Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin. © 2012 Elsevier B.V.

Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: A natural model of neuroinflammation during systemic parasitic infection

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1β, IL-6, IL-10, IL-12p40, IFN-γ, TGF-β and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1β, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-β and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1β and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation. © 2012 Elsevier B.V.

Ki-67 labeling in canine perianal glands neoplasms: A novel approach for immunohistological diagnostic and prognostic

Background: The antibody Ki-67 is a reliable and easy tool to accurately assess the growth fraction of neoplasms in humans and animals, and it has been used to predict the clinical outcome. Therefore, the aim of the present study was to investigate the immunohistochemical expression pattern of Ki-67 in normal and neoplastic perianal glands of dogs to evaluate the possible use of this proliferation marker as an ancillary method of perianal tumor diagnosis. We studied 42 cases of perianal gland neoplasms including adenomas (n = 15), epitheliomas (n = 15), and carcinomas (n = 12). As controls, 13 tissue samples from normal perianal glands were used. A Ki-67 index was established by a computer-assisted image analysis and compared with manual counting. Results: Out of the 42 cases of perianal gland neoplasms, 34 were from males and eight from females. Recurrence was reported in 14 cases, being higher (8/12) in carcinomas. Immunostaining for Ki-67 revealed that the carcinomas showed a higher proliferation rate (9.87%) compared to groups of epitheliomas (2.66%) and adenomas (0.36%). For adenomas and epitheliomas of the perianal glands the computer-assisted counting and the manual counting gave similar results; however, only the computer-assisted image analysis was efficient to predict the perianal gland carcinoma recurrence.Conclusion: Since there were significant differences in the number of Ki-67-positive nuclei, this marker proved to be effective in helping the classification of perianal gland neoplasms and to refine the diagnosis criteria, especially in those samples with high variation in morphology/area. Also, higher Ki-67 index is related to recurrence in cases of perianal gland carcinomas. Further, the computer-assisted image analysis proved to be a fast and reliable method to assess the Ki-67 index in perianal gland neoplasms. © 2013 Pereira et al.; licensee BioMed Central Ltd.

Orbifloxacin: A Review of Properties, Its Antibacterial Activities, Pharmacokinetic/Pharmacodynamic Characteristics, Therapeutic Use, and Analytical Methods

Orbifloxacin is a third generation of fluoroquinolone that exhibits increased antibacterial activity against the Enterobacteriaceae, gram-negative and gram-positive bacteria, anaerobes, and mycobacteria. This drug was synthesized in 1987 and developed as a veterinary chemotherapeutic to use for livestock and domestic pets. Orbifloxacin is labeled for the treatment of skin, soft tissue, and urinary tract infections in dogs, and skin and soft tissue infections in cats, but in some countries, orbifloxacin has been given for the treatment of gastrointestinal and respiratory infections in cattle and swine and other animals. The in vitro activity and clinical efficacy of orbifloxacin against naturally occurring bacterial infections of the skin, ear, soft tissue, udder, and gastrointestinal and respiratory systems in different animals have been evaluated and good responses have been found. The minimum inhibitory concentration of orbifloxacin has been determined in various different pathogens and the results found in the literature are shown in this work. The pharmacokinetics of orbifloxacin has been evaluated by different routes of administration in goats, horses, pigs, rabbits, dogs, cats, camels, cattle, sheep, and fish. Orbifloxacin exhibits excellent pharmacokinetic parameters that suggest that this drug may have good clinical effects on various bacterial infections in these species. All methods described in the scientific literature for determination of orbifloxacin in different matrices were collected and discussed. © 2013 Copyright Taylor and Francis Group, LLC.

Identificação de espécies fúngicas nas onicomicoses do idoso institucionalizado

BACKGROUND: Superficial fungal infections are caused by dermatophytes, yeasts or filamentous fungi. They are correlated to the etiologic agent, the level of integrity of the host immune response, the site of the lesion and also the injured tissue. OBJECTIVE: The purpose of this study is to isolate and to identify onychomycosis agents in institutionalized elderly (60 years old +). METHODS: The identification of the fungi relied upon the com-bined results of mycological examination, culture isolation and micro cultures observation under light microscopy from nail and interdigital scales, which were collected from 35 elderly with a clinical suspicion of onychomycosis and a control group (9 elderly with healthy interdigital space and nails). Both groups were insti-tutionalized in two nursing homes in Sao Bernardo do Campo, SP, Brazil. RESULTS: The nail scrapings showed 51.40% positivity. Of these, dermatophytes were found in 44.40% isolates, 27.78% identified as Trichophyton rubrum and 5.56% each as Trichophyton tonsurans, Trichophyton mentagrophytes and Microsporum gypseum. The second more conspicuous group showed 38.89% yeasts: 16.67% Candida guilliermondii, 11.11% Candida parapsilosis, 5.56% Candida glabrata, and 5.56% Trichosporon asahii. A third group displayed 16.70% filamen-tous fungi, like Fusarium sp, Aspergillus sp and Neoscytalidium sp (5.56% each). The interdigital scrapings pre-sented a positivity rate of 14.29%. The agents were coincident with the fungi that caused the onychomycosis. In the control group, Candida guilliermondii was found at interdigital space in one person. CONCLUSION: Employing a combination of those identification methods, we found no difference between the etiology of the institutional-ized elderly onychomycosis from that reported in the literature for the general population. © 2013 by Anais Brasileiros de Dermatologia.

