A block in glycoprotein processing correlates with small plaque morphology and virion targetting to cell-cell junctions for an oral and an anal strain of herpes simplex virus type-1.

The characteristics of two clinical isolates of HSV-1 obtained from an oral (424) and an anal (490) lesion were compared with the highly passaged KOS strain. In contrast to KOS, the clinical isolates produced small plaques, were more cell-associated and the predominant viral glycoprotein species for gC and gD in infected cell lysates was the precursor, high mannose glycoform. Total virus production in Vero cells was equivalent for the three virus strains in one-step growths. Pulse-chase studies of glycoprotein C processing showed a reduction in rate at 7.5 h post infection and a significant block in processing at 10.5 h post infection for 424 and 490 but not KOS. Similar results were obtained for gD. The significant reduction in glycoprotein processing for 424 and 490 suggests a block in transport of viral glycoproteins or virions to and through the Golgi apparatus. Extracellular virions and the cell surface, prior to cell lysis, contained the processed gC glycoform suggesting a competent cellular glycan processing system. Upon co-infection of 424 or 490 with KOS or a gC- KOS strain, gC was processed to levels equivalent to KOS indicating that 424 and 490 are not inhibitory but that an activity(s) encoded by KOS facilitates maturation of gC from 424 and 490. Unlike KOS infected Vero cells, virion-containing vacuoles were observed in the cytoplasm at 12 h p.i. and extracellular virions were concentrated at cell-cell junctions of 424 or 490 infected cells but not in the perinuclear region. These results suggest that intracellular transport of viral glycoproteins and virions in 424 and 490 infected cells is different from KOS infected cells. The reduced level of viral glycoprotein maturation, virus release, cell surface presence and presence of virions at cell-cell junctions are consistent with small plaque production in tissue culture cells.

Effect of three commercially available adjuvants on the production of antibodies to Pasteurella multocida in broilers.

Three commercially available adjuvants--incomplete Freund's, Ribi adjuvant system, and Hunter's TiterMax--were used to compare antibody responses of broilers to Pasteurella multocida antigen. Differences among treatments over the 9-week study were determined by measuring antibody responses of twice-vaccinated birds using an enzyme-linked immunoassay. The antibody level induced by the vaccine with incomplete Freund's adjuvant was the highest, followed by the vaccines with TiterMax and Ribi.

A comparison of three isolation methods for obtaining immunoglobulin A from turkey bile.

Turkey IgA was isolated from bile by three methods: ammonium sulfate precipitation, polyethylene glycol (PEG) extraction, and lambda-carrageenan extraction. The isolated immunoglobulin fractions were compared using double diffusion, immunoelectrophoresis (IE), and enzyme-linked immunosorbent assay (ELISA). Results indicated that all three methods of isolation are sufficient for the initial isolation step for purification of the immunoglobulin fraction in turkey bile. Because of contaminating IgG, IgM, and other high-molecular-weight proteins, further purification by column chromatography is needed to isolate pure IgA. The lambda-carrageenan extraction method appears to be the method of choice for precipitating the immunoglobulin fraction in bile, because of the high antibody activity after extraction. Like ammonium sulfate precipitation, lambda-carrageenan and PEG extraction are not sufficient as single-step purification methods and should be used as the initial step in the purification of IgA.

Response of broiler chickens to fowl cholera vaccination at 1 to 6 weeks of age.

Broiler chickens, in groups of 10, received a single vaccination by the stick-wing route at 1, 2, 3, 4, 5, 6, or 11 weeks of age with live Clemson University strain of Pasteurella multocida. Twenty non-vaccinates kept in isolation served as controls. Cholera serum antibody titers in all chickens were determined by enzyme-linked immunosorbent assay at weekly intervals. Chickens vaccinated once at 1, 2, 3, 4, 5, and 6 weeks, respectively, attained 25.2%, 28.7%, 34.7%, 46.2%, 51.8%, and 64.6% of the titers of those vaccinated once at 11 weeks of age. Variation in antibody response was greatest in chickens vaccinated at 1 or 2 weeks of age. Additionally, chickens vaccinated at 1 or 2 weeks of age showed the longest response time (5 weeks) to reach maximum antibody titers after a single vaccination. When the original vaccinates were revaccinated at 11 weeks of age, all showed a secondary response equal to or greater than that seen in chickens vaccinated once at 11 weeks of age. Age of the chickens at the time of vaccination and antibody titer were positively correlated (r = 0.997). Overall antibody responses to vaccination were higher and much more uniform as birds increased in age.

