Background The optimal microscopic magnification and number of optical fields of adhesive tape strip cytological slides that should be examined when searching for Malassezia yeasts on canine skin are unknown. Objectives To determine the optimal magnification and the minimum number of optical fields that should be examined to maximise intraobserver repeatability and interobserver reproducibility. Materials and Methods Seven experienced examiners counted, twice, the number of yeasts in 10, 20, 30, 40 and 50 optical fields of 40 slides at ×400 and ×1000 magnification. Results The number of yeasts per unit surface area was significantly higher at ×1000 compared to ×400 magnification. Repeatability and reproducibility for counting the yeasts was very poor. Conclusions and clinical relevance Adhesive tape strip cytological slides should be examined microscopically for Malassezia spp. at ×1000 magnification. The repeatability of this examination for counting the yeasts is poor.

The aim of this 6-month, randomized, blinded, controlled clinical trial was to compare the efficacy and safety of aminosidine-allopurinol combination with that of meglumine antimoniate-allopurinol combination for the treatment of leishmaniosis in dogs without stage III or IV chronic kidney disease. Forty client-owned dogs were randomly assigned to group A [n = 20; aminosidine (15 mg/kg, subcutaneously, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)] or group B [(n = 20; meglumine antimoniate (100 mg/kg SC, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)]. Clinical and clinicopathological evaluations, parasitic load measurement (lymph node and bone marrow microscopy, bone marrow real-time PCR), specific serology and leishmanin skin test (LST) were performed at baseline (time 1) and after 14 (time 2), 28 (time 3), 60 (time 4) and 180 (time 5) days. Both treatments were safe and resulted in significant clinical and clinicopathological improvement, reduction of parasitic load and of indirect immunofluorescence antibody test (IFAT) titer and induction of positive LST. There was no significant difference between groups with regards to the primary outcome measures of the trial that included the proportion of dogs that presented severe treatment-related side effects, were cured and were parasitologically negative at time 5. However, some (proportion of dogs that presented no clinical signs, no hyperglobulinemia and negative serology at time 5) secondary outcome measures showed significant differences in favor of the meglumine antimoniate-allopurinol treatment arm. Treatment-related death occurred in one dog in each group, while injection site reactions appeared at a similar frequency in both groups. Due to the differences in some secondary outcome measures in association with the low power of this trial, it cannot be definitively concluded that the two treatments are equally effective. Therefore, the aminisodine-allopurinol combination cannot be proposed as a first-line treatment of CanL but rather as a second-line treatment that may be particularly useful to avoid repeated administration of meglumine antimoniate and in countries where the latter is not available or registered.

Canine leishmaniosis due to Leishmania infantum is a widespread zoonotic disease. Although aminosidine can be an effective treatment, current therapeutic recommendations do not advocate its use, mainly due to concerns regarding the potential nephrotoxicity and ototoxicity of this drug. The aim of this randomized, blinded, controlled study was to evaluate the nephrotoxicity and ototoxicity of aminosidine-allopurinol combination and compare it with that of meglumine antimonate-allopurinol combination in non-azotemic dogs with leishmaniosis. Forty dogs with leishmaniosis were randomly assigned to be treated with either aminosidine at 15 mg/kg, subcutaneously, once daily for 28 days (group A) or with meglumine antimonate at 100 mg/kg, subcutaneously, once daily for 28 days (group B). In addition to either drug, dogs in both groups were administered allopurinol at 10 mg/kg per os twice daily for 2 months. Kidney function was evaluated through measurement of serum creatinine, urea nitrogen, inorganic phosphorus, and cystatin-c concentrations and complete urinalysis, including protein-to-creatinine ratio, at baseline and after 14, 28, and 60 days from the beginning of the treatment. At the same time points, vestibular and auditory functions were evaluated through neurological examination and brainstem auditory evoked response (BAER) recordings of wave I, wave V, inter-wave I-V latencies, and minimum hearing thresholds. None of the dogs developed clinicopathological evidence of kidney disease during the study. Serum creatinine concentration increased > 0.3 mg/dl over baseline in 2 dogs in group A and in 5 dogs in group B. Parameters of kidney function were not significantly different or were improved compared to baseline and the only difference between the two groups was the lower concentration of serum creatinine in group A. None of the dogs developed peripheral vestibular syndrome or hearing impairment. At the end of the study, parameters of auditory function were not significantly different or were improved compared to baseline and there were no differences between the two groups. The results of this study show that the nephrotoxicity and ototoxicity of aminosidine, when administered to non-azotemic dogs with leishmaniosis at 15 mg/kg subcutaneously once daily for 28 days along with allopurinol, is minimal and does not differ from that of meglumine antimonate.

The purpose of this retrospective study was to describe the historical and clinical findings, the clinicopathological abnormalities, the occurrence and nature of concurrent diseases, the treatment and outcome of 23 dogs with diabetic ketosisketoacidosis (DK-DKA). Inclusion criteria consisted of the presence of clinical signs suggestive of diabetes mellitus (DM) along with persistent hyperglycemia, glycosuria and ketonuria during the first 24 hours of hospitalization. In nineteen dogs (83%) DM had not been previously diagnosed. Common presenting complaints were polyuria/polydipsia (100%), partial or complete loss of appetite (87%), depression (87%), vomiting (65 %) and weight loss (30 %). The most frequent physical examination findings included dehydration (61%), depression (61%), hypotrichosis-alopecia (39%), palpable cranial abdominal organomegaly (26%), pendulous abdomen (26%), lesions compatible with superficial pyoderma (17%), thin and hypotonic abdominal skin (17%), and hypothermia (17%). The most important clinicopathological abnormalities, apart from hyperglycemia, glucosuria, and ketonuria, included anemia (48%), leukocytosis (39%), increased activities of alkaline phosphatase (100%), lipase (56%) and alanine aminotransferase (52%), hypertriglyceridemia (90%) and hypercholesterolemia (84%). Also, 12 dogs demonstrated hypokalemia on admission or during hospitalization. A concurrent disease was identified in 74% of the cases while 26% had two or more comorbidities. The latter included pancreatitis (30%), urinary tract infections (17%), superficial pyoderma (17%), urolithiasis (13%) and hyperadrenocorticism (13%). Twenty two dogs were treated with short-acting insulin (regular or lispro) and one with intermediate-acting (lente) insulin, whereas intravenous fluid therapy was instituted in 78% of them with potassium and phosphorus supplementation in 65% and 9%, respectively. Seventeen (81%) dogs survived to be discharged, three (13%) died during hospitalization, one (4%) was euthanized and on two (9%) occasions owners declined hospitalization after the first 24 hours due to financial constrains or a poor prognosis. Mean duration of hospitalization for the survivors was 5.7 ± 2.4 days, mean time to resolution of ketonuria was 4.2 ± 1.9 days and median time of rapid–acting insulin administration was 4 days (range 2-8 days).

