Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis).

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at -20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen's Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

Clinical and Clinicopathologic Discriminators Between Canine Acute Monocytic Ehrlichiosis and Primary Immune Thrombocytopenia.

Acute canine monocytic ehrlichiosis due to Ehrlichia canis (aCME), and primary immune thrombocytopenia (pITP) are major differentials for dogs presented with thrombocytopenia, and the two diseases may clinically overlap. The aim of this study was to compare dogs diagnosed with naturally occurring aCME and pITP, to establish potentially useful clinical and clinicopathologic discriminators. A clinical record-based retrospective study was performed in 35 dogs diagnosed with aCME and 29 dogs with pITP. Dogs with aCME were significantly younger, and were more likely to experience depression or lethargy, anorexia, body weight loss, fever, lymphadenomegaly, tick infestation, and ocular discharge on admission, compared to dogs with pITP. In contrast, dogs with pITP presented more frequently with overt bleeding and had a significantly higher bleeding score compared to dogs with aCME. Dogs with aCME were more likely to be anemic and hypoalbuminemic on presentation compared to dogs with pITP. Dogs with pITP had higher white blood cell and neutrophil counts as well as lower platelet counts than dogs with aCME and were more likely to present with leukocytosis, neutrophilia and monocytosis. These clinical, hematological, and biochemical findings may be helpful discriminators between aCME and pITP, on the understanding that they will be interpreted in the context of disease-specific testing.

Prevalence of antinuclear antibodies and rheumatoid factor titers in dogs with arthritis secondary to leishmaniosis (Leishmania infantum).

Dogs with infectious arthritis may occasionally exhibit positive serum antinuclear antibody (ANA) and rheumatoid factor (RF) titers; however, relevant data are sparse for arthritis secondary to canine leishmaniosis (CanL) caused by Leishmania infantum. We determined the prevalence of positive serum ANA and RF titers in dogs with arthritis secondary to CanL. Blood samples from adult, client-owned dogs with purulent arthritis secondary to CanL, without any comorbidities, were collected for diagnostic purposes. Serum ANA titers were measured by immunoperoxidase test and RF titers by the Rose-Waaler latex test. Twelve of 23 dogs enrolled prospectively in our study had clinical arthritis, and 11 of 23 had subclinical arthritis. Based on LeishVet clinical staging, 7 dogs had clinical stage II disease, 11 had clinical stage III disease, and 5 had stage IV. None of the 23 dogs was seropositive for ANA; 3 of 23 were positive for RF. ANA and/or RF seropositivity, in dogs with CanL-associated arthritis, appears to be weak, if present at all. Based on our results, positive serum ANA and RF titers should not be expected in dogs with arthritis secondary to CanL.

Evaluation of serum symmetric dimethylarginine as a biomarker of kidney disease in canine leishmaniosis due to Leishmania infantum.

Canine leishmaniosis (CanL)-associated chronic kidney disease is a leading cause of morbidity and mortality in Mediterranean countries. Novel renal biomarkers, such as serum symmetric dimethylarginine (sSDMA), may be useful surrogates for the detection of renal functional impairment. The objectives of this study were to investigate sSDMA concentrations in dogs with CanL, with and without azotemia, and to establish any potential association with the prevalence and severity of proteinuria, with the prevalence of decreased urine specific gravity and with the LeishVet clinical stages of CanL. Serum samples from 68 dogs with CanL (50 nonazotemic and 18 azotemic) and 17 healthy dogs were retrospectively examined. Increased sSDMA was documented in 26 % of dogs with CanL without azotemia and in 83.3 % of dogs with azotemia. Serum SDMA was significantly higher in azotemic compared to nonazotemic dogs and was associated with the presence and severity of proteinuria, the decreased urine specific gravity and the advanced clinical stages of CanL. The results of the present study indicate that sSDMA may be a useful adjunct to serum creatinine and urine protein/creatinine ratio for the detection of CanL-associated nephropathy, but it is of limited value for distinguishing among the LeishVet clinical stages of CanL.

Cardiac troponin I concentrations, electrocardiographic and echocardiographic variables remained unchanged in dogs experimentally infected with Ehrlichia canis.

