Histopathological differences between canine idiopathic sebaceous adenitis and canine leishmaniosis with sebaceous adenitis

Sebaceous adenitis (SA) may be idiopathic (ISA) or associated with other disorders. The purpose of the present study was to compare the cutaneous histopathology of SA in cases in which Leishmania organisms were detected by immunohistochemistry (IHC) with that of cases diagnosed as ISA. Skin sections of 29 patients were evaluated histologically and divided into two groups, one characterized by several epidermal and subepidermal lesions, a granulomatous to pyogranulomatous nodular to diffuse dermatitis involving the sebaceous glands and a positive IHC for Leishmania spp. The other group was characterized by orthokeratotic hyperkeratosis, follicular keratosis with different degrees of pyogranulomatous to granulomatous SA, lack of nodular dermatitis and a negative IHC for Leishmania spp. Hidradenitis was present in both groups. From these results it can be concluded that SA in canine Leishmaniosis (CL) is usually present together with a nodular to diffuse dermal infiltrate and epidermal and subepidermal lesions, and that SA in the absence of dermal inflammation is probably not associated with or suggestive of CL, even in regions where the disease is endemic.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.     

Serum cobalamin concentrations in dogs with leishmaniosis before and during treatment

Hypocobalaminemia in dogs is most commonly associated with gastrointestinal disorders leading to impaired absorption and utilization of cobalamin. The objectives of this study were to compare serum cobalamin concentrations between dogs with leishmaniosis and clinically healthy dogs, and to assess possible alterations of serum cobalamin concentrations in dogs with leishmaniosis at different timepoints during treatment. Fifty-five dogs with leishmaniosis and 129 clinically healthy dogs were prospectively enrolled. Diagnosis of leishmaniosis was based on clinical presentation, positive serology and microscopic detection of Leishmania amastigotes in lymph node aspiration smears. Twenty of the dogs with leishmaniosis were treated with a combination of meglumine antimonate and allopurinol for 28 days and serum cobalamin concentrations were measured in blood samples that were collected before initiation of treatment (timepoint 0) and on days 14 and 28. In order to estimate alterations of serum cobalamin concentrations during treatment, cobalamin concentrations were measured in blood samples from 20 out of 55 dogs with leishmaniosis at all timepoints. Serum cobalamin concentrations were significantly lower in dogs with leishmaniosis before treatment (median: 362 ng/L; IQR: 277−477 ng/L) compared to clinically healthy dogs (median: 470 ng/L; IQR: 367−632 ng/L; P = 0.0035). Serum cobalamin concentrations increased significantly in dogs with leishmaniosis on day 14 of treatment compared to timepoint 0 (P = 0.02).In the present study, serum cobalamin concentrations were significantly lower in dogs with leishmaniosis compared to clinically healthy dogs. In addition, there was an increase in serum cobalamin concentrations during treatment. The clinical significance of hypocobalaminemia in dogs with leishmaniosis remains to be determined.     

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.     

Canine vector-borne disease in travelled dogs in Germany--a retrospective evaluation of laboratory data from the years 2004-2008

When importing dogs from various Mediterranean countries into Western Europe canine vector-borne infections are often considered as a major issue. Several diseases including babesiosis, leishmaniosis, hepatozoonosis, canine heartworm disease or ehrlichiosis can potentially be endemic in this region and pose a potential health risk for travelling dogs. Information on such infections in travelled dogs is scarce and therefore this study has been undertaken to examine the frequency of vector-borne infections in travelled dogs from the years 2004-2008. A total of 997 samples were screened by direct and/or indirect methods. Total seroprevalence was 7.5% with individual seroprevalence for the 3 species Leishmania spp., Ehrlichia canis and Babesia canis spp. ranging from 3.1 to 4.9%. Total detection rate for pathogens by direct methods was 3.5%. Ninteen Giemsa-stained blood smears were positive for large Babesia. None of the samples screened for microfilariae by Knott's test or for Dirofilaria immitis antigen by DiroChek^® were positive. Using PCR methods Leishmania-DNA was detected in 1/42 samples but none of 59 animals screened for E. canis-DNA was positive. The prevalence values as established by indirect and direct pathogen detection are considered as rather low.     

Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses

The aim of this study was to investigate the evolution of the abundance of cytochrome oxidase c subunit IV (NCOIV) and beta subunit of ATP synthase (β-ATP) during the last third of gestation in bovine skeletal muscles. Semitendinosus, longissimus thoracis and rectus abdominis muscles were chosen for the immunoblotting of the respective protein levels. Muscle and blood samples from bovine fetuses of randomly selected breeds were collected at 180, 210, and 260 days post-conception (dpc). The muscle tissue expressions of NCOIV, β-ATP were compared to blood glucose and insulin. At 260 dpc, protein levels of NCOIV raised in skeletal muscles. Additionally, β-ATP in semitendinosus and longissimus thoracis were elevated and paralleled by higher concentrations of blood serum insulin. It corroborates our previous observations indicating that accelerated metabolic differentiation of bovine skeletal muscles is associated with elevated blood insulin and occurs during the last trimester of gestation. Our observations point to the connection between insulin-sensitivity and the molecular mechanisms of mitochondrial contribution to ontogenesis of skeletal muscles.     

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.     

An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (São Paulo, Brazil)

Blood and bone marrow samples were taken from 112 Didelphis spp., collected between March 2005 and February 2006, from urban and peri-urban areas of Bauru, São Paulo State, Brazil, to evaluate the hypothesis that these animals might constitute a reservoir of Leishmania spp. Anti-Leishmania ssp. antibodies were screened in the serum samples using an enzyme-linked immuno-sorbent assay (ELISA) and the polymerase chain reaction (PCR). PCR was performed on fragments of DNA samples from Leishmania spp. using primers 13A and 13B, and showed a positive outcome in 91.6% of the 112 samples tested. Of the 107 samples analyzed by ELISA, 71% were positive. Evidence of epidemiological risk factors such as a circulating parasite and freely moving vectors suggests that Didelphis spp. may participate in the transmission cycle of Leishmania spp. in Bauru.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Nodular granulomatous glossitis as the sole clinical sign in canine leishmaniosis

A 5.5‐year‐old, intact male Rottweiler dog was admitted with a history of multifocal nodular tongue lesions which progressively deteriorated during the previous year. Physical examination revealed several reddish nodules with central depression on the surface of the tongue in an otherwise healthy dog. Clinicopathologic abnormalities included eosinophilia and hyperproteinemia. Lingual nodule cytopathology, histopathology, and immunohistochemistry revealed Leishmania spp. amastigotes and a severe granulomatous glossitis. The dog was also seroreactive to L infantum antigens by an indirect immunofluorescence assay. Clinical reevaluation 3 months after the institution of treatment with allopurinol and miltefosine indicated that the nodular lesions had completely regressed. In endemic areas, lingual nodular lesions may rarely be the sole clinical sign of canine leishmaniosis. Standard medical treatment may provide an excellent prognosis.     

Inflammatory myopathies.

Inflammatory myopathies are the result of infiltration of inflammatory cells into striated muscle, with or without an association with an underlying cause. Two broad classifications are IIMs and secondary inflammatory myopathies associated with other diseases. Standard diagnostic criteria for inflammatory myopathy include the presence of weakness or loss of specific muscle group function, an increase in CK, EMG changes associated with muscle membrane instability, and histologic evidence of inflammation. Not all these criteria, however, must be present. Fresh-frozen biopsy from two proximal muscles is recommended for biopsy confirmation. IIM can either focally affect head or neck muscles or be more diffuse. MMM is an immune-mediated disease characterized by a humoral antibody produced against the unique type IIM and type I variant mvofibers of masticatory muscles of dogs, which causes inflammation and loss of function of the muscles of mastication. Idiopathic polymyositis can affect focal muscle groups (extraocular, laryngeal) or present as multifocal or diffuse involvement of skeletal muscle in the cat and dog. Familial canine DM is an inflammatory disease of the striated muscle, skin, and vasculature in young Collies, Shetland Sheepdogs (Shelties), and, rarely, Collie-crossbred dogs. Immunosuppressive therapy is the key to successful treatment. Protozoal parasitic myopathies are the most common cause of clinically relevant secondary inflammatory myopathies. The degree of systemic involvement is often the limiting factor to successful treatment. Early recognition of the clinical signs for proper diagnostic testing and institution of appropriate therapy can result in a rewarding outcome in treating inflammatory myopathies in the cat and dog.     

