Rapid coagglutination test for detection of rotaviruses in turkeys

Avian rotaviruses present in fecal samples were readily detected using a staphylococcal protein-A coagglutination test on a white porcelain plate. Staphylococci, which produced large amounts of protein-A, were coated with rabbit anti-avian rotavirus serum. The antibody-coated staphylococci were agglutinated specifically by rotavirus present in the fecal sample. The macroscopic agglutination reaction occurred within a few minutes. A total of 40 fecal samples were tested by the coagglutination test. The sensitivity and specificity of the coagglutination test were compared with those of electron microscopy, enzyme-linked immunosorbent assay, and tissue-culture virus-isolation methods. Of the 31 fecal samples positive for rotavirus on electron microscopy, 27 (87%) were positive on coagglutination test. Of the nine electron-microscopy-negative samples, seven (78%) were also negative on coagglutination test. It was concluded that the staphylococcal protein-A coagglutination test can be used as a simple, rapid screening test for avian rotavirus.     

Latex test for rapid rotavirus diagnosis in calves

A latex agglutination test (LA) was compared with direct electron microscopy (EM) for detection of rotavirus infection in calves. A total of 375 samples from 62 calves were collected. Samples were taken when the calves were 1, 3, 5, 7, 10 and 20 days old and some scours samples were collected as well. Altogether 45/375 (12%) specimens were positive in LA and 10/375 (2.7%) were positive in EM. Samples positive in EM were also positive in LA. Out of the 62 calves studied 26/62 (42%) were positive in LA and 8/62 (13%) in EM. We found the LA very easy to perform, to be more sensitive than the EM method and probably a rather specific method for detection of rotavirus infection.     

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: Comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen.

A sandwich ELISA procedure using the chosen monoclonal as “capture and detecting” antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.     

Comparison of the diagnostic methods for studying parvovirus and rotavirus infections of dogs

79 feces samples of dogs between 7 weeks till 13.5 years of age, showing clinical signs of a hemorrhagic gastroenteritis, were tested by a commercial ELISA (DiaSystems Canine Parvo, Tech-America) for Canine Parvovirus (CPV) and by two Latex-Agglutination tests for Rotavirus (30 probes were tested by Rota Screen, 49 samples by Slidex Rota-Kit 2, both tests from BioMerieux). All samples were also examined by electron microscopy. The results of the simultaneous investigations showed 28 times positive and 28 times negative for CPV (70.9%). In 93.7% the investigations for Rotavirus-infection showed identical results by the Latex-Agglutination and electron microscopy: 73 samples were negative, one case showed a positive reaction. In 4 feces samples Rotavirus could only be detected by the Latex-test. In one sample a double-infection (CPV/Rotavirus) could be observed by all methods, in two cases the double-infection was only found by using the Latex-Agglutination. No other viruses could be found by the electron microscope than those described above. The results and the performance of the methods are discussed and compared with the data of other authors.     

Routine isolation and cultivation of bovine rotaviruses in cell culture

Using the bovine embryonic kidney cell line, Aubek, bovine rotaviruses were routinely isolated from fecal samples of calves with diarrhea. Of 125 fecal samples positive for rotavirus by immune electron microscopy and the enzyme-linked immunosorbent assay, 61 isolates were recovered and cultivated continuously.     

Comparison of polyacrylamide gel electrophoresis, an enzyme-linked-immunosorbent assay, and an agglutination test for the direct identification of bovine rotavirus from feces and coelectrophoresis of viral RNAs

The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the "standard," the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGe, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic "super-short" migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.     

