Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum.

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis. After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role. The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis. Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.

Pathologic mechanisms underlying the clinical findings in canine leishmaniasis due to Leishmania infantum/chagasi.

In dogs with symptomatic or asymptomatic leishmaniasis, Leishmania infantum appears to induce a mixed Th1/Th2 immune response that in the sick dog may eventually result in tissue damage via different pathomechanisms, notably granulomatous inflammation (eg, nodular dermatitis, osteomyelitis), immune complex deposition (eg, glomerulonephritis), and/or autoantibody production (eg, polymyositis). This is a compensatory but detrimental mechanism generated mainly because of the insufficient killing capacity of macrophages against the parasite in the susceptible dog. Clinical disease is typically exemplified as exfoliative and/or ulcerative dermatitis, with or without nasodigital hyperkeratosis and onychogryphosis, glomerulonephritis, atrophic myositis of masticatory muscles, anterior uveitis, keratoconjunctivitis sicca, epistaxis, and/or polyarthritis, appearing alone or in various combinations. The pathogenesis of these clinical conditions has recently been highlighted, to a greater or lesser extent. The usually subclinical conditions expressed as chronic colitis, chronic hepatitis, vasculitis, myocarditis, osteomyelitis, orchiepididymitis, and meningoencephalomyelitis, though uncommon, are of pathologic importance from a differential point of view. The leading cause of death among canine leishmaniasis patients is chronic proteinuric nephritis that may progress to end-stage kidney disease, nephrotic syndrome, and/or systemic hypertension. However, even the asymptomatic proteinuria, when profuse, may be a serious problem because it predisposes to arterial thromboembolism and eventually contributes to the deterioration of the body condition.

Basal cell carcinoma in a dog with chronic solar dermatitis.

A seven-year-old, entire male, American Staffordshire bull terrier was diagnosed with chronic solar dermatitis and basal cell carcinoma, based on physical examination, cutaneous cytology and histopathology. Immunohistochemistry revealed that the tumour cells did not express p53. To the authors' knowledge this is the first reported case of canine basal cell carcinoma developing as a complication of chronic solar dermatitis.

Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis.

BACKGROUND: Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick-borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. HYPOTHESIS: APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). ANIMALS: Fifty-six dogs naturally infected with E. canis and 7 clinically healthy control dogs. METHODS: C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis-specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. RESULTS: Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). CONCLUSIONS AND CLINICAL IMPORTANCE: CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.

Absence of myelofibrosis in dogs with myelosuppression induced by Ehrlichia canis infection.

Bone marrow (BM) pathology was assessed in 10 dogs with Ehrlichia canis-induced aplastic pancytopenia. BM core biopsy sections were stained with haematoxylin and eosin and with haematoxylin/van Gieson and Gordon and Sweets' reticulin stain for the detection of collagen and reticulin fibres, respectively. Iron stores were assessed by Perls' Prussian blue staining. There was no significant deposition of collagen or reticulin in any sample, but in seven dogs the BM was depleted of haemosiderin. These findings suggest that myelofibrosis does not play a significant role in the development of BM failure in canine monocytic ehrlichiosis and that iron deficiency may exacerbate the anaemia in the myelosuppressive phase of the disease.

Molecular documentation of Bartonella infection in dogs in Greece and Italy.

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.

Factors associated with the occurrence of epistaxis in natural canine leishmaniasis (Leishmania infantum).

BACKGROUND: Canine leishmaniasis (CanL) is a common cause of epistaxis in dogs residing in endemic areas. The pathogenesis of CanL-associated epistaxis has not been fully explored because of the limited number of cases reported so far. HYPOTHESIS: Epistaxis in CanL could be attributed to more than 1 pathomechanism such as hemostatic dysfunction, biochemical abnormalities, chronic rhinitis, and coinfections occurring in various combinations. ANIMALS: Fifty-one dogs with natural CanL. METHODS: The allocation of 51 dogs in this cross-sectional study was based on the presence (n = 24) or absence (n = 27) of epistaxis. The potential associations among epistaxis and concurrent infections (Ehrlichia canis, Bartonella spp., and Aspergillus spp.), biochemical and hemostatic abnormalities, and nasal histopathology were investigated. RESULTS: Hypergammaglobulinemia (P= .044), increased serum viscosity (P= .038), decreased platelet aggregation response to collagen (P= .042), and nasal mucosa ulceration (P= .039) were more common in the dogs with epistaxis than in those without epistaxis. The other significant differences between the 2 groups involved total serum protein (P= .029) and gamma-globulin (P= .013) concentrations, which were higher, and the percentage platelet aggregation to collagen, which was lower (P= .012) in the epistaxis dogs. CLINICAL IMPORTANCE: CanL-associated epistaxis appears to be the result of multiple and variable pathogenetic factors such as thrombocytopathy, hyperglobulinemia-induced serum hyperviscosity, and nasal mucosa ulceration.