Immunity to Pasteurella multocida in protein-deficient chickens.

Studies were conducted to determine the effects of dietary protein restriction on the humoral immunity (HI) and cell-mediated immunity (CMI) of chickens. New Hampshire chickens were separated into two dietary treatment groups: basal, containing 3,200 kcal/kg and 21% protein; or protein restricted (PR), containing 3,200 kcal/kg and 7% protein. In studies involving HI, half of the birds in each dietary treatment were vaccinated against fowl cholera at 4 and 8 wk of age. Blood samples were collected weekly beginning at 4 wk of age. Overall, unvaccinated birds had lower titers than vaccinated birds and PR groups generally showed lower titers than basal groups. All birds were challenged by palatine cleft inoculation of live, virulent Strain X-73 of Pasteurella multocida. The vaccinated PR group survived live challenge as well as the vaccinated basal group, but all unvaccinated birds died as a result of the challenge, regardless of antibody titer. In studies involving CMI, half of the birds in each dietary treatment were vaccinated at 5 wk of age. At 2 to 3 wk postvaccination, representative birds from each treatment were bled for total and differential blood counts. Also, birds were sacrificed and spleen cells collected. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium with phytohemagglutinin-M (PHA-M), sonicated P. multocida (X-73), or RPMI only.(ABSTRACT TRUNCATED AT 250 WORDS)

Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay.

Broiler minibreeder hens were vaccinated for protection against fowl cholera at 12 and 21 weeks of age using several vaccination schemes, which included a live Pasteurella multocida (CU strain) vaccine, two commercial polyvalent fowl cholera oil-based bacterins, and two experimentally prepared polyvalent oil-based bacterins. Some treatment groups received only live or killed vaccines, whereas others received a live vaccine at 12 weeks followed by a killed product at 21 weeks. At 42 weeks of age, all birds that received the live CU vaccine twice or once followed by a bacterin survived challenge. Birds that received killed vaccines only were significantly less protected but still showed a respectable survival rate of 86%. All unvaccinated controls died within 72 hr after challenge. At 72 weeks of age, overall protection was lower than that at 42 weeks, regardless of vaccination treatment. Antibody titers were usually higher in birds that received bacterins than in those receiving live vaccines, yet overall protection was still greater in those birds that received the live cholera vaccine twice.

Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay.

Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.

Comparison of various antigens and their ability to detect protective antibodies against Pasteurella multocida using enzyme-linked immunosorbent assay.

Various antigenic extracts of the CU strain of Pasteurella multocida were prepared to determine their suitability as plate antigens for use in the enzyme-linked immunosorbent assay (ELISA) for the detection of fowl cholera antibodies. Antisera from two separate broiler breeder flocks with known fowl-cholera-vaccination histories were collected just before the birds were challenged with virulent strain X-73 P. multocida. A potassium thiocyanate (KSCN)-extracted antigen, a capsular (CAP) antigen, a lipopolysaccharide-protein antigen, and heat-stable, salt-soluble antigen were all suitable as ELISA plate-coating antigens. Filtered and unfiltered sonicates of the CU strain of P. multocida were also suitable ELISA plate antigens. The results suggested that different plate antigens were detecting different populations of antibodies formed in response to fowl cholera vaccinations. When antibody titers were correlated with survival after challenge, the KSCN and the CAP plate antigens placed more nonsurvivors into low-antibody-titer ranges and more survivors (protected birds) into the high-antibody-titer ranges than the other plate antigens.

Comparison of filter-paper-eluted whole blood with serum in fowl cholera serology using the enzyme-linked immunosorbent assay.

Two methods for collecting blood for measuring antibody activity of Pasteurella multocida were compared. Whole blood was collected on filter-paper strips, dried for 48 hr at room temperature, and then stored in sealed plastic bags at 4 C. Blood was also collected in the usual manner with a needle and syringe, and serum was harvested and stored at -20 C until tested. Eluates of whole blood, obtained by overnight elution of two 4.8-mm discs in 200 microliters of buffered saline at 4 C, were compared with conventionally harvested serum for antibody activity by enzyme-linked immunosorbent assay (ELISA). Paired samples, taken from the same bird at the same time, showed no significant difference (P less than 0.05) in antibody activity as measured by absorbance when the disc-elution process itself was considered to be a 1:20 dilution. It was concluded that eluates of blood, derived from whole blood dried on filter-paper strips, may be used as an alternative to sera in ELISA for measuring P. multocida antibody activity.

Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in broiler breeders vaccinated (wing web) with the CU fowl cholera vaccine. Birds were bled weekly from 9 to 26 weeks, every other week from 26 to 40 weeks, and every 4 weeks from 40 to 56 weeks of age. Overall mean ELISA antibody titers (9 to 56 weeks) reported as log10 values and survivability of the vaccinates after virulent challenge were as follows: unvaccinated--5.75, 48%; birds vaccinated once at 8 weeks--5.91, 78%; birds vaccinated twice at 8 and 14 weeks--6.11, 100%; birds vaccinated thrice at 8, 14, and 20 weeks--6.23, 100%; birds vaccinated twice at 8 and 20 weeks--6.12, 100%; and birds vaccinated twice at 8 and 20 weeks (plus fowl pox at 8 weeks)--6.08, 95%. Survivability in the vaccinates after virulent challenge with strain X-73 Pasteurella multocida was 100% in birds with ELISA antibody titers (log10) greater than 6.02. Under the conditions of this experiment, birds vaccinated two or three times between 8 and 20 weeks proved to be sufficiently immune at 56 weeks of age to withstand a virulent fowl cholera challenge. Birds not vaccinated or vaccinated only once at 8 weeks were not sufficiently immunized to withstand virulent challenge.

Pathogenicity of coccidia in Japanese quail (Coturnix coturnix japonica).

Coccidia were recovered from a field outbreak in commercially raised Japanese quail from South Carolina. After propagation in unmedicated quail, the culture was identified as a mixture of approximately 65% Eimeria uzura, 33% E. tsunodai, and 2% E. taldykurganica. Several pure cultures of E. uzura were obtained by single oocyst isolation. A micropyle was not present in all oocysts; thus, it is not a reliable taxonomic characteristic for identification of E. uzura. Neither the mixed culture nor the E. uzura isolates were infective for Bobwhite quail, Chukar partridge, pheasants, chickens, or turkeys. Seventeen-day-old quail were inoculated with various doses of sporulated oocysts ranging from 5 x 10(2) to 5 x 10(5) of the mixed culture and 1 x 10(3) to 1 x 10(6) of E. uzura. The rate of weight gain was depressed at 3 or 4 days postinoculation (DPT) with as few as 5 x 10(2) oocysts/quail of the mixed culture. As few as 1 x 10(3) oocysts of E. uzura produced a weight loss. Some individual quail had depressed packed cell volume and plasma pigment compared with levels in the uninoculated controls. Plasma protein was not affected. Young quail (3 days old at inoculation) were more susceptible than 17-day-old quail. Infection did not adversely affect body weights of adult quail, although egg production was reduced. Mortality was seen only with the mixed culture (100 and 8% in 3- and 17-day-old quail given 5 x 10(5) oocysts, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary aflatoxin and ammonia-inactivated aflatoxin on Newcastle disease antibody titers in layer-breeders.

Corn containing aflatoxin and the same corn ammoniated to inactivate aflatoxin was incorporated into layer diets to supply 500 ppb and 2.3 ppb aflatoxin, respectively, to determine whether such diets interfere with immunity to Newcastle disease vaccination. Control diets containing uncontaminated corn, both with and without ammoniation, were also fed. A trial 3 months long with 12 birds per treatment was conducted. Vaccination treatments included no vaccination, a single vaccination at the initiation of each trial, and monthly vaccinations for three months. Serum samples, for determination of Newcastle disease hemagglutination-inhibition titers by the microtiter method, were collected just before vaccination and seven days postvaccination, as well as at trial termination. Birds receiving a single initial vaccination and fed a diet containing 500 ppb aflatoxin showed a significant (P less than 0.05) decrease in HI titers, whereas birds similarly vaccinated and fed a diet containing inactivated (ammoniated) aflatoxin showed no reduction in titers regardless of dietary treatment.

The response of broiler breeder chickens to parenteral administration of avirulent Pasteurella multocida.

Broiler breeder chickens were exposed to avirulent Pasteurella multocida at 14, 22, and 34 weeks of age either by stick wing 1 to 3 times or subcutaneously 3 times. Fowl pox vaccine was mixed with the first P. multocida exposure in some groups. Exposure did not impair egg production or hatch of fertile eggs. Challenge with pathogenic P. multocida serotype 1 at 68 weeks indicated that exposure to avirulent P. multocida 2 or 3 times provided better protection than 1 exposure. Mixing fowl pox vaccine with the avirulent P. multocida did not reduce immunity to fowl cholera or fowl pox.

