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Biomedical subjects

J Hoorfar

Publications and source records attributed to J Hoorfar.

35 records · Page 2Linked to original sources
PubMed2000

Automated 5' nuclease PCR assay for identification of Salmonella enterica.

A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [DeltaRn], >0. 6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (DeltaRn, < or =0.5). All 100 rough Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification of Salmonella, particularly in large reference laboratories.

Exodeoxyribonuclease V↗
PubMed2000

Improved culture methods for isolation of Salmonella organisms from swine feces.

OBJECTIVE: To compare 3 alternative culture techniques for the detection of Salmonella organisms in swine feces with a modification of the International Standard Organization (ISO) 6579 standard protocol. SAMPLE POPULATION: Fecal samples from swine herds suspected of having Salmonella infections. PROCEDURE: 4 experiments were performed to evaluate the following: 1) diagnostic sensitivity of the selective preenrichment and rapid isolation novel technology (SPRINT) protocol, compared with that of the modified ISO protocol; 2) detection limit of the SPRINT protocol for Salmonella organisms; 3) use of tetrathionate-novobiocin (TTN) broth, compared with selenite cysteine (SC) broth for selective enrichment; and 4) use of universal preenrichment (UPE) broth, compared with buffered peptone water (BPW) for preenrichment of samples prior to the use of modified semisolid Rappaport-Vassiliadis (MSRV) plates. RESULTS: Comparing the Salmonella culture results of 183 swine fecal samples, the diagnostic sensitivity of the SPRINT protocol (0.86) was not significantly different than the diagnostic sensitivity of the modified ISO protocol (0.80), although it was 24 hours faster. The SPRINT protocol could detect 5 of the 6 investigated Salmonella serotypes at inoculation concentrations of < 10 colony-forming units (CFU)/25 g of uncontaminated feces. The TTN broth performed significantly better than the SC broth for selective enrichment of Salmonella organisms. There was no significant difference in results of preenrichment of samples between the use of UPE broth or BPW. CONCLUSIONS AND CLINICAL RELEVANCE: The SPRINT protocol may provide a faster alternative for isolation of Salmonella organisms from swine fecal samples. Furthermore, the use of TTN broth instead of SC broth may increase the sensitivity of the modified ISO 6579 protocol.

Animals↗
PubMed1999

A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates.

The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories. The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads. All strains belonging to the serogroups B, C1, C2-C3, and D were grouped correctly. Among the 32 rough presumptive isolates identified, 19 isolates resulted in a band of 882 bp (serogroup B), 11 isolates resulted in a band of 471 bp (serogroup C1), and two isolates showed a band of 720 bp (serogroup D). In conclusion, rough presumptive Salmonella isolates can be conveniently confirmed to the serogroup-level, using the pre-mixed PCR tests. The system can be easily implemented in accredited laboratories with limited experience in molecular biology.

Bacterial Typing Techniques↗
PubMed1999

Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine.

We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was the definitive standard. The primary isolation both for the culture and the EIA methods was carried out by overnight selective enrichment in Preston broth. The results showed good sensitivities for both EIA methods in cattle (95% and 84%) and swine (88% and 69%) samples. However, when testing cattle samples, EIA-2 method resulted in a rather low specificity (32%). This seemed to be partially due to the isolation of nonthermophilic species. In conclusion, EIA-1 method may provide a simple and fast tool with good accuracy in cattle and swine samples for automated screening of large number of samples.

Animals↗
PubMed1999

Evaluation of culture methods for rapid screening of swine faecal samples for Yersinia enterocolitica O:3/biotype 4.

In two studies, seven different culture protocols were compared to test naturally contaminated faecal samples from pigs for isolation of Y. enterocolitica serotype O:3/biotype 4 (n = 70 and n = 79). Four of the protocols were based on the Nordic Committee on Food Analysis (NMKL, protocols), while three protocols were based on a rapid and selective method (here called ITC protocols). The protocols differed mainly in time of pre-enrichment (1, 10 and 24 d) and enrichment (2, 10, 24 d) and the type of selective enrichment media (ITC vs. MRB). The sensitivity of the rapid ITC protocol (24% and 9%) was comparable with the lengthy NMKL-protocols (16% and 11%), while the results of direct plating after 3 h (4%) and the extended enrichment in ITC-broth (4%) were very low. In addition, there was a marked reduction in the number of false positive plates in the short selective protocol (62% vs. 12%). The results indicate possibilities of shortening the culture methods by replacing most of the biochemical tests with an agglutination test based on a monoclonal antibody.