Lectin-Histochemistry: Glycogenosis in Cattle

Ten out of 47 calves that were born in a small Brahman herd from southern Brazil developed progressive muscular weakness and tremors, lethargy and poor body condition. Necropsy was performed on three affected animals. The only gross lesion detected was paleness of the muscles of the trunk and limbs. Multiple cytoplasm vacuoles located in different tissues were the principal microscopic lesions. Vacuoles were particularly evident in skeletal muscles and myocardium. PAS-positive granules were numerous in skeletal muscle fibres and Purkinje fibres of the myocardium, but were also observed in the neurons of the brain and spinal cord, and in the vascular smooth muscle fibres from all the examined tissues. Pretreatment with diastase completely abolished the PAS reactivity. The vacuoles reacted strongly to Griffonia simplicifolia II and Concanavalia ensiformes lectins, whose biding pattern has been reported as useful for demonstration of glycogen. Examination of the electron micrographs revealed that glycogen was free within the cytoplasm or accumulated in membrane-bound granules of several tissues, especially in striated muscle, liver and neurons of the CNS. These findings were consistent with generalized glycogenosis.     

Characterization of lacrimal gland lesions and possible pathogenic mechanisms of keratoconjunctivitis sicca in dogs with leishmaniosis

In a previous study, it was found that 2.8% of dogs with leishmaniosis had keratoconjunctivitis sicca (KCS). The aim of this study was to characterize the lesions present in the lacrimal glands of dogs with leishmaniosis and to determine the presence of the parasite by means of immunohistochemistry. The inflammatory infiltrate was described as granulomatous or pyogranulomatous and was located around the ductal component of the glands. Immunoperoxidase staining localized the parasites following the same pattern. Samples from eyes that had clinical signs compatible with KCS presented inflammatory infiltrate and parasite more commonly than those from eyes without clinical signs. One of the mechanisms of KCS in dogs with leishmaniosis may be the inflammatory infiltrate located around the ducts of lacrimal glands, producing retrograde accumulation and retention of secretion. Meibomian gland was the most commonly affected by the infiltrate, highlighting the possibility of a qualitative KCS in these dogs.     

Recent advances in leishmaniosis in pet animals: epidemiology, diagnostics and anti-vectorial prophylaxis

The leishmanioses are diseases caused by protozoa of the genus Leishmania, parasites infecting numerous mammal species, including humans, and transmitted by the bite of phlebotomine sand flies. They are a large group of diseases ranging over inter-tropical zones of America and Africa, and extend into temperate regions of Latin America, Europe and Asia. Pet animals are found infected with different Leishmania species but Leishmania infantum is the most widespread being dogs the main reservoir of zoonotic visceral leishmaniosis (ZVL). Dogs are very susceptible to this parasite and may suffer from a complex syndrome, canine leishmaniosis (CanL), one of the major zoonoses globally causing severe fatal disease in this animal. Infections in cats and horses have also been reported in areas where CanL is diagnosed. In Europe dogs and cats are common companion animals and their health is of great concern, therefore management of leishmaniosis in pets generally follows that of human ZVL. The recent spread of Leishmania infections in non-endemic territories has been monitored by means of canine surveys, which represent a suitable approach because of the dog's role as a sentinel host. New tools have been developed for the surveillance and control of ZVL. A number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites, with elevated efficacy for both individual and mass protection.     

