Pigmented-anaerobic bacteria associated with canine periodontitis

The etiology of human periodontal disease has been the focus of considerable research, yet relatively little is known about the causative agents of companion animal periodontitis. In humans, Porphyromonas gingivalis, a black-pigmented anaerobic bacteria (BPAB), has been implicated as the primary periopathogen. It has been demonstrated that BPAB are also found in companion animal periodontal pockets. While some animal BPAB have been individually identified, a study to identify the most frequently isolated subgingival BPAB has not been completed using genetic tools. The objective of this work was to identify the types and relative frequencies of pigmented anaerobic bacteria found in the periodontal pockets of dogs. Porphyromonas salivosa, Porphyromonas denticanis (a novel species) and Porphyromonas gulae were found to be the most frequently isolated BPAB associated with canine periodontitis.     

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.     

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.     

Risk of infection with Leishmania spp. in the canine population in the Netherlands

The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania-endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey. The questionnaire was sent to 1478 at random chosen families in the Netherlands. Of the 59.0% responders 28.0% had one or more dogs and 4.8% of these dogs had visited Southern Europe during the summer period of that year. On a total population of 1,200,000 dogs in the Netherlands, this means that each year some 58,000 dogs are at risk of being exposed to a Leishmania infection in Southern Europe. During the period 1990-1992 blood was collected for serology in 1911 dogs presented to the Utrecht University Clinic because of clinical problems not related to leishmaniasis, of which 434 had been in Southern Europe in the foregoing years. None was serologically positive. From these data it can be deduced that the highest chance to obtain leishmaniasis during a vacation in Southern Europe is mathematically less than 1/434 or less than 0.23%. Serology was also performed during the period 1989-1993 in 597 dogs that had been in Southern Europe and were suspected of leishmaniasis. Titers were positive in 145 of these samples. Sixty-four of these dogs were born in the Mediterranean and had been imported into the Netherlands. Excluding these imported dogs, it was calculated that at least 0.027% of the 58,000 dogs yearly taken to Southern Europe during holidays become infected with Leishmania. In order to establish the risk of disease transmission for people in close contact with an infected dog, serum samples of owners and house mates of 37 dogs with leishmaniasis were tested. All 112 sera tested negative. It was concluded that the risk to get leishmaniasis was between 0.027% and 0.23% for the dog when taken to Southern Europe during vacation, and that the risk for owners in non-endemic areas to get leishmaniasis from an infected dog is minimal.     

Association of canine obesity with reduced serum levels of C-reactive protein.

The prevalence of obesity is increasing in dogs as well as in humans. C-reactive protein (CRP) is an important tool for the detection of inflammation and/or early tissue damage and is linked to obesity in humans. The objective of the present study was to determine if serum CRP levels are altered in obese dogs. Fifteen lean (control group) and 16 overweight (obese group) dogs were examined. Blood samples were collected under fasted conditions for serum determination of CRP, glucose, insulin, cholesterol, triglyceride, and fructosamine. Results indicated that obese dogs were insulin resistant because serum insulin and insulin/glucose ratios were higher than in lean dogs (P < or = 0.05). Serum CRP concentrations were lower in obese dogs than in controls (P < or = 0.001). C-reactive protein was negatively correlated with insulin/glucose ratio (R = -0.42) and cholesterol (R = -0.39; P < or = 0.05). Furthermore, levels of cholesterol, triglycerides, and fructosamine were increased in the obese group compared with the control group. Based on these results, it can be postulated that CRP production is inhibited by obesity and insulin resistance in dogs.     

Moprhological diversity of cultured canine gastric Helicobacter spp.