The aim of this study was to compare the risk of seropositivity between stray dogs and privately-owned dogs living in the same endemic area. Sixty-seven stray (group A) and 115 privately-owned (group B) dogs were enrolled. History, physical examination findings and results of serology, haematology and total solids were compared between groups and between seropositive group A and seropositive group B dogs. Variables with p value <= 0.2 were used to build two multivariable models using results of serology as the dependent variable: the first model included the group and historical information, whereas the second included the group, physical examination and results of haematology and total solid measurement. Stray dogs had 4.59-fold and 3.72-fold higher risk of seropositivity compared with privately-owned dogs in the first and second model, respectively. Additional factors associated with positive serology included female sex, not sampling during transmission period, masticatory muscle atrophy, footpad hyperkeratosis, low haematocrit and low platelet count. In conclusion, stray dogs were at higher risk of seropositivity.

Η ηπατοζωόνωση στο σκύλο προκαλeίται από δυο eίδη πρωτόζωων του γένους Hepatozoon. Το παλαιότeρα γνωστό eίδος Η. canis μeταδίδeται μe τον κρότωνα Rhipicephalus  sanguineus και έχeι σχeδόν παγκόσμια γeωγραφική eξάπλωση, eνώ το σχeτικά πρόσφατα ταυτοποιημένο Η. americanum μeταδίδeται μe τον Amblyomma maculatum και μέχρι σήμeρα έχeι διαπιστωθeί  μόνο στις Ηνωμένeς Πολιτeίeς. Το Η. canis, έχοντας  προσαρμοστeί στο σκύλο, προκαλeί συνήθως υποκλινική ή ήπια  κλινική νόσο. Στην τeλeυταία πeρίπτωση, σημαντικό ρόλο φαίνeται  να παίζeι η χορήγηση ανοσοκατασταλτικών ουσιών και τα συνυπάρχοντα  νοσήματα. Η ανορeξία, η κατάπτωση, ο λήθαργος, ο πυρeτός, η λeμφαδeνοπάθeια και η απώλeια του σωματικού βάρους  αποτeλούν τις συνηθέστeρeς κλινικές eκδηλώσeις. Η διάγνωση  της ηπατοζωόνωσης, που οφeίλeται στο Η. canis, βασίζeται στην  ανeύρeση των γαμeτοκυττάρωντου τeλeυταίου, στο κυτταρόπλασμα των ουδeτeρόφιλων κυττάρων (συνήθως 1-5% των ουδeτeρόφιλων), σe eπιχρίσματα αίματος υστέρα από χρώση Giemsa. Η χορήγηση  διπροπιονικής ιμιδοκάρβης, σe συνδυασμό ή όχι μe τη δοξυκυκλίνη, οδηγeί συνήθως σe κλινική ύφeση και eξαφάνιση της  παρασιταιμίας, αν και οι υποτροπές δeν eίναι σπάνιeς. Το Η. americanum, αντίθeτα, προeρχόμeνο πιθανότατα από κάποιο άλλο  eίδος ξeνιστή και έχοντας eλάχιστα προσαρμοστeί στο σκύλο, προκαλeί  ένα χρόνιο καχeξιογόνο και συχνά θανατηφόρο νόσημα. Ο  πυρeτός, το γeνικeυμένο άλγος, η μυϊκή ατροφία και η απροθυμία  ή η αδυναμία μeτακίνησης του ζώου, που αποτeλούν τις τυπικές  κλινικές eκδηλώσeις, eίναι το αποτέλeσμα της σοβαρής κοκκιωματώδους πολυμυίτιδας και των eκτeταμένων πeριοστικών αλλοιώσeων.  Η διάγνωση της ηπατοζωόνωσης, που οφeίλeται στο Η. americanum, θα βασιστeί στην ιστοπαθολογική eξέταση των προσβeβλημένων  μυών ή/και στην ανίχνeυση των eιδικών αντισωμάτων  στον ορό του αίματος, αφού η παρουσία των γαμeτοκυττάρων στα ουδeτeρόφιλα λeυκοκύτταρα eίναι πολύ σπάνια. Ο συνδυασμός  τριμeθοπρίμης-σουλφαδιαζίνης, κλινδαμυκίνης και πυριμeθαμίνης  και στη συνέχeια η μακροχρόνια χορήγηση της αντικοκκιδιακής ουσίας  δeκοκινάτης eπιτυγχάνeι την κλινική ίαση και τη σημαντική αύξηση του χρόνου eπιβίωσης, αν και οι υποτροπές eίναι μάλλον συχνές.

