Diagnosis of Helicobacter spp. infection in canine stomach

A total of 75 biopsied samples of cardia, fundus, body, and pyloric antrum from necropsied dogs that were submitted to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University from April 2003 to June 2004 were investigated. The objectives of this study were to determine the prevalence of Helicobacter spp. in canine stomach by polymerase chain reaction (PCR) in comparison to histochemistry versus immunohistochemistry (IHC), and to correlate these diagnostic methods with the clinical significance in infected dogs. Histopathological results revealed 60.0% (45/75) of samples to be positive, and consisted of mild gastritis in 64.44% (29/45), moderate gastritis in 11.11% (5/45), and severe gastritis in 24.44% (11/45). The proportion showing no histopathological lesions was 40.0% (30/75). Helicobacter spp. were localized to the luminal crypt in 18.67% (14/75), gastric pit in 22.67% (17/75), gastric gland in 21.33% (16/75), and gastric epithelium in 8% (6/75). The percentages of positive samples of Helicobacter spp. diagnosed by hematoxylin and eosin stain (H&E), Warthin Starry stain (WSS), IHC with rabbit polyclonal anti-H. pylori antibody, and PCR were 17.3% (13/75), 46.7% (35/75), 30.7% (23/75), and 10.7% (8/75), respectively. No significant differences weree observed in histopathological changes in portions of the stomach (p>0.05). The diagnosis of Helicobacter spp. by PCR in comparison to that by WSS and IHC was not significantly different (p>0.05). There were no relationships between pathological studies using H&E, WSS, and IHC, and especially between PCR and clinical signs of Helicobacter spp. infections in canine stomachs (p>0.05). The present study revealed significantly different levels of correlation for Helicobacter spp. detection between H&E and WSS (p<0.001). Results indicate that the method of choice for diagnosis of Helicobacter spp. infection in canine stomach is dependent on the purpose of study and appropriate specimen collection.     

PCR technique detection of Leishmania spp. but not Mycobacterium spp. in canine cutaneous 'sterile' pyogranuloma/granuloma syndrome

Cutaneous 'sterile' pyogranuloma/granuloma syndrome (SPGS) is an uncommon canine skin disorder of unknown aetiopathogenesis. Histopathological findings and failure to demonstrate an aetiologic agent are suggestive of this syndrome. Nevertheless, it has been hypothesized that SPGS may be related to an immune response against persistent endogenous or exogenous antigens. The presence of Leishmania and Mycobacterium organisms was investigated by polymerase chain reaction (PCR) techniques in 46 canine skin samples histopathologically diagnosed as SPGS. Concomitantly, an immunohistochemical technique for Leishmania detection was applied on the same samples and the results were compared with those from PCR. The PCR technique yielded positive results for Leishmania spp. in 21 out of 46 skin samples. The results of immunohistochemical techniques were identical to those obtained by PCR. The PCR technique gave negative results for Mycobacterium spp. in all the samples examined. These results suggest the importance of looking for Leishmania spp. in skin biopsies with histopathological findings consistent with the diagnosis of SPGS.     

Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia

Data on Leishmania spp. infection in dogs in Bolivia is scarce. Dogs from an area where 90% of human cutaneous leishmaniasis (CL) cases are due to Leishmania (Viannia) braziliensis were screened for Leishmania infection using established enzyme-linked immunosorbent antibody test (ELISA) protocols. Although none of the 51 dogs surveyed had clinical lesions indicative of CL, 6 out of 51 (11.8%) sampled dogs tested positive by ELISA.     

Papular dermatitis due to Leishmania spp. infection in dogs with parasite-specific cellular immune responses

Papular dermatitis due to Leishmania spp. infection was diagnosed in three boxers and two Rottweilers with Leishmania-specific cellular immunity. Diagnosis was based on histological and immunohistochemical examination of papules in four dogs and on cytological examination in one dog. Serum protein electrophoresis was within reference ranges and low antibody levels to Leishmania infantum were detected. Delayed-type hypersensitivity (DTH) reaction to leishmanin was evaluated before treatment in three dogs with positive results. After meglumine antimoniate therapy for 3 to 4 weeks and allopurinol treatment for 6 to 10 months, all dogs were clinically normal, had positive DTH reactions to leishmanin and reduced antibody titres. In conclusion, we suggest that this previously unreported cutaneous presentation of canine leishmaniosis appears to be associated with specific immunocompetence and, consequently, with a favourable prognosis.     

