Biologics industry challenges for developing diagnostic tests for the National Veterinary Stockpile.

Veterinary diagnostic products generated ~$3 billion US dollars in global sales in 2010. This industry is poised to undergo tremendous changes in the next decade as technological advances move diagnostic products from the traditional laboratory-based and handheld immunologic assays towards highly technical, point of care devices with increased sensitivity, specificity, and complexity. Despite these opportunities for advancing diagnostic products, the industry continues to face numerous challenges in developing diagnostic products for emerging and foreign animal diseases. Because of the need to deliver a return on the investment, research and development dollars continue to be focused on infectious diseases that have a negative impact on current domestic herd health, production systems, or companion animal health. Overcoming the administrative, legal, fiscal, and technological barriers to provide veterinary diagnostic products for the National Veterinary Stockpile will reduce the threat of natural or intentional spread of foreign diseases and increase the security of the food supply in the US.

Application of recombinant fimbrial protein for the specific detection of Salmonella enteritidis infection in poultry.

A number of disease outbreaks of Salmonella enterica serotype enteritidis (SE) in humans have been traced to the consumption of SE-contaminated egg and egg products. A rapid, specific, and inexpensive method of detecting SE infection in poultry is necessary to reduce human outbreaks. We evaluated rSEF14 fimbrial antigen of SE for specific detection of SE-infected birds in latex agglutination test and enzyme-linked immunosorbent assay. rSEF14 antigen was highly specific in identifying birds infected with SE. The sera from birds infected with closely related serogroup-D Salmonella and other avian pathogens did not react with rSEF14 antigen. The rSEF14 antigen identified antibodies in serum of 88% of birds during the first 2 weeks of infection, and 100% of the birds subsequently. The SE-specific antibodies were detected in egg yolk as early as 6 days post-infection in rSEF14-enzyme-linked immunosorbent assay. Our results suggest that rSEF14-based assays could be used as screening tests for detection of SE antibodies and would overcome the cross reactions observed with existing serological tests.

Development and comparison of serologic methods for diagnosing chicken anemia virus infection.

This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.

Relationship of the enzyme-linked immunosorbent assay to indirect immunofluorescent antibody test for the detection of so-called chicken anemia agent antibodies in serum from broiler breeders.

Serum samples collected from breeder chickens ranging in age from 1 day to 55 weeks were tested for CAA antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA) test. The relationship of ELISA to IFA test was determined. The sensitivity of the ELISA relative to the IFA test was 82.64%, and the specificity of the ELISA relative to the IFA test was 56.25%. Agreement between the ELISA and the IFA test was highly significant (Kappa = 0.74, Z = 5.78). We concluded that the ELISA is as good as the IFA test for detecting CAA antibody in sera from chickens.

Pathogenicity of CL-1 chicken anemia agent.

Twelve-day-old broiler-type chickens had hemorrhagic necrotic wing tips. After 10 blind subcultures in an MDCC-MSB1 cell line, a virus (so-called chick anemia agent [CAA]) was isolated and designated CL-1 CAA. Five-day-old specific-pathogen-free chicken embryos from a commercial breeder flock that were found not to possess antibody against CAA were infected with CL-1 virus via yolk-sac injection. Many (49%) infected embryos were small and apparently had died from severe systemic hemorrhage. Hatched chicks were small and had pale feathers, skin, skeletal muscles, bone marrow, and viscera. All infected chicks had small thymuses. These thymuses often were so small that they could not be found grossly (P = 0.002). Anemia occurred within 4 days post-hatch. Microscopically, all hematopoietic organs were markedly atrophic. Septic necrotizing lesions were seen only in organs from CL-1-injected chicks. Physicochemical and pathological characteristics of this virus indicate that it is similar to other isolates of CAA found in Europe and Japan.

Immunosuppression and intracellular calcium signaling in splenocytes from chicks infected with chicken anemia virus, CL-1 isolate.

Hematocrits, histopathology, concanavalin A-induced lymphocyte proliferation, intracellular calcium signaling, and lymphocyte subpopulations were analyzed over a 6-week period in individual chicks inoculated with the CL-1 isolate of chicken anemia virus. Lymphoid depletion/atrophy was present in the thymus and bone marrow by 11 days post-infection (PI). Anemia was present at 14 days PI. The mean lymphocyte proliferation stimulation index (SI) of the inoculated group was significantly lower than that of the control group at 11 days PI. This response was reversed at 18 days PI, when the SI of the inoculated group was significantly higher than that of the controls; values subsequently returned to baseline. The increase in intracellular calcium levels in CAV-infected chicks and controls paralleled the proliferative response. Percentages of CD3-, CD4-, CD8-, and natural-killer-positive-staining cells decreased significantly at 18 and 25 days PI. The most dramatic decrease occurred in the CD8-positive-staining cell population at 18 and 25 days PI.

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes.

E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 +/- 1 degree C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3',5,5'-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.