A retrospective study of 61 cases of spontaneous canine epistaxis (1998 to 2001).

OBJECTIVES: To determine the prevalence and identify possible clinicopathologic indicators of the diseases associated with canine epistaxis. METHODS: The medical records of 61 dogs with epistaxis were reviewed. RESULTS: Systemic diseases, diagnosed in fifty-six dogs, included canine leishmaniasis in twenty-three dogs, canine monocytic ehrlichiosis in twenty-two, concurrent canine leishmaniasis and canine monocytic ehrlichiosis in six, rodenticide toxicity in two and primary immune-mediated thrombocytopenia, suspected oestrogen toxicity and systemic arterial hypertension in one dog each. Intranasal diseases were documented in the remaining five dogs, including transmissible venereal tumour in three dogs, and nasal adenocarcinoma and nasal aspergillosis in one dog each. Mucosal pallor and a generalised bleeding tendency were significantly more common among dogs with canine monocytic ehrlichiosis compared with those with canine leishmaniasis, whereas the opposite was true for peripheral lymphadenomegaly. Also, dogs with canine monocytic ehrlichiosis presented with pancytopenia more frequently compared with those with canine leishmaniasis; in the latter dogs, the median values of haematocrit, leucocyte and platelet counts and serum total protein concentrations were higher. CLINICAL SIGNIFICANCE: Canine leishmaniasis and canine monocytic ehrlichiosis are the leading causes of canine epistaxis in Greece. Mucosal pallor, bleeding tendency and pancytopenia are more likely to be indicative of canine monocytic ehrlichiosis, as opposed to peripheral lymphadenomegaly and hyperproteinaemia in canine leishmaniasis.

Acute tumour lysis syndrome in a dog with B-Cell multicentric lymphoma.

A 5-year-old, spayed female German Shepherd dog was admitted to hospital with marked generalised lymphadenomegaly and splenomegaly. A stage Va B-cell multicentric lymphoma was diagnosed on clinical, cytological (lymph node, bone marrow), histological-immunohistochemical (lymph node excision) and imaging grounds. Since no satisfactory remission was achieved using a multi-drug chemotherapy protocol that included cyclophosphamide, vincristine, cytosine arabinoside, prednisolone, and subsequently supplemented by L-asparaginase, it was replaced by another protocol combining vincristine, L-asparaginase, prednisolone, cyclophosphamide and doxorubicin. Soon after the third weekly session of the second protocol, the clinical status of the animal deteriorated suddenly and severely, with a bleeding tendency, jaundice, hyperuricaemia, hyperphosphataemia, azotaemia, hyperbilirubinaemia and, presumptive disseminated intravascular coagulation. There was also complete regression of lymphadenomegaly. This report emphasises the clinicopathological features and the diagnostic peculiarities of the acute tumour lysis syndrome, which occurs uncommonly in dogs.

Blood alpha-Tocopherol, selenium, and glutathione peroxidase changes and adipose tissue fatty acid changes in kittens with experimental steatitis (yellow fat disease): a comparative study between the domestic shorthaired and Siamese breed.

Twenty domestic shorthaired (DSH) and 20 Siamese (S) kittens were allocated into 4 breed-specific groups, of 10 kittens each, that were fed exclusively cooked sardines (F groups) or commercial feline canned food based on oily fish (C groups) for a 4-month period. Clinical signs were scored every 15 d along with body weight recording and blood sampling for the measurement of alpha-tocopherol and selenium (Se) concentrations and glutathione peroxidase (GSH-Px) activity. Subcutaneous adipose tissue samples were obtained per month to determine its fatty acid composition. Steatitis, reproduced in all 20 F-group kittens, was accompanied by systemic signs in 5 DSH and 6 S animals. The severity of the disease reached its zenith at the second week in the DSH-F-group kittens and the fourth and sixth week in the S-F-group kittens. alpha-Tocopherol plasma level was significantly lower in F groups compared to their corresponding controls, whereas the opposite was true for Se and red blood cell GSH-Px activity. In conclusion, the results of this study have shown that although the morbidity rate is not different between the two breeds, the delay of Siamese cats to develop symptomatic steatitis is presumably attributed to an inherent resistance as a result of the long-standing evolution of more efficient antioxidant mechanisms. Also, the changes in fatty acid composition of the adipose tissue lipids are associated with the progression of the age, breed, and diet and probably with the inflammatory changes of the adipose tissue.

Serum alkaline phosphatase isoenzyme activities in canine malignant mammary neoplasms with and without osseous transformation.