Canine monocytic ehrlichiosis (CME, Ehrlichia canis) has occasionally been associated with myocardial injury. The aim of the present study was to serially measure and evaluate cardiac troponin I (cTnI) concentrations in dogs with experimentally induced acute and subclinical CME and to evaluate potential associations between cTnI concentration and an array of echocardiographic and electrocardiographic parameters. Serum cTnI concentration and simultaneous echocardiographic and electrocardiographic recordings were evaluated in 12 healthy Beagle dogs prior to experimental infection and on days 20 and 90 post-inoculation with E. canis. Almost all serum cTnI concentrations were below the limit of detection and selected electrocardiographic and echocardiographic parameters remained unchanged throughout the study.

Quantitative and qualitative leukocyte abnormalities in dogs with experimental and naturally occurring acute canine monocytic ehrlichiosis.

BACKGROUND: Canine monocytic ehrlichiosis (CME) is one of the most important tick-borne diseases worldwide. Cytopenias have been observed in both acute (nonmyelosuppressive) and chronic (myelosuppressive) CME; however, leukocyte abnormalities and indices have been incompletely described in dogs with acute CME. OBJECTIVES: The aims of this study were to analyze temporal changes in differential leukocyte counts, leukocyte morphology, myeloperoxidase index (MPXI), and lobularity index (LI) in dogs with experimental and naturally occurring acute CME. METHODS: Differential leukocyte counts and morphology were evaluated in archived blood smears from 13 Beagle dogs experimentally infected with Ehrlichia canis and evaluated weekly for 42 days postinfection (DPI); 20 dogs with naturally occurring acute CME also were evaluated. MPXI and LI were obtained from ADVIA reports. Wilcoxon tests were used to assess changes over time; leukogram results in natural cases were assessed in comparison with reference intervals. RESULTS: In experimental dogs, significant decreases in neutrophil, monocyte, lymphocyte, and eosinophil counts, and a mild left shift occurred within 14 DPI. The MPXI decreased significantly between 14 and 21 DPI and remained low, while LI increased from 14 to 35 DPI. Lymphocyte counts rebounded at 21 DPI, normalizing total WBC counts. Neutrophil toxicity was seen rarely, but reactive lymphocytes were observed frequently. Dogs with natural infection had variable patterns of leukocyte changes. CONCLUSIONS: Acute CME is associated with several discrete quantitative and qualitative leukogram changes indicative of concurrent inflammation, antigenic stimulation, and stress. Changes in MPXI and LI warrant further investigation in dogs with CME and other diseases.

Evaluation of the Therapeutic Potential of Anti-TLR4-Antibody MTS510 in Experimental Stroke and Significance of Different Routes of Application.

Toll-like receptors (TLRs) are central sensors for the inflammatory response in ischemia-reperfusion injury. We therefore investigated whether TLR4 inhibition could be used to treat stroke in a standard model of focal cerebral ischemia. Anti-TLR4/MD2-antibody (mAb clone MTS510) blocked TLR4-induced cell activation in vitro, as reported previously. Here, different routes of MTS510 application in vivo were used to study the effects on stroke outcome up to 2d after occlusion of the middle cerebral artery (MCAO) for 45 min in adult male C57Bl/6 wild-type mice. Improved neurological performance, reduced infarct volumes, and reduced brain swelling showed that intravascular application of MTS510 had a protective effect in the model of 45 min MCAO. Evaluation of potential long-term adverse effects of anti-TLR4-mAb-treament revealed no significant deleterious effect on infarct volumes nor neurological deficit after 14d of reperfusion in a mild model of stroke (15 min MCAO). Interestingly, inhibition of TLR4 resulted in an altered adaptive immune response at 48 hours after reperfusion. We conclude that blocking TLR4 by the use of specific mAb is a promising strategy for stroke therapy. However, long-term studies with increased functional sensitivity, larger sampling sizes and use of other species are required before a clinical use could be envisaged.

Acute phase protein and antioxidant responses in dogs with experimental acute monocytic ehrlichiosis treated with rifampicin.

There is currently lack of information on the changes of acute phase proteins (APP) and antioxidant markers and their clinical relevance as treatment response indicators in canine monocytic ehrlichiosis (CME). The objective of this study was to investigate the patterns of C-reactive protein (CRP), haptoglobin (Hp), ferritin and paraoxonase-1 (PON-1) during treatment of dogs with acute CME with rifampicin. Blood serum samples from ten Beagle dogs with experimental acute CME were retrospectively examined. Five dogs (Group A) were treated with rifampicin (10mg/Kg/24h), per os, for 3 weeks and 5 dogs (Group B) received no treatment (infected controls). Two Beagle dogs served as uninfected controls. Blood serum samples were serially examined prior to Ehrlichia canis inoculation and on post-inoculation days 14, 21, 28, 35 and 42. Significant changes of CRP, Hp, ferritin and PON-1 values were found in the majority of infected dogs. However, their concentrations did not differ between the two groups during the treatment observation period. The results of this study indicate that although several APP and PON-1 tend to significantly change in the majority of dogs with acute CME, they were of limited clinical relevance as treatment response indicators in this experimental setting.

