Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge.

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.

Epidemic West Nile encephalitis, New York, 1999: results of a household-based seroepidemiological survey.

BACKGROUND: In the summer of 1999, West Nile virus was recognised in the western hemisphere for the first time when it caused an epidemic of encephalitis and meningitis in the metropolitan area of New York City, NY, USA. Intensive hospital-based surveillance identified 59 cases, including seven deaths in the region. We did a household-based seroepidemiological survey to assess more clearly the public-health impact of the epidemic, its range of illness, and risk factors associated with infection. METHODS: We used cluster sampling to select a representative sample of households in an area of about 7.3 km(2) at the outbreak epicentre. All individuals aged 5 years or older were eligible for interviews and phlebotomy. Serum samples were tested for IgM and IgG antibodies specific for West Nile virus. FINDINGS: 677 individuals from 459 households participated. 19 were seropositive (weighted seroprevalence 2.6% [95% CI 1.2-4.1). Six (32%) of the seropositive individuals reported a recent febrile illness compared with 70 of 648 (11%) seronegative participants (difference 21% [0-47]). A febrile syndrome with fatigue, headache, myalgia, and arthralgia was highly associated with seropositivity (prevalence ratio 7.4 [1.5-36.6]). By extrapolation from the 59 diagnosed meningoencephalitis cases, we conservatively estimated that the New York outbreak consisted of 8200 (range 3500-13000) West Nile viral infections, including about 1700 febrile infections. INTERPRETATION: During the 1999 West Nile virus outbreak, thousands of symptomless and symptomatic West Nile viral infections probably occurred, with fewer than 1% resulting in severe neurological disease.

Antibiotic residues in milk following bulbar subconjunctival injection of procaine penicillin G in dairy cows.

OBJECTIVE: To determine whether, and at what time, penicillin enters milk at a concentration that is detectable following bulbar subconjunctival injection in lactating dairy cows. DESIGN: Randomized clinical trial. ANIMALS: 66 Holstein cows that were at least 2 weeks past calving and had not been treated with antibiotics in the preceding 30 days. PROCEDURE: Cows were randomly assigned to receive a treatment of 1 ml (300,000 units) procaine penicillin G by bulbar subconjunctival injection or remain untreated. Composite milk samples were collected immediately before treatment and 4, 10, 16, 22, 28, and 40 hours after treatment. Milk samples were tested by use of a commercial test for beta-lactam antibiotics. RESULTS: Among penicillin-treated cows, the first positive test results were observed 4 hours after treatment, and the last positive result was observed 22 hours after treatment. The percentages of positive test results before treatment and at 4, 10, 16, 22, 28, and 40 hours after treatment were 0, 9, 87, 42, 8, 0, and 0%, respectively. None of the untreated cows had positive test results for beta-lactam antibiotics at any sampling time. CONCLUSIONS AND CLINICAL RELEVANCE: Penicillin was detected in milk for up to 22 hours after a single subconjunctival injection of procaine penicillin G in cows. This result should be considered when recommending milk withholding periods following the administration of penicillin by this route in lactating dairy cows.