A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis.

A total of 45 non-uremic dogs, with clinical signs indicating leishmaniosis, entered the study. Diagnosis was confirmed by indirect immunofluorescence assay (IFA) on serum and polymerase chain reaction (PCR) on bone marrow samples. The dogs were randomly allocated into Group A (n=37) that received allopurinol (10mg/kg B.W., per os, twice daily) for 4 consecutive months, and Group B (n=8) that were placebo-treated. Clinical signs were scored just before and at monthly intervals throughout the study period, in a blinded and independent fashion. Complete blood count, serum biochemistry profile, urinalysis, lymph node and bone marrow parasitology, IFA and enzyme-linked immunosorbent assay (ELISA) serology and bone marrow PCR were carried out at the beginning and at the end of the trial. A total of three Group A and one Group B dogs died of end stage kidney disease that developed during the trial. In Group A animals that endured the trial there was a significant improvement in the general body condition, conjunctivitis, peripheral lymphadenopathy, splenomegaly, masticatory muscle atrophy, ulcerative stomatitis, epistaxis, exfoliative dermatitis, cutaneous ulcerations, blepharitis and nasodigital hyperkeratosis. The same observation was made for anemia, lymphopenia, hyperproteinemia, hyperglobulinemia, hyperphosphatemia, increased alkaline phosphatase activity and the low albumin/globulin ratio. By contrast, no improvement of any kind was seen in Group B dogs. Lymph node and bone marrow parasite numbers were significantly decreased in Group A animals. In Group B, that occurred only in the lymph nodes. Apart from remission of clinical signs and restoration to normal of clinicopathological abnormalities, allopurinol did not eliminate Leishmania organisms, as the PCR result on bone marrow was still positive in all the dogs that finished the trial.     

Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from dogs with natural clinical leishmaniosis

The aim of this study was to detect Leishmania infantum DNA by real-time PCR in urine from different groups of dogs with clinical leishmaniosis. Urine from 10 clinically healthy dogs and 43 dogs with clinical leishmaniosis diagnosed by positive serology and/or bone marrow PCR were studied. The group of 43 dogs with clinical leishmaniosis was divided into three subgroups: 13 dogs with renal insufficiency and proteinuria (urine protein-creatinine ratio greater than one), 13 dogs with only proteinuria, and 17 dogs with neither renal insufficiency nor proteinuria. The detection of Leishmania DNA was performed by light cycler real-time PCR using hybridization probes in each urine sample. Leishmania positive PCR was found in 47% (20/43) of the urine from leishmaniotic dogs, while all urine from clinically healthy dogs were negative. The percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency and proteinuria, 23% (3/13) in dogs with proteinuria and 35% (6/17) in dogs with neither renal insufficiency nor proteinuria. Dogs with renal insufficiency and proteinuria presented a statistical significant greater percentage of positive Leishmania PCR in urine when compared with the other subgroups (P<0.02). This study demonstrates the presence of Leishmania DNA in urine of dogs with leishmaniosis. Those dogs with severe renal damage present a greater number of Leishmania parasites in urine.     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

Canine Visceral Leishmaniosis in Anastácio,Mato Grosso do Sul State,Brazil

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed as Leishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.     

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.     

A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea)