Chemical characterization and acaricide potential of essential oil from aerial parts of Tagetes patula L. (Asteraceae) against engorged adult females of Rhipicephalus sanguineus (Latreille, 1806)

Rhipicephalus sanguineus, commonly known as the brown dog tick, is one of the most widely distributed species of tick. In dogs, it can cause anemia and provide the transmission of pathogenic microorganisms such as Babesia canis, Ehrlichia canis, Hepatozoon canis, Anaplasma platys, and Mycoplasma haemocanis. To man, it can transmit the intracellular parasites Rickettsia rickettsii and Rickettsia conorii, the causative agents of the Rocky Mountain spotted fever in the Americas and Mediterranean and spotted fever in Europe and North Africa. Its control is performed by applying synthetic formulations composed of pyrethroids; however, continued use of these products results in environmental damage and acquisition of resistance. Alternatively, studies with botanical insecticides have been increasingly recurrent. Therefore, this study aimed to test the efficacy of essential oil of Tagetes patula, a ruderal species widely described in the literature for its insecticidal properties, in engorged females of R. sanguineus by the adults immersion test (AIT) and impregnated paper disk test (IPDT). The essential oil used, through gas chromatography coupled to mass spectrometry, revealed the presence of 55 compounds, being the 4-vinyl guaiacol and gamma terpinene the majority ones. The AIT compared to the IPDT was more efficient in inhibiting oviposition of tick; however, the eggs laid by the females submitted to saturated atmosphere with essential oil, from IPDT, not hatched, interrupted their development cycle. Besides being a pioneer work, the results presented here contributes to new researches, aiming the incorporation of essential oil in an acaricide for use in the environment. © 2013 Springer-Verlag Berlin Heidelberg.

Evaluation of von willebrand factor during pregnancy, lactation and oestrous cycle in bitches affected and unaffected by von willebrand disease

Plasmatic concentrations of von Willebrand Factor (vWF) increase during pregnancy in humans and dogs; however the mechanism of such increase is still not well defined. The aims of this study were: (i) to evaluate changes in vWF concentration during pregnancy and during the subsequent oestrous cycle in bitches affected and unaffected by von Willebrand Disease (vWD); (ii) to correlate the vWF levels and cortisol levels in both groups. Seven vWD affected (GI) and nine unaffected (GII) bitches were used. The animals were assessed during pregnancy, parturition, lactation and non-gestational oestrous cycle in 11 moments (Pregnancy 1, Pregnancy 2, Parturition, Lactation 1, Lactation 2, Lactation 3, Anestrus, Proestrus, Oestrus, Diestrus 1, and Diestrus 2). The following tests were performed; measurement of von Willebrand factor antigen (vWF:Ag), albumin and cortisol. In both groups, vWF concentration remained stable during the non-gestational oestrous cycle, but increased during pregnancy, with the highest value observed at parturition. Increases of 70% and 124% in vWF were seen in GI and GII, respectively, compared to anestrus. No correlation was found between vWF and cortisol. Values of vWF:Ag changed during pregnancy, with a peak at parturition, both in vWD affected and unaffected animals. Values of vWF were not altered in the different phases of the oestrous cycle following pregnancy in both groups. Evaluation of vWF during pregnancy can cause false negative results for vWD, but assessment can be performed at any point in the oestrous cycle of non-pregnant bitches. © 2012 Blackwell Verlag GmbH.

T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd.

