A compared histopathological study on kidneys and eye bulbs in distinct clinical presentations of canine leishmaniasis by Leishmania infantum.

Canine leishmaniasis (CanL) caused by Leishmania infantum commonly progresses with renal and ophthalmic lesions associated with active systemic disease. As chronic inflammation related to immune complex deposits is a pathophysiological factor in the development of both glomerulonephritis and uveitis, we aimed to evaluate renal and ocular histopathological lesions and analyze whether they were related to each other and the clinical degree of the disease. For that, we evaluated 15 dogs from CanL-endemic areas. L. infantum PCR-positive dogs were studied according to disease severity into two different groups: Group-1 (G1) had data from seven dogs with mild to moderate CanL and no history of treatment, and G2 was formed with eight dogs with severe to terminal disease that had not responded to CanL treatment. Histopathological analysis of kidneys showed higher frequencies and intensities of glomerular basement membrane thickening (p = 0.026), deposits in glomeruli (p = 0.016), epithelial necrosis (p = 0.020), tubular dilatation (p = 0.003) and interstitial fibrosis (p = 0.04) in G2 dogs than in G1 dogs. Surprisingly, the histopathology of eye bulbs showed a higher frequency and intensity of retinitis (p = 0.019) in G1 dogs than in G2 dogs. The comparative analysis showed that there was no correspondence between histopathological findings in kidneys versus eyes in milder or more severe CanL. Our findings suggested that (1) clinically undetectable eye alterations can be more precocious than those in kidneys in the development of CanL, and (2) the lower frequency of eye lesions and higher frequency of renal lesions in dogs with terminal disease even after treatment indicate that therapy may have been effective in reducing CanL-associated ophthalmic disease but not proportionally in reducing kidney disease.

Nutritional adjuvants with antioxidant properties in the treatment of canine leishmaniasis.

Clinical improvement of dogs treated for canine leishmaniasis (CanL) requires reducing Leishmania infantum loads, which depend on intracellular oxidant compounds to destroy the parasite. However, oxidative species' excess and antioxidants consumption can culminate in oxidative stress, resulting in increased, widespread inflammation. We aimed to evaluate if early or late addition of nutritional adjuvants (NAs) - omega-3 polyunsaturated fatty acids and B vitamins - to anti-Leishmania drugs (ALDs) in the treatment of CanL would be clinically beneficial. For that, serum biomarkers including oxidative stress parameters were analyzed during 12 months in dogs allocated to two treatment groups: (G1) NAs administered from 30 days prior to the beginning of ALDs; and (G2) NAs administered from 61 days after the beginning of ALDs. Both G1 and G2 continued to receive NAs until the 12th month. The ALDs administered were metronidazole associated with ketoconazole (40 days), followed by allopurinol from day 41 until the 12th month. G1 exhibited superior inflammation control, with reduced globulins (p = 0.025), specific anti-Leishmania immunoglobulins (p = 0.016), total protein (p = 0.031), and an increased serum albumin/globulin ratio (p = 0.033), compared to G2. The early use of NAs associated with ALDs is clinically beneficial in treating dogs with CanL.

Vincristine and ivermectin combination chemotherapy in dogs with natural transmissible venereal tumor of different cyto-morphological patterns: A prospective outcome evaluation.

Vincristine is the first-line drug for the chemotherapy of canine transmissible venereal tumor (CTVT). Drug resistance is related to tumor cyto-morphological patterns of CTVT. There are anti-cancer properties of ivermectin, thus, a combination of ivermectin and vincristine could be an effective chemo-therapeutic treatment regimen for CTVT. Study aims, therefore, were to (1) assess the frequency of CTVT cyto-morphologies, and (2) evaluate treatment efficacy and possible adverse reactions to vincristine compared with a combination vincristine and ivermectin. Dogs (n = 41) with CTVT were characterized by tumor cyto-morphology and disease severity and of those, 20 were randomly allocated into two groups. There was a control group (G-Vin; n = 10) in which there was treatment with vincristine; and an experimental group (G-Iv/Vin; n = 10) in which there was treatment with the ivermectin/vincristine combination. Although dogs in the G-Iv/Vin group had more severe disease at the beginning of the study (P = 0.0031), the number of weeks and chemotherapy sessions until tumor remission were similar among dogs of the two groups, indicating both treatments were effective. There was a decrease in the leukocyte counts (P = 0.0020), related to neutropenia (P = 0.0371) in the G-Vin but not the G-Iv/Vin treatment group. There was no tumor resistance that developed during the study regardless of the treatment regimen used or tumor cytomorphology. In summary, the use of the vincristine/ivermectin combination was well tolerated and efficacious for CTVT treatment.

