A turbidimetric method for fibronectin assay in the dog

An immunoturbidimetric method, using spectrophotometry, for the assay of canine plasma fibronectin concentration was compared with the immunoelectrophoretic method. The spectrophotometric method (S) correlated positively (r = 0.7) and significantly (P less than 0.01) with the immunoelectrophoretic method (I). The regression equation was S = 0.37I + 53. Ninety-five percent confidence levels for the regression line were calculated to allow detection, by spectrophotometry, of plasma fibronectin concentrations outside the normal range.     

Application of the fluorometric DNA quantitative assay using 4'6-diamidine-2-phenylindole for the evaluation of lymphocyte blastogenic response

A fluorometric DNA microassay using the fluorescent reagent 4'6-diamidine-2-phenylindole dihydrochloride (DAPI) was developed for evaluating a lymphocyte DNA synthesis assay. Triton X-100 (0.05 per cent) was added to the cultured lymphocytes, then DAPI (0.8 micrograms ml-1) was added as a DNA-specific fluorescent intercalating agent. Results are expressed as fluorescence intensity (FI) and stimulation index. FIs measured by DAPI fluorometric assay were highly correlated with [3H]-thymidine incorporation (r = 0.868, P less than 0.01, n = 28). The method is simple, reliable and widely applicable for evaluating blastogenic responses.     

Use of laparoscopic-assisted jejunostomy for fecal diversion in the management of a rectocutaneous fistula in a dog

A 2-year-old female Siberian Husky was referred for evaluation of a rectocutaneous fistula of unknown etiology. On evaluation, a rectal tear and an associated perivulvar abscess and draining tract were identified. Several attempts were made to repair the rectocutaneous fistula and associated rectal tear. Primary repair and fascia lata graft repair failed. Successful management was achieved via a laparoscopic-assisted end-on jejunostomy for fecal diversion, and the wound healed readily by second intention. During the period of hospitalization, the dog lost a considerable amount of weight. Particular care should be taken regarding fluid therapy, administration of antimicrobials, and adequate nutrition in patients with rectocutaneous fistulas. Overall, the use of laparoscopic-assisted end-on jejunostomy for fecal diversion in the management of rectocutaneous fistulas in dogs appears to be feasible; end-on or loop jejunostomy may also be an option for the treatment of other diseases of the distal portion of the gastrointestinal tract.     

Diagnosis of systemic cryptococcosis by fecal cytology in a dog

A 3-year-old Boxer was presented with progressive diarrhea, vomiting, and lethargy of 5-months duration. The dog had watery black feces, a mature neutrophilia, and microcytic anemia. Cytologic evaluation of a direct fecal smear stained with Wright's-Giemsa revealed numerous encapsulated, narrow-based, budding organisms consistent with Cryptococcus sp. Pyogranulomatous inflammation and Cryptococcus organisms also were observed in ultrasound-guided fine-needle aspirates of the small intestine and mesenteric lymph nodes, and in histologic sections of colonic biopsies obtained by endoscopy. Multifocal chorioretinitis by fundic examination was consistent with systemic mycosis, and the reciprocal antigen titer (1600) on a cryptococcal antigen latex agglutination test for Cryptococcus neoformans was markedly increased. Using immunohistochemistry, the organism was identified further as C neoformans var. grubii (C neoformans var. neoformans serotype A). After 3 weeks of antifungal treatment, ultrasound examination revealed urinary bladder wall thickening, and Cryptococcus organisms were found in a urine sediment preparation. After 4 months of treatment, the dog was clinically normal and had no abnormal findings on CBC, serum biochemistry, urinalysis, or fecal cytology; however, the antigen titer remained unchanged, mesenteric lymphadenomegaly and jejunal wall thickening were still evident, and cytologic evaluation of fine-needles aspirates of the jejunal wall revealed budding Cryptococcus organisms. Intestinal involvement in dogs with cryptococcosis is rare, and diagnosis by fecal cytology has not been documented previously.     

