Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins.

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm-raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive-sequence-mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep-PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep-PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.

A novel Henneguya species from channel catfish described by morphological, histological, and molecular characterization.

Channel catfish Ictalurus punctatus from a commercial farming operation in the Mississippi Delta were submitted for examination for the presence of infection by the trematode Bolbophorus damnificus. The fish were instead found to possess skin nodules suggestive of Henneguya pellis, a species previously described in the blue catfish I. furcatus. Despite the dermal location and distribution of lesions, morphological characteristics of the myxospores were inconsistent with H. pellis. Spores possessed a lanceolate spore body 15.4 +/- 1.5 microm (mean +/- SD; range = 12.2-19.3 microm) in length and 5.5 +/- 0.6 microm (range = 4.5-6.8 microm) in width in valvular view, and 4.7 +/- 0.2 microm (range = 4.2-5.0 microm) in width in sutural view. Polar capsules were pyriform and unequal in both length and width and contained polar filaments with six coils. Polar capsules measured 6.1 +/- 0.8 microm (range = 4.0-7.9 microm) long and 1.7 +/- 0.3 microm (range = 1.0-2.2 microm) wide. The caudal appendages were 50.5 +/- 8.3 microm (range = 34.8-71.4 micorm) long and the total length of the spore was 65.9 +/- 8.6 microm (range = 48.2-90.0 microm). The "blister like" plasmodia were round or ovoid, up to 2 mm in diameter, and randomly distributed throughout the epidermis of the fish. Histologically, plasmodia were confined to the dermis and elicited no inflammatory reaction from the fish. A blast search of the 18S small subunit rDNA sequence obtained by polymerase chain reaction amplification resulted in no identical sequence matches but indicated a close relationship to H. gurlei, H. ictaluri, and H. exilis. The unique host record, spore morphology, and novel genetic sequence derived from this isolate lead us to propose this isolate as a novel species, H. sutherlandi.

Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen.

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 microg of free or liposomal Streptococcus mutans crude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P < 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P < 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.

Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase I.

Glucosyltransferase (GTF) enzymes of mutans streptococci are considered virulence factors due to their ability to synthesize adhesive glucans, which facilitate cell-to-cell adherence and accumulation. In this study we report the cloning, expression, and characterization of the catalytic (CAT) and glucan-binding (GLU) domains of S. mutans GTF-I encoded by gtfB. The CAT and GLU polypeptides represent amino acid residues 253 to 628 and 1183 to 1473, respectively, of S. mutans GTF-I. Antibodies to recombinant CAT and GLU were generated in rabbits and purified by affinity chromatography. Purified anti-CAT antibodies significantly inhibited water-insoluble glucan synthesis by S. mutans and S. sobrinus GTFs (P < 0.0001 and P < 0.05, respectively). The purified anti-GLU antibodies significantly inhibited both water-insoluble and water-soluble glucan synthesis by S. mutans GTFs (P < 0.0001 and P < 0.05, respectively). These results demonstrate that anti-CAT and anti-GLU antibodies are capable of inhibiting a variety of GTF activities. Since antibodies to S. mutans in saliva are implicated in protection against disease, we next assessed the ability of CAT and GLU polypeptides to induce mucosal antibody responses in mice. Intranasal (i.n.) immunization of mice with CAT showed significantly (P < 0.005) elevated levels of specific immunoglobulin G (IgG) antibody activity in serum and specific IgA antibody activity in serum, saliva, vaginal washes, and fecal samples. GLU immunized animals showed significantly (P < 0.005) elevated levels of specific IgA antibody activity in serum and vaginal secretions. Taken together, these results demonstrate that the recombinant CAT and GLU polypeptides are effective in inducing both mucosal and systemic immune responses. The ability of these polypeptides to induce a mucosal IgA immune response in mice after i.n. immunization supports their use as subunit vaccine candidates in the development of an anticaries vaccine.

Induction of protective immune responses against Venezuelan equine encephalitis (VEE) virus aerosol challenge with microencapsulated VEE virus vaccine.