Histopathology of the tegument of rabbits infested by Rhipicephalus sanguineus (ACARI: IXODIDAE) ticks and exposed to selamectin (active principle of acaricide Revolution®, Pfizer)

Ticks are hematophagous ectoparasites which can transmit several diseases to the host during their feeding process. When ticks mechanically damage the tissue, they eventually induce inflammatory responses on the skin spot where they are fixed. One of the alternatives to control these ectoparasites is the use of chemical substances like selamectin - the active principle of Pfizer's antiparasitic Revolution® - a macrocyclic lactone capable of doing neurotoxic damage to the tick and eventually eliminating infestation in dogs and cats. The purpose of this study was to analyze, using histological and histochemical techniques, the occurrence of morphophysiological alterations in the skin of the host rabbits exposed to selamectin and infested with Rhipicephalus sanguineus (Acari: Ixodidae). Histologically, the exposed and infested rabbits showed a partial and/or total decrease in the stratum corneum and the epithelium decreased in the number of cell layers, consequently reducing the stratification (thinning) and quite pronounced formations of sub-epidermal edemas with consequent disorganization of collagen fibers in the dermal layer's connective tissue. Histochemical tests showed strong periodic acid-Schiff-positive reaction in the hair follicle and some regions of the dermis, besides resynthesis of collagen fibers detected by Mallory's trichrome technique. The obtained results showed that selamectin acts like a toxicant agent when in contact with the skin of the rabbit infested with ticks, inducing morphophysiological alterations in the acute inflammatory process in the animal's tegument. Selamectin is a chemical substance which has a dose-dependent action since higher concentrations cause greater morphophysiological damage in the skin of rabbits. © 2013 Springer-Verlag Berlin Heidelberg.

Zymographic patterns of MMP-2 and MMP-9 in the CSF and cerebellum of dogs with subacute distemper leukoencephalitis

Distemper leukoencephalitis is a disease caused by the canine distemper virus (CDV) infection. It is a demyelinating disease affecting mainly the white matter of the cerebellum and areas adjacent to the fourth ventricle; the enzymes of the matrix metalloproteinases (MMPs) group, especially MMP-2 and MMP-9 have a key role in the myelin basic protein fragmentation and in demyelination, as well as in leukocyte traffic into the nervous milieu. To evaluate the involvement of MMPs during subacute distemper leukoencephalitis, we measured the levels of MMP-2 and MMP-9 by zymography in the cerebrospinal fluid (CSF) and in the cerebellum of 14 dogs naturally infected with CDV and 10 uninfected dogs. The infected dogs presented high levels of pro-MMP-2 in the CSF and elevated levels of pro-MMP-2 and pro-MMP-9 in the cerebellar tissue. Active MMP-2 was detected in the CSF of some infected dogs. As active MMP-2 and MMP-9 are required for cellular migration across the blood-brain barrier and any interference between MMPs and their inhibitors may result in an amplification of demyelination, this study gives additional support to the involvement of MMPs during subacute distemper leukoencephalitis and suggests that MMP-2 and MMP-9 may take part in the brain inflammatory changes of this disease. © 2013 Elsevier B.V.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. © 2013 Elsevier B.V.

Neutrophil dysfunction varies with the stage of canine visceral leishmaniosis

Canine visceral leishmaniosis (CVL) causes a dependent-stage alteration in neutrophil oxidative metabolism. When production of reactive oxygen species (ROS) exceeds the antioxidant capacity of neutrophils, apoptosis is triggered, impairing the viability and function of these cells, which can predispose dogs to infection. However, the uremic condition observed in late-stage CVL can also alter the viability and function of human neutrophils. To more clearly understand this relationship, the apoptosis rate and oxidative metabolism of neutrophils from control dogs (n= 20) were compared to dogs in moderate (n= 15) and very severe (n= 15) stage CVL, classified according to LeishVet Consensus. To assess neutrophil oxidative metabolism, superoxide production was measured using the nitroblue tetrazolium reduction test (NBT) in isolated neutrophils. The apoptosis rate of neutrophils was estimated using the morphological method. Moderate-stage dogs presented increased superoxide production, while dogs with very severe stage CVL presented decreased superoxide production and an increase neutrophil apoptosis rate. Leishmaniosis causes differential neutrophil dysfunction according to disease stage. In moderate stage CVL, increased superoxide production is observed with no change in neutrophil viability. However, in very severe stage CVL, decreased superoxide production and increased apoptosis occur associated with uremia. © 2013 Elsevier B.V.