The cell morphology, the number of flagella, the occurrence of periplasmic fibrils and ultrastructural structures of five groups of cultured canine gastric Helicobacter spp. were compared. The study included four strains of Helicobacter felis, four strains of Helicobacter bizzozeronii, one strain of ‘Flexispira’, six strains of an unnamed spiral organism 2 and one strain of an unnamed spiral orgaism 3 which were isolated from gastric biopsies. Cultures were studied with negative staining, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Bacterial dimensions were measured from the negative staining samples and values were tested with ANOVA and Bonferroni tests. The organisms studied differed from each other morphologicall. H. felis was a slightly spiraled organism with periplasmic fibrils. ‘Flexispira’ was a thin and straight organism with periplasmic fibrils. H. bizzozeronii was a tightly spiraled organism. Spiral organism 2 was loosely spiraled and thicker than the other organisms. Spiral organism 3 was a short curved rod having a single bipolar flagellum. The other species had multiple flagella. As a conclusion the canine gastric Helicobacter spp. can be differentiated from each other morphologically with an electron microscope. The morphological differences were mainly found in the structures involved in motility. The importance of the differences may lie in their impact on the colonization in a gastric mucous environment.     

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.     

Clinical, radiological and bacteriological findings in canine periodontitis

The clinical and radiological features and bacterial flora were studied in 16 small dogs with periodontitis. Gingival retraction, bleeding and alveolar bone loss were the most typical findings, whereas deep periodontal pockets were infrequently found. Periodontitis was frequently localised to certain regions of the dentition, most often in premolars or incisors. However, the deepest periodontal pockets were found in canine teeth. The mean pocket depth was 2·0 ± 0·4 mm (mean ± SD). The mean percentage of the sites with a pocket depth of more than 3 mm was 10·5 per cent. The mean occurrence of gingival bleeding after probing was 22·7 ± 12·7 per cent and the mean percentage of furcation lesions in multirooted teeth per dog was 46·0 ± 23·5 per cent. Tooth mobility was seen in 26·7 ± 13·3 per cent of the teeth. In each case subgingival plaque samples were taken for microbiological examination from two teeth with periodontitis and one healthy tooth. There was a clear difference between the diseased and healthy pockets in the detection frequency of the following Gram-negative anaerobes: pigmented, non-pigmented slime producing and fusiform rods. The counts of Gram-negative pigmented, other non-pigmented and fusiform rods as well as Gram-positive cocci were clearly higher in the diseased pockets. Pigmented Gram-negative rods (mainly asaccharolytic, Porphyromonas-like species) were the most common finding in both diseased and healthy pockets.     

Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease‐free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm² (4.6‐607.6 cells/mm²) and 8.5 mm² (0‐163.1 cells/mm²), respectively. The median area of CD204+ macrophages was 4.7% (1.3%‐23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.     

Parenteral immunization of pigs against infection with Treponema hyodysenteriae.

Six intravenous injections of formalin-inactivated Treponema hyodysenteriae were given to 8 specific-pathogen-free pigs at 6-day intervals. The 8 vaccinated and 8 control pigs were challenged intragastrically with pure cultures of T hyodysenteriae 7 and 8 days after the last intravenous injection. Clinical signs of swine dysentery were observed in all 8 control pigs, but was observed in only 1 of the immunized pigs. Three control pigs died. These findings suggest that parenteral immunization with T hyodysenteriae provided a marked degree of protection against subsequent intragastric challenge exposure with the homologous isolate of T hyodysenteriae.     

Association between contamination of public squares and seropositivity for Toxocara spp. in children

A concomitant study was carried out, of the association of positive serology for Toxocara spp. in 90 children who played in public squares used for leisure, with the frequency with which each child used these areas, and the presence of eggs of Toxocara spp. in the sand or grass in these locations. The sand and grass of their peridomiciles and school playgrounds, as well as the feces of their dogs were also analyzed for Toxocara. Serum samples were tested for IgG antibodies to Toxocara canis excreted-secreted larval antigens by ELISA, and blood samples for eosinophilia. The water-sedimentation technique was used to evaluate the presence of parasite eggs in the sand and grass turfs, and in feces of the dogs that also frequented these locations. 16/90 (17.8%) of the children were seropositive for Toxocara spp. There was a positive association between seropositivity in children who played in the public squares six or seven times a week, with a parasite load above 1.1 eggs/g of sand, as well as with contamination of the peridomicile, even at less than 1.0 egg/g of sand. Eosinophilia, the habit of geophagy, age from one to four years, and the presence of parasitized pet dogs were also positively correlated with seropositivity in the children. Eggs were found in 15/15 (100%) of the public squares, 17/90 (18.9%) of the peridomiciles, 3/13 (23.1%) of the schools, and 12/41 (29.3%) of the dogs living in the peridomiciles investigated.     

