Aurélien Grellet

Prevalence and risk factors of astrovirus infection in puppies from French breeding kennels

Aiming at determining the prevalence and the risk factors associated to astrovirus infection in puppy, fecal samples were collected in 316 puppies (age from 5 to 14 weeks of age) from 33 French breeding kennels. Data were registered for each puppy, including age, breed, gender, origin of the dog, and feces quality. The samples were tested by specific RT-PCR for the presence of canine astrovirus. Astroviruses were identified in 20.9% (66/316) of the puppies and in 42% (14/33) of the breeding kennels. Young puppies (i.e. <7 weeks of age) and puppies from large breeding kennels were more likely to be infected by the astrovirus. No association between the quality of feces and astrovirus infection could be determined in this survey.

Association between fecal S100A12 concentration and histologic, endoscopic, and clinical disease severity in dogs with idiopathic inflammatory bowel disease

Idiopathic inflammatory bowel disease (IBD) in dogs can be challenging to diagnose and fecal markers of disease that correlate with its severity could potentially be clinically useful. Surrogate inflammatory markers, such as the concentration of fecal S100A12, are used to detect active IBD in humans. The aim of this study was to determine the relationship between fecal canine S100A12 concentrations and clinical, endoscopic, and histologic disease severity. Twenty-six dogs with IBD and 90 healthy control dogs were enrolled. Spot fecal samples were collected and fecal canine S100A12 concentrations measured by an in-house ELISA. The correlation of fecal canine S100A12 concentrations with clinical disease activity (using the canine chronic enteropathy clinical activity index scoring system) and with endoscopic and histologic disease severity (using semi-quantitative grading systems) was assessed in dogs with IBD. Concentrations of fecal canine S100A12 were significantly higher in dogs with IBD (median [interquartile range]: 223 [21-3477]ng/g) than in healthy controls (median [interquartile range]: 9 [5-31]ng/g; P<0.0001). Fecal canine S100A12 concentrations correlated with the CCECAI score (ρ=0.4778; P=0.0408) and the severity of endoscopic lesions in the duodenum (ρ=0.4703; P=0.0354) and colon (ρ=0.9747; P=0.0144), but not with the severity of histopathologic changes except for inflammatory lesions in the colon (ρ=0.8669; P=0.0230). A concentration of 273ng fecal canine S100A12/g feces or greater distinguished (a) dogs with moderate to severe endoscopic disease in any GI section from dogs with at most mild endoscopic disease, and (b) dogs with very severe clinical disease (i.e., a CCECAI score of ≥12) from dogs with a CCECAI score of <12, with a sensitivity of 71% and 90%, respectively, and a specificity of 89% and 75%, respectively. This study showed that fecal canine S100A12 concentrations are increased in dogs with IBD. Further, this study showed that fecal canine S100A12 is associated with the clinical disease activity, the severity of endoscopic lesions, and the severity of colonic inflammation in dogs with IBD. Fecal S100A12 concentrations are potentially useful as a biomarker of inflammation in dogs with IBD.

