A colony lift immunoassay for the specific identification and quantification of Listeria monocytogenes.

A colony lift immunoassay (CLI) has been developed to detect Listeria monocytogenes after the organisms have been cultured on filter membranes or agar plates. Polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA), used in the CLI, were prewet with methanol and used to imprint colonies that were grown on the filter or agar plates. A positive control was applied to the edge of each membrane. The imprinted membranes were subsequently air dried, peroxidase neutralized, blocked, and reacted for 20 min with a 2-microg/ml unconjugated Mab EM-7G1 solution. The membranes were washed briefly and reacted for 30 min with a 1:2000 dilution of a commercially prepared peroxidase-labeled goat anti-mouse secondary antibody (Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD). After a second wash step, the membranes were exposed to a 3,3',5, 5'-tetramethylbenzidine membrane substrate (KPL), rinsed in deionized water, and allowed to dry. Colonies of L. monocytogenes were identified by a blue color reaction on the membrane, which could be used to reference the colonies either on the filter membranes or agar plates. The CLI was tested against a wide range of Listeria species as well as several non-Listeria species and was shown to have a high degree of sensitivity (96%) and specificity (90%). We have shown that it is useful as a simple and rapid method to detect and identify L. monocytogenes.

An enzyme-linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens.

A model enzyme-linked immunosorbent assay (ELISA) system was developed which permits the user to evaluate replicate single sera dilutions of 46 test and control serum samples for the presence of specific antibody against up to eight different antigens in one assay. The system makes use of a transplanting device for the simultaneous transfer of aliquots of independently diluted replicate test samples along with conjugate and substrate from a serum reservoir plate or from a reagent reservoir onto different antigen coated target plates. Kinetically read, raw absorbance data from tests are transmitted to a microcomputer, where absorbance values from all tests are quickly reduced to predicted titer levels by computer analysis. All titer computations involve the use of a single prescribed standard curve for predicting antibody titer against the different antigens. Between assay repeatability of this procedure is high and its potential for replacing a number of different conventional assays for epidemiological studies has been evaluated.

Improved detection of nontyphoid and typhoid Salmonellae with balanced agar formulations.

A strategically balanced medium was developed for the improved detection of nontyphoid and typhoid salmonellae. Its balanced sugar (cellobiose, lactose, mannitol, and trehalose) and protein (beef extract and polypeptone peptone) formulation provided Salmonella with a selective growth advantage over non-Salmonella enteric organisms. The formulations promoted the production and detection of H2S production levels that otherwise might be missed with traditional agar formulations. In combination, these advantages contributed to increased sensitivity without the loss of specificity. In comparative studies using 86 samples of meat products (beef, pork, and chicken), the new media, Miller-Mallinson (MM) agar and xylose lysine tergitol (Niaproof) 4 agar, possessed significantly higher sensitivity (P < 0.001) and an improved specificity over bismuth sulfite, hektoen enteric, and xylose lysine desoxycholate agars. However, these samples did not contain nontyphoid salmonellae with weak to ultraweak H2S production characteristics. Modified formulations of MM agar were generally similar to bismuth sulfite and hektoen enteric agars in the identification of four of seven globally diverse strains of Salmonella serotype Typhi. Two of these seven strains were found to produce more readily identifiable black (H2S-positive) colonies on MM agar, whereas one of the seven was not readily detected by any of the media. The improved detection of nontyphoid and typhoid salmonellae attests to the sensitivity of MM agar and to its potentially broad utility in both clinical and food quality laboratories.

Development and evaluation of a 24-hour method for the detection and quantification of Listeria monocytogenes in meat products.

A 24-h filter monitor-based test, Listeria-SELeCT, has been developed to quantify Listeria monocytogenes organisms in meat samples with a sensitivity of < or = 1.0 CFU/g. The technique comprises a filter monitor-based system and a colony lift immunoassay to identify and enumerate the target organism. Meat homogenates were centrifuged and the eluate was filtered to trap and immobilize the microorganisms on the filter. Fraser broth was then added to the filter apparatus to allow the organisms to become established overnight and to inhibit contaminants, after which the filters were transferred onto Modified Oxford medium agar, a selective medium for L. monocytogenes. After 10 to 12 h, a colony lift immunoassay was used to confirm and enumerate suspect colonies on the filter. A correlation study between the Listeria-SELeCT method and the most probable number technique showed the Listeria-SELeCT to be considerably more accurate than the most probable number for quantitatively determining the number of viable organisms in meat samples. Because of ease and speed of testing, the Listeria-SELeCT system also provided major advantages over the most probable number technology.

