Evaluation of methods to diagnose Clostridium perfringens-associated diarrhea in dogs

OBJECTIVE: To assess the prevalence of Clostridium perfringens enterotoxin in feces of dogs with and without diarrhea, and to compare the use of microbial cultures from fecal specimens and evaluation of stained fecal smears for endospores with the presence of enterotoxin as tools for diagnosing C perfringens-associated diarrhea. DESIGN: Prospective study. ANIMALS: 144 dogs representing hospitalized dogs with (n = 41) or without (50) diarrhea, and clinically normal dogs treated as outpatients (53). PROCEDURE: Fresh fecal specimens from all dogs were examined as Gram-stained fecal smears to determine numbers of Gram-positive spore-forming rods/100x objective field. Enterotoxin was assayed directly by use of a reverse passive latex agglutination assay. Fecal specimens were plated directly to prereduced egg yolk agar plates and incubated overnight at 37 C in an anaerobic chamber. At 24 hours, up to 3 lecithinase-positive colonies were subcultured to Brucella blood agar to evaluate for double zone hemolysis. Colonies with double zone hemolysis were tested for aerotolerance and Gram-stained. RESULTS: A significant difference was not detected among groups with respect to the presence of C perfringens as determined by culture, the presence of endospores, and the reaction patterns of fecal enterotoxin assays. An association was not found between number of endospores and the presence of fecal enterotoxin. CLINICAL IMPLICATIONS: The presence of C perfringens enterotoxin in feces of dogs, as detected by the latex agglutination assay used in this study, correlates poorly with the number of fecal endospores, regardless of the dog's clinical status.     

Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias.     

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea

Background

This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden.

Findings

Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole.

Conclusions

This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs.     

Evaluation of two enzyme immunoassays for detection of Clostridium difficile toxins A and B in swine

Diagnosis of Clostridium difficile-associated disease (CDAD) in neonatal pigs is accomplished, in part, by detection of toxins A (TcdA) and B (TcdB) in feces or colonic contents. Samples (n=115) were tested simultaneously with two toxin assays (Clostridium difficile Tox A/B II, TechLab, Blacksburg, VA; Gastro-tect Clostridium difficile Toxin A+B, Medical Chemical Corporation). Previous comparison of the Tox A/B II assay to the reference method (toxicity in CHO cell monolayers) revealed an overall correlation of 88%, with 91% sensitivity and 86% specificity, a positive predictive value of 86 and a negative predictive value of 84. In comparing the two EIAs, a group of nine samples were positive in both assays and a group of 92 were negative in both. However, 14 samples positive in the Tox A/B II were negative in the Gastro-tect assay. Thus, in comparison to the Tox A/B II assay, the Gastro-tect assay was 100% specific but only 39% sensitive. Its negative predictive value was 87, but its positive predictive value was 100. Thus, the Tox A/B II kit is apparently superior to the Gastro-tect Toxin A+B test for diagnosis of CDAD in neonatal pigs.     

Nosocomial diarrhea associated with enterotoxigenic Clostridium perfringens infection in dogs

A retrospective analysis of the medical records of 30 consecutive cases of diarrhea occurring in dogs that were hospitalized in a teaching hospital was performed. A prospective analysis of culture results for Clostridium perfringens of dogs with diarrhea were compared with those of a control nondiarrheal group. Hospital-acquired diarrhea in dogs was found to be associated with multiple serotypes of enterotoxigenic Clostridium perfringens. Other potential etiologic agents could not be isolated. Clinical signs were variable, and included mild depression, anorexia, and soft to watery diarrhea with or without frank blood, mucus, and tenesmus. Fever was not present. There were no hematologic or serum biochemical abnormalities, nor were there any consistent virologic or parasitologic findings. Salmonella spp or Campylobacter spp were not identified by fecal culture. No risk factors could be identified. A dog that was euthanatized on the day it developed diarrhea had intestinal histologic findings suggestive of clostridial enteritis. Dogs with diarrhea had significantly higher fecal clostridial counts than did dogs without diarrhea (mean log10 counts +/- SD = 6.34 +/- 1.79 vs 4.75 +/- 2.07). Enterotoxin was found in the feces of 41% of diarrheic dogs but in only 7% of dogs without diarrhea.     

Recurrent diarrhea associated with enterotoxigenic Clostridium perfringens in 2 dogs

Two dogs were diagnosed with enterotoxigenic Clostridium perfringens-associated diarrhea. Diarrhea was responsive to antimicrobial therapy, but recurred after treatment was ceased. Clostridium perfringens enterotoxin was present in feces during diarrheic episodes but not when feces were normal. Both dogs responded to a prolonged course of oral cephalexin and dietary modification.     

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.     