Fecal calprotectin concentrations in adult dogs with chronic diarrhea

OBJECTIVE To evaluate fecal calprotectin concentrations in healthy dogs and dogs with chronic diarrhea, to identify cutoff values for fecal calprotectin concentrations for use in differentiating dogs with chronic diarrhea and a canine chronic enteropathy clinical activity index (CCECAI) < 12 from dogs with chronic diarrhea and a CCECAI ≥ 12, and to evaluate the association between histologic evidence of intestinal mucosal changes and fecal calprotectin concentrations in dogs with chronic diarrhea. SAMPLE Fecal samples from 96 adult dogs (27 dogs with chronic diarrhea and 69 healthy control dogs). PROCEDURES Severity of clinical signs was evaluated on the basis of the CCECAI scoring system. Endoscopy was performed in all dogs with chronic diarrhea, and mucosal biopsy specimens were evaluated histologically. Fecal calprotectin concentration was quantified via radioimmunoassay. RESULTS Fecal calprotectin concentrations were significantly higher in dogs with chronic diarrhea than in healthy control dogs. Fecal calprotectin concentrations were also significantly higher in dogs with a CCECAI ≥ 12, compared with concentrations for dogs with a CCECAI between 4 and 11. Fecal calprotectin concentrations were significantly higher in dogs with chronic diarrhea associated with histologic lesions, compared with concentrations in control dogs, and were significantly correlated with the severity of histologic intestinal lesions. Among dogs with chronic diarrhea, the best cutoff fecal calprotectin concentration for predicting a CCECAI ≥ 12 was 48.9 μg/g (sensitivity, 53.3%; specificity, 91.7%). CONCLUSIONS AND CLINICAL RELEVANCE Fecal calprotectin may be a useful biomarker in dogs with chronic diarrhea, especially dogs with histologic lesions.

Prevalence, risk factors of infection and molecular characterization of trichomonads in puppies from French breeding kennels

Abstract The trichomonad species Tritrichomonas fetus and Pentatrichomonas hominis were recently identified in the feces of dogs with diarrhea. However the prevalence and pathogenicity of these parasites in the canine population still remained poorly resolved. Therefore the aim of the present study was (1) to determine the prevalence of trichomonads infecting puppies living in French breeding kennels, (2) to confirm the predominance of P. hominis in dogs, (3) to investigate the genetic diversity of P. hominis isolates identified in the French canine population and (4) to evaluate the risk factors for infection by P. hominis and the influence of the parasite on feces consistency. A total of 215 both diarrheic and non-diarrheic puppies from 25 French breeding kennels were included in this epidemiological survey. Fecal samples from each puppy were examined for 6 gastrointestinal pathogens: parvovirus type 2 (CPV2), coronavirus, Toxocara canis, Cystoisospora ohioensis-complex, Cystoisospora canis, and Giardia intestinalis. A part of each collected stool was also tested for the presence of motile trichomonads by microscopy after culturing. The prevalence of trichomonad infection was 15.8% (34/215) among puppies and 20% (5/25) among breeding kennels. DNA from 26 of the 34 positive samples was successfully amplified using a trichomonad-specific primer pair. Analysis of the sequences of PCR products indicated that P. hominis was the only trichomonad infecting the canine population. All the puppies infected with P. hominis belonged to large breed dogs. Moreover, puppies from large breeding kennels, excreting a high level of G. intestinalis and/or excreting a high level of C. canis oocysts showed a higher probability of being positive for P. hominis infection. Univariate analysis also revealed an increased risk for P. hominis infection in puppies with abnormal feces. However, in a multivariate analysis, CPV2 was the only gastrointestinal pathogen associated with abnormal feces. Since enteropathogens were commonly found in dogs infected by P. hominis, the pathogenic potential of this trichomonad species remained uncertain and has to be further evaluated by experimental infection studies.

Inadequate passive immune transfer in puppies: definition, risk factors and prevention in a large multi-breed kennel.

The prevalence of neonatal mortality is high in the canine species and far from well-studied. In most domestic neonates, an appropriate colostrum intake is a key element of the control of neonatal mortality. The aim of this study was to evaluate the impact of passive immune transfer on puppy mortality, assessed through serum immunoglobulin G (IgG) concentration at 2 days of age. Factors impacting passive immune transfer and the value of an oral immunoglobulin supplementation to prevent it were also analyzed. A total of 149 puppies from 34 litters (12 breeds) within one breeding kennel were included. Blood samples were collected at 2 days of age and colostrum was collected from their dams 1 day after whelping to assay IgG concentration. Puppies were weighed at birth and at 2 days of age for calculation of growth rate. Mortality was recorded until 3 weeks of age. Seventy randomly assigned puppies were orally supplemented with hyper-immunized adult plasma twice within the first 8h of life. IgG concentration at 2 days of age was significantly correlated with weight gain during the first 2 days of life. The multivariable model with litter as a random effect demonstrated that neonatal mortality was not influenced by breed size, sex, supplementation, litter size, nor colostrum IgG concentration, but by puppy IgG concentration at 2 days of age. According to the ROC curve, the minimal IgG concentration at and below which puppies were at higher risk of death was determined at 230 mg/dl. Puppy IgG concentration was significantly associated with growth rate, but not with breed size, sex, supplementation, litter size or colostrum IgG concentration in a multivariable model with litter as a random effect. This study demonstrates that neonatal mortality in puppies is related to the quality of passive immune transfer. The oral supplementation with hyper-immunized canine plasma neither decreased risk of mortality, nor improved serum IgG concentration at 2 days of age in puppies. Attention must thus be paid to early colostrum intake to control the neonatal mortality in puppies.