Improved plating media for the detection of Salmonella species with typical and atypical hydrogen sulfide production.

The Salmonella detection ability of 2 surfactant-supplemental media, xylose-lysine-tergitol (Nia-proof) 4 (XLT4) and Miller-Mallinson (MM) agar, was compared against that of several commonly used plating media. XLT4 and MM appeared to be the most efficient in detecting Salmonella in meat products and food animal environments. MM was superior to XLT4 in detecting those increasingly more prevalent strains of Salmonella possessing weak to ultraweak H2S production characteristics.

Isolation of Salmonella from poultry tissue and environmental samples: a nationwide survey.

A nationwide survey of veterinary laboratories culturing poultry tissue and environmental samples for Salmonella found a large variation in isolation procedures. There were 17 different selective enrichment media or combinations of enrichment media being used for poultry tissue samples. Variations were found in how long the selective enrichments were incubated and in the temperature of incubation. There were 14 different plating media being used. Many laboratories screen and identify only one colony from the plating media. For the protection of poultry breeding and hatchery organizations and with the formation of official poultry monitoring programs, with their legal and public health implications, it becomes increasingly important that diagnostic laboratories adopt minimal standardized protocols for isolating Salmonella.

Clinical, cultural, and serologic observations of avian mycoplasmosis in two chicken breeder flocks.

Two chicken breeding flocks from different breeding lines were studied serologically and culturally for Mycoplasma gallisepticum (MG) throughout their growing and laying period. Infection was proven by successful isolation of MG from both breeders and progeny originating from these two flocks. Observations of these flocks which were serologically and culturally negative for Mycoplasma synoviae (MS) further disclosed that: 1) negative plate tests of large numbers of day-old progeny may sometimes be found in flocks known to be infected with MG; 2) it may be very difficult to isolate MG consistently from some infected flocks; 3) overgrowth of M. gallinarum may interfere with successful cultivation of MG; 4) a persistent breeder flock reactor rate of greater than 10-20% but less than 80-100% for a 4-to-12-week period is a strong indication of MG infection despite weak or negative MG hemagglutination-inhibition (HI) test results; and 5) antibodies for all strains of MG may not react equally to the standard USDA MG-HI antigen.

Occurrence of duck virus enteritis (duck plague) in Pennsylvania, 1968-74.

During the 7-year period 1968-74 cases of duck virus enteritis (duck plague) were diagnosed in waterfowl in Pennsylvania. Muscovy ducks were affected in 8 cases, geese in 3 cases, and mallard ducks in 1 case. In 5 of these cases either domestic or wild ducks were closely associated with infected waterfowl but were unaffected.

Rapid serological profiling by enzyme-linked immunosorbent assay. I. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure specific antibody activity in sera of chickens exposed to Newcastle disease virus (NDV). A near-linear relationship existed between the log of the corrected absorbance of antisera at a single working dilution and the corresponding observed serum titers as determined by a standard serial-dilution method. Regression analysis was used to construct a standard curve and extract an equation from this relationship. The equation was used to convert corrected absorbance readings of the single working dilution directly into predicted ELISA antibody activity titers. In a comparative study, a correlation (P less than 0.01) was found between ELISA and hemagglutination-inhibition (HI) antibody titers to NDV. ELISA titers were as much as 160 times greater than the HI titers. ELISA was also able to detect much lower levels of antibody activity than the HI test.

The use of the drag-swab technique and improved selective plating media in the recovery of Salmonella arizona (7:1,7,8) from turkey breeder hens.

The litter of six turkey hen flocks was sampled using the drag-swab technique to determine the effectiveness of this method in detecting Salmonella arizona. Two flocks with the lowest biosecurity standards were found to have S. arizona. Results showed that the drag-swab technique can provide a sensitive and cost-effective measure of S. arizona infection within a flock.

Monitoring poultry farms for Salmonella by drag-swab sampling and antigen-capture immunoassay.

Drag-swab (DS) sampling, at the rate of four DS gauze pads per flock (house); modified culture procedures (novobiocin-supplemented plating media and delayed secondary selective enrichment); and Salmonella antigen-capture (SAC) technology were combined in screening one layer flock and 38 market-age broiler flocks. The results showed that low (negative) SAC sample-to-positive control (S/P) ratios were related to the negative culture recovery of Salmonella. Similarly, high (positive) S/P ratios were related to and indicative of positive culture recoveries. Extensive sampling and testing of 18 of the 39 flocks disclosed A) that five flocks with negative culture recoveries from feathers and freshly voided feces had essentially no positive DS-SAC values, and B) that 13 flocks with positive culture recoveries from feathers and/or fresh feces all had positive DS-SAC values.