BACKGROUND: Increased serum activity of total alkaline phosphatase (TALP) has been found in dogs with mammary neoplasms, especially malignant mixed tumors. We hypothesized that the bone isoenzyme of alkaline phosphatase (BALP), a specific indicator of osteoblastic activity and bone formation, may contribute to increased TALP in dogs with mammary neoplasms with osseous transformation. OBJECTIVE: The purpose of this study was to compare serum TALP, BALP, and other ALP isoenzyme activities in dogs with mammary malignant neoplasms with and without osseous transformation. METHODS: Twenty-one female dogs with malignant mammary neoplasms were compared with 21 clinically healthy, age-matched female control dogs. Physical, clinicopathologic (including preprandial and postprandial serum bile acids, ACTH stimulation, and low-dose dexamethasone suppression tests), radiographic, and ultrasonographic examinations were performed on all dogs with tumors to assess coexisting conditions. On the basis of histologic examination of excised tumors, dogs were further classified as having epithelial (n = 11) or mesenchymal/mixed (epithelial-mesenchymal) (n = 10) neoplasms, the latter of which had histologic and radiologic evidence of bone formation. Serum TALP, BALP, liver alkaline phosphatase (LALP), and corticosteroid-induced alkaline phosphatase (CALP) activities were measured using biochemical methods. RESULTS: Dogs with malignant mammary tumors had significantly higher (P < .05) median serum TALP (170 U/L), BALP (59 U/L), LALP (49 U/L), and CALP (24 U/L) activities, compared with control dogs (81, 32, 37, and 5 U/L, respectively). Significantly higher activities of BALP and LALP were found in dogs with epithelial neoplasms; whereas, only CALP activity was higher in dogs with mesenchymal/mixed neoplasms. There was no significant difference in TALP or isoenzyme activitities between epithelial and mesenchymal/mixed groups. CONCLUSION: BALP activity is increased in some dogs with malignant mammary tumors but does not account for the increase in TALP in dogs with neoplasms that have osseous transformation.

Effects of allopurinol treatment on the progression of chronic nephritis in Canine leishmaniosis (Leishmania infantum).

Forty dogs with canine leishmaniosis (CL) participated in this study, which was designed to investigate the effect of allopurinol on the progression of the renal lesions associated with this disease. The animals were allocated into 5 groups. Group A dogs (n = 12) had neither proteinuria nor renal insufficiency, group B dogs (n= 10) had asymptomatic proteinuria, and group C dogs (n = 8) were proteinuric and azotemic. Two more groups, CA and CB, comprising 5 dogs each, served as controls for groups A and B, respectively. Group A, B, and C dogs received allopurinol PO (10 mg/kg q12h) for 6 months, whereas group CA and CB dogs were placebo-treated. Serum biochemistry profile, urinalysis, urine protein/creatinine ratio, and glomerular filtration rate (GFR) measurements were carried out at the beginning of the study, the 3rd month, and the 6th month, whereas renal biopsies were carried out only at the beginning and the end of the trial. Membranoproliferative glomerulonephritis was the most common cause of chronic renal failure. Mesangioproliferative and tubulointerstitial nephritis were detected even in group A and CA dogs. Allopurinol not only lowered proteinuria in group B dogs but also prevented the deterioration of GFR and improved the tubulointerstitial, but not the glomerular, lesions in both group A and group B dogs. Further, it resolved the azotemia in 5 of the 8 dogs admitted with 2nd stage chronic renal failure (group C). Consequently, treatment with allopurinol is advisable in CL cases with asymptomatic proteinuria or 1st-2nd stage chronic renal failure.

Chronic hepatitis associated with canine leishmaniosis (Leishmania infantum): a clinicopathological study of 26 cases.

Hepatic tissue samples were obtained from 26 dogs humanely destroyed because of naturally occurring leishmaniosis (Leishmania infantum). None of the animals had palpable hepatomegaly or any other physical finding or historical evidence indicative of liver failure. However, serum biochemistry revealed hypoalbuminaemia (6/26), increased alkaline phosphatase (ALP) activity (15/26), and increased concentrations of total bilirubin (2/26) and post-prandial bile acids (4/26). Three main histological patterns were identified. In pattern 1 (3/26), the liver microarchitecture remained unchanged apart from the presence of individual or clustered macrophages in the sinusoids. In pattern 2 (20/26), there was multifocal, mild to moderate, granulomatous to pyogranulomatous infiltration of the hepatic parenchyma, particularly in the portal areas. Pattern 3 (3/26), which was the most severe form, was characterized by marked portal lymphoplasmacytic infiltration with occasional broaching of the limiting plate and extension into the adjacent parenchyma. In this pattern there was also mild portal fibrosis, together with lymphoplasmacytic aggregates within the parenchyma and small clusters of lymphocytes and plasma cells within the sinusoids. All three patterns were associated with hepatocyte vacuolation (15/26 dogs), and haemosiderin accumulation within the hepatocyte cytoplasm. Congestion was present in the liver of five dogs. No correlation was found between histopathological pattern and breed, sex, age, clinical manifestations, serum biochemical profile or parasite load in the hepatic tissue; patterns 1-3 may, however, represent sequential stages of hepatic leishmania infection during the chronic course of the disease.