Risk factors of weaning diarrhea in puppies housed in breeding kennels

Abstract Diarrhea represents one of the most frequent disorders in dogs. In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death. Prevention of diarrhea in puppies requires a global approach encompassing enteropathogens, environment and management practices especially when housed in groups. The purpose of this study was to determine prevalence of enteropathogens in puppies in breeding kennels and to identify risk factors of diarrhea. Two hundred and sixty six puppies (between 5 and 14weeks of age) from 29 French breeding kennels were included. For each kennel, data about environment, management of the kennel and puppies’ characteristics (age, sex and breed) were collected. For each puppy, fecal consistency and fecal excretion of enteropathogens (viruses and parasites) was evaluated. At least one enteropathogen was identified in 77.1% of puppies and 24.8% of puppies presented abnormal feces. The main risk factor of weaning diarrhea was fecal excretion of canine parvovirus type 2 (odds ratio=5; confidence interval 95%: 1.7–14.7). A targeted sanitary and medical prophylaxis against canine parvovirus type 2 should be implemented to decrease risk of weaning diarrhea.

Validation of a fecal scoring scale in puppies during the weaning period.

Abstract In puppies weaning is a high risk period. Fecal changes are frequent and can be signs of infection by digestive pathogens (bacteria, viruses, parasites) and indicators of nutritional and environmental stress. The aim of this study was to define a pathological fecal score for weaning puppies, and to study the impact on that score of two intestinal viruses (canine parvovirus type 2 and canine coronavirus). For this, the quality of stools was evaluated on 154 puppies between 4 and 8weeks of age (100 from small breeds and 54 from large breeds). The scoring was performed immediately after a spontaneous defecation based on a 13-point scale (from 1; liquid to 13; dry and hard feces). Fecal samples were frozen for further viral analysis. Each puppy was weighed once a week during the study period. The fecal score regarded as pathological was the highest score associated with a significant reduction in average daily gain (ADG). Fecal samples were checked by semi-quantitative PCR or RT-PCR for canine parvovirus type 2 and canine coronavirus identification, respectively. The quality of feces was affected by both age and breed size. In small breeds, the ADG was significantly reduced under a fecal score of 6 and 7 for puppies at 4–5 and 6–8weeks of age, respectively. In large breeds, the ADG was significantly reduced under a fecal score of 5 whatever the age of the puppy. Whereas a high viral load of canine parvovirus type 2 significantly impacted feces quality, no effect was recorded for canine coronavirus. This study provides an objective threshold for evaluation of fecal quality in weaning puppies. It also emphasizes the importance to be given to age and breed size in that evaluation.