Vaccination of chickens through the drinking water with a live, Pasteurella multocida, vaccine.

Broiler-type chickens were vaccinated orally at several ages with various dilutions of the Clemson University (CU) strain of Pasteurella multocida vaccine and then challenged orally with virulent strain P-1059 or strain X-73 of P. multocida to determine the degree of protection produced by vaccination. When vaccinated at 8 and 12 weeks of age for 2 consecutive days with a 1:133 dilution of the vaccine and then challenged at 17 and 19 weeks of age with strain X-73 and P-1059 P. multocida, respectively, no significant levels of protection were elicited in the vaccinates. Significant levels of protection were elicited in the vaccinates for 2 consecutive days at 9 weeks and at 13 weeks with 1:133 and 1:50 dilutions of the vaccine respectively, and then challenged at 16 weeks with strain X-73 and at 20 weeks with strain P-1059. Broilers vaccinated at 12 and 16 weeks for 3 consecutive days and then challenged at 20 weeks with strain X-73 showed a highly significant level of protection upon challenge when compared to the controls.

Subcutaneous vaccination of chickens with a live, avirulent Pasteurella multocida, Vaccine.

The immunogenic responses to various routes of vaccination of broiler-type chickens to the Clemson University strain of Pasteurella multocida were evaluated. Broilers were vaccinated at 9 or 10 weeks of age using the oral (drinking water), palatine cleft, ocular, or subcutaneous injection routes. All birds were challenged at 11 or 12 weeks of age with virulent X-73 strain of P. multocida by the palatine cleft method. The degree of efficacy of the various vaccination routes differed among three experiments. Nevertheless, the subcutaneous route produced the greatest degree of protection in all experiments and in two experiments differences were highly significant (P less than .01). Protection levels as high as 95% and 97.5% were attained in broilers vaccinated subcutaneously and no undesirable lesions or cheesy masses formed under the skin in the back of the necks of broilers.

Separation of gametocytes of Leucocytozoon smithi from whole peripheral turkey blood.

A technique that uses a Ficoll density-gradient system was devised for isolating gametocytes of the parasitic blood protozoan Leucocytozoon smithi from whole turkey blood. Turkey blood containing gametocytes was layered onto a 35% solution of Ficoll and centrifuged. A white band, containing gametocytes and leukocytes, was observed just below the Ficoll-plasma interphase, clearly separated from the underlying packed erythrocytes. The gametocyte-leukocyte suspension was then subjected to a second centrifugation on a continuous gradient of Ficoll ranging from 12% at the top of the tube to 35% at the bottom. The second centrifugation separated the white cells containing the gametocytes into 5 distinct layers: 1) thrombocytes; 2) gametocytes of L. smithi; 3) lymphocytes; 4) a band of monocytes; and 5) granulocytes. Recovered gametocytes, stored at 4 C in a sterile balanced salt solution, retained their morphological integrity for a minimum of 4 weeks.

Effect of cholera preventative treatments on reproductive performance of turkey hens.

Turkey hens were treated with either the Clemson University (CU) strain of P. multocida, chlortetracycline, bacterin, or no cholera preventative treatment during the growing period. This was followed by CU vaccination during the production period in all except an untreated control group and resulted in no significant differences (P less than 0.05) in egg production, egg weight, fertility, hatchability, body weight, or feed consumption. Best liveability after post-production challenge with P-1059 strain of P. multocida was 92% for those treated with CU vaccine in both the grower and production periods. Hens receiving no cholera preventative treatment had only 8% liveability. These data indicate no adverse effect from treating turkey hens with CU vaccine during the production period.

Leucocytozoon smithi: persistence of gametocytes in peripheral turkey blood.

Ten-week-old Broad-breasted White turkeys were exposed for 2 weeks to a natural infection of Leucocytozoon smithi in Marlboro and Sumter Counties in South Carolina. The birds were then returned to Clemson, South Carolina, and housed in pens proofed against black flies (Simulium sp.) to prevent any reinfection. Blood smears, stained with Giemsa's stain, were prepared at regular intervals to determine the presence of gametocytes of L. smithi after a single natural infectional exposure. Gametocytes were observed in the blood smears for 13 consecutive months, after which the experiment was terminated.