Evaluation of ultrasound as a method of assessing renal size in the dog

The aim of this study was first to determine the reproducibility and accuracy of ultrasonographic measurement of linear renal parameters in the dog. It was determined that renal length, width and depth could all be measured with a high degree of reproducibility, assuming a careful scanning technique. These measurements were found to be an accurate reflection of the true dimensions of the kidney, although there was a tendency for renal length to be underestimated. A simple method of renal volumetry was then evaluated, assuming that the kidney approaches the shape of an ellipsoid. This technique proved to be quick and simple to perform. The volume estimated in this way proved to be a good indicator of the true renal volume, although the estimated volume tended to be lower than the true volume measured by water displacement.     

Food sensitivity in the dog: a quantitative study

Over a period of one year, 251 dogs were presented to a UK-based dermatology referral clinic. Eighty-five of these were either diagnosed as having symptoms compatible with atopy (58 dogs), or suffered from chronic otitis or recurrent pyoderma. All 85 were placed on a carefully restricted diet for eight to nine weeks in an attempt to establish whether the symptoms were due to food sensitivity. In total, 19 were shown to have food sensitivity, representing 7.6 per cent of all dogs presented to the clinic, and one-third (32.7 per cent) of those dogs with signs compatible with a diagnosis of atopy. In five dogs with proven food sensitivity, otitis was the principal clinical sign and, in two others, recurrent pyoderma. In the population studied, labradors appeared to be predisposed to the condition. Improvement was monitored by asking owners to assess their dog's symptoms on an ordinal scale of pruritus. In those cases in which food sensitivity was confirmed, significant reduction in pruritus occurred. Most of these could be maintained long term on a commercial restricted-component diet. Particular effort was made to ensure owner compliance with the diet trials, using an explanation and model based upon a Venn diagram showing assumed links between atopy and several 'flare factors'. It was found that this approach significantly enhanced client understanding and cooperation. It is concluded that a careful approach, monitored by active clinical audit, will help to establish the true incidence of food sensitivity.     

Evaluation of the anaerobic method for the analysis of fecal microflora of beagle dogs.

The plate-in-bottle method of Mitsuoka et al. (1969) for the counting of fecal bacteria in beagle dogs was superior to an anaerobic jar method. Comparisons of three nonselective media, such as medium 10 supplemented with 10% cecal extract of dogs (designated as M10C), M10 with 10% fecal extract of dogs (M10F), and M10, by the plate-in-bottle method indicated that the visible bacterial counts for M10C were higher than those for M10 and M10F. The high percentage (18.4%) of numbers of the extremely oxygen-sensitive anaerobes to the fecal total counts by using the plate-in-bottle method with M10C was also observed.     

Comparison of methods for assay of fecal proteolytic activity

Fecal proteolytic activity (FPA) in ten normal dogs was readily detected either calorimetrically using azocasein substrate or by radial enzyme diffusion into agar gels containing casein substrate. Daily FPA ranged from 17 to 207 azocasein units/g (ACUIG) or 4 to 18 mm of casein hydrolysis, while mean 3-day FPA ranged from 58 to 10 1 ACUIG or 7 to 15 mm. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0% to 71% of proteolytic activity. Proteolytic activity decreased steadily in fecal specimens stored at room temperature or above, but there was only slight loss in activity during storage for up to 5 days at 4 degrees C. Proteolytic activity was unaffected by repeated freezing and thawing and samples could be stored for long periods at -2 degrees C without noticeable loss of activity. It is concluded that assays of FPA using either azoprotein substrate or radial enzyme diffusion into agar gels containing casein substrate allow evaluation of exocrine pancreatic function in dogs, provided that several samples are tested. These methods are suitable for application in a variety of species.     