Abattoirs↗
PubMed1998

Importance of pre-enrichment media for isolation of Salmonella spp. from swine and poultry.

The performance of two new (1-day) culture methods, Salmonella Enrichment Broth (SEB) and Revive, and an alternative pre-enrichment broth, designated Universal pre-enrichment broth (UB), was compared to the internationally accepted buffered peptone water (BPW). The study was directed towards detection of Salmonella in 100 faecal samples from porcine and 100 neck-skin samples from poultry. The sensitivity (number of positive cases per method among all the positive cases) of the conventional pre-enrichment in BPW was found to be 0.77 for swine and 0.66 for poultry samples, while a combination of the BPW method with parallel pre-enrichment of the same sample in UB resulted in high sensitivity for swine (0.92) and poultry (0.95) samples. A 2-h pre-enrichment in the non-selective Revive, followed by overnight enrichment in selective broth, resulted in a low sensitivity, particularly for the neck-skin samples (0.16, P = 0.001). The SEB method in the porcine samples resulted in a sensitivity (0.71) comparable to the standard method (P = 0.31). In conclusion, additional pre-enrichment of samples in UB may substantially increase the culture sensitivity. During routine screening of large numbers of samples, it may be advantageous to use SEB rather than standard culturing.

Abattoirs↗
PubMed1997

Detection of antibodies to Salmonella lipopolysaccharide in muscle fluid from cattle.

OBJECTIVE: To compare muscle fluid with serum samples for detection of antibodies to Salmonella lipopolysaccharide. SAMPLE POPULATION: Muscle fluid and serum samples from 2 cattle populations: 1 from the island of Bornholm with no history of salmonellosis (n = 39), and the other from the S dublin-enzootic areas of Jutland (n = 144). PROCEDURE: Salmonella dublin (O:1,9,12), S typhimurium (O:1,4,5,12), and Salmonella O:9-blocking ELISA were used for testing the samples. RESULTS: In the S dublin ELISA, all serum and muscle fluid samples from cattle on the island of Bornholm had OD450 values well below the cutoff value (0.5). For samples obtained from cattle in the enzootic areas of Jutland, high correlation was found between serum and muscle fluid samples (rs = 0.89, P < 0.001). In addition, 19% (28/144) of the cattle had ELISA-positive muscle fluid and serum samples; 2% (3/144) had positive results for muscle fluid only, whereas 1 animal had positive results for serum only (kappa = 0.91, P < 0.0001; sensitivity and specificity of 97%). The same samples had similar significant correlation in the S typhimurium ELISA (rs = 0.88, P < 0.001, kappa = 0.7, P < 0.001; sensitivity of 73% and specificity of 98%) and the O:9-blocking ELISA (rs = 0.49, P < 0.001). CONCLUSION AND CLINICAL RELEVANCE: Muscle fluid samples taken at slaughter can be used as a practical alternative to serum samples for surveillance of Salmonella infections in cattle.

Animals↗
PubMed1996

Comparison between persisting anti-lipopolysaccharide antibodies and culture at postmortem in salmonella-infected cattle herds.

Herds with recent clinical outbreaks of Salmonella dublin (7 herds) and S. typhimurium (4 herds) infections were followed serologically in O-antigen ELISAs over about one year, divided in four equal sampling phases. Animals found to be persistent high-reactors or seronegative at the end of the study were slaughtered and subsequently cultured for salmonella in a selected number of organ samples. Approximately 3% of all animals had high seroreactions up to 17 months after the outbreaks, and less than half of the seropositive animals in the S. dublin-infected herds were salmonella culture positive at slaughter (14/31). However, one persistently seronegative animal was also culture positive. Furthermore, as much as 70% of the male calves investigated at postmortem in the S. dublin-infected herds were high-reactors, among which approx. 56% were culture positive. Surprisingly, 2 of the 14 animals found culture positive turned out to be culture positive for S. typhimurium only. In the S. typhimurium study, none of the 17 animals investigated at postmortem were salmonella culture positive. All sera from these animals were negative in the O:9 blocking ELISA, and no serum sample was positive in the S. dublin ELISA, alone. In conclusion, although serology based on the O-antigens appears to be useful to identify salmonella-infected herds, it seems to be insufficient for identification of persistently infected animals.

Animals↗
PubMed1996

Seroreactivity of Salmonella-infected cattle herds against a fimbrial antigen in comparison with lipopolysaccharide antigens.