The possibility of the serological differentiation of Clostridium species using enzyme immunoassays

A method for serotyping and diagnosis of 30 selected Clostridia strains of 12 different species by an enzyme-immuno-assay using polystyrene balls as a solid phase is described and tested for its efficiency. The reference strains and the homologous test strains could be differentiated from the heterologous in principle. A reliable detection of the several serotypes of Cl. perfringens was not perfectly to achieve with the conjugates used here, cross-reaction with Cl. chauvoei could be removed by absorption. The conjugate of Cl. bifermentans reacted with Cl. sordellii and Cl. difficile too, and must be absorbed prior any diagnostic applications with vegetative cells of the corresponding strains. Concerning the antigenic relationships between the Clostridia strains used here, the results of Ellner and Green (7, 8) as well as of Poxton and Byrne (14) could be reconfirmed. Moreover the applied technique offers the possibility to come to a more detailed result concerning the degree of antigenic relationship. The developed method is a valuable tool for the differentiation of pathogenic Clostridia and within the group Cl. bifermentans/Cl. sordellii/Cl. difficile a rapid diagnosis at the species level could be achieved. Some limitations by cross-reactions are given, but results can be improved by absorption of the conjugates.     

Evaluation of an enzyme linked-immunosorbent assay for the diagnosis of Brucella ovis infection in rams

A simple enzyme linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Brucella ovis infections in rams. Serums from brucellosis accredited-free flocks and flocks known to be infected with B. ovis were tested and the results correlated with warm complement fixation (CF) test and bacteriological examination of semen. Both the ELISA and the CF test detected 0.5% false positive reactions in rams from clinically negative flocks. However the ELISA detected significantly more positive reactors in infected flocks and the CF test failed to detect some rams excreting B. ovis. The ELISA proved to be a valuable test in eradicating brucellosis from infected flocks.     

Evaluation of a routine diagnostic fecal panel for dogs with diarrhea

OBJECTIVES: To assess the diagnostic yield of a routine fecal panel and determine whether Clostridium perfringens or C difficile toxin production is associated with acute hemorrhagic diarrheal syndrome (AHDS) in dogs. DESIGN: Case-control study. ANIMALS: 260 dogs with diarrhea and 177 dogs with normal feces. PROCEDURE: Medical records were reviewed for results of culture for C difficile, Campylobacterspp, and Salmonella spp; C perfringens fecal enterotoxin (CPE) assay via ELISA or reverse passive latex agglutination (RPLA) assay; fecal endospore enumeration; C difficile toxin A assay; and parasite evaluation. RESULTS: Prevalence of CPE in dogs with diarrhea was 22/154 (14.3%) via ELISA and 47/104 (45.2%) via RPLA assay, versus 9/74 (12%) via ELISA and 26/103 (25%) via RPLA assay in control dogs. Prevalence of C difficile was 47/260 (18%) in dogs with diarrhea and 41/74 (55%) in control dogs. Prevalence of C difficile toxin A was 26/254 (10.2%) in dogs with diarrhea and 0/74 in control dogs. Diagnosis of AHDS was made in 27 dogs; 8 had positive results for CPE, 7 had positive results for toxin A, and 1 had positive results for both toxins. Campylobacter spp were isolated from 13 of 260 (5%) dogs with diarrhea and 21 of 74 (28.4%) control dogs. Salmonella spp were isolated from 3 (1.2%) dogs with diarrhea. CONCLUSIONS AND CLINICAL RELEVANCE: Diagnostic value of a fecal panel in dogs with diarrhea appears to below.     

Comparison of polymerase chain reaction-restriction fragment length polymorphism assay and enzyme assay for diagnosis of G(M1)-gangliosidosis in Shiba dogs.

In the present study, diagnostic methods for canine G(M1)-gangliosidosis were examined by comparing a DNA mutation assay with an enzyme assay. Sixty-two Shiba dogs of a pedigree with G(M1)-gangliosidosis were differentiated into 3 genotypes, i.e., normal, heterozygous, and homozygous affected dogs, using a DNA mutation assay, which consists of polymerase chain reaction amplification and the determination of restriction fragment length polymorphisms. The beta-galactosidase activity in leukocytes, umbilical cords, and plasma was measured using 4-methylumbelliferyl beta-D-galactoside and p-nitrophenyl beta-D-galactoside as artificial substrates and compared among the 3 genotypes. The results showed that it was possible to identify homozygous dogs with the enzyme assay using leukocytes and umbilical cords. When using leukocytes, heterozygous carriers could be differentiated from normal dogs in many cases. However, the use of the DNA mutation assay is essential for a complete determination of heterozygous carriers because of the overlap in the distribution of enzyme activity between these 2 groups. When umbilical cords were used, heterozygous carriers could not be differentiated from normal dogs because of no significant difference in enzyme activity between these 2 groups. The beta-galactosidase activity in plasma was not applicable to the diagnosis and genotyping of G(M1)-gangliosidosis in Shiba dogs.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs

Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies.     