Enzyme-linked immunosorbent assay, using monoclonal antibody, to detect enterotoxic Escherichia coli K99 antigen in feces of dairy calves

A modified, double-antibody, enzyme-linked immunosorbent assay (ELISA) was developed to detect the K99 pilus antigen of enterotoxic Escherichia coli (ETEC) in feces of calves. Extremely high positive to negative ratios (greater than 200) were obtained by using monoclonal antisera as the primary antibody. Strong positive reactions were obtained with strains of E coli known to produce the K99 antigen; however, non-enteropathogenic E coli (strains not producing the K99 antigen), Salmonella, Proteus, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, and rotavirus produced negative results. Seventy-five fecal samples, 8 from healthy calves and 67 from calves with neonatal calf diarrhea were examined with the K99 ELISA for the presence of ETEC. Rotavirus test and fecal culture results were available on feces from calves with diarrhea and were used with the K99 ELISA results to determine the specific cause of the disease. Enterotoxic E coli was the predominant agent detected in the feces of 29 diarrheal calves less than 5 days of age. Mixed infections of rotavirus and ETEC were also common in these calves, but rotavirus infections alone were not detected. In 38 calves greater than or equal to 5 days, rotavirus was detected without ETEC. Of these calves, only 2 produced positive tests with the K99 ELISA. Salmonella sp and Proteus sp were detected from 5 of 67 calves with diarrhea.     

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

Evaluation of two rapid assays for detecting Cryptosporidium parvum in calf feces

OBJECTIVE: To evaluate 2 rapid, patient-side assays for detection of Cryptosporidium parvum in feces from neonatal calves with diarrhea. DESIGN: Diagnostic test evaluation Sample Population-Fecal samples from 96 neonatal (1 to 30 days old) calves with diarrhea. PROCEDURE: Results of the rapid assays were compared with results of microscopic examination of fecal smears that had been stained with diamant fuchsin stain. RESULTS: One of the rapid assays correctly identified 56 of 62 (90%) fecal samples positive for C. parvum oocysts and 33 of 34 (97%) fecal samples negative for oocysts. The other assay correctly identified 53 of 62 (85%) fecal samples positive for oocysts and 33 of 34 (97%) fecal samples negative for oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that these 2 rapid assays are accurate when used to detect C. parvum in fecal samples from neonatal calves with diarrhea.     

Prevalence of bovine group A rotavirus shedding among dairy calves in Ohio.

Fecal samples were collected from 450 neonatal calves, ranging from 1 to 30 days old, between May, 1988 and May, 1989 to estimate the prevalence of bovine group A rotavirus in a stratified random sample of Ohio dairy herds. Calves were from 47 dairy herds chosen to be representative of Ohio herds. Bovine group A rotavirus was detected in fecal samples by a cell culture immunofluorescence test (CCIF) and ELISA. Of 450 samples tested, 46 (10%) were positive by CCIF and 67 (15%) were positive by ELISA. The agreement beyond chance between the 2 assays was good (kappa = 0.65). The overall prevalence rate of rotavirus shedding was 16.4% (74/450). Forty-three percent (29/67) of the samples positive by ELISA were subgroup 1, none were subgroup 2, and the remaining 57% (38/67) could not be assigned to either subgroups 1 or 2. Thirty herds (62.5%) had at least 1 group A rotavirus-positive calf (mean number of samples per positive herd = 12.4), and 17 herds (37.5%) had no rotavirus-positive calves (mean number of samples per negative herd = 6.0). A live oral rotacoronavirus vaccine was used in neonatal calves of only 1 herd and 3 of 17 (17.6%) calves from this herd were positive for group A rotavirus. The percentage of the rotavirus-positive fecal samples from all calves (n = 450) when stratified by fecal consistency was as follows: 28.3% (13/46) had liquid feces; 25.6% (10/39) had semiliquid feces; 23.4% (22/94) had pasty feces; and 10.7% (29/271) had firm feces.(ABSTRACT TRUNCATED AT 250 WORDS)     

A latex agglutination test for the detection of Echinococcus multilocularis coproantigen in the definitive hosts

A latex agglutination test for detecting Echinococcus multilocularis coproantigen in definitive hosts was developed using latex beads sensitized with EmA9 monoclonal antibody raised against somatic antigens of adult E. multilocularis. A primary test (LA 1) was performed on 82 fecal samples of necropsied foxes, of which 46 were infected, and resulted in 61% sensitivity and 86% specificity. To increase the sensitivity, 4 ng/mL of excretory/secretory antigens of adult worms was added to the samples in a secondary test (LA 2), resulting in 91% sensitivity and 61% specificity. The positive predictive value of the LA 1 test and the negative predictive value of the LA 2 test were both 85%. The combination of the LA 1 and LA 2 tests is applicable and practical for use in situations that require quick diagnosis or screening based on the following interpretation: the samples that are positive in the LA 1 test are positive; the samples that are negative in the LA 2 test are negative; and the samples that are negative in the LA 1 test and positive in the LA 2 test are classified as suspicious.     