The IgG seroreaction of Salmonella-infected cattle herds against a fimbrial antigen (SEF14) was compared with that against lipopolysaccharide (LPS) antigens. Sera from 23 dairy herds (n = 205) from an island with no occurrence of salmonellosis, four herds (n = 303) with recent outbreaks of S. dublin and four herds (n = 168) with recent outbreaks of S. typhimurium, were tested in a SEF14-ELISA, S. dublin LPS (0:1, 9, 12) ELISA and S. typhimurium LPS (0:1, 4, 5, 12) ELISA. At a cut-off OD of 0.5, only one of the animals tested from the salmonellosis-free island showed significant seroreaction against the SEF14 antigen, which was confirmed in a Western-blot analysis. Three out of the four S. dublin-infected herds had several seroreactors in the SEF14-ELISA, whereas all the four herds were positive in the 0:1, 9, 12-ELISA. All but two samples (both from the same herd) in the four S. typhimurium-infected herds, positive in the 0:1, 4, 5, 12-ELISA, had OD values below 0.5 in the SEF14-ELISA. The results indicate that cattle can produce detectable specific antibodies against fimbrial antigens which may be used for screening of S. dublin-infected herds, particularly in areas with low prevalence of salmonellosis, increasing the predictive value of serology.

Animals↗
PubMed1995

Evaluation of an O-antigen ELISA for screening cattle herds for Salmonella typhimurium.

A total of 2585 serum samples from 62 dairy herds located in four different regions of Denmark were tested in an O-antigen (0:1,4,5,12)-based ELISA for the detection of antibodies against Salmonella typhimurium. Ten closed herds from an island with no reported occurrence of salmonellosis for several years, and 12 herds from a salmonella enzootic area which had had clinical outbreaks of S typhimurium were used to define a herd ELISA cut-off value. When herds with at least 5 per cent of the serum samples having an optical density of > 0.5 were considered ELISA-positive, all 10 herds from the salmonellosis-free island were ELISA-negative, and all but one of the 12 S typhimurium-infected herds were ELISA-positive, which resulted in a herd test sensitivity of 0.92 and herd test specificity of 1.0. Eleven of the 12 S typhimurium-infected herds were negative in a blocking ELISA based on a monoclonal antibody to the 0:9 antigen of the serogroup D salmonellas, indicating the possibility of rapid serogroup-specific screening of herds by means of these two tests. Ten other randomly selected herds with clinical outbreaks of S dublin were all, to a large extent, positive in the 0:1,4,5,12-ELISA, whereas a S dublin (0:1,9,12)-ELISA described previously appeared to be more serogroup D-specific. Thus, the 0:1,4,5,12-ELISA appears to be useful for detecting herd infections with S typhimurium, and positive reactions may be further discriminated by the serogroup D-specific ELISA.

Animals↗
PubMed1995

Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Enzyme-linked immunosorbent assay for screening of milk samples for Salmonella typhimurium in dairy herds.

We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive herd specificity, 0.9 and herd sensitivity, 1.0). A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S typhimurium infections, but further modifications are needed to test bulk tank milk samples.

Animals↗
PubMed1994

Serodiagnosis of Salmonella dublin infection in Danish dairy herds using O-antigen based enzyme-linked immunosorbent assay.

Usefulness of two enzyme-linked immunosorbent assays (ELISA) for screening of dairy herds for antibodies to lipopolysaccharide (LPS) of Salmonella dublin (O:1,9,12) was investigated. Sera (3097) were collected from 40 dairy herds located in three areas of Denmark with different prevalence of salmonellosis: ten salmonellosis-free herds from the island of Samsø where there is no history of salmonellosis, ten salmonellosis-free herds from the island of Sealand where outbreaks are infrequent, and 20 salmonella infected herds from Jutland where salmonellosis is enzootic. The samples were analyzed for antibodies to S. dublin LPS using an indirect (O:9,12) and a blocking (O:9) ELISA. Using herd history of salmonellosis, herd location and clinical state of the herds as reference, the herd sensitivity and herd specificity of the tests were 100% and 100% in the indirect ELISA and 95% and 100% in the blocking ELISA, respectively. A significant correlation was found between the two tests (rs = 0.46, p < 0.001). However, the indirect ELISA detected more seropositive animals than the blocking ELISA (17% vs. 7%, respectively). In calves from Sealand, level of background reaction was significantly lower (p < 0.001) compared to the heifers and the cows. The percentages of seropositive calves in both tests were higher (p < 0.01) in comparison to cows (19 vs. 8 in indirect ELISA, and 14 vs. 6 in blocking ELISA, respectively). Results of the study indicated that it is possible to apply LPS ELISA in serological screening for salmonellosis.