Validation of a chemiluminescent enzyme immunometric assay for plasma adrenocorticotropic hormone in the dog

Background: The concentration of canine adrenocorticotropic hormone (ACTH) is usually determined by radioimmunoassay. However, chemiluminescent assay techniques have many advantages for clinical endocrine testing. Objectives: The objectives of this study were to validate a commercially available chemiluminescent assay for determination of canine ACTH concentration and to determine whether protease inhibitors are appropriate for use in the chemiluminescent assay system. Methods: Biological specificity was evaluated by treatment of 3 dogs with ovine corticotropin‐releasing hormone (CRH) followed by serial measurements of ACTH and by comparison with a previously validated immunoradiometric assay. All samples were collected both in the presence and absence of aprotinin, a protease inhibitor. The assay was further evaluated by measurement of intra‐assay precision, interassay precision, and recovery after dilution. Results: Baseline ACTH concentrations ranged from 5.6 to 15.3 pg/mL, and maximum ACTH concentrations of 158 to 1240 pg/mL were observed 30–60 minutes after CRH administration. Plasma samples collected with aprotinin had significantly lower ACTH concentrations than did samples collected without aprotinin. The intra‐assay coefficients of variance (CVs) ranged from 4.1 to 8.2%, and interassay CVs ranged from 4.6 to 14.8%. Recovery after dilution with canine plasma ranged from 93.4 to 103.0% of predicted concentration; however, inadequate recovery was observed with other diluents. There was a high correlation with the immunoradiometric assay (r= .925) but a significant negative bias (‐32.9, 95% confidence interval ?50.8 to ?14.9). Conclusions: This chemiluminescent assay is a valid technique for measurement of ACTH in canine plasma. ACTH concentration measured by chemiluminescence is lower than that measured by immunoradiometry. Aprotinin decreases the measured concentration of ACTH, and this effect should be taken into account when interpreting results. Diluents supplied with the kit should not be used for dilution of canine samples.     

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.     

仔猪粪便DNA中产气荚膜梭菌主要毒素基因检测及荧光定量PCR计数方法的建立

为探究产气荚膜梭菌(Cp)及其产生的β2毒素对7日龄内仔猪腹泻的影响,本试验利用PCR技术对江西不同地区96份仔猪粪样DNA进行分析。首先检测产气荚膜梭菌α、β、β2、ε、ι、CPE毒素基因来推断Cp存在和基因型,然后对不同地区的β2毒素基因全长进行测序比对、MEGA7构建进化树,最后采用荧光定量PCR对携带CpA(β2+)的健康、腹泻样品中的Cp菌数进行测定。结果显示:α毒素基因在健康、腹泻仔猪的检测率分别为85.4%(41/48),81.3%(39/48),β2毒素基因则分别为79.2%(38/48),77.1%(37/48);β、ε、CPE及ι毒素基因未能检测到,综合各分型毒素检测结果,阳性粪样存在的Cp均为A型。β2基因全长除九江地区得到的β2基因在第706～733bp缺失28个碱基,其他地区β2全长序列不多于3个碱基的差异,与GenBank登录号AJ537530.1对应序列同源性最高。携带CpA(β2+)的健康、腹泻粪样同浓度DNA中Cp菌数为104～105 CFU,经SPSS17分析,差异不显著(P0.05)。结果表明,江西地区的猪源β2毒素基因保守性较高,但仔猪腹泻与β2毒素基因存在与否和Cp菌数没有明显的相关关系,是否与毒素表达有关有待进一步的研究,本试验结果为研究Cp致病机制提供了依据。     

Dyserythropoeisis, sideroblasts/siderocytes and hemoglobin crystallization in a dog

Dyserythropoiesis characterized by enhanced intramedullary destruction, pathologic sideroblasts and siderocytes, and hemoglobin crystallization was detected in a female Cocker Spaniel presented for poor exercise tolerance. Examination of peripheral blood revealed intraerythrocytic crystals, granulation of erythrocytes, nucleated erythroid cells, reticulocytosis and marked variation in erythrocyte morphology in the absence of anemia. Bone marrow examination revealed sideroblasts, a low M:E ratio and evidence of enhanced intramedullary destruction of erythroid cells. Electron microscopy of peripheral blood and bone marrow confirmed pathologic mitochondrial iron accumulation in erythroid cells and the presence of intraerythrocytic hemoglobin crystals. A cause for the hematologic changes was not identified. After the animal became clinically normal, siderocytes disappeared from peripheral blood but intraerythrocytic crystals and reticulocytosis persisted.     