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

The phenology and population dynamics of Culicoides spp. in different ecosystems in The Netherlands

The Netherlands has enjoyed a relatively free state of vector-borne diseases of economic importance for more than one century. Emerging infectious diseases may change this situation, threatening the health of humans, domestic livestock and wildlife. In order to be prepared for the potential outbreak of vector-borne diseases, a study was undertaken to investigate the distribution and seasonal dynamics of candidate vectors of infectious diseases with emphasis on bluetongue vectors (Culicoides spp.). The study focused primarily on the relationship between characteristic ecosystems suitable for bluetongue vectors and climate, as well as on the phenology and population dynamics of these vectors. Twelve locations were selected, distributed over four distinct habitats: a wetland area, three riverine systems, four peat land areas and four livestock farms. Culicoides populations were sampled continuously using CO(2)-baited counterflow traps from July 2005 until August 2006, with an interruption from November 2005 to March 2006. All vectors were identified to species level. Meteorological and environmental data were collected at each location. Culicoides species were found in all four different habitat types studied. Wetland areas and peat bogs were rich in Culicoides spp. The taxonomic groups Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) were strongly associated with farms. Eighty-eight percent of all Culicoides consisted of the taxon C. obsoletus/Culicoides scoticus. On the livestock farms, 3% of Culicoides existed of the alleged bluetongue vector Culicoides dewulfi Goetghebuer. Culicoides impunctatus Goetghebuer was strongly associated with wetland and peat bog. Many Culicoides species were found until late in the phenological season and their activity was strongly associated with climate throughout the year. High annual variations in population dynamics were observed within the same study areas, which were probably caused by annual variations in environmental conditions. The study demonstrates that candidate vectors of bluetongue virus are present in natural and livestock-farm habitats in the Netherlands, distributed widely across the country. Under favourable climatic conditions, following virus introduction, bluetongue can spread among livestock (cattle, sheep and goats), depending on the nature of the viral serotype. The question now arises whether the virus can survive the winter conditions in north-western Europe and whether measures can be taken that effectively halt further spread of the disease.     

Association of Treponema spp. with canine periodontitis

To evaluate the association of oral Treponema (T.) spp. with severity of canine periodontitis, subgingival plaque samples of dogs of various breeds undergoing surgery were investigated. A wide range of oral Treponema spp. was analysed by a molecular and culture-independent approach applying DNA-DNA dot blot hybridization analysis and fluorescence in situ hybridization using Treponema specific oligonucleotide probes specific for phylogenetic groups I-VII of oral treponemes as well as probes specific for T. socranskii and T. denticola. To assess the periodontal status of affected dogs clinical parameters were measured and the periodontal status was classified from grade 0 (physiological periodont) to 3 (severe periodontitis). The periodontal status correlated significantly with an increasing concentration of volatile sulfur compounds (VSC, r=0.854) determined with a Halimeter, indicating a positive correlation between the presence of VSC-producing bacteria and periodontitis. In this study Treponema spp. of phylogenetic groups III, V-VII were not detected in any sample, whereas T. denticola-like treponemes were found only in 2 of 51 animals. However, treponemes belonging to phylogenetic groups I, II and IV of oral treponemes or T. socranskii were found in up to 64.84% of the dogs. The detection rate of Treponema spp. was significantly associated with an increased periodontal status. Treponemes present in periodontal lesions were also visualized by fluorescence in situ hybridization of gingival biopsies showing Treponema spp. not only in the microbial biofilm but also within the gingival tissue. The data presented here indicate that oral Treponema spp. are associated with canine periodontitis. Similar to human periodontitis, treponemes of groups I, II and IV and T. socranskii were found more frequently the higher the degree of periodontitis was.     

Risk factors for Campylobacter spp. infection in Senegalese broiler-chicken flocks

Our objective was to identify the risk factors for Campylobacter infection in Senegalese broiler flocks. Seventy broiler farms were studied around Dakar from January 2000 to December 2001 around Dakar. A questionnaire was administered to the farmers, and samples of fresh droppings were taken to assess the flocks' Campylobacter status. About 63% of the flocks were infected by Campylobacter spp.; Campylobacter jejuni was the most-prevalent species (P < 0.05). An elevated risk of Campylobacter infection was associated with other animals (mainly laying hens, cattle and sheep) being bred in the farm, the farm staff not wearing their work clothing exclusively in the poultry houses, uncemented poultry-house floors and the use of cartons that transport chicks from the hatchery to the farm as feed plates (rather than specifically designed feed plates). Alternatively, thorough cleaning and disinfection of poultry-house surroundings and manure disposal outside the farm were associated with decreased flock risk.     