Immunoglobulin G concentration in canine colostrum: Evaluation and variability

Canine neonates are born hypogammaglobulinemic, and colostrum is their main source of immunoglobulins. The purpose of this study was to evaluate the immune quality of canine colostrum and its variability both among bitches and among mammary glands. The immune quality was estimated from immunoglobulin G (IgG) concentration (ELISA test). The correlation of IgG concentration with refractometry was evaluated. From a total of 44 bitches from 13 different breeds from a single breeding kennel, samples of colostrum and blood were collected one day after the parturition onset. Colostrum was collected separately from each pair of mammary glands (180 pairs). The mean colostrum IgG concentration in our population was 20.8 ± 8.1g/L (ranging from 8.0 to 41.7 g/L) with no influence of breed size, litter size, age of dam or serum IgG concentration. Colostrum IgG concentration varied widely among pairs of mammary glands within one bitch (variation coefficient: 42 ± 32.1%). Nevertheless, no single pair of mammary glands was found to produce regularly a secretion of higher quality. No difference in IgG concentration was recorded between anterior and posterior pairs either. The BRIX index and the refractive index were significantly, but moderately correlated with colostrum IgG concentration (r=0.53 and 0.42, respectively). This study demonstrates a great variability in immune quality of colostrum among bitches and among mammary glands within one bitch. Further studies on the suckling behavior of puppies and on determination of the minimal immune quality of colostrum are required to evaluate their impact of this high variability on neonatal mortality in dogs.

Effect of age, gestation and lactation on faecal IgA and calprotectin concentrations in dogs

Faecal calprotectin and IgA have been suggested as non-invasive markers of gut health. Faecal calprotectin is a marker of intestinal inflammation in adults, whereas IgA has been suggested as a marker of intestinal immunity. The purpose of the present study was to evaluate the effect of gestation, lactation and age on faecal concentrations of these biomarkers. Thirty puppies, nineteen pregnant or lactating bitches and eighty-nine healthy control adult dogs were included in the study. Faeces were collected from the fourth week of gestation until the eighth week of lactation in pregnant and lactating bitches, and between 4 and 9 weeks of age in puppies. Faeces from the eighty-nine healthy control adult dogs were also collected. Faecal calprotectin and IgA concentrations were measured. Faecal calprotectin concentrations in control dogs were significantly lower than faecal calprotectin concentrations in puppies between 4 and 6 weeks of age (P < 0·001) or between 7 and 9 weeks of age (P = 0·004). Puppies between 4 and 6 weeks of age had significantly higher faecal IgA concentrations compared with puppies between 7 and 9 weeks of age (P = 0·001). Bitches during their second month of lactation had significantly lower faecal IgA concentrations compared with their first month of lactation (P = 0·049). Faecal calprotectin and IgA have been suggested as non-invasive and easily measured biomarkers of gut health in adults. However, the present study underlines that faecal IgA and calprotectin concentrations vary markedly depending of physiologic factors such as gestation, lactation and age. These factors need to be considered when these faecal biomarkers are used for evaluation of intestinal immunity or inflammation.

Protection against canine parvovirus type 2 infection in puppies by colostrum-derived antibodies.

During the first weeks of life puppies remain protected against canine parvovirus type 2 (CPV2) infection thanks to maternally derived antibodies (MDA) absorbed with colostrum after birth. The objective of the present study was to present the variability in CPV2-specific passive immune transfer and its consequences in puppies naturally exposed to the parvovirus. Seventy-nine puppies from one breeding kennel were included in the study at birth and followed until 56 d of age. Once per week the MDA titre for CPV2 specific antibodies was determined in blood. Viral excretion was also evaluated on a rectal swab by CPV2 PCR assay and puppies were weighed to determine growth rate. At 2 d of age, thirty-four out of seventy-nine puppies (43 %) had MDA ≤1:160 (designed group A) and forty-five puppies (57 %) had greater MDA titres (designed group B). The level of absorbed maternal antibodies was shown to be associated with breed size and growth rate during the first 48 h of life. The MDA level declined with age in all cases; however, the proportion of puppies with the antibody level considered as protective against CPV2 infection was significantly higher in group B compared with A from day 2 until 42. Among all puppies surviving until 56 d of age, sixty-seven out of seventy (95·7 %) underwent CPV2 infection. However, puppies from group A excreted CPV2 significantly earlier than puppies from group B. The present study demonstrates the link between passive immune transfer, in terms of level of specific MDA absorbed, and length of the protection period against parvovirus infection in weaning puppies.