Longitudinal study to investigate variation in results of repeated ELISA and culture of fecal samples for Mycobacterium avium subsp paratuberculosis in commercial dairy herds

OBJECTIVE: To determine sources and amounts of variation in a kinetics ELISA (KELA) and results of culture of fecal samples for Mycobacterium avium subsp paratuberculosis (MAP) in repeated tests of individual cows. ANIMALS: 112 cows on 6 commercial dairy farms in New York. PROCEDURE: A nonrandom longitudinal study was conducted from January 2001 to March 2002. A KELA was performed monthly, and MAP culture was performed bimonthly. Cow- and herd-level data were collected. The KELA and culture results were analyzed by use of models that corrected for clustering within herds and repeated measures on cows. RESULTS: Cows of second or higher lactation had increased KELA values, compared with values for first-lactation cows. Cows had lowest KELA values during the first 15 days in milk; KELA values increased until 60 days in milk and then stabilized. Moderate and heavy shedders had significantly higher KELA values than culture-negative cows, and KELA values of shedders progressively increased over time. On average, the KELA value was significantly increased 132 days after a cow was first detected to be a moderate shedder and 236 days after a cow was first detected to be a low shedder. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis suggests that KELA results vary on a cow-level on the basis of lactation number and stage of lactation. High KELA values indicate heavy fecal shedding, but the KELA is not useful in identifying low and moderate shedders that can require up to 236 days to have a significant increase in KELA value.     

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.     

RT-PCR检测犬粪便中的冠状病毒

根据犬冠状病毒(caninecoronavirus, CCV) 的S基因序列, 用计算机设计并合成了1对引物P1、P2, 以此引物及以从美国进口的CCV疫苗的反转录产物为模板, 初步建立了检测CCV的RT PCR。将纯化的PCR产物成功地克隆到pGEM T easy载体中, 鉴定、测序并进行同源性分析。结果表明, 与1 71和UCD 1株同源性分别为91 5%和94 3%。该RT PCR能对疫苗中CCV及CCV参考株USCV B1的S基因进行特异性地扩增,长度为575bp, 对犬的其他病毒及CRFK细胞未能扩增出任何片段, 该RT PCR能检测出0 5μLCCV培养液/2g粪便。用建立的RT PCR在上海对50条犬粪便进行检测, 结果表明CCV阳性6例。本研究建立了检测犬粪便中的CCV的RT PCR, 能用于犬冠状病毒流行病学调查。     

Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA

A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.6%. Serum and ear tissue samples from 11 persistently infected (PI) BVDV calves were tested. While serum samples were negative after intake of colostrum, the ear tissue samples could be detected positive for BVDV all the time. Testing multiple samples derived from the same ear from PI cattle yielded positive results and low variation. Using cattle ear tags combining the ear tag application with sampling of a small ear tissue plug and testing those tissue samples with an ACE could be a reliable and economic way of BVDV testing.     

Detection of Salmonella in faecal, tissue, and feed samples by conventional culture methods and VIDAS Salmonella Test

The VIDAS Salmonella Test (VST) is an enzyme-linked fluorescent immunoassay for the detection of Salmonella-antigens. The suitability of VST for the detection of Salmonella in faecal, tissue, and feed samples was evaluated by the comparison with routine culture methods. From 312 naturally contaminated samples 17 were classified as Salmonella positive by routine methods and 28 by VST. Salmonella were isolated from 15 VST positive samples by the routine method and from eight samples only by an extended culture method. Five positive VST results could not be proved by culture. Two samples were classified as positive by the routine method and as false-negative by VST. The sensitivity varied between 88% and 100% and the specifity between 92% and 100%, depending on the kind of sample. Matrix or serovar specific factors resulting in a false VST result could not be determined. The performance of VST was easy and did not require special experiences. Mostly, samples with Salmonella negative results were faster detected than by culture methods. VST is suitable for the detection of Salmonella in the studied kind of samples especially as a screening method.     

Detection of sarcocystosis in heifers and pigs using the indirect immunofluorescence method and trypsinization of muscle tissue

The incidence of sarcocystosis was studied in cattle and pigs of different age categories by means of the methods of muscular tissue trypsinization and indirect immunofluorescence reaction (IFR). On the whole, 173 head of cattle and 107 pigs were examined. In the category of calves, a positive serological reaction was proved in 75% of cases, and positivity in the trypsinization of muscular tissue was found only in 15% of cases. In the category of beef bulls 64% of cases were serologically positive and 96% were positive in the trypsinization test. In the category of cows 70.84% of cases were positive serologically and 85.42% by trypsinization. In fattened pigs serological positivity was demonstrated in 10.53% of cases but the direct determination of sarcocysts failed in all cases. On the other hand, trypsinization of sows was positive in 8% and IFR was negative in all cases. The discussion deals with the correlations of all results obtained by the above diagnostic methods.     