Clinical, cytologic, and histologic features of a mammary micropapillary carcinoma in a dog

Mammary invasive micropapillary carcinoma is a rare variant of mammary carcinoma that was recently recognized in dogs. The cytologic features and biologic behavior of such neoplasms in dogs have not yet been widely discussed in the veterinary literature. We report the clinical, cytologic, and histologic features of a canine micropapillary carcinoma in a 13-year-old female mongrel dog. The mammary region presented with extreme local pain, severe edema and erythema, and multifocal epidermal ulceration, which is typical for an inflammatory mammary carcinoma. Fine-needle aspirates were highly cellular and consisted of individual cells and papillary cell clusters with characteristics of malignant epithelial cells. Histologic examination revealed neoplastic cells arranged in small papillae without fibrovascular cores, sometimes inside clear lymphatic spaces, indicating lymphovascular invasion. Regional lymph node evaluation revealed metastatic cells. Due to deteriorating clinical condition the dog was euthanatized 5 months after mastectomy. At necropsy, metastatic neoplastic mammary cells were found in popliteal and mediastinal lymph nodes, the right femoral biceps muscle, liver, heart, lungs, and urinary bladder. © 2013 American Society for Veterinary Clinical Pathology.

Investigação sorológica de espécies de Ehrlichia em cães, equinos e humanos de um assentamento rural do sul do Brasil

The aims of this study were to determine the seroprevalence of Ehrlichia spp. and risk factors for exposure in a restricted population of dogs, horses, and humans highly exposed to tick bites in a Brazilian rural settlement using a commercial ELISA rapid test and two indirect immunofluorescent assays (IFA) with E. canis and E. chaffeensis crude antigens. Serum samples from 132 dogs, 16 horses and 100 humans were used. Fifty-six out of 132 (42.4%) dogs were seropositive for E. canis. Dogs > one year were more likely to be seropositive for E. canis than dogs ≤ one year (p = 0.0051). Ten/16 (62.5%) and 8/16 (50%) horses were seropositive by the commercial ELISA and IFA, respectively. Five out of 100 (5%) humans were seropositive for E. canis and E. chaffeensis. Rhipicephalus sanguineus (n = 291, 97.98%) on dogs and Amblyomma cajennense (n = 25, 96.15%) on horses were the most common ticks found. In conclusion, anti-Ehrlichia spp. antibodies were found in horses; however, the lack of a molecular characterization precludes any conclusion regarding the agent involved. Additionally, the higher seroprevalence of E. canis in dogs and the evidence of anti-Ehrlichia spp. antibodies in humans suggest that human cases of ehrlichiosis in Brazil might be caused by E. canis, or other closely related species.

Perioperative evaluation of tumescent anaesthesia technique in bitches submitted to unilateral mastectomy

Background: Tumescent anaesthesia (TA) is a widely used technique in oncologic surgeries necessitating large resection margins. This technique produces transoperative and postoperative analgesia, reduces surgical bleeding, and facilitates tissue divulsion. This prospective, randomised, blind study evaluated the use of TA in bitches submitted to mastectomy and compared the effect of TA with an intravenous fentanyl bolus. A 2.5-mcg/kg intravenous fentanyl bolus (n = 10) was compared with TA using 0.275% lidocaine (n = 10) in bitches submitted to unilateral mastectomy. Sedation was performed by intramuscular (IM) injection of 0.05 mg/kg of acepromazine combined with 2 mg/kg of meperidine. Anaesthesia was induced with 5 mg/kg of intravenous propofol and maintained with isoflurane/O2. Heart and respiratory rates; systolic, mean, and diastolic arterial blood pressures; central venous pressure; SpO2; ETCO2; inspired and expired isoflurane concentrations; and temperature were measured transoperatively. Visual analogue scales for sedation and pain and the Glasgow composite and Melbourne pain scales were used for postoperative assessment. The surgeon investigated the quality of the surgical approach, considering bleeding and resection ability, and the incidence of postoperative wound complications.Results: The heart rate was lower and the end-tidal isoflurane concentration was higher in dogs treated with fentanyl than in dogs treated with TA. A fentanyl bolus was administered to 8 of 10 dogs treated with fentanyl and to none treated with TA. Intraoperative bleeding and the mammary gland excision time were lower in dogs treated with TA. The maximal mean and individual plasma lidocaine concentrations were 1426 ± 502 ng/ml and 2443 ng/ml at 90 minutes after infiltration, respectively. The Glasgow Composite Pain Scale scores were higher in dogs treated with fentanyl than in dogs treated with TA until 2 hours after extubation.Conclusions: Compared with intravenous fentanyl, TA in bitches: may be easily performed in non-inflamed, ulcerated, adhered mammary tumours; has an isoflurane-sparing effect; improves transoperative and immediate postoperative analgesia; is apparently safe for use in clinical conditions as evidenced by the fact that it did not produce any adverse signs or lidocaine plasma concentrations compatible with toxicity; does not modify the recovery time; and facilitates the surgical procedure without interfering with wound healing. © 2013 Credie et al.; licensee BioMed Central Ltd.