Evaluation of anthelmintic activity in captive wild ruminants by fecal egg reduction tests and a larval development assay.

The effectiveness of anthelmintics was evaluated in four herds of captive ruminants, wapiti (Cervus elaphus), Armenian red sheep (Ovis orientalis), giraffe (Giraffa camelopardalis), and pronghorn (Antilocapra americana), by the use of fecal egg reduction tests (FERTs) and a commercial larval development assay (LDA) designed to evaluate susceptibility or resistance of nematodes to anthelmintics. Haemonchus sp. was the predominant nematode in the red sheep, giraffe, and pronghorn herds, whereas Ostertagia sp. and Trichostrongylus sp. were predominant in the wapiti. The LDA data indicated susceptibility by the worms to benzimidazoles except in the red sheep flock, which showed a high level of resistance. High levels of resistance to levamisole were seen in the worm populations from the wapiti and red sheep, moderate resistance in the pronghorn herd, and susceptibility in the giraffe herd. Worms were susceptible in all four herds to a combination of benzimidazole/levamisole. There was suspected avermectin resistance by Trichostrongylus sp. in the wapiti herd and by Haemonchus sp. in the giraffe. The FERTs agreed with the LDA in showing the Haemonchus in the giraffe was susceptible to fenbendazole and had suspected resistance to ivermectin, whereas Haemonchus in the red sheep and pronghorn were susceptible to ivermectin. There was correlation between the tests evaluating anthelmintics. The LDA is useful as a screening test in the selection of an anthelmintic for use in grazing ruminants, but the effectiveness of a drug in a host species may depend as much on the dose used, and the method of administration, as it does on the parasite's sensitivity to the anthelmintic.     

Evaluation of a radioimmunoassay for type III procollagen peptide in the dog

Type III procollagen peptide (P-3-P) is a serum marker for hepatic fibrosis in humans. The utility of a commercially available radioimmunoassay for P-3-P was evaluated in the dog. The specificity of the assay was assessed by polyacrylamide gel electrophoresis (PAGE) of canine serum and purified bovine P-3-P, followed by Western immunoblotting with rabbit aniti-P-3-P serum. The sensitivity was assessed by performing the radioimmunoassay on dilutions of sera from 22 dogs. Polyacrylamide gel electrophoresis of purified bovine P-3-P and sera from two dogs suspected of having elevated P-3-P concentrations revealed no homologous bands of staining. Western immunoblotting showed marked cross-reactivity of the high antisera concentrations with several components of the serum proteins, but none corresponding to the purified P-3-P. All tested sera from dogs had minimal competitive binding with radiolabeled P-3-P in the radioimmunoassay. Dilution curves of dog sera did not parallel either the standard curve or the dilution curve of a known test human serum. There were no statistically different P-3-P concentrations in any of the groups of dogs studied. It was concluded that currently available radioimmunoassay kits for the measurement of P-3-P in the human are not applicable in the dog. Seemingly, the structure or metabolism of canine P-3-P may vary significantly from that of the bovine or human, limiting the sensitivity and specificity of this assay in the dog.     

Use of the D-dimer assay for diagnosing thromboembolic disease in the dog

Although the exact incidence of pulmonary thromboembolism (PTE) in small animals is unknown, it is thought that PTE is a substantial, under-diagnosed complication. The difficulty in diagnosing PTE in small animals is confounded by its subtle symptomatic presentation and a lack of clinical suspicion, coexisting disease states, and lack of noninvasive tests that are sensitive and specific for the diagnosis of PTE. Although numerous laboratory markers of coagulation have been studied, only the D-dimer assay has shown clinical utility in detecting early embolism in humans. This paper examines the use of D-dimer assays and other clinical modalities in the diagnostic approach to thromboembolic disease in small animals.     

微波消解DAN荧光法测定饲料中的微量硒

本文采用微波消解2,3-二氨基萘(DAN)荧光法测定饲料中的微量硒,加标回收率平均值为105.5%,相对标准差为3.33%,此方法具有准确可靠,操作简便易行等优点。     

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.