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Ferritin and paraoxonase-1 (PON-1) were measured in dogs experimentally infected by Leishmania infantum (during experimental infection and following treatment) and also in naturally-infected dogs which presented different degrees of proteinuria. Experimentally-infected dogs were monitored for 7 months post-infection, then treated for 3 months with allopurinol, and their response to therapy was followed for 11 additional months. Naturally-infected dogs were staged based on the urine protein/creatinine (UPC) ratio into three groups as follows: group 1 (non-proteinuric; UPC ratio:0.2), group 2 (borderline proteinuric; UPC ratio: 0.2-0.5) and group 3 (proteinuric; UPC ratio0.5). An increase in serum ferritin values and a decrease in PON-1 activity were observed 2 months after infection. Both analytes returned to preinfection values following treatment. Significantly higher concentrations of ferritin were observed in dogs classified as either borderline or proteinuric when compared with non-proteinuric dogs whereas serum PON-1 activity was decreased only in proteinuric dogs.

Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.

Immunoglobulin (Ig) G subclasses were measured in dogs naturally and experimentally infected with Ehrlichia canis using enzyme-linked immunosorbant assay (ELISA). In this study, a higher IgG2 subclass response was noticed to natural and experimental E. canis infection in dogs. Anti-E. canis-IgG2 optic density (OD) values were found to be significantly higher than anti-E. canis-IgG1 during the different phases of the disease, and no differences in the IgG subclass responses to E. canis infection were found between symptomatic and asymptomatic dogs. Doxycycline treatment, which eliminated the rickettsia in three of four persistently infected dogs, had no noticeable influence on the E. canis-IgG subclass OD values during the treatment period. In order to facilitate the study, an ELISA for the detection of anti-E. canis IgG was developed and was shown to be sensitive and specific for E. canis-IgG, and in a significant correlation with the indirect immunofluorescence antibody test.

The association of Helicobacter pylori with gastritis, peptic ulcers, and gastric neoplasia has led to fundamental changes in the understanding of gastric disease in humans. The relationship of Helicobacter spp. infection to gastric disease in dogs is unclear. The objective of this study was to determine if Helicobacter infection affects the gastric secretory axis of dogs. Eight Beagle dogs with naturally acquired Helicobacter spp. infection were studied before and after (4 and 29 days) the attempted eradication of Helicobacter spp. with a combination of amoxicillin, metronidazole, and famotidine (AMF). Six specific-pathogen-free, Helico-bacter-free Beagle dogs served as controls. The electron microscopic appearance of spiral organisms in infected dogs indicated coinfection with Helicobacter felis- and H bizzozeronii-liks organisms. Unstimulated gastric pH and fasting, postprandial, and bombesin-stimulated plasma gastrin were similar in both infected and uninfected dogs, although a trend (P= .09) toward higher meal-stimulated gastrin was observed in infected dogs at 60 minutes. Pentagastrin-stimulated maximal acid output (mmol HC1/ kg0 75/hour) and titratable acidity (mmol HCl/mL) were similar in both infected and uninfected dogs, but gastric pH during maximal acid output was lower (P < .01) in uninfected dogs. Mild gastric inflammation was present in both infected and uninfected dogs. Gastric spiral organisms were undetectable in 6/8 infected dogs 4 days after AMF but had recurred in 8/8 dogs 29 days after AMF. Analysis of gastric DNA with Helicobacter-specific primers indicated persistence of Helicobacter DNA at 4 and 29 days after antibiotic therapy. Acid secretion, plasma gastrin, and mucosal inflammation were not affected by the transient suppression of Helicobacter spp. by AMF. These findings suggest that gastric secretory function in dogs is not markedly perturbed by naturally acquired Helicobacter spp. infection and that treatment with amoxicillin, metronidazole, and famotidine causes suppression rather than eradication of gastric Helicobacter spp. in dogs.

Acute phase proteins (APPs) have been proposed as useful markers for the diagnosis and monitoring of treatment of dogs infected by Leishmania infantum. However, the kinetics and behavior of these proteins in canine leishmaniasis is still unknown. The aim of this study was to monitor the kinetics of APPs in dogs experimentally infected with L. infantum, before, during and after therapy against canine leishmaniasis. Levels of serum haptoglobin, serum amyloid A and C-reactive protein from 6 infected beagles, positive by both PCR and parasite culture, were monitored for 7 months post-infection. The dogs were then treated for 3 months with allopurinol (20 mg mg/kg/day PO), and their response to therapy was followed for 11 additional months. Levels of Immunoglobulins G and M were recorded during these 21 months and compared. Experimental infection with L. infantum amastigotes induced an increase in all APPs studied which was statistically significant 2 months after infection for all proteins. Clinical recovery was accompanied by a significant decrease of all APPs 1 month after the beginning of treatment. However, differences were found between the APPs in both magnitude and duration of serum level elevations. The increase in total IgG and IgM was delayed in comparison to APPs and contrarily to the APPs, these immunoglobulins did not significantly decrease with treatment. In conclusion, the results of this study suggest that APPs could be used as early markers for disease as well as for monitoring the response to treatment in canine leishmaniasis.