Microscopic lesions of naturally occurring and experimental "spiking mortality" in young broiler chickens.

Tissues from five chickens from each of 44 flocks affected with "spiking mortality" were examined histologically. Tissues from these chickens were characterized by: multifocal necrosis of hepatocytes with congestion and blood lake formation; severe lymphocyte necrosis in the bursal medulla, thymic cortex, and gut-associated lymphoid tissues; acute necrotic vasculitis in the alimentary serosa and liver; rickets; and air-sac disease in survivors. No specific etiology was identified in sections examined.

Rapid serological profiling by enzyme-linked immunosorbent assay. III. Simultaneous measurements of antibody titers to infectious bronchitis, infectious bursal disease, and Newcastle disease viruses in a single serum dilution.

The present paper describes a method for measuring enzyme-linked immunosorbent assay (ELISA) antibody titers to infectious bronchitis (IB), infectious bursal disease (IBD), and Newcastle disease (ND) viruses from a single serum dilution using the same assay procedures and analyses. A regression line equation generated for predicting NDV ELISA antibody titers at a single serum dilution was successfully used to predict ELISA antibody titers to IB and IBD antigen preparations at the same test sera dilutions. Duplicate samples of 46 field and control sera were transferred simultaneously, with the aid of a replica plating device, from a reservoir plate to three different test plates, which were coated separately with IB, IBD, and ND antigens. After similar indirect ELISA parameters were conducted for each antigen, raw absorbance values from duplicate tests and controls were transmitted directly from an ELISA reader to a microcomputer, which subsequently processed average corrected absorbance values directly into individual ELISA antibody titer for each test antigen. The computer output listed individual field and control sample titers to each antigen, as well as graphing the relative ELISA titer distribution of the entire test group to each antigen. In a comparative study, test sera were evaluated for ELISA antibody titer to NDV, IBV, and IBDV by this method in three separate assays. Consistent titer values were obtained in all assays, as 96% of the 414 replicate titers evaluated varied less than twofold.

Vaccination with F-strain Mycoplasma gallisepticum to reduce production losses in layer chickens.

The effect of Mycoplasma gallisepticum (MG) infection or vaccination of Conn. F-strain MG on 45 weeks of egg production was analyzed using production records from 132 flocks of commercial layer hens. The flocks were located in Pennsylvania, and the data were collected for two years. On the average, layers maintained free from infection with MG laid 15.7 more eggs/hen housed than the MG-infected layers; figures were adjusted for layer-strain effect. This adjusted advantage decreased to 8.7 eggs/hen housed when uninfected flocks were compared with vaccinated flocks. Adjusted average production of vaccinated flocks was 7.0 eggs/hens housed more than production of MG-infected flocks. Egg production of four layer strains was observed with respect to vaccination or natural infection with MG. The four strains responded similarly whether vaccinated or infected. Route of vaccination and age of layer at time of vaccination influenced egg production of vaccinated birds. The adjusted average production/hen housed was 4.9 eggs greater for birds vaccinated via drinking water than for birds vaccinated via spray. The adjusted average was 10.3 eggs/hen housed greater for birds vaccinated between 8 and 18 weeks of age than for birds vaccinated after 18 weeks.

Vaccination of ring-necked pheasant for marble spleen disease.

Laboratory evidence indicates that a safe and effective procedure was developed for vaccinating pheasants against marble spleen disease. Field trials confirm the safety of the vaccine and suggest that vaccination will prevent marble spleen disease. Vaccination is by drinking-water administration of turkey-spleen-propagated turkey-origin live avirulent virus to pheasants 4 1/2 weeks old or older. The effect of field vaccination was studied in 39,000 pheasants in pens where recurrent annual mortality had been 5-15%.

Prevalence of Salmonella in broiler flocks: effect of litter water activity, house construction, and watering devices.