Medical records of six dogs admitted with acute hypoadrenocorticism were reviewed. All 6 animals were bradycardic and had prolonged capillary refilling time. Hypothermia was detected in 5/6 animals. Clinicopathological evaluation on admission revealed anemia (3/6 dogs), increased (2/6) and normal (2/6) lymphocyte and eosinophil counts, azotemia and hyperkalemia (6/6), hyponatremia, in 5/6 dogs in which sodium was measured with a sodium (Na) : potassium (K) ratio lower than 24, hypoglycemia (2/6) and hypercalcemia, hypocholosterolemia, increased serum alkaline phosphatase and alaninoaminotransferase activities, one dog each. Urine specific gravity was lower than 1025 in 4 dogs. Thoracic radiographs and abdominal ultrasonography disclosed microcardia (2/6), pleural effusion (2/6) and ascites (1/6). Two dogs (2/6), also, presented atrial standstill or atrioventricular block, detected on electrocardiograms. In all 6 animals emergency treatment included the use of intravenous normal saline and glucocorticoids (dexamethasone) immediately after the completion of an ACTH stimulation test with tetracosactide. Two dogs died, one during the 1st day and the other on the 4th day of hospitalization, the latter after the sudden appearance of severe hemorrhagic gastroenteritis. Hospitalization time in the remaining 4 dogs ranged from 3 to 10 days. Prolongation of hospitalization was associated with worsening of anemia, hypoalbuminemia, neurologic complications and non-responsive azotemia. Four dogs (4/6) were discharged on oral prednisolone and fluodrocortisone, but one died after a week for unknown reasons. Acute hypoadrenocorticism is a life threatening condition requiring emergency treatment for a successful outcome. Its inclusion in the differential diagnosis of canine emergencies, presented with acute weakness and gastrointestinal signs, is mandatory, but for its confirmation a complete laboratory and, most importantly, an ACTH stimulation test are always required.

Leukogram, an important part of complete blood count, includes the total count, absolute differential counts and leukocyte morphology. For a valid interpretation of the leukogram, proper blood sampling and post-sampling handling is considered a prerequisite. Total leukocyte counts in the clinically healthy adult dog and cat usually range from 6000 to 17000 /μΐ and from 5500 to 19500 /μΐ, respectively. Neutrophils, lymphocytes, monocytes, eosinophils and rarely basophils are the leukocytes recognized in the peripheral blood of these animal species. Leukocytosis and leukopenia may reflect pathological or physiologic responses to various endogenous or exogenous factors. Morphologic leukocyte changes may indicate an underlying inflammatory process (i.e. toxic changes), congenital and/or hereditary hematological abnormalities (i.e. Pelger-Hüet anomaly, Chediak-Higashi syndrome) and immune-mediated (i.e. systemic lupus erythematus) or neoplastic (i.e. lymphoid/myeloid leukemias) diseases. Detection of canine distemper virus inclusion bodies, Ehrlichia sp. morulae, Hepatozoon canis gamonts etc. in the cytoplasm of the various types of leukocytes is the most objective way to diagnose the relevant infectious diseases in the domestic carnivores.

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.

BACKGROUND:Three flea species, Pulex irritans, Ctenocephalides canis and C. felis parasitize shepherd dogs living on sheep farms in Greece. The aim of this randomized, blinded, placebo-controlled trial was to examine the efficacy of spinosad, when administered three times every 4 weeks, as the only intervention to treat and prevent flea infestations in shepherd dogs living on sheep farms.METHODS:Thirty dogs living on sheep farms and infested by at least 24 fleas were randomly allocated into equal groups. Group A dogs received spinosad (45-70 mg/kg body weight), every 4 weeks for three administrations, whereas Group B dogs were placebo-treated. Flea counting was performed at the beginning of the trial (day 0) and after 14, 28, 56 and 84 days. The first five fleas from each dog and 2-6 fleas collected from 5-11 sheep were used for species identification.RESULTS:The percentage of dogs with zero flea counts was significantly higher in group A than in group B at days 14, 28, 56 and 84 and flea counts were significantly lower in group A than in group B at days 14, 28, 56 and 84. In group A, flea counts were significantly lower at days 14, 28, 56 and 84 compared to day 0 whereas there were no changes in flea counts of group B dogs. The percent efficacy of spinosad for the treatment and prevention of flea infestation was higher than 98% (arithmetic means) or higher than 99% (geometric means) throughout the study. No adverse reactions were recorded. C. canis was the predominant flea species of dogs at day 0. In group A the relative abundance of C. felis increased at day14 whereas in group B the relative abundance of P. irritans increased at days 14, 28, 56 and 84.CONCLUSIONS:Spinosad is safe and effective for the treatment of C. canis and C. felis infestations and for the prevention of P. irritans, C. canis and C. felis infestations in shepherd dogs living in close proximity to sheep.

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05-95.54%) and 86.15% (95% confidence interval: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

A 5-month old Caucasian dog was presented with a 20-day history of abdominal distention along with inappetance, depression and vomiting of 24-hour duration. Physical examination findings included depression, ascites, mild inspiratory dyspnea and dehydration. Clinicopathological evaluation revealed hyperammonemia, hypoalbuminemia, hyperbilirubinemia, hypoglycemia and hyponatremia. Μicrohepatia and free abdominal fluid was detected with abdominal ultrasonography. During exploratory laparotomy, multiple acquired portosystemic collateral vessels were found, indicative of portal hypertension, along with a small liver of normal color and texture. Liver histopathology included features consistent with liver hypoperfusion. These findings supported the diagnosis of primary portal vein hypoplasia with portal hypertension. The animal recovered uneventfully postoperatively and was discharged with diuretics, hepatoprotectants and a low-protein diet and remains healthy two years after diagnosis. This case underscores that a favorable prognosis may be anticipated in cases of primary portal vein hypoplasia with portal hypertension, thus, justifying the long-term conservative management instead of considering euthanasia.