Mathematical Modelling Using Predictive Biomarkers for the Outcome of Canine Leishmaniasis upon Chemotherapy.

Prediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient's prognosis.

Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 µm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 µg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 µg/mL, and oxypurinol at 10-200 µg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

Leishmania infantum-derived lipophosphoglycan as an antigen in the accurate serodiagnosis of canine leishmaniasis.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage.

Incidence of skin tumors in dogs in Salvador, Bahia state, Brazil (2007-2016) / Frequência de dermatopatias tumorais em cães da cidade de Salvador, Bahia (2007-2016)

This study aimed to establish the incidence of skin tumors (cutaneous proliferative lesions of neoplastic or non-neoplastic nature) in dogs diagnosed by histopathological evaluation at the Veterinary Pathology Laboratory (LPV) of the Federal University of Bahia (UFBA) in a 10-year (2007-2016) historical series. Of the 1945 histopathological diagnoses made in this period, 503 were skin biopsies, and 617 dermatological problems (87 dogs, 17.3%, presented more than one positive diagnosis) were found. Of the 617 diagnoses of dermatopathy, 546 (88.49%) were tumors and 71 (11.51%) were non-tumorous alterations. The 546 conditions more profoundly studied were from 453 dogs, 468 (85.7%) neoplastic and 78 (14.3%) non-neoplastic tumors. The 468 neoplasms were classified as follows: 230 benign (49.14%), 215 malignant (45.94%), 23 borderline (epitheliomas) (4.91%), 51.92% (243/468) mesenchymal, 42.74% (200/468) epithelial, 4.91% (23/468) melanocytic, and 0.43% (2/468) metastatic (mammary gland). The most commonly diagnosed neoplastic dermatopathies were mastocytoma (14.7%) and lipoma (7.48%). Among the 78 non-neoplastic conditions (14.3%), epidermal inclusion cyst (39.74%) and trichogranuloma (15.38%) were the most frequent. Canine dermatopathies accounted for 26% of the biopsy files of the LPV-UFBA. Distinct simultaneous dermatological problems were frequently found in the dogs assessed (one in six). Considering that these conditions can present with different cellular origin and biological behavior, it is crucial that histopathological evaluation be performed in fragments from the different cutaneous lesions.(AU)

Objetivou-se com esse estudo determinar a frequência de dermatopatias tumorais (lesões proliferativas cutâneas que cursam com aumento de volume de natureza neoplásicas ou não neoplásicas) em cães, diagnosticadas por exame histopatológico no Laboratório de Patologia Veterinária (LPV) da Universidade Federal da Bahia (UFBA) na série histórica de 10 anos (2007-2016). Dos 1.945 exames histopatológicos realizados no período, 503 tratava-se de biópsias cutâneas, dentre os quais, foram diagnosticados 617 dermatopatias (87 cães, 17,3%, apresentavam mais de um diagnóstico). Dos 617 diagnósticos de dermatopatias 546 (88,49%) foram tumorais e 71 (11,51%) não tumorais. As 546 dermatopatias tumorais, estudadas com mais ênfase, foram diagnosticadas em 453 cães, 468 (85,7%) eram neoplásicas e 78 (14,3%) não neoplásicas. Das 468 dermatopatias tumorais neoplásicas encontradas 230 foram benignas (49,14%), 215 malignas (45,94%), 23 borderline/epiteliomas (4,91%), 51,92% (243/468) de origem mesenquimal, 42,74% (200/468) epiteliais, 4,91% (23/468) melanocíticas e 0,43% (2/468) metastáticas para a pele (primárias de glândula mamária). As dermatopatias neoplásicas mais diagnosticadas foram o mastocitoma (14,7%) e o lipoma (7,48%). Dentre as 78 dermatopatias tumorais não neoplásicas (14,3%), os cistos de inclusão epidermal (39,74%) e o tricogranuloma (15,38%) foram os mais frequentes. As dermatopatias caninas representaram 26% da casuística no LPV/UFBA. A ocorrência de dermatopatias tumorais simultâneas distintas foi comum nos cães desse estudo (um a cada seis); como podem ter origens celulares e comportamentos biológicos diferentes, enfatiza-se a importância da coleta e envio para exame histopatológico de fragmentos das diferentes lesões cutâneas.(AU)

Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species.

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog's body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.

Avaliação da infectividade parasitária a Lutzomyia longipalpis por xenodiagnóstico em cães tratados para leishmaniose visceral naturalmente adquirida / Evaluation of parasite infectivity for Lutzomyia longipalpis by xenodiagnosis in dogs treated for natural visceral leishmaniasis

O efeito de um protocolo quimioterápico multidrogas contra a leishmaniose visceral (LV) canina, sobre a capacidade de transmissão de Leishmania infantum ao vetor, foi analisado por meio de xenodiagnóstico. Trinta e cinco cães naturalmente infectados foram avaliados antes e durante o tratamento com a combinação de metronidazol, cetoconazol e alopurinol a cada três meses por até um ano. Em cada avaliação, os cães foram individualmente submetidos ao xenodiagnóstico e quantificação da carga parasitária por PCR quantitativa. O tratamento foi eficaz em bloquear a transmissibilidade parasitária do cão para o flebotomíneo (p= 0,011) nos cães avaliados. Houve significante correlação entre recuperação clínica e infectividade: cães com melhora clínica mais evidente apresentaram menores chances de transferir L. infantum ao Lutzomyia longipalpis via xenodiagnóstico (r=0,528, p= 0,002). Esses resultados demonstram que o tratamento canino com o protocolo proposto pode representar uma alternativa ao sacrifício de cães no Brasil como medida de controle da doença, uma vez que as drogas utilizadas não são aplicadas ao tratamento da LV humana em áreas endêmicas.(AU)

The outcome of a multidrug chemotherapeutic protocol against canine visceral leishmaniasis (VL) has been evaluated for its effect on dogs' capacity of transferring Leishmania infantum to sand flies by xenodiagnosis. Thirty-five naturally infected dogs were examined before and during treatment with a combination of metronidazole, ketoconazole, and allopurinol, at every three months up to one year. For each evaluation, treated dogs were individually submitted to xenodiagnosis and quantitative PCR to quantify parasite load in sand flies. The treatment was effective in blocking parasite transmission from host to sand flies (p=0.011) in the assessed dogs. There was a significant correlation between clinical improvement and sand fly infectivity: dogs that achieved better clinical conditions showed a lower chance of L. infantum transference to vector by xenodiagnosis (r=0.528, p=0.002). These results demonstrate that the treatment of dogs with the proposed protocol may represent an alternative to dog culling in Brazil for disease control, since these drugs are not used for treating human VL in endemic areas.(AU)

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors.

The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector.

Association between Leishmania infantum DNA in the hair of dogs and their infectiousness to Lutzomyia longipalpis.

Diagnosis of infection with Leishmania infantum by DNA detection in the hair has been recently demonstrated in dogs and wild animals. Our objective was to investigate if polymerase chain reaction (PCR) in hair might be used to identify infectious dogs. Thus, we assessed the infectiousness to Lutzomyia longipalpis by xenodiagnosis in comparison with the detection of L. infantum DNA by PCR in the hair, and with serology for anti-Leishmania IgG by ELISA in 15 positive dogs for L. infantum infection. Eight healthy dogs were included as negative controls. Among the 15 infected dogs, 13 were found positive in the ELISA (87%), 12 were PCR positive in the hair (80%), and 10 were positive in xenodiagnosis (67%). Positivity in the hair was associated with positivity in spleen (p=0.0003), seropositivity for antibodies (p=0.0006) and parasite transmission to L. longipalpis (p=0.0028). Considering the benefits to animal welfare and feasibility of hair sampling method, studies in larger and more diverse populations of naturally infected dogs from endemic areas should be conducted to evaluate the sensitivity, specificity, and predictive values of PCR using hair as a possible biomarker of infectiousness in dogs.