Litter samples from 24 flocks of broilers and four flocks of broiler breeders were evaluated for Salmonella contamination, water activity (Aw), and total moisture content (MC). The drag swab (DS) monitoring system was used to collect samples to detect Salmonella contamination. Simultaneously, representative samples of the uppermost surfaces of dry (loose) litter and wet (caked) litter were collected for Aw and MC analyses. On dry litter surfaces, high Aw values (0.90-0.95) were associated with flocks Salmonella-positive using DS; low Aw values (0.79-0.84) were associated with flocks Salmonella-negative by DS; and transition Aw values (0.85-0.89) were associated with flocks having an increased risk for the presence of Salmonella. The association of high Aw values with Salmonella risk was not observed for wet (caked) litter surfaces. Observations suggest that limiting Aw in the litter base of broiler houses may create a less favorable environment for the multiplication of Salmonella and thus a more hygienic environment for broiler production.

Evaluation of possible alternatives to double-strength skim milk used to saturate drag swabs for Salmonella detection.

The drag-swab Salmonella screening technique was evaluated using less expensive alternatives to double-strength skim milk (2 x SM) as a saturating medium for drag swabs. Ten pre-determined Salmonella-positive poultry houses were studied. In the first phase, Salmonella screening efficiency of drag swabs impregnated with 2 x SM and commercially available canned Carnation evaporated skim milk (CESM) were compared. Results showed CESM to be a less efficient alternative. In the second phase of the study, the Salmonella screening efficiency of drag swabs impregnated with 2% buffered peptone water (BPW), physiological saline (PS), and distilled water (DW) were evaluated along with an unimpregnated drag swab (dry drag swab) (DD) as possible alternatives to 2 x SM. The efficiency of Salmonella detection using various impregnation treatments were in the following order: 2 x SM > PS > BPW > DW > DD.

Evaluation of radiolabeled and colorimetric DNA probes in comparison with an antigen screening assay for the detection of Salmonella from poultry farms.

A total of 48 environmental drag-swab samples from various poultry farms were tested for the presence of Salmonella spp. by culture, an enzyme-linked immunosorbent assay-based Salmonella antigen screening (SAS) assay, and two DNA probes (radiolabeled and colorimetric). The radiolabeled DNA probe was allowed to hybridize with culture-positive samples (n = 8) and was found to detect Salmonella spp. in all cases (100%). Both of the probes, subsequently hybridized with culture-negative samples (n = 8), were observed to yield good agreement (91%) with the culture findings. The remaining samples (n = 32) were tested by the SAS assay, and where there was no agreement between the culture and SAS, samples were further examined by the DNA probes. Results using both probes agreed with those obtained by culturing the samples but did not agree with the SAS assay result when the ratio of samples tested to samples positive (S/P) cutoff value used was 0.5.

Epizootiology, pathology, and microbiology of an outbreak of urolithiasis in chickens.

An outbreak of urolithiasis that doubled the annual mortality rate of chickens in a large flock of table-egg-layers is described. Despite the presence of a large unilateral urolith and/or severe renal atrophy, the layers often maintained active egg production and apparent homeostasis until a small urolith blocked the ureteral flow from the contralateral kidney. This terminal episode appeared to produce acute obstructive renal failure, rapidly developing visceral gout (visceral urate deposition), uremia, and death. The atrophy observed appeared to be acquired and progressive. Histologic features in the kidneys were acute to chronic glomerulonephritis, interstitial nephritis, and pyelonephritis. Epizootiologic and microbiologic studies indicated that a combination of infectious and noninfectious mechanisms may have been involved. Causative roles for calcium-phosphate imbalance, infectious bronchitis (IB), Newcastle disease (ND), and adenovirus or reovirus infections could be neither excluded nor confirmed. Contributory factors may have been spray ND-IB and other vaccinations of 15-week-old ND-IB-susceptible pullets, water deprivation, shipping stress, Mycoplasma synoviae infection, immune complex disease, and mycotoxins.

The effect of exposure, storage times, and types of holding media on the drag-swab monitoring technique for Salmonella.

Four maintenance media were compared for the preservation of the sensitivity of drag swabs and to assess the survival of Salmonella spp. on drag swabs at reduced temperatures. The effects of Difco double-strength skim milk (2 x SM), 2% buffered peptone water (BPW), a modified liquid Cary-Blair transport medium (LCB), and lactose broth holding medium were compared, as were storage periods of various lengths. The results with enzyme-linked immunosorbent antigen capture assay and highly selective plating media detection systems showed that 2 x SM had the highest level of recovery of salmonellae after prolonged storage, both under refrigeration at 4 C for 3 days and under frozen conditions of -15 C for 2 weeks.