Effect of pooling bovine fecal samples on the sensitivity of detection of E. coli O157:H7

To assess the effect of pooling fecal samples on the sensitivity of detection of E. coli O157:H7, 12 calves, inoculated orally with 10(8)cfu per calf of nalidixic acid resistant E. coli O157:H7, were used to provide positive fecal samples. After inoculation, calves were sampled twice weekly. Negative fecal samples were from calves at a local dairy. Samples from inoculated calves were incubated without pooling or were mixed with known negative fecal samples in a 1:4 ratio or a 2:3 ratio (positive:negative) for detection of E. coli O157:H7. Samples were enriched 6h in Gram negative broth with vancomycin, cefixime, and cefsoludin, underwent immunomagnetic separation with Dynabeads, and were plated onto sorbitol MacConkey agar with cefixime, and tellurite (SMACct). Morphologically typical colonies were plated onto blood agar, incubated overnight at 37 degrees C and an indole test was performed on each colony. Indole positives colonies were plated on SMAC agar with 20 microg/ml nalidixic acid (SMACnal). Colonies that grew on SMACnal were confirmed by O157 agglutination. Sensitivity of detection in non-pooled samples was 77%. Samples pooled 1:4 and 2:3 with negative samples were 55 and 52% sensitive, respectively. Pooling decreased sensitivity of detection for E. coli O157:H7 in bovine fecal samples (P<0.01). A deterministic binomial probability model was developed to assess the probability of detecting pens of cattle shedding E. coli O157 using a pooling protocol or individual samples. Pooling decreased sensitivity of detection at low pen prevalence compared to individual samples but was similar at high prevalence.     

Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods.

The use of direct electron microscopy, enzyme-linked immunosorbent assay, and protein A-gold immunoelectron microscopy for the identification of bovine coronavirus and type A rotavirus were examined. Two hundred and forty-nine samples from diarrheic calves and winter dysenteric cattle from seven geographic areas in Quebec were examined for the presence of viruses by direct electron microscopy of negatively stained preparations. In addition, all the samples were analyzed by enzyme-linked immunosorbent assay, and a random selection of 47 samples were also analyzed by protein A-gold immunoelectron microscopy. Thirty-nine percent of samples examined by direct electron microscopy contained viral particles; bovine coronavirus and type A rotavirus were the most common viruses involved. Overall agreement between any two of the methods used compared favorably with results obtained by others using similar methods. The presence of coronavirus and rotavirus in fecal samples obtained from neonatal calves and the presence of coronavirus in samples from winter dysenteric adult cattle suggested their etiological roles in the respective diseases. Furthermore, results from protein A-gold immunoelectron microscopy of coronavirus-like particles implied that a different coronavirus or some other viruses might be involved in these diseases. Finally, the efficiency of direct electron microscopy, enzyme-linked immunosorbent assay and protein A-gold immunoelectron microscopy as diagnostic tools is discussed.     

Detection of rotavirus in horses with and without diarrhea by electron microscopy and Rotazyme test

A total of 142 equine fecal samples (93 field fecal and 49 experimental fecal specimens) were examined for rotavirus using direct electron microscopy (EM) and the Rotazyme test. Eighty-six stool specimens were diarrhea samples. The Rotazyme test sensitivity and accuracy as compared to EM was determined by the visual (color reaction) and spectrophotometric methods. The overall agreement was 94.8% and 92.3% between EM and Rotazyme visual and spectrophotometric methods, respectively when suspect reactions (1 + color reaction or net absorbance between 0.05 and 0.1) were not included. The Rotazyme test is a quick, simple, and accurate diagnostic test for detection of rotavirus in equine fecal samples. It could be used by the equine practitioner with a minimum of laboratory facilities and by diagnostic and research laboratories.     