International audience; Dogs are the main reservoir host for zoonotic visceral leishmaniasis caused by Leishmania infantum. In this study we investigated the immune response in spleens of L. infantum-infected dogs by measuring the mRNA expression levels for a wide panel of cytokines, transcription factors and chemokines. mRNA levels and parasite load were followed during 7 months of experimental infection and 14 months post-treatment, and were compared to naturally-infected (NI) dogs. Similarly, serum anti-Leishmania IgG and IgG subclass levels were measured during experimental infection. An increase in IFN-$\gamma$, T-bet, IP-10 and RANTES was found in the experimentally and NI dogs, implicating a substantial type-1 immune response during canine visceral leishmaniasis. IL-4, a type-2 associated cytokine, increased as early as one month after experimental infection, while IL-5 was high at later stages. Interestingly, the expression levels of the Treg-associated cytokines, IL-10 and TGF-$\beta$, did not change during the infection. Total anti-Leishmania IgG and IgG subclasses increased during the experimental infection. However, no association with specific cytokine patterns was observed. Parasite load in the spleens increased as early as one month post-infection and remained high until treatment. The load was higher in the polysymptomatic NI dogs than in the experimentally-infected dogs. This study indicates that both type-1 and type-2 immune responses occur in the spleen during canine L. infantum infection, and suggests that the early elevation of IL-4 might have a role in the persistence of parasites in the presence of high IFN-$\gamma$ expression.

Detailed histopathological evaluation of the gastric mucosa of Helicobacter-infected cats is complicated by the difficulty of recognizing Helicobacter organisms on hematoxylin and eosin (HE)-stained sections and the ability of multiple Helicobacter species to infect cats. In this study, the presence and localization of different species of Helicobacter in the stomachs of cats was investigated using silver staining and immuno-histochemistry. Five groups containing 5 cats each were established (group 1: urease negative and Helicobacter free; groups 2, 3, 4, and 5: urease positive and infected with Helicobacter heilmannii, unclassified Helicobacter spp., Helicobacter felis, and Helicobacter pylori, respectively). Gastric samples were evaluated by HE and silver staining and by immunohistochemistry with 3 different anti- Helicobacter primary antibodies. Helicobacter were detected by Steiner stain in all infected cats at the mucosal surface, in the lumen of gastric glands, and in the cytoplasm of parietal cells. In silver-stained sections, H. pylori was easily differentiated from H. felis, H. heilmannii, and unclassified Helicobacter spp., which were larger and more tightly coiled. No organisms were seen in uninfected cats. Helicobacter antigen paralleled the distribution of organisms observed in Steiner-stained sections for 2 of the 3 primary antibodies tested. The antisera were not able to discriminate between the different Helicobacter species examined. A small amount of Helicobacter antigen was present in the lamina propria of 3 H. pylori-, 3 H. felis-, and 1 H. heilmannii-infected cat. Minimal mononuclear inflammation was present in uninfected cats and in those infected with unclassified Helicobacter spp. and H. heilmannii cats. In H. felis-infected cats, lymphoid follicular hyperplasia with mild pangastric mononuclear inflammation and eosinophilic infiltrates were present. The H. pylori-infected cats had severe lymphoid follicular hyperplasia and mild to moderate mononuclear inflammation accompanied by the presence of neutrophils and eosinophils. These findings indicate that Steiner staining and immunohistochemistry are useful for detecting Helicobacter infections, particularly when different Helicobacter species can be present. Monoclonal antibodies specific for the different Helicobacter species could be important diagnostic aids. There appear to be differences in the severity of gastritis in cats infected with different Helicobacter species.