Cellular immunophenotyping of exfoliative dermatitis in canine leishmaniosis (Leishmania infantum).

Lymphocyte subsets, major histocompatibility complex (MHC)-II expressing cells and number of amastigotes in the epidermis and dermis were investigated immunohistochemically in 48 dogs with patent leishmaniosis, with or without exfoliative dermatitis (ED) to study the immunopathogenesis of this common cutaneous form of the disease. Skin biopsies were obtained and compared for ED sites (group A, n = 26), normal-appearing skin from the same animals (group B, n = 24), and leishmanial dogs not exhibiting ED (group C, n = 22), and normal controls (group D, n = 22). The CD3+, CD45RA+, CD4+, CD8+ (CD8a+), CD21+, and MHC-II+ cells and leishmania amastigotes were identified immunohistochemically and counted with the aid of an image analysis system. Pyogranulomatous to granulomatous dermatitis, expressed in various histopathological patterns, was noticed in all groups A and B and in half of group C dogs. In the epidermis, the low number of T-cells and their subsets did not differ significantly between groups A and B, but CD8+ outnumbered CD4+ lymphocytes in both groups. MHC-II+ expression on epidermal keratinocytes was intense in the skin with and without lesions from dogs with ED but not in group C dogs. CD3+, CD8+ and MHC-II+ cells were fewer in group C compared to group A and B dogs. In the dermis, CD3+ cells in group A animals were mainly represented by the CD8+. CD45RA+ and CD21+ cells were also seen in high numbers. MHC-II expression, potentially in lymphocytes, fibroblasts, dendritic cells, and macrophages was intense. The numbers of all cellular subpopulations in the dermis were significantly different between the groups, being highest in group A and lowest in group D. In sebaceous adenitis sites, CD4+ outnumbered CD8+ cells in contrast to the neighbouring dermis and the epidermis. The number of CD21+ and CD45RA+ cells was much lower in the inflamed sebaceous glands compared to the dermis. Finally, the number of amastigotes in the normal-appearing skin was significantly higher in the ED dogs (group B) than in those not exhibiting this cutaneous form of the disease (group C).

Short-term exogenous glucocorticosteroidal effect on iron and copper status in canine leishmaniasis (Leishmania infantum).

Prednisolone was administered as an anti-inflammatory for 7 consecutive days in 11 dogs with leishmaniasis (CL group) and 5 clinically normal dogs (control group). After a 15-day wash-out phase, the same medication was given as an immunosuppressive for another 7-day period. In both animal groups and experimental periods an overall significant increase of serum iron and transferrin saturation was noted. Serum copper showed a significant increase during the anti-inflammatory period in the control group and a significant decrease during the immunosuppressive period in the CL group. No differences or changes of any kind regarding bone marrow hemosiderin were found between the 2 groups either before or after the end of both experimental periods. The only change noticed in the hematocrit values was a significant decrease in the control group after the end of the anti-inflammatory period. Based on these findings the use of prednisolone cannot be recommended and, if contemplated, should be carefully monitored, especially at an immunosuppressive dosage, because it may promote parasite replication through the induction of increased serum iron levels and hypocupremia.

Diagnostic correlation of CT-MRI and histopathology in 10 dogs with brain neoplasms.

Ten dogs with primary (n = 8) and metastatic (n = 2) brain tumours were studied in an attempt to evaluate the diagnostic sensitivity of computed tomography (CT) or magnetic resonance imaging (MRI). Of the clinical signs noticed, seizures (seven of 10), behavioural abnormalities and cognition dysfunction (seven of 10), compulsive walking and circling (six of 10), sensorimotor (five of 10) and neuro-opthalmological (two of 10) dysfunction were the most common. In all 10 animals that finally died of the disease or were killed, the histopathological diagnosis that followed necropsy was taken as a golden standard in the CT or MRI prediction of the histological type of brain neoplasms. In every instance, tumour detection, morphology and histological differentiation were possible with the aid of either CT (seven of 10) or MRI (three of 10) imaging especially after contrast enhancement. Only one CT-evaluated dog, diagnosed as meningioma, was found to be astrocytoma on histopathology. Interestingly, a rare case of cerebellar medulloblastoma was correctly identified in MRI scans.