Urinary Aldosterone/Creatinine Ratio After Fludrocortisone Suppression Consistent with PHA in a Cat.

A 9 yr old cat was presented with clinical signs and laboratory abnormalities attributed to arterial hypertension (mean systolic arterial pressure, 290 mm Hg). Plasma aldosterone concentration was increased at the time of admission (651 pmol/L), but serum creatinine and potassium concentrations were within the reference range. A second increased aldosterone (879 pmol/L) and normal plasma renin activity (1.85 ng/mL/hr) resulted in an increased aldosterone/renin ratio, which was suggestive of primary hyperaldosteronism (PHA). To further support the diagnosis of PHA, the urinary aldosterone/creatinine ratio was calculated both before and after oral administration of fludrocortisone acetate (0.05 mg/kg q 12 hr for 4 consecutive days). The urinary aldosterone/creatinine ratio was 92.6 × 10(-9) before fludrocortisone administration and 155.8 × 10(-9) 4 days later. Absence of suppression was typical of PHA. The cat had a limited response to antihypertensive medication and died before treatment for PHA could be instituted. A necropsy was not permitted by the owner.

Synovial fluid cytology in experimental acute canine monocytic ehrlichiosis (Ehrlichia canis).

Evidence-based information of a cause-and-effect relationship between Ehrlichia canis infection and polyarthritis in naturally- or experimentally-infected dogs is currently lacking. The aim of this prospective study was to investigate whether synovial fluid cytological evidence of arthritis could be documented in dogs with acute monocytic ehrlichiosis. Direct synovial fluid cytology smears from eight Beagle dogs experimentally infected with E. canis were examined prior to, and on 21, 35 and 63 days post-inoculation. The cytological variables assessed included cellularity, percentages of mononuclear cells and neutrophils, macrophage reactivity and evidence of E. canis morulae. The median cellularity and percentages of mononuclear cells and neutrophils prior to inoculation did not differ when compared to post-inoculation cytological evaluation. Increased cellularity, E. canis morulae or cytological evidence of arthritis or macrophage reactivity were not observed throughout the course of the study. In the present study, no cytological evidence of arthritis was found in dogs with experimental acute canine monocytic ehrlichiosis, suggesting that E. canis infection should be considered a rather uncommon cause of arthritis in dogs.

Molecular identification of Bartonella species in dogs with leishmaniosis (leishmania infantum) with or without cytological evidence of arthritis.

Recent evidence suggest that Bartonella species may cause polyarthritis and lameness in dogs. Canine leishmaniosis (CanL) due to Leishmania infantum is a multi-systemic disease often occurring in association with arthritis. We hypothesized that concurrent Bartonella infection may be a contributing factor for the development of arthritis in dogs with CanL. Hence the primary objective of this study was to investigate the molecular prevalence of Bartonella spp. in dogs with naturally occurring CanL, with or without cytologically documented arthritis. Thirty-eight dogs with CanL (31 with neutrophilic arthritis and 7 without arthritis) were retrospectively studied. Seventy-four archived clinical specimens from these 38 dogs, including 33 blood samples, 19 bone marrow (BM) samples and synovial fluid (SF) aspirates from 22 dogs were tested for Bartonella spp. DNA using the Bartonella alpha proteobacteria growth medium (BAPGM) diagnostic platform. Overall, eight (21.1%) dogs were infected with one or two Bartonella species; however, Bartonella spp. infection was not associated with arthritis in dogs with CanL. Further prospective studies are warranted to determine if there is a correlation between Bartonella spp. infection and the development of arthritis in dogs with CanL.

Serum canine pancreatic lipase immunoreactivity in experimentally induced and naturally occurring canine monocytic ehrlichiosis (Ehrlichia canis).