Venezuelan equine encephalomyelitis (VEE) virus, a member of the family Togaviridae, genus Alphavirus, causes disease in humans and equids. The virus is normally transmitted by the bite of an infected mosquito however, it can also be highly infectious by aerosol. The purpose of the present study was to determine the effectiveness of formalin-fixed, 60Co-irradiated VEE virus microencapsulated in poly DL-lactide-co-glycolide in inducing immune responses protective against aerosol challenge with virulent VEE virus. Balb/c mice were primed by subcutaneous injection of microencapsulated VEE virus vaccine, followed 30 days later by a single immunization with the same vaccine given via the oral, intratracheal (i.t.) or subcutaneous (s.c.) route. Mice boosted by the i.t. or s.c. route had higher plasma IgG anti-VEE virus levels than orally immunized animals. The responses in the former groups were similar in magnitude to those seen in mice primed and boosted by the i.t. route. Antibody activity was detected in bronchial-alveolar and intestinal washes, fecal extracts and saliva from immunized animals. The levels of IgG and IgA antibody activity in bronchial-alveolar wash fluids from mice boosted by the i.t. route were higher than those seen in animals immunized by the oral or s.c. route with the microsphere vaccine. Mice immunized with the microencapsulated VEE virus vaccine were protected from lethal VEE virus infection following aerosol challenge at approximately three months after the initial immunization. Mucosal immunization via the i.t. route appeared to be the most effective regimen, since 100% of the mice resisted aerosol challenge.

Mucosal immunogenicity of a recombinant Salmonella typhimurium-cloned heterologous antigen in the absence or presence of coexpressed cholera toxin A2 and B subunits.

An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors. These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 [Th1]/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites. CTA2/B may thus be useful as an S. typhimurium-cloned adjuvant for coexpressed protein antigens.

Immunization with a plasmid expressing pneumococcal surface protein A (PspA) can elicit protection against fatal infection with Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a protectioneliciting protein of Streptococcus pneumoniae. We observed that immunization of BALB/c mice with a plasmid expressing PspA significantly protected the mice from lethal challenge with S. pneumoniae when compared to control mice that received injections of the plasmid vector alone. The plasmid construct expressing PspA has been designated pKSD2601. Mice immunized intramuscularly with pKSD2601 had a mean log of colony-forming units of 2.67 +/- 0.25 pneumococci circulating in their blood at 24 h after challenge as compared with control mice that had a mean log of colony-forming units of 4.95 +/- 0.59. Those mice with lower numbers of pneumococci subsequently survived the challenge. Given the quantitative nature and ultimate end point (ie live versus dead) our mouse model should be useful in working out optimum expression of bacterial genes for DNA immunization.

Intratracheal gene delivery with adenoviral vector induces elevated systemic IgG and mucosal IgA antibodies to adenovirus and beta-galactosidase.

One major concern about using adenoviral vectors for repetitive gene delivery to lung epithelial cells is the induction of an immune response to the vector, thus, impeding effective gene transduction. To assess the immune response to the adenoviral vector, repetitive intratracheal (i.t.) gene dosing was performed in CD-1 mice using the replication-deficient adenovirus 5 (Ade5) vector carrying the lacZ gene, and compared to the antibody responses induced by conventional intranasal (i.n.) and intraperitoneal (i.p.) routes of immunization. Kinetics of serum IgG, IgA, and IgM antibody responses to the adenoviral vector and to beta-galactosidase (beta-Gal) were evaluated. Two or three adenoviral vector doses given by i.t., i.n., or i.p. routes resulted in serum IgG titers in excess of 1:200,000, whereas serum IgM and IgA were moderately induced. Analysis of the predominant murine IgG subclass was determined to be IgG2b and IgG2a. To determine the localization of this antibody response, the ELISPOT assay was employed. Lymphocytes were isolated from the lung, the lower respiratory lymph nodes (LRLN), the nasal passages (NP), and the spleen. For i.t- and i.n.-administered mice, the highest IgA spot-forming cell (SFC) response to Ade5 and beta-Gal was located in the NP and in the lung. Both the lung and the LRLN showed elevated numbers of IgG SFCs (4- to 12-fold greater than splenic IgG SFC response) for Ade5 and beta-Gal. This evidence suggests that the lung and associated lymphoid tissues were the source for serum antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of protective immune responses to Venezuelan equine encephalitis (VEE) virus with microencapsulated vaccine.