Xenodiagnosis on dogs with visceral leishmaniasis: Canine and sand fly aspects related to the parasite transmission.

One of the main limitations for the effective control of canine leishmaniasis in endemic areas is the difficulty in identifying infectious dogs. The objective of this study was to determine factors, related to dogs and to parasite detection in sand flies, which are associated with the positive xenodiagnosis of Leishmania infantum using the vector Lutzomyia longipalpis. The xenodiagnosis was performed in 50 owned dogs residing in endemic areas, which were divided into three different groups: G1-26 dogs proved to be infected and classified by severity of VL clinical signs on physical examination; G2-15 dogs proved to be infected and classified by severity of clinical signs and intensity of laboratory abnormalities; G3-nine dogs that were seropositive for anti-Leishmania IgG in ELISA tests. Parasite search in the sand flies after having fed on dogs in the xenodiagnosis was performed by both methodologies, PCR and dissection followed by microscopy. In G1, 58% (15/26) of dogs were able to transmit Leishmania to the vector, when parasite detection in sand flies were performed by PCR technique, 5 days after blood meal, whereas in G2, 53% (8/15) transmitted the parasite to the vector, however, confirmation was performed by direct observation of parasite through optical miscroscopy held 10 days after blood meal. Rate of infectiousness of dogs to sand flies was positively associated to severity of disease (p=0.042 and p=0.040), regardless the method used for clinical classification or for parasite detection in sand flies after xenodiagnosis. In G1 30% (3/10) of dogs with subclinical infection were infectious to the vector, while 80% (12/16) of dogs with clinical disease were also infectious. Even more, 17% (1/6) of dogs that had moderate disease were infectious to the sand flies, while 78% (7/9) of dogs with severe disease were infectious in G2. Still in G2, the proportion of sand flies infected (grade of infectiousness) was significantly lower (p=0.0098) when they fed on dogs with moderate disease (1%) in comparison with dogs with severe disease (38%). The dogs from G3 presented a rate of infectiousness of 11% (1/9), demonstrating that the indirect ELISA is not a good indicator of infectiousness and, therefore, should not be used as a confirmatory test for the euthanasia of dogs, as it is currently done in Brazil.

Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis.

Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4-100) in splenic aspirate; 79.2% (CI 68-90.3) in lymph nodes; 77.3% (CI 64.5-90.1) in skin; 75% (CI 63.1-86.9) in blood; 50% (CI 30-70) in bone marrow; 37.5% (CI 24.2-50.8) in left-eye; and 29.2% (CI 16.7-41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7-79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5-96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.

Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp.

In this work, we investigated the association between the disruption of splenic lymphoid tissue and the severity of visceral leishmaniasis in dogs. Clinical and laboratory data from 206 dogs were reviewed. Spleen sections collected during the euthanasia of these animals were analyzed, and the splenic lymphoid tissue samples were classified as well organized (spleen type 1), slightly disorganized (spleen type 2), or moderately to extensively disorganized (spleen type 3). Of 199 dogs with evidence of Leishmania infection, 54 (27%) had spleen type 1, 99 (50%) had spleen type 2, and 46 (23%) had spleen type 3. The number of clinical signs associated with visceral leishmaniasis was significantly higher in the animals with evidence of Leishmania infection and spleen type 2 or 3 than in the animals with spleen type 1. Alopecia, anemia, dehydration, dermatitis, lymphadenopathy, and onychogryphosis were all more frequent among animals with evidence of Leishmania infection and spleen type 3 than among the dogs with evidence of Leishmania infection and spleen type 1. The association between the severity of canine visceral leishmaniasis and the disorganization of the splenic lymphoid tissue was even more evident in the group of animals with positive spleen culture. Conjunctivitis and ulceration were also more common in the animals with spleen type 3 than in the animals with spleen type 1. The serum levels (median, interquartile range) of albumin (1.8, 1.4-2.3 g/dL) and creatinine (0.7, 0.4-0.8 mg/dL) were significantly lower and the serum levels of aspartate aminotransferase were significantly higher (57, 39-95 U) in animals with spleen type 3 than in animals with spleen type 1 (2.8, 2.4-3.4 g/dL; 0.9, 0.7-1.2 mg/dL and 23, 20-32 U, respectively). Our data confirm the hypothesis that disruption of the splenic lymphoid tissue is associated with a more severe clinical presentation of canine visceral leishmaniasis.