Anoplocephala perfoliata (Cestoda, Cyclophyllidea), the commonest intestinal tapeworm of horses, can cause colic, intussusceptions, ileal impactions and intestinal perforations. Common diagnostic techniques for A. perfoliata infection, i.e. coprology and serology, show inherent limitations in terms of sensitivity and specificity and new approaches are thus required. Hence, the present study compared the reliability of coprological, serological (i.e. ELISA) and molecular (i.e. nested PCR) methods in detecting A. perfoliata infection in naturally infected horses and in horses treated with a combination of ivermectin and praziquantel. Of 42 horses subjected to coprological examination, 16 and 26 resulted negative and positive, respectively for the presence of A. perfoliata eggs at the coprological examination. The 26 coprologically positive animals were also positive by nested PCR. Fifteen out of the 16 horses coprologically negative were negative at the molecular assay, while one yielded a PCR product detectable on an agarose gel. Eighteen out of 26 positive horses were treated with a combination of ivermectin 18.7 mg/g and praziquantel 140.3mg/g and resulted subsequently negative by coprology and nested PCR performed 2 weeks after treatment. All infected and untreated animals had a high ELISA test optical density indicating high infection intensity and associated risk of colic. However, high optical density values were also obtained in four horses post-treatment and in three horses that were negative on molecular and coprological analysis. The results of the present work indicate that the nested PCR assay represents a valid method for the specific molecular detection of A. perfoliata in faecal samples collected from naturally infected horses and may have advantages over coprological and serological approaches for diagnosing A. perfoliata infection.     

Canine leishmaniosis

Canine visceral leishmaniosis (CVL) is an infectious disease of zoonotic potential, caused by protozoan parasite of the genus Leishmania. Common clinical manifestations of canine visceral leishmaniosis include decrease of appetite, progressive weight loss, exercise intolerance, peripheral lymph node and spleen enlargement, chronic renal and liver disease, muscle, atrophy, polyarthritis and others. Because the Polish literature in the field contains no information on leishmaniosis in animals the recognised case of this disease is presented. Homeless mongrel, intact female dog, 3 years of age was brought to a veterinary clinic because of apathy, and generalised dermatologic lesions to perform routine examination. Because therapeutic effect of primarily recognised scabies was unsatisfactory, the skin samples from ear margins, trunk and lesion of the area of the left gluteal region for histopatologic examination were taken. Due to suspicion of leishmaniosis, fine-needle aspiration biopsy of lymph nodes, skin lesions, ocular discharge and imprint samples from skin lesion were performed, and tissue collected were examined under optical microscopy for identification of Leishmania amastigotes. To confirm cytologic diagnosis, blood samples for serological tests (enzyme-linked immunoabsorbent assay-ELISA; indirect immunofluorescence assay test-IFAT) were taken. Based on physical examination, histopatology, cytopathology and serology, canine visceral leishmaniosis was finally diagnosed.     

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of S?o Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical signs of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Ara?atuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick-stained for optical microscopy. Direct immunofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum

BACKGROUND: Lymphadenopathy in canine leishmaniosis has been reported as reactive lymphoid hyperplasia or granulomatous (histiocytic) lymphadenitis. However, we are unaware of information on the effect of latent Leishmania infection on lymph node cytology compared with clinically affected dogs. OBJECTIVES: The aim of the present study was to investigate cytologic patterns of lymphadenopathy in dogs with clinical and subclinical forms of leishmaniosis and to correlate cytologic findings with the density of Leishmania amastigotes in fine needle aspiration (FNA) smears. METHODS: FNA cytology of prescapular or popliteal lymph nodes was evaluated on 32 dogs with clinical evidence of leishmaniosis (group A), 24 subclinically infected dogs (group B), and 17 clinically healthy noninfected dogs (group C); groups were based on the results of serologic and PCR tests for Leishmania sp. Differential nucleated cell counts (based on 300 cells) and amastigote density were determined microscopically. Cytologic findings were categorized and compared among groups. RESULTS: Cytologic abnormalities were found in 19 of 32 (59.4%) dogs in group A, 1 of 24 (4.2%) dogs in group B, and 2 of 17 (11.8%) dogs in group C and were significantly more frequent in group A than group B (P <.001) or C (P = .001). In group A, 68.7% of the dogs had lymphoid hyperplasia, 12.5% had lymphoid hyperplasia and histiocytic lymphadenitis, 6.3% had histiocytic lymphadenitis, and 3.1% had lymphoid hyperplasia and neutrophilic lymphadenitis. Lymphoid hyperplasia was also noted in 1 dog in group B, and lymphoid hyperplasia and eosinophilic lymphadenitis were each found in 1 dog in group C. Lymph node smears from 31 (96.9%) dogs in group A and 6 (25%) dogs in group B were positive for Leishmania amastigotes; however, no correlation was found between the density of amastigotes and cytopathologic patterns of lymphadenopathy. CONCLUSION: Abnormal lymph node cytology is much more common in dogs with clinical leishmaniosis than in dogs with subclinical infection, and primarily involves lymphoid hyperplasia. Despite finding no association between the density of amastigotes and type of lymphadenopathy, lymph node cytology still is a valuable diagnostic tool for diagnosing canine leishmaniosis.     