Venezuelan equine encephalomyelitis (VEE) virus is a mosquito-borne arbovirus of major human health significance in the New World. Currently two forms of VEE virus are used for immunization of humans and horses, i.e. a live attenuated and a formalin-inactivated vaccine. Clinical evidence suggests that these vaccines are not fully efficacious and may produce certain undesirable side-effects. In the present study, microspheres composed of biocompatible and biodegradable poly (DL-lactide-co-glycolide) (DL-PLG) were evaluated for their effectiveness as a delivery system of whole, inactivated VEE virus vaccine for the induction of protective immune responses. Mice receiving 50 micrograms VEE virus in microspheres composed of an equimolar ratio of DL-lactide and glycolide (50:50 DL-PLG) exhibited a primary circulating IgG antibody response which was approximately 32-times higher than the response induced with the same dose of unencapsulated (free) virus. A similar difference in responses was seen with antigen doses ranging from 3.1 to 50 micrograms. A rapid increase in antibody activity was seen after the secondary immunization (day 50). Formalin fixation of inactivated VEE virus was important for immunogenicity since the circulating anti-VEE virus antibody response induced with microencapsulated nonformalin-fixed virus vaccine was lower than that induced with microencapsulated formalin-fixed virus vaccine. Furthermore, at low antigen concentrations, DL-PLG microsphere vaccines prepared with the solvent methylene chloride induced higher antibody responses than those prepared using ethyl acetate as the solvent. Microencapsulated vaccine also induced higher VEE virus neutralization titers than did free virus vaccine. Finally, the microencapsulated virus was more effective than the free virus in inducing immune responses protective against systemic challenge with virulent VEE virus. These results demonstrate that DL-PLG microspheres containing formalin-fixed, inactivated VEE virus were effective in augmenting circulating IgG antibody levels and neutralization titers to the VEE virus following systemic immunization and in affording enhanced protection against systemic challenge with virulent VEE virus. The effects of antigen form and the microsphere processing solvent on the immunogenicity of the vaccine are discussed.

Effect of temperature on the immune system of channel catfish (Ictalurus punctatus)--I. Leucocyte distribution and phagocyte function in the anterior kidney at 10 degrees C.

1. Temperatures of 18 degrees C for acclimation or assay had minimal or no effect on channel catfish phagocyte function. Significant suppression was observed at 10 degrees C acclimation and assay temperature. 2. According to the results of a multiple acclimation/assay temperature combination study, the primary impact of temperature on phagocyte function was due to the assay temperature. 3. The only functional change caused by acclimation temperature was the possible adaptation of the respiratory burst. However, 10 degrees C acclimation did cause a decline in the number of lymphocytes in the anterior kidney but not the number of neutrophils. In a temperature-kinetic study, the suppressive effect of 10 degrees C assay temperature was confirmed. 4. Results of our study indicated that phagocytosis in channel catfish is temperature-mediated. However, phagocytes appeared to be more resistant to low temperature than lymphocytes, which implies the importance of phagocytosis in the defense mechanisms of channel catfish at low temperatures.

Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes.

Four monoclonal antibodies (MAbs) were evaluated for specific reactions to various catfish peripheral blood leucocytes and anterior kidney cells. The MAb 9E1 served as a standard and positive control for all test reactions because of its defined reactivity with channel catfish immunoglobulin and immunoglobulin bearing cells. Of the four MAbs, two have been characterized as being specific for a non-immunoglobulin marker on lymphocytes, thus marking T lymphocytes and two were specific for catfish neutrophils. Morphological, flow cytometric and functional analysis of the reactive cells verified these findings.