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG, and subclasses, parasitism, and parasite transmission by xenodiagnosis.

BACKGROUND: The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use. PURPOSE: To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL). METHODS: From 2010 to 2012, healthy dogs were vaccinated with Leishmune(®) (50 animals) or Leish-Tec(®) (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis. RESULTS: Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune(®) and Leish-Tec(®) groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune(®) and on the 21st day after the second dose of Leish-Tec(®). The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune(®) and Leish-Tec(®) groups, respectively. The Leishmune(®) group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec(®) group (p<0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p<0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune(®) group, and 7.9% (3/38) of the Leish-Tec(®) group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively. CONCLUSIONS: No significant differences were observed in dogs vaccinated with Leishmune(®) or Leish-Tec(®), with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune(®)-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec(®) exhibited adverse reactions with greater frequency and severity.

Uso do plasma rico em plaquetas no tratamento da doença articular degenerativa em cão: relato de caso / Use of platelet-rich plasma in the treatment of degenerative joint disease in dog: case report / Uso de plasma rico en plaquetas en el tratamiento de la enfermedad degenerativa de las articulaciones en perro: relato de caso

No presente trabalho é descrito o uso do plasma rico em plaquetas (PRP) no tratamento de um caso de osteoartrose naturalmente adquirida em um cão. O PRP foi obtido a partir de sangue venoso autólogo coletado em citrato de sódio. A separação do precipitado celular do sobrenadante foi realizada de forma estéril. Após adição de cloreto de cálcio e liberação dos fatores de crescimento, o PRP foi infiltrado no joelho do animal em estudo. Após o tratamento, os parâmetros de apoio, crepitação e dor articular apresentaram melhora, não havendo intolerância do animal à infiltração. Preliminarmente, pode-se dizer que é esta é uma técnica minimamente invasiva e de alta aplicabilidade. O emprego do PRP para infiltração mostrou ser simples, rápido, barato e seguro para o tratamento da osteoartrose no animal em questão. Estudos com maior número de casos em cães são necessários, a fim de validar este procedimento como prática terapêutica na rotina clínica...

The use of platelet-rich plasma (PRP) in the treatment of a natural case of osteoarthrosis in a dog is reported in the present work. The PRP was obtained through autologous venous blood collected in sodium citrate. The cellular precipitate was separated from the supernatant in a sterile condition. After the addition of calcium chloride and the release of growth factors, the PRP was infiltrated in the knee of the dog under study. After the treatment, weight-bearing, crackle and joint pain parameters presented improvement without intolerance to the infiltration by the animal. Preliminarily, it can be concluded that this is a minimally invasive method with high applicability, as well as being simple, quick, cheap and safe for the treatment of degenerative joint disease to the described animal. The study of a larger number of cases in dogs is necessary in order to validate the procedure for clinical routine...

En este estudio se ha descrito el uso del plasma rico en plaquetas (PRP) en el tratamiento de un caso de osteoartritis, adquirido naturalmente por un perro. El PRP ha sido obtenido a partir de la sangre venosa autóloga recogido en citrato de sodio. La separación del precipitado celular del sobrenadante se ha realizado de forma estéril. Después de la adición de cloruro de calcio y liberación de los factores de crecimiento, el PRP ha sido infiltrado en la rodilla del animal en estudio. Tras el tratamiento, los parámetros de apoyo, crepitación y dolor articular presentaron mejora, no ocurriendo intolerancia del animal a la infiltración. Preliminarmente, se puede decir que esta es una técnica poco invasiva y de alta aplicabilidad. El empleo del PRP para infiltración ha mostrado ser sencillo, rápido, barato y seguro para el tratamiento de osteoartritis en el animal estudiado. Estudios con mayor número de casos en perros son necesarios, para que se pueda validar este procedimiento como práctica terapéutica en la rutina clínica...