Conditions associated with canine hypothyroidism.

Careful review of the literature regarding clinical signs caused by hypothyroidism in dogs has shown that some assumptions regarding the relation of hypothyroidism to other conditions are based on anecdotal evidence. Cutaneous manifestations are present in most hypothyroid dogs, but the specific abnormalities and breed variations remain to be clearly defined. Decreased metabolic rate manifested by obesity and lethargy is also common. Neurologic manifestations, although uncommon, clearly occur in hypothyroid dogs. Cardiac abnormalities seem to be common, but their clinical significance is questionable. The only consistent hematologic abnormality that occurs in hypothyroid dogs is anemia; evidence for acquired von Willebrand's disease or other bleeding disorders is negligible. Reproductive dysfunction secondary to hypothyroidism is unlikely to occur in male dogs, and there is no evidence to support abnormalities in female dogs. The relation of megaesophagus, laryngeal paralysis, ocular abnormalities, and gastrointestinal disorders with hypothyroidism remains to be established. Future research into canine hypothyroidism may serve to convert dogma into a more clear understanding of the manifestations and pathophysiologic findings of this common endocrinopathy.     

Morphological variants developing in L-form cultures of two strains of Actinomyces spp. of canine origin

The present study has shown that a catalase-positive Actinomyces-like organism, UCD 19-480, failed to grom from its clinical fluid origin as normal colonies on three types of L-form media, although all three media were later shown to support it from a blood agar plate source. The growth of 19-480 from a fresh sulfur granule into a droplet of modified Barile Yaguchi Eveland (BYE) L-form broth was studied in a sealed slide incubated at 25–28°C for 11 weeks. Within 7 days, very long, bizarre branched filaments were detected near the crushed granule mass. Microcolonies were present at 5 weeks with radial filaments extending 0.5 cm from a core. The core consisted of pleomorphic vesicles arranged in a ring. Crystals appeared within and around the granule, but not elsewhere. After repeated passage on blood agar plates, 19-480 and a second, non-granule strain of recent origin, 339-180, were inoculated into BYE L-form broths as well as broths of new design. Unique membranous structures exhibiting actinomycete morphology were detected at 3 weeks in the 19-480 trypticase soya cultures containing putrescine and at 9 weeks in BYE L-form broths. They were restricted to the deep portions of certain broth types at predictable times and were not present in incubated controls. They fluoresced intensely with acridine orange and were weakly Gram-positive. ‘Pseudocolony’-like formations were detected at 8 weeks in the 3% NaCl + 5% sucrose BYE broth culture of UCD 19-480. It is suggested that the absence of normal colonies of these agents of L-form plate cultures of clinical fluids is due to growth of spreading variants unique to the actinomycete group.     

Ultrastructural characterization of colonic lesions in pigs inoculated with Treponema hyodysenteriae.

Twelve pigs were inoculated orally with pure cultures of Treponema hyodysenteriae. Pigs were necropsied at different time intervals postinoculation; colonic specimens were collected and prepared for light and electron microscopy. The earliest colonic lesion detected by electron microscopy consisted of superficial vascular congestion and dilatation, edema of the lamina propria and intercellular separation of the epithelial cells at the crypt shoulders. This lesion progressed to epithelial cell necrosis and extrusion into the lumen and extravasation of red cells. Large numbers of spirochetes were present and free, between, over and under necrotic epithelial cells whether in place or partially extruded. Spirochetal penetration of colonic enterocytes and intracytoplasmic multiplication were confirmed in this study. The spirochetes were found to invade the epithelial cells only from their lateral borders. The relationship between T. hyodysenteriae and the colonic anaerobes was not determined.