Electron microscopic detection rate of enteral viruses in diarrhea of dogs, cats, calves, swine and foals in the year 1988--electron microscopic study results

During 1988 fecal and gut samples of 641 dogs, 198 cats, 576 calves, 108 piglets and 64 foals with diarrhoea were investigated for virus infections by electron microscopy. In samples of dogs and cats parvovirus was detected at a proportion of 21.9% and 16.7%, respectively; rotavirus alone or together with coronavirus was found only in 0.3-1.5% of the specimens. In samples of calves rotavirus, as well as coronavirus dominated with a detection rate amounting to 17.4% and 26.6% respectively (including 4.5% of mixed infections); parvovirus was present in a ratio of 0.5%. Specimens of piglets mainly contained coronavirus (25.0%), and in lower percentages rotavirus (2.8%), rota- and coronaviruses (0.9%) and parvovirus (0.9%). In feces of foals rotavirus was detected in 6.3% and particles resembling picornavirus in 4.7% of cases. Not identifiable virus particles resembling corona-or picornaviruses were rarely found (between 0.6-2.5) also in specimens of the other animal species.     

Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.     

Canine serum C-reactive protein detected by means of a near-patient test for human C-reactive protein

OBJECTIVES: The aim of the study was to evaluate the reliability of a rapid human C-reactive protein near-patient slide reversed passive latex agglutination test (Randox) for the semi-quantitative determination of canine serum C-reactive protein. METHODS: The concentration of C-reactive protein was determined in 244 canine serum samples by an established automated immunoturbidimetric method and in various predilutions by a commercially available reversed passive latex agglutination test for human C-reactive protein. The results were compared to assess if the reversed passive latex agglutination test reflected the results of the established method with special emphasis on the reversed passive latex agglutination test's ability to identify samples characterised as positive or negative by the established method. RESULTS: The reversed passive latex agglutination test reflected the C-reactive protein concentration in canine serum samples at all the tested predilutions (undiluted, 1:4, 1:8 and 1:16). When applying a predilution of 1:8, the positive and negative analytical predictive values for discriminating between positive and negative samples (according to the established quantitative method) were high (0.94 [0.82 to 0.99] and 0.97 [0.93 to 0.99], respectively). CLINICAL SIGNIFICANCE: In conclusion, this near-patient test was able to reflect the serum C-reactive protein concentration in canine samples in a reliable and clinically useful manner and could be applicable for general practice for evaluating C-reactive protein levels in canine serum.     

Viruses and virus-like particles detected during examination of feces from calves and piglets with diarrhea

A total of 763 fecal or intestinal samples from diarrheic calves and piglets were examined for viral content by immunofluorescence, electron microscopy or cell culture. Routine fluorescent antibody and cultural tests detected rotavirus (n=126), coronavirus (n=80) and bovine viral diarrhea virus (n=13). Electron microscopy detected rotaviruses (n=24) and coronaviruses (n=17) not identified by standard fluorescent antibody tests. Other viruses detected by electron microscopy included Breda virus-like particles (n=49), astroviruses (n=1), caliciviruses (n=1), rhabdoviruses (n=1), parvoviruses (n=2), enteroviruses (n=3), togavirus-like particles (n=2), and “chained” particles (n=5). Mixtures of several of the viruses were detected in a number of fecal samples.

The survey emphasized the value of electron microscopy as a broad-spectrum diagnostic tool.

     

Evaluation of quantitative latex agglutination for detection of Cryptosporidium parvum, E. coli K99, and rotavirus in calf feces.

A new methodology for detection of rotavirus, Escherichia coli K99, and Cryptosporidium parvum in bovine fecal samples was developed based on a quantitative latex agglutination technique (QLAT). Calibrated microspheres coated with specific antibodies to 1 of the enteric pathogens are quantitatively agglutinated by the antigens present in diluted fecal sample. The test is performed in a 96-well flat-bottom plate. The samples were tested with a commercial enzyme-linked immunosorbent assay kit prior to being analyzed by QLAT. The calculated sensitivity and specificity are adequate for field conditions, because the amount of the pathogenic agents is generally high. The overall time to perform the test was about 20 minutes.