Detection of Helicobacter spp. in gastric, fecal and saliva samples from swine affected by gastric ulceration

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.     

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds.

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.     

Detection of serum adiponectin in the cat, bear, and horse using antibody from a commercial ELISA kit

Adiponectin is a hormone secreted from adipose tissue that closely associates with insulin resistance. In contrast to other adipokines, adiponectin levels decrease as fat mass increases. The ability to measure adiponectin in veterinary species will allow researchers to assess hormone function and may lead to improved screening tests and treatments for insulin resistance and type II diabetes. A commercial ELISA kit (B-bridge International) is available for adiponectin quantification in mice and rats. To test this assay's ability to bind adiponectin in several veterinary species known to develop insulin resistance with obesity, we conducted a Western blot analysis comparing mouse, cat, bear, and horse serum using the antibody provided with the assay. The proteins were subjected to denaturing SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed in 5% milk solution. The membrane was then incubated with the rabbit anti-mouse adiponectin antibody provided with the ELISA kit, followed by incubation with peroxidase-labeled goat anti-rabbit IgG. Detection of bound antibody was performed using enhanced chemoluminescence. Bands corresponding to the adiponectin monomer (30 kDa) were apparent for each sample tested. Therefore, the antibody provided in the ELISA kit appears to bind with adiponectin in all species tested. Our laboratory has previously validated the ELISA mouse/rat adiponectin kit for use in cats. High DNA homology across species and strong antibody binding at the 30 kDa site warrant validation of the kit's accuracy in other species at risk for insulin resistance (eg. horses and bears).     

Comparison of an indirect immunofluorescence assay, western blot analysis, and a commercially available ELISA for detection of Ehrlichia canis antibodies in canine sera

OBJECTIVE: To examine the correlation between results for an indirect immunofluorescence assay (IFA) that uses Ehrlichia canis antigen as a substrate (ie, E canis-IFA), 2 western blot (WB) analyses, and a commercially available ELISA in the detection of E canis antibody in dog sera. SAMPLE POPULATION: 54 canine serum samples that were reactive on E canis-IFA and 16 canine serum samples that were E canis-IFA nonreactive. PROCEDURE: Serum samples were evaluated by use of 2 WB analyses and a commercially available ELISA. Correlation between results of the 3 testing modalities (ie, IFA, WB analyses, and the ELISA) was examined by use of nonreactive (E canis-IFA reciprocal titer, < 20), low-titer (reciprocal titer, 80 to 160), medium-titer (reciprocal titer, 320 to 2,560), and high-titer (reciprocal titer, 5,120 to > 20,480) serum samples. RESULTS: For all serum samples in the nonreactive (n = 16), medium-titer (17), and high-titer (18) groups, correlation of results among IFA, WB analyses, and the commercially available ELISA was excellent. A poor correlation was found between IFA results and those of WB analyses and the ELISA for serum samples in the low-titer group (19), with only 4 of the 19 serum samples having positive results on both WB analyses and the commercially available ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: The discrepancy between E canis-IFA, WB analyses, and the commercially available ELISA results for the low-titer serum samples may be related to a high IFA sensitivity or, more likely, a lack of specificity associated with cross-reactivity among Ehrlichia spp.     

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniosis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.     

Comparative studies of the detection of rotavirus in fecal samples of calves with diarrhea with the latex test "Slidex Rota-Kit 2" and electron microscopy

Of 68 fecal samples from calves with diarrhoea which were tested for rotavirus with the latex agglutination test "Slidex Rota-Kit2" and by electron microscopy 33 samples were positive and 33 were negative with both tests respectively. Divergent results (latex test positive/EM negative and vice versa) were observed in one specimen only, respectively. Cross reactions with other viruses diagnosed by electron microscopy were not observed with the latex agglutination test. The "Slidex Rota-Kit2" is another suitable test for the diagnostic laboratory as well as for the veterinary practitioner for the detection of rotavirus in fecal samples of calves.     

Rapid detection of avian pneumovirus in tissue culture by microindirect immunofluorescence test.

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.     