Eletrocardiografia computadorizada em cães: estudo comparativo / Computerized electrocardiography in dogs: a comparative study

Computerized electrocardiography (C-EKG) has been more frequently used in Veterinary Medicine. Many equipment models are available for this purpose. Due to possible device sensitivity and reproducibility differences during examination, the main goal of this study was to compare electrocardiographic parameters of dogs using two different C-EKG systems: Wincardio Micromed® (WIN) and TEB ECGPC® (TEB). Forty two healthy male and female dogs of different breeds (Cocker Spaniel, Dachshund, Labrador, Pinscher, Pitbull Terrier, Poodle, Schnauzer, Shih Tzu, Yorkshire and mongrel dogs), with age between 4 months and 16 years old were grouped according to weight and evaluated by both systems. The electrocardiographic measurements were performed on DII lead for both systems. The study showed that the TEB system was more sensitive for measurement of P wave and QRS complex duration, while the WIN system showed more sensitivity for the measurements of amplitude of the same parameters. The larger animals (26-37kg) showed greater variance in the measurements of P wave and QRS complex amplitude and duration than the groups of medium (14-25kg) or smaller (3-13kg) dogs. These differences must be considered when using diverse computerized electrocardiography systems to perform measurements due to the possibility of erratic interpretation of the results between veterinary medicine services.

O método de eletrocardiografia computadorizada (ECG-C) vem sendo crescentemente difundido na medicina veterinária, havendo atualmente diversas marcas e modelos de eletrocardiógrafos disponíveis no mercado. Diante da possibilidade de diferenças na sensibilidade e na reprodutibilidade das medidas obtidas nos traçados, o presente estudo teve como objetivo comparar os parâmetros eletrocardiográficos de cães, obtidos por dois sistemas. Foram avaliados dois diferentes softwares computadorizados, o Wincardio Micromed® (WIN) e o modelo TEB ECGPC® (TEB). Quarenta e dois cães hígidos, de diferentes raças (Cocker Spaniel, Daschund, Labrador, Pinscher, Pit Bull Terrier Poodle, Schnauzer, Shit Tzu, Yorkshire e sem raça definida), machos e fêmeas e com idade entre 4 meses e 16 anos foram agrupados segundo o peso e examinados pelos dois sistemas. As medidas eletrocardiográficas dos diferentes traçados foram analisadas na derivação DII. Os resultados indicaram que o sistema TEB apresentou maior sensibilidade na obtenção das medidas de duração da onda P e do complexo QRS, enquanto o sistema WIN foi mais sensível para determinar as medidas de amplitude dos mesmos parâmetros. Os animais de maior porte (26-37kg) apresentaram maior variância nas medidas de duração e amplitude de onda P e duração do complexo QRS em comparação aos cães de médio (14-25kg) e pequeno (1-13kg) porte. O achado de diferenças entre os sistemas testados deve ser levado em consideração ao se empregar os diversos equipamentos para diagnóstico por meio de ECG-C na rotina clínica, de modo a evitarem-se divergências na interpretação dos exames entre diferentes prestadores de serviços veterinários.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres / Indirect ELISA for the serological diagnosis of visceral leishmaniasis in wild canids

Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.

An assessment of the genetic diversity of Leishmania infantum isolates from infected dogs in Brazil.

Correlations between the genetic diversity of Leishmania infantum (syn. L. chagasi) isolates and their respective geographic origins support the theoretic assumption that visceral leishmaniasis probably originated in the Old World. Because dogs are widely considered to be the main reservoir of this disease, the present study aimed to investigate the degree of genetic divergence among 44 leishmanial canine isolates from two Brazilian cities, Jequié and Campo Grande, located approximately 2,028 km from each other. We hypothesized that a low degree of genetic divergence would be observed among these isolates. In fact, statistical analyses found no significant differences between the isolates using both random amplified polymorphic DNA and multilocus microsatellite typing genotyping techniques with three and seven markers, respectively. These findings provide support for the recent introduction of L. infantum into the New World.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens.

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.