Autumn acquisition of nematodes by calves given a morantel sustained-release bolus the preceding spring.

Reinfection with nematodes late in the grazing season was assessed in calves treated the preceding spring with a morantel sustained-release bolus (MSRB). During an initial 155-day grazing period, MSRB-treated calves (n = 15) grazed a pasture used the preceding year for identically treated calves (MSRB pasture). Control calves (n = 15) were not given anthelmintic treatment in the spring and grazed heavily contaminated herbage for the initial 155-day period (control pasture). At the end of the initial grazing period, 3 calves from each group were killed for parasite recovery and counting, with control calves found to harbor 9.2 times more nematodes, compared with the MSRB-treated calves. Nematode counts from tracers killed periodically during the initial grazing period were of similar proportions, reflecting the much greater nematode exposure experienced by the control calves, compared with the MSRB-treated calves. At the end of the initial grazing period, 10 calves (5/group) were placed on a common, contaminated pasture after all were treated twice with fenbendazole (10 mg/kg of body weight, 7 days apart) while on concrete. The second grazing period was for 29 days, followed by a 3-day confinement on concrete. Then, the calves were killed and necropsied. During the 29-day grazing period, the MSRB-treated calves maintained their weight advantage over the control calves, and significant differences in nematode egg counts were not found between the 2 groups of calves. At necropsy, the MSRB-treated calves harbored 27.9% fewer nematodes than did the controls, indicating that prior therapeutic and prophylactic anthelmintic activities of the MSRB did not predispose the animals to enhanced acquisition of nematodes after MSRB protection.

Efficacy of morantel against nematode populations in calves exposed on a pasture stocked for two years with morantel sustained-release bolus-treated calves.

Two groups of 10 parasite-free calves were maintained either for 2 weeks on a pasture grazed by nonmedicated cattle (pasture A) or for 3 weeks on a pasture grazed by morantel sustained-release bolus-treated cattle (pasture B) for the preceding 2 years. After a 4-week holding period to allow for maturation of acquired gastrointestinal nematodes, 5 calves from each group were administered a therapeutic dose (10 mg/kg of body weight) of morantel tartrate. All calves were necropsied 1 week later, and the abomasal and small intestinal nematodes were isolated, identified, and enumerated. A comparison of efficacies between nonmedicated and morantel tartrate-treated calves of each pasture demonstrated that morantel was equally effective against the gastrointestinal nematode infections, regardless of infection source (ie, pasture A vs pasture B). The overall nematode reductions due to morantel tartrate treatment of calves that grazed pastures A and B were 98% and 96%, respectively. It was concluded that the sensitivity of gastrointestinal nematodes to morantel tartrate was not diminished in calves maintained on pasture B, which had been stocked with morantel sustained-release bolus-treated calves for the preceding 2 grazing seasons.

Efficacy of oral clorsulon in the treatment of Fasciola hepatica infections in calves.

Twenty calves at each of 2 Arkansas locations were inoculated with infective Fasciola hepatica metacercariae. After 56 days, the calves at each site were randomly assigned by weight to 2 treatment groups of 10 calves/group; vehicle control or clorsulon at the rate of 7 mg/kg of body weight. All treatments were given orally as a suspension. Calves were killed 6 weeks after treatment and F hepatica counts were performed for all animals. At the 2 sites, mean levels of efficacy were 96% and 91%. Adverse reactions to clorsulon or the vehicle were not observed in the calves.

Controlled field trial on the anthelmintic effectiveness of the morantel sustained-release bolus in grazing calves.