Ehrlichia canis infection causes multisystemic disease in dogs (canine monocytic ehrlichiosis, CME) which is associated with variable morbidity and mortality. Atypical clinical manifestations, including gastrointestinal signs, may occasionally occur in CME and approximately 10-15% of dogs are presented with historical or clinical evidence of vomiting, diarrhea, and/or abdominal discomfort. The objective of this study was to investigate if there are any alterations in serum canine pancreatic lipase immunoreactivity (cPLI) in dogs with experimentally induced or naturally occurring monocytic ehrlichiosis. Serum samples from 10 Beagle dogs experimentally infected with E. canis and two healthy uninfected Beagles were serially examined; samples from 20 naturally infected dogs (10 with non-myelosuppressive [NME] and 10 with myelosuppressive [ME] ehrlichiosis) were also examined at a given point in time (cross-sectional sampling). None of the experimentally infected Beagles showed gastrointestinal signs or increased cPLI concentrations prior to or following the artificial infection. Three naturally infected dogs with NME and one with ME demonstrated serum cPLI concentrations in the diagnostic range for pancreatitis (>400 µg/L) without showing gastrointestinal signs. The results of the present study indicated that 4/20 (20%) of dogs naturally infected with E. canis demonstrated increased serum cPLI concentrations consistent with mild and clinically inapparent pancreatitis.

Efficacy of rifampicin in the treatment of experimental acute canine monocytic ehrlichiosis.

OBJECTIVES: To assess the efficacy of rifampicin in achieving clinical and haematological recovery and clearing infection in dogs with experimentally induced acute monocytic ehrlichiosis. METHODS: Five Ehrlichia canis-infected Beagle dogs were treated with rifampicin (10 mg/kg/24 h orally for 3 weeks), nine E. canis-infected dogs received no treatment (infected untreated dogs) and two dogs served as uninfected controls. Clinical score, platelet counts, immunofluorescent antibody titres and PCR detection of E. canis-specific DNA in blood, bone marrow and spleen aspirates were evaluated on post-inoculation days 21 (start of rifampicin), 42 (end of rifampicin) and 98 (end of the study). RESULTS: By day 21 post-inoculation, all infected dogs became clinically ill and thrombocytopenic, seroconverted and were PCR positive in at least one tissue. Clinical scores and antibody titres did not differ between the treated and infected untreated dogs throughout the study. The rifampicin-treated dogs experienced an earlier resolution of their thrombocytopenia (Kaplan-Meier survival plot, P=0.048), and the median platelet counts were significantly higher in the treated compared with the infected untreated dogs on post-inoculation days 42 (P=0.0233) and 98 (P=0.0195). At the end of the study, three treated and six untreated infected dogs remained PCR positive in one tissue each. CONCLUSIONS: The rifampicin treatment regimen applied in this study hastened haematological recovery, but was inconsistent in eliminating the acute E. canis infection.

Cytologic patterns of lymphadenopathy in canine monocytic ehrlichiosis.

BACKGROUND: Recognition of different cytologic patterns in lymph nodes (LNs) from dogs with canine monocytic ehrlichiosis (CME) and noninfectious causes of lymphoid reactivity may have diagnostic utility. OBJECTIVES: The aims of the present study were to compare cytologic patterns in LNs of dogs with different phases of CME, to investigate the association of cytologic pattern and presence of Ehrlichia spp. morulae, and to compare patterns of lymphoid reactivity between dogs with CME and those with noninfectious causes of lymphoid hyperplasia. METHODS: Cytologic preparations of LNs from 35 dogs with nonmyelosuppressive CME (group A), 16 dogs with myelosuppressive CME (group B), 26 dogs with noninfectious diseases (group C), and 15 healthy dogs (group D) were evaluated. Percentages of lymphocyte types, plasma cells, macrophages, neutrophils, and eosinophils were determined. Samples from dogs in groups A and B were evaluated for the presence of morulae. RESULTS: Cytologic abnormalities in LNs were recorded in 54% of dogs in group A, 88% in group B, 39% in group C, and 0% in group D and were more frequent (P=.02) in dogs with myelosuppressive CME than those with nonmyelosuppressive CME. Plasma cell hyperplasia was more frequent in CME than in noninfectious diseases (P=.03). An association between the presence of cytologic abnormalities and morulae in group A dogs was not found. CONCLUSIONS: Dogs with myelosuppressive CME have more lymphoid cytologic abnormalities than dogs with nonmyelosuppressive CME. LN plasmacytosis is the major pattern of lymphadenopathy in dogs with CME and is found more frequently in dogs with CME than in dogs with noninfectious causes of lymphadenopathy.