Urban Prevalence of Listeria spp. and Listeria monocytogenes in Public Lavatories and on Shoe Soles of Facility Patrons in the European Capital City Vienna

The aim of this study was to determine the prevalence of Listeria spp. and Listeria monocytogenes (L. monocytogenes) in urban public lavatories and on shoe soles of facility patrons in a European capital city. More than 91% of all municipal public lavatories in Vienna close to public hubs were included in this study. Overall, 373 swab samples of public lavatories and shoes of facility patrons were enriched, according to ISO 11290‐1. Listeria monocytogenes isolates were subtyped using pulsed‐field gel electrophoresis. A total of 24 samples were positive for Listeria spp., yielding an overall prevalence of 6.4% (24/373). Listeria monocytogenes was found in 2.1% (8/373) of all samples. Swabs from lavatories in parks, container lavatories and lavatories at markets had the highest prevalences of 20.7% (6/29), 20% (2/10) and 12.5% (1/8) Listeria spp., respectively. These detection rates were statistically significantly higher than those associated with lavatories in shopping centres (P = 0.003, P = 0.002, P = 0.02) and at public transport locations (P = 0.0004, P = 0.005, P = 0.02). Shoes sampled at Christmas markets showed the highest Listeria spp. and L. monocytogenes prevalences of 80% (4/5) and 40% (2/5), respectively. With regard to shoe type, Listeria spp. detection rates were 14.3% (3/21; winter boots), 13.3% (2/15; hiking boots), sport shoes (5.9%; 2/34) and brogues (5.1%; 4/79). No Listeria spp. were found on shoe soles that had smooth treads (0/76), while Listeria spp. were detected on 19.5% (8/41) of medium depth tread shoe types and on 9.4% (3/32) of deep tread shoes. These data suggest that soil environment is still one of the most important reservoirs for the foodborne pathogen L. monocytogenes.     

Bovine Haematological Values during Fattening with Possible Implications for Muscle Growth

Haemoglobin concentration (Hb), haematocrit (PCV) and mean corpuscular haemoglobin concentration (MCHC) were determined as indicators of oxygen-carrying capacity in 25 Simmental bulls during intensive fattening. Their possible relations to muscle growth were also investigated. The animals were the progeny of two sires. Blood samples were taken at 6, 9, 12 and 15 months of age. After slaughter, the right thoracic rib cut (7–9) was dissected into the longissimus dorsi muscle (LD), other muscles (OM) and total muscles (TM) (LD+OM). There was a significant increase in the mean PCV and Hb values during fattening. The PCV and Hb values were significantly correlated with the tissue share of OM or TM only at 12 and 15 months of age. LD muscle share was not significantly correlated with any of the parameters at any fattening time. It would appear from these results that muscle growth proceeds in two phases. The first is until the age of 12 months and the other from then to maturity.     

Hypertrophy of intestinal smooth muscle in cats

Pathological findings of four cats with severe and diffuse smooth muscle hypertrophy of the small intestine (MHSI) are reported and compared to those of five cats with segmental MHSI secondary to neoplastic obstruction and four controls. Histology demonstrated a constant association between idiopathic MHSI and submucosal fibrosis and chronic lymphoplasmacytic enteritis. Morphometry (gut diameter, thickness and area of muscular layers, number and density of smooth muscle nuclei) and MIB-1-immunolabelling showed that the thickness increase was mostly due to hypertrophy, but hyperplasia was also evident. Microbiology from ileal content samples was performed in two cats with primary MHSI, and Campylobacter spp. were isolated, which were also demonstrated by immunohistochemistry and ultrastructure. The association of chronic enteritis with idiopathic MHSI suggests that factors released in intestinal inflammation may also act as hypertrophy stimuli for smooth muscle cells.     

Leishmania infantum in wild animals in endemic areas of southern Italy

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears.This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.