Pathogenic and fecal Escherichia coil strains from turkeys in a commercial operation

The biochemical phenotypes and antimicrobial susceptibility patterns of 105 clinical Escherichia coli isolates from flocks with colibacillosis in a turkey operation were compared with 1104 fecal E. coli isolates from 20 flocks in that operation. Clinical isolates and 194 fecal isolates with biochemical phenotypes or minimum inhibitory concentrations for gentamicin and sulfamethoxazole similar to clinical isolates were tested for somatic antigens and the potential virulence genes hylE, iss, tsh, and K1. The predominant biochemical phenotype of clinical isolates contained 21 isolates including 14 isolates belonging to serogroup 078 with barely detectable beta-D-glucuronidase activity. Thirty-five fecal isolates had biochemical phenotypes matching common phenotypes of clinical isolates. Sixty-six (63%) clinical isolates exhibited intermediate susceptibility or resistance to gentamicin and sulfamethoxazole compared with 265 (24%) fecal isolates (P < 0.001). Seventy-seven clinical isolates reacted with O-antisera, of which 51 (66%) belonged to the following serogroups: O1, O2, O8, O25, O78, O114, and O119. In comparison, 8 of 35 (23%) fecal isolates subtyped on the basis of biochemical phenotype belonged to these serogroups and four of 167 (2%) fecal isolates subtyped on the basis of their antimicrobial resistance patterns belonged to these serogroups. Iss, K1, and tsh genes were detected more often among clinical isolates than these fecal isolates (P < 0.05). In summary, a small subgroup of E. coli strains caused most colibacillosis infections in this operation. These strains existed at low concentration in normal fecal flora of healthy turkeys in intensively raised flocks. The data suggest that colibacillosis in turkey operations may be due to endogenous infections caused by specialized pathogens.     

Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5 + 11] (15.4%). G10P[11] and G6 + G10P[5] were each detected at a rate of 7.7%, and G6 + G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.     

Isolation of coronaviruses in cell culture from nasal swab samples of calves with respiratory tract diseases in the Federal Republic of Germany

Coronavirus was isolated in HRT-18 tissue culture from nasal swabs of 4 from 15 calves showing respiratory symptoms. The strains showed cytopathic effects and could be identified as coronaviruses of bovine origin by their mouse erythrocyte agglutinating activity as well as serological specific hemagglutination inhibition and electron microscopical methods. This is the first report on the isolation of coronaviruses in tissue culture from the respiratory tract of calves in the FRG.     

LAMP方法快速检测实验猴粪便样品中志贺菌

为了缩短检测周期,加快通关速度,根据基因库中志贺菌属特异性基因(ipa H)的保守序列,试验设计1套特异性环介导等温扩增(LAMP)引物,建立了实验猴粪便中志贺菌的LAMP检测方法。对志贺菌纯培养的敏感性可达7.5×100CFU/m L,对临床样品的敏感性可达到7.5×103CFU/m L;整个检测过程仅需12 h,且不需要昂贵的实验设备;该方法对12株其他菌株无扩增反应,且试验结果可通过肉眼观察颜色变化直接判定。试验表明,LAMP方法简便、灵敏、特异,适用于实验猴粪便样品中志贺菌的快速检测。与PCR相比,LAMP方法更加适合在基层推广使用。     

Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples.

The performance of flow cytometry (FC) was compared with immunofluorescence microscopy (IM) for detection of Giardia duodenalis in bovine feces. Samples from 36 adult dairy cows and 208 dairy calves were collected. Flow cytometry test characteristics were calculated using continuous, ordinal, and dichotomized results. Spearman correlation coefficients comparing the results of the 2 tests were 0.47 and 0.68 for cows and calves, respectively. Using IM as indicative of presence or absence of G. duodenalis cysts in each sample, likelihood ratios of FC results with 0, 1, and > or = 2 gated events indicated that samples with 1 gated event were likely to be positive in the cows but not in the calves. Immunofluorescence microscopy detected G. duodenalis in 69.7% and 48.1% of cows and calves, respectively. When dichotomizing the FC results at a cut-off point of 1 or 2 gated events, 46.3% and 19.9% of the cow and 51.9% and 35.1% of the calf samples, respectively, were classified as G. duodenalis-positive. Relative to IM, the sensitivity in the cows was 0.59 and 0.28, respectively, and 0.76 and 0.64, respectively, in the calves. At a cut-off point of 1, 65.7% and 73.1% of the cow and calf samples, respectively, were correctly classified in FC, and at a cut-off point of 2, 49.3% and 78.4% were correctly classified in the cows and calves, respectively. Flow cytometry was less sensitive than IM. Possible reasons and research needed to improve FC for G. duodenalis detection are discussed.