common clinical finding.8 It has also been suggested that hepatic disease may occur later in the course of infection (after azotemia). Given that the dogs in the present study did not have long-term follow-up, it is possible that some dogs were azotemic prior to evaluation and consequently their disease involvement was misclassified. However, this seems less likely given that the dogs classified as having hepatic-only disease involvement did not have any urinalysis findings indicative of renal damage. Additionally, in 5 of 7 of these dogs, diagnosis was made on the basis of a convalescent titer. This suggested that hepatic-only involvement was present at initial evaluation. The present study identified 7 of 51 dogs as having hepatic-only involvement without evidence of azotemia or glucosuria. This should alert clinicians to the possibility of leptospirosis with hepatic changes alone.

Radiographic evidence of respiratory tract disease was found in 10 of 23 dogs, with 1 dog evaluated because of respiratory tract signs alone (increased respiratory effort and rate). Thoracic radiography revealed a diffuse dense interstitial pattern. There was no evidence of pneumonia on CBC results, and the patient was afebrile. Coagulation values were within reference ranges. During hospitalization, the dog later developed acute renal failure, leading to diagnostic testing confirming leptospirosis. This dog survived with treatment; consequently, no necropsy was performed to identify possible leptospirosis-associated pulmonary hemorrhage. Had the dog not developed acute renal failure, a diagnosis of leptospirosis may have not been made. This should alert clinicians to the possibility of leptospirosis with respiratory tract signs alone.

Although the retrospective nature of this study limited the ability to definitively identify pulmonary changes as being caused by leptospirosis, necropsy findings in 4 of 5 dogs were consistent with previous human reports10–12 of leptospirosis-induced pulmonary changes (fibrosis, vasculitis, and thromboemboli). It is possible that some of the radiographic changes observed in the present study may have been caused by fluid therapy because 23 of the 51 dogs were referred and may have received IV fluids prior to our evaluation. Although most thoracic radiographic findings in this study were consistent with leptospirosis-associated pulmonary hemorrhage (interstitial and alveolar pattern),6 3 dogs had pleural effusion and 1 dog had sternal lymphadenopathy. Two of the 3 dogs with pleural effusion had hypoalbuminemia, radiographic evidence of pleural effusion, and ultrasonographic evidence of peritoneal effusion, making effusion secondary to hypoalbuminemia most likely. The third dog had albumin concentration of 2.7 g/dL (reference range, 2.5 to 3.7 g/dL) and was referred from another veterinary hospital, so it is possible that this dog received IV fluid therapy that caused the pleural effusion. The single dog with sternal lymphadenopathy did not have other thoracic radiographic abnormalities, and an abdominal ultrasonographic examination was not performed. The cause of the lymphadenopathy was unknown, but may have resulted from hepatitis. Given that leptospirosis is not a common differential diagnosis for respiratory distress, it is also possible that some dogs evaluated because of respiratory tract disease alone were not tested and consequently not included in this study. Clinicians should consider the possibility of leptospirosis in dogs with signs of respiratory tract disease.

Abdominal ultrasonographic findings associated with leptospirosis have not been clearly defined. The renal changes identified in the present study were consistent with a previous report6 and included increased renal cortical echogenicity (n = 16), renomegaly (12), pyelectasia (8), and abdominal effusion (9). Specific hepatic changes have not been previously described. Most of the hepatic changes identified in the present study were heterogeneous hepatic parenchyma (n = 7) and gallbladder sediment (16). It was unclear whether these were incidental findings or related to leptospirosis.

Limitations of this study included its retrospective nature. Although every effort was made to exclude dogs with concurrent disease, it is possible that some were included. Dogs were classified via organ involvement on the basis of serum biochemical and urinalysis results. It is important to note that USG was not considered in identifying dogs with renal involvement; therefore, it is possible that some dogs with prerenal azotemia had their disease involvement misclassified. Additionally, organ involvement could not be classified in 1 dog; that dog was referred for leptospirosis testing because of a known exposure and was seropositive. The dog had concentrated urine with 3+ proteinuria and therefore may have had renal involvement. It is possible that the dog was evaluated early in the disease process before azotemia developed or values of liver-associated variables increased. It is also possible that evidence of organ dysfunction was missed because of IV fluid therapy administered prior to referral. Retrospective review also made it difficult to determine the underlying cause of pulmonary changes. Although necropsy findings were consistent with leptospirosis,12 necropsies were only performed in 5 dogs. The large number of dogs with atypical clinical signs may have been influenced by the referral nature of this hospital. Many dogs with typical signs of leptospirosis may not have been referred and consequently not included in review. Vaccination history was based on owner reports and referral veterinarian records, so there was a possibility for errors in that information. Finally, the low number of dogs in this study and the basis of diagnosis on MAT results made it difficult to determine significant associations between serogroups with the highest antibody titers and various clinical signs.

In general, clinical signs of canine leptospirosis in the present study were similar to those previously reported. The most commonly identified antibody titers were against serogroups Grippotyphosa and Icterohemorrhagiae. Of the 51 dogs, 23 (45%) had negative initial (acute) MAT results, but positive results at convalescence, reinforcing the importance of obtaining both acute and convalescent titers. Atypical clinical signs were identified, and leptospirosis should be considered as a differential diagnosis in cases of primary hepatic disease and pulmonary disease. Leptospirosis should not be excluded in dogs up to date on annual leptospirosis vaccination.

ABBREVIATIONS

ALP

Alkaline phosphatase

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

GGT

γ-Glutamyltransferase

MAT

Microscopic agglutination test

PT

Prothrombin time

PTT

Partial thromboplastin time

UPC

Urine protein-to-creatinine concentration ratio

USG

Urine specific gravity

a.

SNAP-4Dx, IDEXX Laboratories, Fremont, Calif.

b.

Protatek Reference Laboratory, Chandler, Ariz.

c.

North Carolina State University Laboratory, Raleigh, NC.

d.

ANTECH Laboratories, Irvine, Calif.

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