Benjamin Lucio

Pathogenesis of Salmonella enteritidis infection in laying chickens. I. Studies on egg transmission, clinical signs, fecal shedding, and serologic responses.

Laying hens were inoculated orally, intracloacally (IC), or intravenously (IV) with Salmonella enteritidis phage type 8 isolates from a human (E700-87) eggs (Y-8P2), or the ovary of a hen (27A). Oral or IV inoculation of 2 x 10(8) to 4 x 10(8) colony-forming units (CFU) of E700-87 caused depression, anorexia, reduced egg production, diarrhea, and some mortality. Lower doses resulted in milder clinical signs. S. enteritidis was cultured from the shells of a few eggs but not from egg contents. Fecal shedding persisted for up to 6 weeks in some birds. Isolate Y-8P2 (10(6) CFU) also caused anorexia, diarrhea, and a drop in egg production. Hens inoculated orally or IC were less severely affected than those inoculated IV. Fecal shedding was intermittent and lasted up to 18 days. Eggshells from the IC-inoculated birds had the highest rate of contamination, and S. enteritidis was isolated from the albumen of 11 and yolk of three of 726 eggs. Oral inoculation of 10(6) CFU of isolate 27A resulted in a bacteremic infection with seeding of the liver, spleen, peritoneum, ovule, and oviduct. However, the birds remained clinically normal with normal egg production. S. enteritidis was cultured from the yolk and albumen of a small number of eggs until 11 days postinfection. Antigen prepared from S. enteritidis detected antibody in more sera than did commercially available S. pullorum antigen in agglutination tests.

Infectious Bursal Disease Emulsified Vaccine: Effect upon Neutralizing-Antibody Levels in the Dam and Subsequent Protection of the Progeny

Two groups of White Leghorn hens, one vaccinated at 2 weeks against infectious bursal disease (IBD), and the other free of antibodies against IBD virus (IBDV), were vaccinated at 16 weeks old with a suspension or a multiple emulsion of killed IBDV. Untreated hens from each flock were kept as controls. All birds were bled periodically for 50 weeks after vaccination. Twenty-five weeks after vaccination fertile eggs were collected. Antibody levels in the yolk were determined. Chickens hatched from the fertile eggs were bled and challenged with IBDV at weekly intervals to determine the persistence of serum antibodies and resistance to bursal atrophy. Hens revaccinated with the emulsified vaccine and the chickens derived from them had the highest neutralizing and most uniform titers, and complete protection in the chickens lasted to 4 weeks. Two of five chickens were still protected against challenge at five weeks of age. In chickens derived from hens revaccinated with a suspension of killed virus, protection was always partial, being in close relation with age; four of five chicks were protected in the first week of age and the rate of protection diminished to only 1 out of 5 by five weeks of age. Chickens obtained from unrevaccinated hens were protected only during the first week of life.

Identification of the chicken anemia agent, reproduction of the disease, and serological survey in the United States.

An agent with antigenic, physicochemical, and pathological characteristics of chicken anemia agent (CAA) was isolated from broiler chickens and was designated chicken infectious anemia (CIA)-1. CIA-1 was resistant to chloroform treatment and passed through 50-nm-diameter-pore membranes. When inoculated in embryonally bursectomized and/or intact chickens, CIA-1 produced signs and lesions characteristic of CIA: low hematocrit values, pale bone marrow, thymic and bursal atrophy, and enlarged liver. Microscopic lesions were a reflection of macroscopic observations. When injected into 4-week-old chickens CIA-1 induced antibodies against the Cux-1 CAA isolate. In CsCl, CIA-1 had a density of 1.36 g/ml. Antibodies against CAA were found in breeder and commercial chicken flocks from Arkansas, Connecticut, Georgia, Kansas, Maine, Michigan, New York, and Pennsylvania. The age of these flocks ranged from 10 to 78 weeks.

Immunosuppression and active response induced by infectious bursal disease virus in chickens with passive antibodies.

SUMMARY A sequential study was carried out on the interaction of passive antibodies and age with infectious bursal disease virus (IBDV) infection. Chickens with high levels of passive antibodies were protected from bursal atrophy and immunosuppression, and their active immune response to IBDV was inhibited. As their passive antibodies waned, their active immune response increased. Chickens with passive antibodies were protected from immunosuppression for up to 4 weeks, while protection against bursal atrophy lasted for only 1 or 2 weeks. Infection with IBDV suppressed the immune response to Salmonella pullorum antigen in chickens 4 weeks old or younger. The response to bovine serum albumin was suppressed only in 1- and 2-week-old chickens.

Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus.

Three virus isolates (ECV-1, -2, and -3) recovered from cloacae of chickens in flocks that experienced drops in egg production were identified as infectious bronchitis virus (IBV), based on characteristic embryo lesions, chloroform sensitivity, coronavirus morphology, and serology. Because these isolates were recovered from the cloacae of the hens, their tissue tropism was compared with the prototype strain of IBV, Massachusetts-41 (M-41), in experimentally inoculated chickens. During the 39-day period postinoculation (PI), virus isolation was attempted from digestive and respiratory tracts, kidney, and cloacal swabs. ECV-1, ECV-2, and M-41 were more frequently recovered from the cecal tonsils than from other tissues. ECV-1, ECV-3, and M-41 were also recovered from kidney for up to 39 days PI. ECV-2 and ECV-3 had a limited distribution in respiratory tissues, being isolated only sporadically from trachea, bronchus, and lung. Surprisingly, ECV-2 was isolated from esophagus at 2, 16, 30, and 39 days PI; otherwise, its distribution in other tissues was sporadic. Results confirmed that IBV, including M-41, can infect a variety of tissues and that some isolates may be recovered frequently from digestive tract tissues, particularly from the cecal tonsils.