A cross-sectional study of Leishmania spp. infection in clinically healthy dogs with polymerase chain reaction and serology in Greece

The prevalence of gastrointestinal parasites in stray dogs, and dogs with owners was investigated by fecal examinations from 271 dogs employing sedimentation, simple flotation and centrifugation-flotation methods. The centrifugation-flotation method, when compared to simple flotation or sedimentation methods was generally more accurate in the diagnosis of all intestinal parasites, but statistical differences were detected only in relation to Giardia spp. and Cystoisospora spp. (synonym Isospora spp.). The following parasites, with their respective prevalence, were diagnosed in the fecal samples: Ancylostoma spp. (23.6%); Toxocara canis (5.5%); Trichuris vulpis (4.8%); Spirocerca lupi (1.9%); Dipylidium caninum (0.7%); Giardia spp. (12.2%); Hammondia heydorni (2.6%); Cystoisospora spp. (8.5%); and Sarcocystis spp. (2.2%). The prevalence of most parasites was similar for dogs of mixed-breed and for dogs of a defined-breed, except for Cystoisospora spp. and T. canis which showed a significantly higher prevalence in mixed-breed dogs. The prevalence of Ancylostoma spp. (17.1%) was significantly lower in stray dogs than in those with an owner (31.9%) and the prevalence of Giardia spp. and Cystoisospora spp. was higher in stray dogs (P < 0.05). No effect of season on the occurrence of the different parasite genera could be observed, except for Ancylostoma spp., for which an increase in the percentage of dogs shedding eggs was observed at the beginning of Summer with a peak occurrence during April and May (Autumn). The prevalence of Ancylostoma spp., T. canis, T. vulpis, Giardia spp. and Cystoisospora spp. was higher in adult males than in adult females, but significant differences between the two groups occurred only with Giardia spp. Young animals were found to more frequently shed Nematode eggs in feces than adult animals.     

The phenology and population dynamics of Culicoides spp. in different ecosystems in The Netherlands

The Netherlands has enjoyed a relatively free state of vector-borne diseases of economic importance for more than one century. Emerging infectious diseases may change this situation, threatening the health of humans, domestic livestock and wildlife. In order to be prepared for the potential outbreak of vector-borne diseases, a study was undertaken to investigate the distribution and seasonal dynamics of candidate vectors of infectious diseases with emphasis on bluetongue vectors (Culicoides spp.). The study focused primarily on the relationship between characteristic ecosystems suitable for bluetongue vectors and climate, as well as on the phenology and population dynamics of these vectors.Twelve locations were selected, distributed over four distinct habitats: a wetland area, three riverine systems, four peat land areas and four livestock farms. Culicoides populations were sampled continuously using CO₂-baited counterflow traps from July 2005 until August 2006, with an interruption from November 2005 to March 2006. All vectors were identified to species level. Meteorological and environmental data were collected at each location.Culicoides species were found in all four different habitat types studied. Wetland areas and peat bogs were rich in Culicoides spp. The taxonomic groups Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) were strongly associated with farms. Eighty-eight percent of all Culicoides consisted of the taxon C. obsoletus/Culicoides scoticus. On the livestock farms, 3% of Culicoides existed of the alleged bluetongue vector Culicoides dewulfi Goetghebuer. Culicoides impunctatus Goetghebuer was strongly associated with wetland and peat bog. Many Culicoides species were found until late in the phenological season and their activity was strongly associated with climate throughout the year. High annual variations in population dynamics were observed within the same study areas, which were probably caused by annual variations in environmental conditions.The study demonstrates that candidate vectors of bluetongue virus are present in natural and livestock-farm habitats in the Netherlands, distributed widely across the country. Under favourable climatic conditions, following virus introduction, bluetongue can spread among livestock (cattle, sheep and goats), depending on the nature of the viral serotype. The question now arises whether the virus can survive the winter conditions in north-western Europe and whether measures can be taken that effectively halt further spread of the disease.     

Pathological changes in kidneys of dogs with natural Leishmania infection.