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonotic disease. One of the most commonly used drugs for the treatment of CanL is meglumine antimonate. Drugs of this class have been associated with pancreatitis and cardiotoxicity in humans infected with Leishmania spp. The aim of this study was to measure serum canine pancreatic lipase immunoreactivity (Spec cPL) and cardiac troponin I (cTnI) concentrations in dogs with leishmaniosis during treatment with meglumine antimonate, and to compare them with those of dogs with leishmaniosis not treated with this drug. A total of 30 non-uremic dogs with leishmaniosis, living in Greece, were prospectively enrolled into the study. Of the 30 dogs, 20 (Group A) were treated with a combination of meglumine antimonate (100 mg/kg, SC, q24h) and allopurinol (10 mg/kg, PO, q12h) for 28 days, while 10 dogs (Group B) were treated with allopurinol alone (10 mg/kg, PO, q12h) for 28 days. Blood samples were collected at timepoint 0 (before treatment) and at 14 and 28 days after the initiation of treatment. None of the dogs treated with meglumine antiomonate had a Spec cPL concentration suggestive of pancreatitis (≥400 μg/L) or clinical signs suggestive of pancreatitis at any of the timepoints. Similarly, none of the dogs treated with meglumine antiomonate had a serum cTnI concentration above the upper limit of the reference range (>0.5 ng/mL) or clinical evidence of cardiotoxicity at any of the 3 timepoints. In the present study, meglumine antimonate treatment in dogs with leishmaniosis did not result in clinical or laboratory evidence of either pancreatitis or cardiotoxicity.

Hyperthyroidism is a common endocrinopathy in cats older than 8 years, with no sex or breed predisposition. Benign adenomas and adenomatous hyperplasia of the thyroid gland is observed in the majority of cases. Symptoms reflect the effect of thyroid hormone excess in various systems, with weight loss, polyphagia, polyuria-polydipsia, cardiovascular and gastrointestinal abnormalities being common clinical manifestations. On clinical examination, there is frequently prominent thyroid enlargement. Common laboratory abnormal findings include increased activity of alkaline phosphatase and alanino-aminotransferase, hyperphosphataemia, azotaemia and decreased concentration of ionized calcium and creatinine. Definite diagnosis of the disease is based on the demonstration of increased blood concentration of thyroid hormones. Measurement of thyroxine concentration, alone or in conjunction with concentration of free thyroxine, is usually sufficient to reach a diagnosis. When diagnosis is uncertain, thyroid stimulating hormone, scintigraphy and dynamic function tests can be used. The possibility of concurrent diseases (e.g., renal failure, diabetes mellitus) must be investigated, as their presence has implications on diagnosis and treatment. Medical therapy, thyroidectomy, radionine therapy and low iodine diet are also valid options for treatment. Each has advantages and disadvantages that a clinician must take into consideration before instigating treatment. Prognosis for hyperthyroidism is favourable if no severe disease exists concurrently.

Hypothyroidism may predispose to the development of canine leishmaniosis or it may appear during the course of the latter due to infiltration and destruction of the thyroid gland by infected macrophages. The main purpose of this study was to evaluate thyroid function through measurement of serum total thyroxin (tT4), free thyroxin (fT4), and canine thyroid stimulating hormone (cTSH) concentrations in 36 dogs with leishmaniosis, before and after 2 and 4 weeks of treatment with allopurinol with or without meglumine antimonate. Before treatment 27/36 (75%) dogs had serum tT4 concentrations below the lower limit of the reference interval but only 2 of them had concurrently serum fT4 concentrations below the lower limit of the reference interval and none had increased serum cTSH concentrations. During treatment there were no significant changes in serum tT4 or fT4 concentrations, whereas a significant increase in serum cTSH was observed. Two dogs had decreased serum tT4 and fT4 but normal cTSH concentrations before treatment and two other dogs had decreased serum tT4 and increased cTSH, but normal fT4 concentrations during the treatment period. Although hypothyroidism could not be definitively excluded in these dogs it is considered unlikely based on their overall hormonal profile, clinical presentation, and response to treatment. Therefore, hypothyroidism does not appear to be an important predisposing disease or a frequent complication of canine leishmaniosis.

Veterinary RecordVolume 169, Issue 18 p. 470-470 Research Bleeding time in healthy dogs sedated with morphine and medetomidine M. E. Mylonakis DVM, PhD, Corresponding Author M. E. Mylonakis DVM, PhD [email protected] Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceE-mail for correspondence [email protected]Search for more papers by this authorG. M. Kazakos DVM, PhD, G. M. Kazakos DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorD. Pardali DVM, PhD, D. Pardali DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorP. Kostoulas DVM, PhD, P. Kostoulas DVM, PhD Department of Epidemiology, Biostatistics and Economics of Animal Production, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon Street, GR-43100 Karditsa, GreeceSearch for more papers by this authorM. Kritsepi-Konstantinou DVM, PhD, M. Kritsepi-Konstantinou DVM, PhD Diagnostic Laboratory, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorT. Petanides DVM, PhD, T. Petanides DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorA. D. Galatos DVM, PhD, A. D. Galatos DVM, PhD Department of Surgery, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon Street, GR-43100 Karditsa, GreeceSearch for more papers by this authorA. F. Koutinas DVM, PhD, A. F. Koutinas DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this author M. E. Mylonakis DVM, PhD, Corresponding Author M. E. Mylonakis DVM, PhD [email protected] Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceE-mail for correspondence [email protected]Search for more papers by this authorG. M. Kazakos DVM, PhD, G. M. Kazakos DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorD. Pardali DVM, PhD, D. Pardali DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorP. Kostoulas DVM, PhD, P. Kostoulas DVM, PhD Department of Epidemiology, Biostatistics and Economics of Animal Production, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon Street, GR-43100 Karditsa, GreeceSearch for more papers by this authorM. Kritsepi-Konstantinou DVM, PhD, M. Kritsepi-Konstantinou DVM, PhD Diagnostic Laboratory, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorT. Petanides DVM, PhD, T. Petanides DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this authorA. D. Galatos DVM, PhD, A. D. Galatos DVM, PhD Department of Surgery, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon Street, GR-43100 Karditsa, GreeceSearch for more papers by this authorA. F. Koutinas DVM, PhD, A. F. Koutinas DVM, PhD Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Street, GR-54627 Thessaloniki, GreeceSearch for more papers by this author First published: 29 October 2011 https://doi.org/10.1136/vr.100265Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume169, Issue18October 2011Pages 470-470 RelatedInformation

A 14-year-old domestic shorthair cat was admitted with a 2-month history of excessive body weight loss and generalized exfoliative dermatitis with erythroderma. Radiographic and ultrasonographic examination revealed a cranioventral cavitary mass within the anterior mediastinum. An ultrasound-guided fine needle aspiration cytology from the mass revealed numerous small mature lymphocytes intermingled with much fewer inflammatory mast cells. These findings, along with a cellpoor interface and mixed cell dermatitis demonstrated on skin histopathology, made the diagnosis of paraneoplastic exfoliative dermatitis straightforward. This uncommon case illustrates the relative value of keratosebborheic skin disease as a useful indicator of an internal malignancy in the cat.