The direct and indirect anthelmintic efficacies of the morantel sustained-release bolus given to calves were assessed in a 154-day controlled field trial. A permanent calf pasture (divided into 2 lots) and naturally parasitized calves were used. The medicated calves were given the bolus at the time they were placed on the pastures. Control calves did not receive anthelmintic therapy. The effectiveness of the bolus to control parasitic gastroenteritis was determined by monitoring various parasitologic determinants. The treated calves had significantly (P less than 0.01) reduced numbers of fecal nematode eggs for every posttreatment sampling period when compared with the control calves. Tracer calves, used periodically during the study to indicate pasture larval infectivities, had equivalent worm burdens at the beginning of the trial (treated vs control pasture). Tracer calves, added later in the study to the lot with treated calves, harbored 83% to 94% fewer nematodes than did their counterparts in the lot with the controls. Plasma pepsinogen concentrations, reflective of abomasal worm burden size and/or activity, were significantly higher (P less than 0.01) in the control than in the treated calves from day 54 until trial termination. At trial termination, the treated calves weighed an average of 27.8 kg more and harbored 80.9% fewer nematodes than the control calves. The morantel sustained-release bolus is an anthelmintic delivery device that has therapeutic and prophylactic antinematode activities. To achieve its optimum performance, the bolus must be used so that the epizootiologic patterns of the predominate parasitic nematodes are effectively disrupted. Generally, internal nematode parasitisms in the calf flourish during the animal's first springtime grazing period.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiparasitic efficacy of ivermectin in naturally parasitized sheep.

Sixteen sheep harboring naturally acquired parasitisms were allocated to 1 of 2 treatment groups: (i) sheep given ivermectin in an oral solution at the dosage rate of 200 micrograms/kg of body weight, and (ii) those given the vehicle at a dosage rate of 0.25 ml/kg. All animals were necropsied at 2 weeks after treatment. Parasites and percentages of parasitic reductions, as demonstrated in this trial, were: Dictyocaulus filaria (99.4%), Oestrus ovis first stage instars (100%), Trichuris ovis (98.9%), Strongyloides papillosus (99.8%), Nematodirus spathiger (100%), arrested 4th stage Nematodirus spp (96.2%), Trichostrongylus colubriformis (100%), T axei (100%), Oster tagia circumcincta (100%), Haemonchus contortus (100%), and arrested Haemonchus spp 4th stage larvae (99.9%). The sheep showed no adverse effects due to ivermectin or vehicle administration.

Antiparasitic effectiveness of ivermectin in the horse.

By way of a controlled trial, the anthelmintic efficacies of the injectable and paste formulations of ivermectin were evaluated in the horse. Treatment was given at the rate of 200 microgram/kg of body weight. Regardless of formulation, 100% removals were demonstrated for Strongylus vulgaris (intestinal), S edentatus, 2nd and 3rd instars of Gastrophilus nasalis and G intestinalis, "small strongyles," Triodontophorus serratus and T tenuicollis. Adult Cylicocyclus insigne populations were eliminated at the rates of 99.9% and 96.3% by the injectable and paste formulations, respectively. Levels of 4th-stage Cylicocyclus spp larvae, as detected in the luminal contents of the colon, were reduced by 88.3% and 33.2% by the injectable and paste formulations, respectively. There were no adverse tissue or behavioral reactions induced by either preparation of ivermectin.

Effectiveness of ivermectin in the treatment of equine Parascaris equorum and Oxyuris equi infections.

Fifteen horses harboring naturally acquired, patent Parascaris equorum and Oxyuris equi infections were equally allotted to 3 treatment groups given (1) injectable vehicle; (2) injectable ivermectin at the dose rate of 200 microgram/kg of body weight; and (3) injectable ivermectin at the rate of 300 microgram/kg. All treatments were given IM in the neck. All animals were killed 14 days after treatment and examined for the targeted nematodes. Regardless of dose rate, ivermectin proved 100% effective in the removal of adult O equi and P equorum infections. Levels of immature P equorum were decreased by 98.5% in both ivermectin-treated groups. Oxyuris equi 4th-stage larval injections were decreased by 95.7% and 99.9% by the 200 and 300 microgram/kg ivermectin treatments, respectively. Adverse reactions to the injections of drug were not seen.