A study was made of the nephropathy in canine leishmaniasis produced in ten adult dogs naturally infected with Leishmania infantum. Renal function analyses were performed (uraemia, creatinaemia, plasma proteins, biochemistry and urinary sediment), the humoral immune response (fluorescent antibodies and levels of serum IgG, IgM and IgA) was assessed and histopathological studies were carried out. Correlation of the results showed acute renal insufficiency which was reversible in two animals (endotheliomesangial glomerulonephritis) and irreversible in four cases corresponding to glomerulonephritis in its Type I and Type II proliferative forms; extensive increase in the glomerular basal membrane, proliferation of mesangial cells and growth of the mesangial matrix were observed, as was a widespread incidence of immune complex deposits. Two animals showed chronic renal insufficiency. Lack of renal changes (minimal-changes glomerulonephritis) in two dogs was accounted for in one animal by an almost complete absence of symptoms and in the other by chronic viscerocutaneous symptoms; neither showed more than a slight immunoglobulin response.     

Autochthonous canine babesiosis in The Netherlands

Outbreaks of autochthonous babesiosis, caused by Babesia canis, occurred in The Netherlands in the spring and autumn of 2004 affecting 23 dogs. Nineteen animals recovered after treatment, whereas four dogs died. Adult Dermacentor reticulatus ticks collected from these dogs indicate that canine babesiosis could become endemic in The Netherlands.     

Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis

Leishmania infantum infection is highly prevalent in endemic areas. Dogs with leishmaniosis may develop keratoconjunctivitis sicca (KCS). The goals of this study were (1) to quantify Leishmania amastigotes in the Meibomian glands (MG), main lacrimal gland (MLG) and nictitating membrane gland (NMG) from dogs with leishmaniosis; (2) to compare these results to immunohistochemistry (IHC), and (3) to explore the association between the Leishmania parasite load and the presence of ocular clinical signs. Twenty-five dogs diagnosed with leishmaniosis were included. MG, MLG and NMG from both eyes were collected. Histopathology, IHC and real-time PCR were performed. All specimens yielded positive real-time PCR results. For all three glands, samples from dogs with ocular clinical signs had mean ΔCt (cycle threshold) values significantly lower (higher parasite loads) than those from dogs without signs. Cut-off values of ΔCt<0, ΔCt<4 and ΔCt<4.9 for MG, MLG and NMG, resulted in a likelihood ratio of positives of 5.9, 6.38 and 6.38, respectively. Samples with ΔCt values below the reported cut-off were significantly more likely to display clinical signs related to KCS than those with results above the cut-off, for all three glands. Similarly, ΔCt values below the cut-off were significantly associated with positive IHC. In this study real-time PCR has been standardised for use in MG, MLG and NMG. A cut-off value established for each of these tissues may aid the clinician in the discrimination between ocular signs related to Leishmania from those associated with other causes of KCS.     

Immunological characteristics of experimental murine infection with Leishmania (Leishmania) amazonensis

The murine models of Leishmania infection are well-studied and suitable models for studying this disease, which, despite its incidence of nearly 2 million new cases worldwide per year and its prevalence of 12 million cases, has been a somewhat neglected disease. Data obtained using such models are important for a better understanding of the disease in humans due to similarities in physiology and the advantage provided by the uniform infection profile within each mouse strain. In this review, we focus on studies of experimental murine infection with Leishmania (Leishmania) amazonensis, a species that has been associated with infections exhibiting various clinical features in humans. Mainly, we point out and discuss reports on: the effects of variations of the inoculum (such as strain, site, and size) in the establishment and development of the infection; characteristics of the infection in distinct mouse strains; and, the effects and subversions of the infection on components of the host innate and adaptive immune responses. The results obtained in these studies show that L. (L.) amazonensis infection in mice presents some unique features and immunoregulatory mechanisms, making it an interesting model for obtaining further knowledge of potential drugs targets and immunotherapy in Leishmania infection.     

Evaluation of 65% permethrin spot-on and deltamethrin-impregnated collars for canine Leishmania infantum infection prevention

During the 2004 and 2005 sand fly seasons, we evaluated the efficacy of a 65% spot-on solution of permethrin (Exspot, Schering & Plough) and deltamethrin-impregnated collar (Scalibor, Intervet) in reducing Leishmania infantum infection, in a canine leishmaniasis (CanL) endemic region (Liguria) in Italy. Immunofluorescent assay (IFA) revealed that three of 120 dogs (2.5%) treated with a 65% spot-on solution of permethrin, as three of 119 dogs (2.5%) treated with deltamethrin-impregnated collar have shown seroconversion after sand fly season. On the contrary, seroconversion was 15% in 188 untreated control dogs. Treatment reduced the risk of infection by 84%. The difference in treated dogs and control ones is highly significant (chi2 = 12.4; P = 0.0004). Our results show that treatment with 65% spot-on solution of permethrin and the deltamethrin-impregnated collar are effective in reducing the risk of acquiring L. infantum infection.