An 8-year-old, intact male German Shepherd dog was referred because of anorexia, intermittent vomiting, and diarrhea of 5 days’ duration. The dog was housed outdoors and was incompletely vaccinated (booster interval in excess of 5 years for parvovirus, distember, adenovirus, and Leptospira spp.). Physical examination (including monocular indirect ophthalmoscopy) identified lethargy, weakness, moderate pain on anterior abdominal palpation, dehydration, halitosis, and fever (40.41C). CBC was unremarkable, whereas the important serum biochemical findings included hypoalbuminemia (2.1 g/ dL; reference range, 2.5–3.5 g/dL), normoglobulinemia (3.9 g/dL; reference range, 3–4.5 g/dL), hyperbilirubinemia (2.1mg/dL; reference range, 0.3–0.8mg/dL), and increased alkaline phosphatase (ALP) (1,300U/L; reference range, 50–210U/L) and alanine aminotransferase (ALT) (160U/L; reference range, 10–34U/L) activities. Analysis of an orange-colored urine sample obtained by cystocentesis disclosed an average of 2 granular casts/low power field and a urine protein-to-creatinine ratio of 2.9 (reference interval, o0.5); urine culture failed to grow bacteria. On admission, a Giemsa-stained buffy coat smear was negative for Ehrlichia sp., Babesia sp., Hepatozoon canis, and Mycoplasma sp. organisms (1,000 microscopy fields). Serum was tested for Leishmania infantum and Babesia canis antibodies by indirect immunofluorescence assays (IFA), whereas Ehrlichia canis antibodies were tested by an in-office ELISA test. All 3 tests were negative. The dog was positive for Dirofilaria immitis antigens but negative on a modified Knott’s test for microfilaria. Thoracic radiography was unremarkable, but moderate hepatomegaly and splenomegaly were evident on abdominal radiographs and confirmed by abdominal ultrasonography (enlarged and diffusely hypoechoic liver). Leptospirosis was suspected, based on epidemiologic (endemic disease in Greece), historical, and clinical data and the fact that all the infectious disease tests described above were negative. Dirofilariasis (stage 1) was not considered a contributory factor for the clinical presentation of this dog. Pending microscopic agglutination test (MAT) serology results for Leptospira spp., the dog was hospitalized and treated with crystalloids (60mL/kg IV daily), ampicillin (20mg/kg IV q8h), enrofloxacin (10mg/kg SC q24h), ursodeoxycholic acid (15mg/kg PO q24h), ranitidine (2mg/kg IV q12h), and sucralfate (1 g/30kg PO q12h). Because no clinical or biochemical improvement was noticed after 5 days of hospitalization, a diagnostic laparotomy was performed to obtain wedge liver biopsies. Apart from mild hepatomegaly and firm consistency of hepatic parenchyma, no other abnormalities were noticed. Imprint smears were made of hepatic tissue and Giemsa-stained for cytological examination. Formalin-fixed and frozen liver tissue was retained for future polymerase chain reaction (PCR) and histopathology. Impression cytology of the liver identified several round-to-oval and occasionally polyhedral hepatocytes with an eccentrically placed round nucleus (occasional cells were binucleated) and slightly basophilic cytoplasm that were distributed individually or in clusters among a slightly hemorrhagic background. Dark green intracytoplasmic granules consistent with bile and solid green to black bile casts were observed between contiguous hepatocytes. There were numerous mature lymphocytes, with fewer plasma cells, macrophages, and nondegenerate neutrophils dispersed among the hepatocytes (Fig 1). Several dark-blue cytoplasmic inclusions consistent with Ehrlichia sp. morulae were observed in lymphocytes and macrophages (Fig 1). There was also mild extramedullary From the Companion Animal Clinic (Mylonakis, Petanides, Koutinas), the Diagnostic Laboratory (Kritsepi-Konstantinou), the Laboratory of Microbiology and Infectious Diseases (Siarkou), the Laboratory of Pathology (Psychas), Faculty of VeterinaryMedicine, Aristotle University of Thessaloniki (AUTh), Greece; the Department of Pathology, Division of Medical Microbiology, The Johns Hopkins University School of Medicine, Baltimore, MD (Dumler); the College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA (Diniz); the Intracellular Pathogens Research Laboratory, College of Veterinary Medicine, North Carolina State University, Raleigh, NC (Breitschwerdt); and the Division of Veterinary Pathology, Infection and Immunity, School of Clinical Veterinary Science, University of Bristol, UK (Day). This work was undertaken in the Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greece. An oral presentation of this case was presented by the first author at the 11th ECVCP Congress, October 7–9, 2009, Thessaloniki, Greece (proceedings p. 166). Corresponding author: Mathios E. Mylonakis, DVM, 18 Kyprou Str., 55133, Thessaloniki, Greece; e-mail: mmylonak@vet.auth.gr. Submitted September 9, 2009; Revised February 2, 2010; Accepted February 18, 2010. Copyright r 2010 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2010.0501.x Abbreviations:

An 8-year-old, intact male German Shepherd dog was referred because of anorexia, intermittent vomiting, and diarrhea of 5 days' duration. The dog was housed outdoors and was incompletely vaccinated (booster interval in excess of 5 years for parvovirus, distember, adenovirus, and Leptospira spp.). Physical examination (including monocular indirect ophthalmoscopy) identified lethargy, weakness, moderate pain on anterior abdominal palpation, dehydration, halitosis, and fever (40.4°C). CBCa was unremarkable, whereas the important serum biochemical findings included hypoalbuminemia (2.1 g/dL; reference range, 2.5–3.5 g/dL), normoglobulinemia (3.9 g/dL; reference range, 3–4.5 g/dL), hyperbilirubinemia (2.1 mg/dL; reference range, 0.3–0.8 mg/dL), and increased alkaline phosphatase (ALP) (1,300 U/L; reference range, 50–210 U/L) and alanine aminotransferase (ALT) (160 U/L; reference range, 10–34 U/L) activities. Analysis of an orange-colored urine sample obtained by cystocentesis disclosed an average of 2 granular casts/low power field and a urine protein-to-creatinine ratio of 2.9 (reference interval, <0.5); urine culture failed to grow bacteria. On admission, a Giemsa-stained buffy coat smear was negative for Ehrlichia sp., Babesia sp., Hepatozoon canis, and Mycoplasma sp. organisms (1,000 microscopy fields). Serum was tested for Leishmania infantum and Babesia canis antibodies by indirect immunofluorescence assays (IFA), whereas Ehrlichia canis antibodies were tested by an in-office ELISAb test. All 3 tests were negative. The dog was positive for Dirofilaria immitis antigensc but negative on a modified Knott's test for microfilaria. Thoracic radiography was unremarkable, but moderate hepatomegaly and splenomegaly were evident on abdominal radiographs and confirmed by abdominal ultrasonography (enlarged and diffusely hypoechoic liver). Leptospirosis was suspected, based on epidemiologic (endemic disease in Greece), historical, and clinical data and the fact that all the infectious disease tests described above were negative. Dirofilariasis (stage 1) was not considered a contributory factor for the clinical presentation of this dog. Pending microscopic agglutination test (MAT) serology results for Leptospira spp., the dog was hospitalized and treated with crystalloidsd (60 mL/kg IV daily), ampicilline (20 mg/kg IV q8h), enrofloxacinf (10 mg/kg SC q24h), ursodeoxycholic acidg (15 mg/kg PO q24h), ranitidineh (2 mg/kg IV q12h), and sucralfatei (1 g/30 kg PO q12h). Because no clinical or biochemical improvement was noticed after 5 days of hospitalization, a diagnostic laparotomy was performed to obtain wedge liver biopsies. Apart from mild hepatomegaly and firm consistency of hepatic parenchyma, no other abnormalities were noticed. Imprint smears were made of hepatic tissue and Giemsa-stained for cytological examination. Formalin-fixed and frozen liver tissue was retained for future polymerase chain reaction (PCR) and histopathology. Impression cytology of the liver identified several round-to-oval and occasionally polyhedral hepatocytes with an eccentrically placed round nucleus (occasional cells were binucleated) and slightly basophilic cytoplasm that were distributed individually or in clusters among a slightly hemorrhagic background. Dark green intracytoplasmic granules consistent with bile and solid green to black bile casts were observed between contiguous hepatocytes. There were numerous mature lymphocytes, with fewer plasma cells, macrophages, and nondegenerate neutrophils dispersed among the hepatocytes (Fig 1). Several dark-blue cytoplasmic inclusions consistent with Ehrlichia sp. morulae were observed in lymphocytes and macrophages (Fig 1). There was also mild extramedullary hematopoiesis, identified by occasional metarubricytes, metamyelocytes, and megakaryocytes. Bone marrow (BM) cytology performed the day after liver cytology was normocellular, with normal appearing hemopoietic lineages, mild plasmacytosis, and no evidence of Ehrlichia inclusions or other infectious agents. Photomicrograph of liver impression cytology. Numerous small lymphocytes (arrows) are seen adjacent to single or clustered hepatocytes (Giemsa stain). The inset in the upper right corner contains lymphocytes and neutrophils adjacent to 2 hepatocytes. A cytoplasmic Ehrlichia sp. morula is seen in a lymphocyte (arrowhead). In the lower right corner inset, an Ehrlichia sp. morula is seen in a lymphocyte adjacent to a hepatocyte. Bar = 10 μm. Histopathology of liver tissue stained with hematoxylin and eosin disclosed moderate portal fibrosis with bile duct proliferation, dilatation of thin-walled vessels and subtle portal to portal bridging fibrosis. There also was a mild mixed inflammatory infiltrate associated with the portal areas that included lymphocytes, plasma cells, macrophages, neutrophils, and eosinophils (Fig 2). Mild portal hemorrhage accompanied by occasional hemosiderophages was present. There was mild diffuse microvesicular vacuolation of the hepatocyte cytoplasm and generalized vascular congestion. Overall, the histopathological findings were indicative of portal hepatitis. Immunohistochemistry was performed on the formalin-fixed liver tissue as previously described1 except that E. canis monospecific polyclonal antibody to E. canis gp36 (36 kDa glycoprotein) antigen was used (The Johns Hopkins University School of Medicine, Baltimore, MD). As positive controls, lung tissue from a known E. canis-infected dog and splenic tissue from a human patient infected with Ehrlichia chaffeensis were used (Fig 3). Several cells in the hepatic tissue, presumably monocytes and lymphocytes, were infected with Ehrlichia spp. (Fig 3). (A) Photomicrograph of liver histopathology showing portal fibrosis, bile duct proliferation, hemorrhage, and inflammation. There is mild microvesicular vacuolation of the cytoplasm of adjacent hepatocytes (hematoxylin and eosin stain). Bar = 200 μm. (B) Higher magnification of affected portal area showing mixed neutrophilic and mononuclear inflammation. Bar = 100 μm. Immunohistological demonstration of Ehrlichia canis from the liver of a dog with acute portal hepatitis and monocytic ehrlichiosis. E. canis morula (arrowhead) within the cytoplasm of a mononuclear cell. Upper right inset: E. canis morula within the cytoplasm of a mononuclear cell