Clinical and serological follow-up in dogs with visceral leishmaniosis treated with allopurinol and sodium stibogluconate

Seven dogs with parasitologically proven clinical visceral leishmaniosis (Leishmania infantum infection) were treated with a combination of allopurinol and sodium stibogluconate. The dogs received first orally 15 mg/kg of allopurinol every 12 h until the clinical signs improved, in the following 1 month period allopurinol at same dose and subcutaneously 30 mg/kg of sodium stibogluconate combination were given daily and at the end of the combined treatment, allopurinol was continued alone at the same dose till the end of 8 months. During the treatment period, dogs were supported by additional proteins, vitamins, and minerals. A long acting insecticide (collar or drop) was also used in order to prevent further parasite transmission. Follow-up was maintained by clinical, clinicopathological evaluation, and parasitological examination of lymph node, serology using the indirect immunofluorescent antibody test (IFAT). Before treatment commenced, the most important clinical signs were exfoliative dermatitis, ulcerations, peripheral lymhadenopathy, pale mucous membranes, weight loss, and ocular lesions. Clinicopathological findings included commonly anaemia, hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. Before the treatment, amastigotes were seen in six of the seven dogs by examination of lymph node aspiration, and IFAT-titers were positive in all dogs. At the end of 8 months treatment, remission of clinical signs, restoration to normal of clinicopathological abnormalities were noticed. Lymph node aspiration was performed on three out of the seven dogs at the end of the treatment because of the very small sizes of the lymph nodes, and no amastigotes were observed. Although the mean IFAT-titer of the dogs were significantly (P < 0.001) lower compared with pretreatment, IFAT-titers of dogs were still positive. No relapses occurred during treatment period and a 6-24-month duration after the end of therapy. Based on the above results, long-term use of allopurinol combined with sodium stibogluconate together with support treatment concluded to have enough therapeutic efficacies in the treatment of dogs with visceral leishmaniosis. Observations of the cases for possible relapses were still going on and insecticide application was carefully carrying on in order preventing a possible re-infection.     

Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens

A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122 bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.     

Utility of polymerase chain reaction for analysis of antigen receptor rearrangement in staging and predicting prognosis in dogs with lymphoma

In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions with unique sequences, resulting from recombination of the V, D, and J genes. The purpose of this study was to determine the utility of polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) for molecular staging and predicting prognosis in canine lymphoma. We hypothesized that the PARR assay would offer a sensitive method for detecting neoplastic cells in blood, and that the presence of such cells would be a negative prognostic finding compared with dogs with no detectable circulating tumor cells. Eighty-six patients with histologically or cytologically confirmed lymphoma were studied from initial diagnosis until death or euthanasia. All patients had PARR assays of a representative tumor-infiltrated lymph node and peripheral whole blood. Sixty-two patients had clonal rearrangements detected in the lymph node and were able to be staged by PARR. Seventeen patients (27%) had no detectable tumor in their blood and 45 (73%) were blood positive. Our findings showed that (1) PARR correlated with clinical stage in that the PARR assay was more likely to detect tumor cells in blood in stage 5 lymphomas, (2) PARR was more sensitive for detecting circulating tumor cells than visual assessment of blood or bone marrow because 80% of stage 3 lymphomas were blood-PARR-positive, and (3) PCR stage was not prognostic for disease-free interval (DFI) or survival.     