Evaluation of a three-axial acceleromyography monitor in dogs compared with mechanomyography

ObjectiveTo compare the ratio of the train-of-four (TOF) and double burst stimulation (DBS) obtained with three-axial acceleromyography (AMG) and mechanomyography (MMG) in dogs during recovery from a rocuronium-induced neuromuscular block.Study designProspective, randomized, experimental study.AnimalsA total of six intact healthy adult male Beagle dogs, weighing 9.1 ± 1.9 kg and aged 3–5 years.MethodsDogs were anesthetized with intravenous (IV) dexmedetomidine and propofol, and isoflurane in oxygen. Neuromuscular function was measured with AMG and MMG in the contralateral thoracic limbs. Rocuronium (0.5 mg kg^–1) was administered IV, and the TOF and DBS ratios measured. During neuromuscular block offset, MMG values were recorded when AMG first reached ratios of 0.9 and 1.0. True recovery from neuromuscular block was determined as MMG ratio ≥ 0.9. The false-positive (AMG ≥ 0.9 or 1.0, and MMG ratio < 0.9) rate was determined. Paired values were compared, and bias and limits of agreement were calculated. Receiver operating characteristic (ROC) curves were created.ResultsWhen AMG first reached 0.9 and 1.0 during recovery, MMG values were lower (p < 0.040). When AMG reached 0.9, the false-positive rate was 29% with TOF and 27% with DBS. It decreased to 12% (TOF) and 11% (DBS) when a ratio of 1.0 was used. AMG values were higher than paired MMG values (p < 0.001). The AMG overestimated MMG by 24% and 22% for TOF and DBS, respectively. Areas under the ROC curves (95% confidence interval) were 0.91 (0.89, 0.94) and 0.86 (0.81, 0.94) for TOF and DBS, respectively.Conclusionsand clinical relevance The three-axial AMG monitor overestimated neuromuscular function and, in some cases, indicated adequate recovery despite the MMG ratio being < 0.9. A TOF or DBS ratio of at least 1.0 should be considered when monitoring recovery of neuromuscular block with this AMG device.     

Evaluation of immunohistochemistry for the diagnosis of sporotrichosis in dogs

The aim of this study was to apply immunohistochemistry (IHC) for the diagnosis of canine sporotrichosis and to compare this method with the Grocott’s silver stain (GSS) and periodic acid Schiff (PAS) techniques. Eighty-seven dogs with sporotrichosis (group 1) and 35 with American tegumentary leishmaniosis (ATL) (group 2) were studied. The fungus was detected in group 1 by GSS, PAS and IHC. IHC was also applied to group 2 to evaluate the occurrence of cross-reactions. PAS, GSS and IHC detected yeast cells in 19.5%, 43.7% and 65.5% of the group 1 cases, respectively. The detection of intracellular antigens of Sporothrix schenckii by IHC increased the sensitivity of the histological diagnosis to 80.5%. No positive reaction was observed in ATL lesions. The results suggest that IHC may be indicated for the diagnosis of sporotrichosis because of its higher diagnostic sensitivity.     

Evaluation of five portable blood glucose meters for use in dogs

OBJECTIVE: To evaluate clinical and analytical accuracy of 5 portable blood glucose meters (PBGM) used to measure blood glucose concentrations in dogs and to determine potential sources of error. DESIGN: Prospective study. ANIMALS: 221 dogs. PROCEDURE: Venous blood samples were obtained, and results of the 5 PBGM were compared with results of a hexokinase reference method. Agreement among methods was determined by use of error grid analysis and statistical methods. RESULTS: Accuracy of the PBGM varied with glucose concentration of the sample. The largest differences between results of the PBGM and results of the reference method were obtained with samples with high glucose concentrations; 4 PBGM tended to underestimate and 1 PBGM tended to overestimate the true glucose concentration. Absolute differences between results of the PBGM and results of the reference method were small for samples with low glucose concentrations and samples with concentrations in the reference range. None of the PBGM yielded measurements that would result in clinically unacceptable errors. Within-run and between-day precision was good for all PBGM, and results were not affected by use of EDTA or heparin to anticoagulate blood. Readings of the PBGM were significantly higher for blood samples with low Hct than for samples with normal Hct. For 3 PBGM, samples < 3 microliters resulted in inaccurate measurements. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that currently available PBGM are sufficiently accurate for use in clinical practice to determine blood glucose concentrations in dogs.     

Evaluation of two test procedures for diagnosis of giardiasis in dogs.

Two commercially available tests, an antigen-capture ELISA for use on fecal samples, and a peroral nylon string test for use in dogs, were compared with a zinc sulfate fecal concentration technique (ZSCT) for detection of giardiasis in dogs. Of 77 dogs and 164 fecal samples (from these dogs), 33 and 52, respectively were found to be Giardia-positive on the basis of results of the ZSCT. The ELISA gave false-negative results for 10 and 14% of ZSCT-positive dogs and fecal samples, respectively, and false-positive results (relative to the ZSCT test results) in 13 and 10% of ZSCT-positive dogs and fecal samples, respectively. Of the 18 string-tested dogs, 14 were positive by results of the ZSCT. Of the 4 dogs that were Giardia-negative by ZSCT, 2 were Giardia-positive by ELISA. Dogs were sedated and given water and metoclopramide to aid passage into the duodenum of the capsule containing a nylon string. Of the 21 string tests performed on the 18 dogs, only 5 strings reached the duodenum, and 0 of the 5 yielded positive results for Giardia sp. Because the string broke in 1 dog (leaving most in the gastrointestinal tract and, therefore, producing a risk of string foreign body) further string tests were not done.     

Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.