from lung tissue of a known E. canis-infected dog (E. canis positive control). Lower right inset: E. chaffeensis in the cytoplasm of a mononuclear cell from the spleen of a human patient (E. chaffeensis positive control). E. canis immunohistochemistry with hematoxylin counterstain. Bar = 10 μm. At day 7 postadmission (PA), a CBC obtained before the institution of doxycycline treatment indicated moderate thrombocytopenia (130,000/μL; reference range 200,000–500,000/μL) and buffy coat smears were negative for Ehrlichia morulae (2,000 microscopy fields). However, E. canis 16S rDNA was amplified by PCR assays from a whole blood sample (Laboratory of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, AUTh) obtained on day 7 PA. In addition, E. canis DNA subsequently was amplified and sequenced from BM aspirate material and frozen-preserved liver tissue (obtained on days 7 and 6 PA, respectively) (Intracellular Pathogens Research Laboratory, College of Veterinary Medicine, NCSU).2,3 The sequences determined by direct sequencing of the approximately 1.4 kb PCR amplicons (nearly entire length of ehrlichial 16S rDNA)3 or those obtained from clones of the 431 bp PCR amplicons from BM (E. canis-specific 16S rDNA segment) into pGem-T Easy vectorj were 99.7% identical with each other (1 base pair different in 364 bp, with primers excluded from analysis), as well as to E. canis Greek strains previously characterized (GenBank accession numbers EF011110 and EF011111).3 DNA sequence of E. canis from the liver was 100% similar to the Greek strains mentioned before. DNA from Bartonella sp. was not amplified from the liver and BM after applying a 1-step conventional PCR.4 Also, MAT for Leptospira spp. was negative. Treatment with doxycyclinek (5 mg/kg PO q12h for 4 weeks) was instituted 24 hours after visualization of morulae in impression smears (day 7 PA). Fever abated within 12 hours and clinical resolution of all clinical signs occurred within 48 hours after initiating doxycycline therapy. Serial serum biochemical analyses showed hypoalbuminemia (2.0 g/dL; reference range 2.5–3.5 g/dL), hyperbilirubinemia (1.1 mg/dL; reference range 0.3–0.8 mg/dL), increased ALP (761 U/L; reference range 50–210 U/L) and ALT (238 U/L; reference range 10–34 U/L) activities at days 8 and 20 (ALP, 322 U/L; ALT, 83 U/L) PA; all biochemical abnormalities had resolved by day 40 PA. CBC results at days 20 and 40 PA were within reference ranges. Serological results with in-office ELISA and IFA tests were negative for E. canis antibodies at 2 and 4 weeks PA, respectively. The dog was seroreactive for E. canis, however, at 6 weeks PA (IFA titer, 1 : 800; laboratory cut-off value, 1 : 100). Retrospectively, IFA for Rickettsia rickettsii, performed in paired serum samples from days 7 and 40 PA, was positive (titers 1 : 256 and 1 : 512, respectively; laboratory cut-off value, ≥ 1 : 64), but DNA from spotted fever group rickettsiae was not amplified from the liver and BM after applying a PCR assay as described previously.5 The split-dose (a single injection of 2.5 mg/kg IM, followed 1 month later, by 2 injections of the same dose, 24 hours apart) melarsominel protocol for D. immitis also was instituted after completion of doxycycline therapy. After the last clinicopathological evaluation (day 40 PA), regular phone communications indicated that the animal has remained clinically healthy during the 4-year follow-up period. An E. canis-associated severe hepatitis was suspected in this dog, based on clinical findings, liver cytology, histopathology, immunohistochemistry, PCR amplification of E. canis DNA from blood, BM, and liver by 2 different laboratories, and the temporal relationship of the clinical response to initiation of doxycycline treatment. Historically, mild-to-moderate increases in liver enzyme activities and liver histopathology frequently have been recognized in canine monocytic ehrlichiosis (CME),6–8 but E. canis-associated hepatopathy is extremely rare as a predominant clinical manifestation, especially in the acute phase of the disease. Although clinical staging in natural CME is not straightforward, the acute onset of illness in this dog in association with the seroconversion (6 weeks PA) and a normocellular BM seem to support the acute nature of E. canis infection.9 Severe hepatic disease has been reported uncommonly in pancytopenic dogs with advanced E. canis-induced BM aplasia, which could be attributed to anemic hypoxia, intrahepatic hemorrhage and occasionally, to secondary bacterial sepsis. Nevertheless, even in these chronic cases, liver is not the sole or target organ.10,11 In this dog, the histological diagnosis was portal hepatitis, based on a mixed inflammatory infiltration, portal fibrosis and cholestasis; the latter was documented by hepatic cytology (visualization of bile casts in canaliculi) along with hyperbilirubinemia and increased ALP activity.12 However, no histopathologic evidence of cholestasis was noticed, probably because of the embedding procedure.13 The moderate fibrosis seen in this dog has not been previously reported in experimentally or naturally infected dogs6 and therefore is difficult to explain in the context of the acute CME; therefore, the possibility that the E. canis-associated hepatopathy in this dog occurred in addition to a preexisting liver insult cannot be ruled out. In 2 previous experimental studies of CME, portal infiltration with lymphocytes, plasma cells, macrophages, and focal accumulations of reticuloendothelial cells compressing adjacent hepatocytes,14 or centrilobular fatty degeneration and mild-to-moderate perivascular with periportal mononuclear cell infiltration have been described.15 Also, in a retrospective pathological study of naturally infected dogs, many of which were assumed to be chronically infected, centrilobular degeneration or necrosis and portal plasmacytosis were found.6 Despite the fact that none of these dogs had overt liver disease, mixed cell or lymphoplasmacytic portal inflammation was a common histopathological finding. Interestingly, severe E. chaffeensis-associated hepatitis in acutely infected people has been associated with similar hepatic pathology (ie, mixed cell portal infiltration, cholestasis, and focal hepatic necrosis), which, similarly to this dog, may occur before seroconversion.1,16,17 