Humoral Immune Responses to Chicken Infectious Anemia Virus in Three Strains of Chickens in a Closed Flock

This is a comparative study on seroconversion to chicken infectious anemia virus (CIAV) in a closed flock of specific-pathogen-free chickens undergoing a natural outbreak and after vaccination of some of these flocks with a commercial, live vaccine. The N2a strain (B21B21 haplotype) had the highest seroconversion after natural infection (94%) or vaccination (100%), followed by the P2a strain (B19B19) at 75%-82% seroconversion after natural infection and 85% seroconversion after vaccination. The S13 (B13B13) chickens were 26% seropositive after natural infection and 75% seropositive after vaccination. N2a chickens with polymerase chain reaction (PCR)-positive tissues were 97% seropositive compared to 80%-83% PCR-positive and seropositive for the P2a chickens and only 8% seropositive and PCR-positive for the S13 chickens. Seroconversion occurred at or near sexual maturity after natural infection in seven flocks studied.

Determination of the Detection Limit of the Polymerase Chain Reaction for Chicken Infectious Anemia Virus

Chicken infectious anemia virus (CIAV) DNA in infected cell cultures and chicken tissues was detected using a polymerase chain reaction (PCR) assay. The complete CIAV genome of several strains was amplified in two segments with two sets of primer pairs. The DNA segments of four CIAV strains and full-length Cux-1 strain DNA were cloned. After amplification, 100 original genome equivalents were detected by Southern hybridization. The sensitivity of the assay was enhanced considerably by performing a reamplification with nested primers. This modification permitted the detection of one molecule of CIAV DNA. Some problems of the assay and its possible application are discussed.

Depletion of CD4+ and CD8+ T Lymphocyte Subpopulations by CIA-1, a Chicken Infectious Anemia Virus

The suppressive effect of chicken infectious anemia virus (CIAV) on T-lymphocyte subpopulation was evaluated in vivo by flow cytometry and dual immunostaining on frozen sections. Between 14 and 21 days postinoculation (PI), the percentage of CD4-, CD8-, and CT1-positive (CD4+, CD8+, and CT1+) cells was significantly lower in chickens infected at 1 day of age with CIA-1 strain of CIAV than in controls. The mean percentage of CD4+ cells in the thymus was only 43%, whereas in the controls it was 77%. The mean percentages of CD8+ cells in the thymus in infected and control chickens was 54% and 90%, respectively, and of CT1+ cells, 44% and 92%, respectively. At 28 days PI, the percentage of CD4+, CD8+, and CT1+ cells was similar in infected and control chickens. Also at 14 and 21 days PI, immunoperoxidase staining demonstrated fewer CD4+, CD8+, and CT1+ cells in the thymus of infected chickens than in controls. In frozen sections of thymus stained with CIA-1 antibodies and CD4, CD8, or CT1, few of the cells positive for CIAV antigen seen in the outer zone of the cortex carried CD4, CD8, or CT1 molecules. These results suggest that CIA-1 infection either destroys cells expressing CD4, CD8, or CT1 molecules on their surface or interferes with the expression of these molecules.

Differentiation and detection of infectious bronchitis virus subtypes by immunofluorescence.

A wide range of cross-reactions among strains was found when immunofluorescence was used as a method for differentiation of IBV strains. Only conjugates prepared against 97 and Massachusetts strains were able to detect all the strains of IBV included in this study. The fluorescence with heterologous strains was weaker than the one present when tested with homologous strains. When the rinsing period after staining was extended from 10 minutes to 2 hours, the cross-reactions were markedly diminished, and only 97 and Holte conjugates gave bright fluorescence with other than their homologous strains. Holte conjugate gave good fluorescence with virus 97, and the 97 and 609 conjugates with Gray virus. Thus, the fluorescent antibodies appear to offer possibilities for the differentiation of IBV isolates. When infected hens were examined, IBV antigen was detected by fluorescent antibodies only in the larynx, trachea, and lung. The conjugates contained antibodies against Mareks Disease agent. In addition they produced some fluorescence which behaved in the bird tissues as a specific reaction but was eliminated by absorption with chicken or rabbit liver tissue powder. Therefore, when diagnosing IBV or any other disease in chicken tissue one should be aware of the possibility of extraneous antibodies or antigens even in chickens which appear healthy and are from a specific pathogenfree flock.

Abrogation of age-related resistance to chicken infectious anemia by embryonal bursectomy.

Embryonally bursectomized (Ebx) chickens developed signs and lesions typical of chicken infectious anemia (CIA) when infected with CIA-1 isolate of chicken infectious anemia virus (CIAV) at 21 or 38 days of age. In both cases, the chickens had low hematocrit values after the 14th day of inoculation, and the percentage of CD4+ and CD8+ cells in the thymus was markedly reduced at 21 days postinoculation. Even though intact chickens became infected, they never developed low hematocrit values. The data support the hypothesis that age-related resistance to CIA is antibody-mediated and is not due to disappearance of the CIAV target cell; the data also suggest that CD4+/CD8+ cells are the target for infection.