The principal aims of this study were to evaluate the humoral immune response (IgG) of cats with gastric Helicobacter spp. infection, and to determine the prevalence of different types of Helicobacter spp. in the stomachs of cats. The Helicobacter infection status of 45 cats (12 healthy spay/neuter cats, 9 sick cats, 24 colony cats) was determined by evaluating endoscopic gastric biopsies for urease activity, presence of Helicobacter-like organisms (HLO) on histopathology, and genus and species-specific PCR. Serum samples were evaluated with a kinetic enzyme linked immunosorbent assay (ELISA) utilizing the high molecular cell-associated protein (HM-CAP) fraction of H. felis ATCC 49179.Seventeen of 45 cats were infected with Helicobacter spp.: "H. heilmannii" 9/17, H. felis 4/17, mixed "H. heilmannii" and H. felis 3/17, unclassified-Helicobacter spp. 7/17. H. pylori was not detected in any cat. Kinetic ELISA results were significantly higher for infected cats, than for uninfected cats. Cats infected with different Helicobacter spp. showed similar distribution of OD/min values. There were no effects of age or clinical signs on the results of kinetic ELISA. No correlation between colonization density and seroconversion was observed. There were statistically significant, but weak correlations between the degree of seroconversion and the degree of inflammation, and the number of lymphoid follicles. Infected cats had more severe inflammation in the pylorus and fundus than uninfected cats. Infected sick cats had a higher degree of pyloric, but not fundic inflammation, than healthy infected cats and uninfected sick cats. The results indicate that naturally acquired infection with gastric Helicobacter spp. is associated with seroconversion (IgG) in cats. The similar ELISA values in cats infected with a variety of Helicobacter spp. suggests substantial antigenic homology between different Helicobacter spp. The higher degree of inflammation in infected than uninfected cats, supports a role for Helicobacter as a cause of gastritis in cats.

The relationship of Helicobacter felis, an organism that is observed in the stomachs of dogs, to gastric disease in dogs is unclear. The objective of this study was to determine if Helicobacter felis infection alters gastric morphology and gastric secretory function in dogs. Five specific-pathogen-free (SPF), Helicobacfer-free Beagle dogs were examined before and for 26 weeks after inoculation with H. felis (ATCC 49179). Three SPF uninfected dogs served as controls. All five dogs became colonized by H. felis as determined by urease activity, histopathology, polymerase chain reaction, and transmission electron microscopic examination of serial gastric biopsies. The degree of colonization ranged from 10 organisms/400 X field. The fundus, body, and cardia were most heavily colonized. Evaluation of gastric biopsies showed mild gastric inflammation and lymphoid follicles in both infected and uninfected dogs. There was no correlation between the number of organisms observed and the degree of gastric inflammation or number of lymphoid follicles. The gastric secretory axis, assessed by fasting and meal-stimulated plasma gastrin, mucosal gastrin and somatostatin immunoreactivity, fasting gastric pH, and pentagastrin-stimulated gastric acid secretion, was similar in both infected and uninfected dogs. Fasting gastric pH was not a reliable indicator of gastric secretory function. These findings suggest that H. felis may not be a gastric pathogen in dogs. However, the density of colonization and limited duration of infection should be considered when interpreting these findings.

The present study sought to determine the spectrum of diseases associated with subnormal concentrations of serum cobalamin in cats undergoing investigation of suspected gastrointestinal problems. The solid-phase boil radioassay (RA) for cobalamin employed in the present study was immunologically specific, precise, and accurate, with a sensitivity of 15 pg/mL. The RA yielded results that strongly correlated with those obtained by bioassay (Spearmann rho = .805; P < .0001), although the absolute values were lower for the RA. Forty-nine of 80 serum samples submitted during the period of January 1996-January 1998 had cobalamin concentrations below the reference range for healthy cats (range 900-2,800 pg/mL; mean +/- SD, 1,775 +/- 535 pg/mL; n = 33). Cats with subnormal cobalamin concentrations (mean +/- SD; 384 +/- 272 pg/mL, range 3-883 pg/mL) were middle-aged or older and were presented for weight loss. diarrhea, vomiting, anorexia, and thickened intestines. Definitive diagnoses in 22 cats included inflammatory bowel disease (IBD), intestinal lymphoma, cholangiohepatitis or cholangits, and pancreatic inflammation. Serum concentrations of cobalamin were particularly low in cats with intestinal lymphoma, three-fifths of whom also had subnormal serum concentrations of folate (< 9 ng/mL). The simultaneous presence of disease in the intestines, pancreas, or hepatobiliary system in many cats made it difficult to determine the cause of subnormal cobalamin concentrations. The circulating half-life of parenteral cyanocobalamin was shorter in 2 cats with IBD (5 days) than in 4 healthy cats (12.75 days). The presence of subnormal serum concentrations of cobalamin in 49 of 80 cats evaluated suggests that the measurement of serum cobalamin may be a useful indirect indicator of enteric or pancreatic disease in cats. The rapid depletion of circulating cobalamin in cats suggests that cats may be highly susceptible to cobalamin deficiency. However, the relationship of subnormal serum cobalamin concentrations to cobalamin deficiency and the effect of cobalamin deficiency on cats remain to be determined.