Detection of Ehrlichia canis by PCR in different tissues obtained during necropsy from dogs surveyed for naturally occurring canine monocytic ehrlichiosis

A molecular study for the detection of Ehrlichia canis was carried out on tissues obtained at necropsy from randomly selected dogs with the intention of investigating naturally-occurring canine ehrlichiosis. The tissues evaluated for the presence of E. canis included lymph nodes, spleen, liver, bone marrow, and blood. Eight of the 18 dogs included were found to be positive for E. canis by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. Two dogs were positive for Anaplasma platys of which one dog was co-infected with E. canis and A. platys. Blood (5/8) and lymph nodes (5/8) were the tissues found to yield the highest number of positive E. canis PCR results with 7/8 dogs positive in the blood or lymph node. E. canis and A. platys DNA could be amplified by PCR when tissue samples were obtained 72h after the time of death.     

Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples

Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis.In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use.The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis.The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre > or =200), and 19% by microscopic examination of lymph node aspirates.     

Evaluation of cell-mediated immune responses and bacterial clearance in 6-10 months old water buffalo (Bubalus bubalis) experimentally vaccinated with four dosages of commercial Brucella abortus strain RB51 vaccine

Thirty water buffalo, obtained from a brucellosis-free farm, were used to evaluate cell-mediated immune responses and bacterial clearance in response to vaccination with Brucella abortus strain RB51 (RB51) in a dose-response study. The animals were randomly divided into five treatment groups. Groups I--V received the recommended dose (RD) of RB51 vaccine once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Cell-mediated immune response to RB51 was assessed by the histological examination of haematoxylin and eosin (H&E) stained sections of lymph nodes draining the sites of inoculation and by comparison of stimulation indices (SI) derived from gamma interferon (IFN-gamma) assay. A mixture of cytoplasmic proteins from B. melitensis B115 (brucellergene) was used as a specific antigenic stimulus to peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) up to 22 post-initial-inoculation week (PIW). Supernatants harvested at 18-24h after the in vitro antigenic stimulus were assayed for their IFN-gamma content by using a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit. Clearance of RB51 was assessed by the sequential immunohistochemical examination of sections of draining lymph nodes post-inoculation. There was no observable expansion of the deep cortex of lymph nodes on H&E sections indicating poor T-cell stimulation. All group V (control) water buffalo PBMC ELISA values were negative (SI<2.2) at all PIW sampling intervals. Overall PBMC IFN-gamma assay detected vaccinates from treatment groups' I--IV 67% (4/6), 83% (5/6), 33% (2/6) and 67% (4/6), respectively. LNMC IFN-gamma assay was unimpressive and there was a negative correlation (--.08) between the results of PBMC and LNMC of IFN-gamma assay. Clearance of RB51 occurred between 4 and 6 PIW in treatment groups I and III and between 6 and 12 PIW in groups II and IV. RB51 was not detected in any of the control animals at sampling intervals post-inoculation.     

Determination of the number of mast cells in lymph node, bone marrow, and buffy coat cytologic specimens from dogs.

Cytologic samples of popliteal lymph node, proximal femoral bone marrow, and the buffy coat fraction of blood were obtained from 56 dogs. The number of mast cells on 1 slide of each sample was determined by microscopic examination. Eleven of 46 slides of lymph node aspirate contained mast cells (range, 1 to 16; mean, 6.4; median, 5 mast cells/slide). Fifty-one bone marrow aspirate slides were evaluated. Two of these contained a single mast cell. None of the 53 buffy coat smear slides examined contained any mast cells. These results indicated that in clinically normal dogs, a few to several mast cells may be encountered in smears of lymph node aspirate, mast cells are rare in smears of bone marrow aspirate, and mast cells are absent from smears of buffy coat.