The fact that hepatitis was disproportionally severe in human patients with few or no E. chaffeensis morulae in leukocytes, hepatocytes, or biliary structures has raised suspicions that pathogenesis of E. chaffeensis hepatitis is related to an overzealous host immune response to the organism, rather than the direct cytopathic effect of the organism itself. A similar pathogenesis also could apply to CME.1 Upon admission, the dog did not show many of the typical clinical and clinicopathological features of CME, such as thrombocytopenic bleeding tendency, peripheral lymphadenomegaly, and positive antibody titer to E. canis. On the other hand, documentation of an acute febrile illness accompanied by hepatic and renal clinicopathologic abnormalities raised the suspicion of leptospirosis.18 However, the ensuing treatment with ampicillin and enrofloxacin failed to improve the clinical condition of the dog. Notably, in a previous study, the lack of efficacy of enrofloxacin in dogs experimentally infected with E. canis also was documented.19 Thrombocytopenia, which is common in CME, appeared only transiently in this dog (at day 7 PA), emphasizing the fact that ehrlichiosis cannot be excluded because of a normal platelet count.6 Hypoalbuminemia, a consistent finding in CME,7,11 was attributed to protein-losing nephropathy, liver disease, vascular injury, or some combination of these. Serum albumin concentrations were normalized by day 20 PA, along with the improvement of liver disease but the proteinuria was not further evaluated. Although seroconversion is used to document acute ehrlichiosis in dogs and humans, it may lag behind clinical expression of the disease9; the same could have applied in this dog because seroconversion did not occur until 6 weeks PA. The first evidence supporting E. canis infection in this dog was obtained after a careful review of liver cytology, in which several morulae were found. In contrast, buffy coat smears (which were shown in 1 study20 to be of high diagnostic sensitivity in acute CME) were repeatedly negative in this dog between days 1 and 7 PA. It is hypothesized that this dog may have been infected with a hepatotropic strain of E. canis. In this respect, use of a quantitative real-time PCR assay could provide a more efficient demonstration of ehrlichial density in the liver, as compared with blood, BM, or other tissues, because the conventional PCR assays used in both laboratories provide only qualitative evidence (DNA detected or not detected) of the presence of E. canis in the various tissues.21 To our knowledge, this is the first case report describing immunohistological demonstration of E. canis morulae similar to what has been described in human monocytic ehrlichiosis and may be useful in establishing a diagnosis of CME.1,22 The numerous morulae found on hepatic cytology and immunohistochemistry of this dog, together with the detection of E. canis DNA in the liver and the observation of morulae in hepatic imprints of acutely infected dogs,14 suggest that the liver should be considered as a potential target organ for application of PCR testing for epidemiological studies and posttreatment monitoring.23,24 The possibility of coinfection with canine leishmaniosis, bartonellosis, and spotted fever group rickettsiosis was ruled out with the aid of serology and BM cytology (leishmaniosis), BM and liver PCR (Bartonella spp.), and BM and liver PCR (spotted fever group rickettsiosis), because these diseases have been associated with liver disease either symptomatic or asymptomatic.25–27 Based on our experience, in canine leishmaniosis the associated liver disease is asymptomatic in most cases,26 with only a few dogs showing chronic hepatitis, liver failure or both which usually responds to antileishmanial and supportive treatment. The different sequential IFA titers for R. rickettsii were interpreted as being identical, as the criterion for seroconversion (ie, a 4-fold rise in antibody titer) was not achieved.27 Interestingly, in a previous study in Greece, 10/19 dogs with CME had serological evidence of exposure to R. conorii (the agent of Mediterranean spotted fever), but PCR failed to amplify the rickettsial DNA from any of the dogs,11 suggesting that Greek dogs with CME frequently are exposed to, but rarely persistently infected by, a rickettsial agent. The authors also cannot rule out that this dog might have been chronically infected by E. canis,28 and that the dramatic response to treatment was the result of eradication of another doxycycline-responsive agent. However, the seroconversion documented in this dog strongly suggests recent infection. Dirofilariosis was considered an incidental finding in this dog, because there was no historical, clinical, or radiographic evidence of clinical disease and most importantly, the marked clinical and biochemical improvement preceded the institution of adulticide treatment with melarsomine. In conclusion, although a cause-and-effect relationship cannot be definitely established, this report provides strong evidence that acute CME may induce symptomatic hepatitis as the predominant clinical manifestation. It is therefore suggested that CME should be included in the differential diagnosis when dogs are admitted with fever and laboratory evidence of hepatic disease, especially in the endemic areas for the disease. aVetABC, Scil Animal Care Company, Viernheim, Germany bImmunoComb, Biogal-Galed, Kibbutz Galed, Israel cSnap Canine Heartworm PF; IDEXX Laboratories Inc, Westbrook, ME dLactated Ringers, Vioser, Trikala, Greece eAmpicillin, Pentrexyl, Bristol-Myers-Squibb, New York, NY fBaytril, Bayer, Leverkusen, Germany gUrsofalk, Galenica, Athens, Greece hZantac, Glaxo-S.K., Athens, Greece iPeptonorm, Uni-Pharma, Athens, Greece jPromega, Madison, WI kRonaxan, Merial, Lyon, France lImmiticide, Merial The authors thank Dr Jere McBride, Department of Pathology, University of Texas Medical Branch, Galveston, for generously providing the polyclonal antibodies to E. canis glycoproteins.

ABSTRACT This study reports the occurrence of “ Bartonella rochalimae ” in Europe and the presence of Bartonella vinsonii subsp. berkhoffii genotypes II and III in dogs in southern Italy and provides DNA sequencing evidence of a potentially new Bartonella sp. infecting dogs in Greece and Italy.