Animals↗
PubMed1993

Prophylactic nutritional modification of the incidence of diabetes in autoimmune non-obese diabetic (NOD) mice.

Experiments in rodent models of insulin-dependent diabetes mellitus (IDDM) suggest that destruction of pancreatic beta cells can be both initiated and inhibited by certain environmental factors such as dietary constituents. We studied nutritional impact of certain protein sources of natural-ingredient, non-purified (NP) rodent diet on diabetes incidence and insulitis severity in the spontaneous diabetic, non-obese diabetic (NOD) mouse. Long-term ad lib. feeding of diets containing wheat flour (800 g/kg), and to a lesser extent soya-bean meal (400 g/kg), were associated with relatively high diabetes incidence (65 and 45% respectively), whereas a diet based on hydrolysed casein (HC; 200 g/kg) as the only source of protein significantly (compared with the wheat-flour diet) inhibited expression of diabetes (22%). Feeding a hypo-allergenic soya-bean-protein hydrolysate resulted in diabetes incidence and insulitis severity similar to that of the soya-bean-meal-fed group. This may indicate that protein hydrolysis per se may not be necessary for dietary modification of diabetes in the NOD mouse. The window of vulnerability to diabetogenic diets was found to be between weaning and about 70 d of age. In the diabetic mice insulitis was less frequent in the HC-fed group when compared with those fed NP (P = 0.04), soybean meal (P = 0.03), soya-bean-protein hydrolysate (P = 0.012) or wheat flour (P = 0.0002). In the non-diabetic mice the wheat-flour diet was associated with a high insulitis severity in comparison with the HC group (P = 0.004). Early avoidance of NP diet was associated with lower degree of insulitis in both diabetic (P = 0.00003) and non-diabetic mice (P = 0.001) when compared with the mice fed on the HC diet later in life. These findings are contributing to further clarification of diabetes-promoting dietary constituents, which may have some nutritional implications for IDDM-susceptible children.

Animals↗
PubMed1992

Impact of dietary protein and fat source on the development of insulin-dependent diabetes in the BB rat.

Epidemiological studies show a remarkable geographical difference in the prevalence of IDDM, suggesting a role for environmental factors such as diet, infection, or stress in the etiology of the disease. Dietary modification has already been shown to be effective in the prevention of autoimmune diabetes in the BB rat and NOD mouse. We studied the effect of protein and fat source in the prophylaxis of diabetes in the BB rat. Natural ingredient rat chow was consistently associated with a high expression of the disease, whereas a casein-based, defined diet significantly inhibited the development of diabetes. Substitution of casein with raw red lentils resulted in a markedly higher incidence. This is the first highly diabetogenic defined diet in the BB rat. Neither fish oil nor soy oil enhanced diabetes expression in the BB rat. Increased amounts of soy oil also did not influence the disease process. These results suggest a central role for dietary protein source in the pathogenesis of BB rat diabetes. We speculate that plant proteins containing anti-nutrients such as chemicals, lectins, enzyme inhibitors, and nonphysiologic amino acids may initiate or hasten the pathogenesis process via beta cell stress or immune response activation.

Animal Feed↗
PubMed1991

Dietary plant materials and development of diabetes in the BB rat.

The present study was designed to examine further the impact of individual plant protein sources found in a diabetogenic, cereal-based, rodent laboratory diet, NIH-07 [open formula, nonpurified rat and mouse diet (positive control)], on the development of diabetes. Diabetes-prone BB rats that were pan-T(OX19+)-lymphopenic were fed a low diabetogenic diet during gestation and lactation. Progeny of these rats were fed a normal or autoclaved NIH-07 diet, or one of eight other diets based on the AIN-76A formulation, with modified protein sources as follows: hydrolyzed casein (HC), soybean meal, HC+ trypsin inhibitor (TI) in water (2 mg/mL, wheat germ, alfalfa seeds, Brewer's yeast, red lentils and a plant protein mixture. Feeding soybean meal increased the incidence of diabetes compared with the negative control, HC diet (47% vs. 12% incidence, P = 0.02). Wheat germ, alfalfa seeds and plant protein mixture resulted in an intermediate incidence of diabetes of 33%; the incidence was lower for Brewer's yeast and lentils (20% and 13%). Autoclaving (121 degrees C, 10 min) the NIH-07 diet or the presence of TI in drinking water had a minimal effect on diabetes frequency, suggesting heat-labile plant toxicants were not directly involved. Thus, certain dietary plant protein sources or associated agents may influence the development of spontaneous diabetes in the BB rat.

Age Factors↗