Terry L. Bowersock

Oral vaccination with alginate microsphere systems

Abstract Oral vaccination is a simple, efficient way of inducing immunity at mucosal surfaces. The slow development of oral vaccines has been mainly due to the lack of suitable delivery systems. We have used hydrogel microspheres to deliver various vaccines to several animal species by oral administration. Oral delivery of vaccines using alginate microspheres elicited the production of secretory IgA (sIgA) at the mucosal surfaces in mice, rabbits, and cattle. Oral vaccination of chicken resulted in an increased delayed-type hypersensitivity, a cell-mediated immune response, indicating a positive response to the vaccine. Our studies have clearly shown that alginate microspheres are effective for the oral administration of vaccines.

Streptococclcs suis infection in swine: a retrospective study of 256 cases. Part II. Clinical signs, gross and microscopic lesions, and coexisting microorganisms

A retrospective study of 256 cases of naturally acquired Streptococcus suis infections in swine submitted to the Indiana Animal Disease Diagnostic Laboratory from 1985 to 1989 was undertaken to describe the clinical signs, lesions, and coexisting organisms associated with S. suis serotypes 1–8 and 1/2. Infected pigs generally had clinical signs and gross lesions referable to either the respiratory system or to the central nervous system (CNS), but not both. Neurologic signs were inversely related to gross lesions in the respiratory tract (R 2 = −0.19, P = 0.003), as were respiratory signs and gross lesions in the CNS (R 2 = −0.19, P = 0.003). Suppurative bronchopneumonia was the most common gross lesion observed (55.2%, overall). Fibrinous and/or suppurative pleuritis, epicarditis, pericarditis, arthritis, peritonitis, and polyserositis were also reported. In 68% of the pigs, other bacteria in addition to S. suis were isolated. Escherichia coli (35.0%) and Pasteurella multocida (30.0%) were the most commonly recovered bacterial agents. Mycoplasma and viral agents were identified less often, and their role in the development of streptococcosis was difficult to assess. In pigs infected with serotypes 2–5, 7, 8, and 1/2, suppurative meningitis with suppurative or nonsuppurative encephalitis, suppurative bronchopneumonia, fibrinopurulent epicarditis, multifocal myocarditis, and cardiac vasculitis were the most common microscopic lesions observed, whereas pigs infected with serotype 1 generally presented with suppurative meningitis and interstitial pneumonia. Microscopic lesions were morphologically similar among serotypes and were also similar to those reported with other pyogenic bacteria. The distribution of clinical signs and the gross and microscopic lesions in pigs infected with S. suis varied among serotypes. However, these differences were not statistically significant and could not be used to distinguish between the various serotypes. These findings suggest that in pigs infected with S. suis, suppurative or fibrinopurulent inflammation in brain, heart, lungs, and serosae predominates and that bacterial culture is needed to confirm a diagnosis of streptococcosis in swine and to differentiate this disease from those caused by other pyogenic bacteria.

Induction of pulmonary immunity in cattle by oral administration of ovalbumin in alginate microspheres

Respiratory infectious diseases are an important cause of economic losses to the cattle industry. There is a need for an effective, easy to administer vaccine to the critical bacterial pathogens that cause pneumonia in cattle. An orally administered vaccine could be given to a large number of animals without significant stress to the animals and with minimal labor. The purpose of this study was to determine whether the oral administration of a model antigen (ovalbumin) in alginate microspheres could induce pulmonary immunity in cattle. Calves were vaccinated orally with ovalbumin (OVA) following either a subcutaneous (s.c.) or oral priming dose of OVA. Calves primed and boostered by oral administration (oral/oral) of OVA encapsulated in alginate microparticles had increased numbers of antigen-specific IgA ASCs (ASCs) in bronchoalveolar lavage (BAL) fluids. Calves that received a s.c. priming followed by an oral booster inoculation (s.c./oral) of OVA in alginate microspheres had a greater number of anti-OVA IgA, IgG1 and IgG2 ASCs in BALF. S.c./oral calves also had increased numbers of anti-OVA IgG1 ASCs in peripheral blood whereas oral/oral calves had none. S.c./oral calves had increased anti-OVA IgG1, IgG2, and IgA titers in BALF, and IgG1 and IgG2 in serum compared to both oral/oral and sham vaccinated calves. These results indicate that oral administration of antigen encapsulated in alginate microspheres results in a mucosal immune response in the respiratory tract of cattle. Furthermore, s.c. priming both enhanced the IgA response and stimulated an IgG1 and IgG2 response not seen in oral/oral calves. The difference in antibody isotype results suggest that design of the vaccination protocol can direct antibody responses as needed for a specific immunization program.

Immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine.

Pasteurella multocida is an important bacterial pathogen of domestic rabbits. To evaluate the ability of a thiocyanate extract (PTE) of P. multocida to stimulate an immune response and protect against infection with P. multocida, rabbits were immunized subcutaneously or intranasally on Days 7, 21 and 35. Cholera toxin, a potent mucosal adjuvant, was included in one treatment group. Rabbits immunized subcutaneously (SC) or intranasally (IN) had significant increases in serum anti-PTE IgG but not IgA. In contrast, only rabbits immunized IN with PTE developed significant titers of nasal lavage anti-PTE IgA and cholera toxin significantly enhanced this response. In a second study rabbits were immunized via the drinking water with PTE incorporated into alginate microparticles on Days 7, 14 and 21. Mild increases in serum IgG were noted in rabbits immunized with PTE in microparticles, with or without cholera toxin, and this increase was significant (P<or=0.05) on Day 21 for rabbits receiving PTE and cholera toxin. Nasal lavage anti-PTE IgA was significantly (P<or=0.05) increased in rabbits immunized orally with PTE, with or without cholera toxin, in microparticles. This effect was not enhanced by cholera toxin. Rabbits orally immunized with PTE in microparticles had significantly fewer colony forming units of homologous P. multocida recovered from the lungs and nasopharynx following an intranasal challenge. These results demonstrate that PTE incorporated into alginate microparticles and administered orally is immunogenic and confers protective immunity.

Oral immunization of rabbits against Pasteurella multocida with an alginate microsphere delivery system.

Oral delivery of microencapsulated antigens is a potential means to vaccinate rabbits against Pasteurella multocida, a common bacterial pathogen. Groups of five rabbits were dosed orally on days 0, 7, and 14 with alginate microspheres prepared to contain no added protein, 5 mg of a potassium thiocyanate extract of P. multocida (PTE), or 5 mg of PTE with 200 micrograms of cholera toxin (CT). In addition, groups were dosed orally with 5 mg of soluble PTE with or without 200 micrograms CT, intranasally (IN) with 1 mg of soluble PTE, or with saline. Serum and nasal lavage samples collected prior to initial immunization and 10, 16, and 21 days later were assayed by ELISA for anti-PTE IgG and IgA. Strong nasal lavage IgA and serum IgG activities were found in samples from rabbits immunized with PTE IN or orally when incorporated into microspheres. Addition of CT did not significantly enhance either response. To examine the development of protective immunity, groups were similarly immunized and challenge-exposed IN on day 16 with 10(6) CFU of P. multocida. One week later, rabbits were euthanized, and specimens from the lungs, nasopharynx, liver, and inner ear were cultured for P. multocida. Less severe infections of the lung and nasopharynx developed in rabbits immunized with PTE IN or orally in microspheres, with or without added CT. In addition, culture of liver and tympanic bullae samples from these rabbits yielded growth of P. multocida less frequently compared to other P. multocida-challenged rabbits. Coadministration of CT and PTE did not significantly improve protective immunity to challenge.

Multiple serotypes and strains of Streptococcus suis in naturally infected swine herds.

We present a brief description of the epidemiology, clinical signs, and lesions in 21 accessions in which multiple serotypes of S. suis or multiple distinct isolates (strains) of the same capsular serotype of S. suis were identified. Isolates of the same capsular serotype that had different antibiogram profiles were considered to be distinct isolates or “strains” for the purposes of this study. Streptococcus suis isolates of the same capsular serotype and identical antibiogram profiles were considered to be identical organisms. These cases were selected to determine whether or not herds infected with multiple serotypes (or strains) of S. suis had unique features that might serve to distinguish these herds from those infected with a single serotype and strain of S. suis and to provide additional information on the development of this disease in swine. Selected cases were limited to those in which isolates were identified by capsular serotyping. Untypeable isolates of S. suis were not included in this study. Case selection criteria, data collection procedures, etc., were as previously described. 12 Of 277 accessions in which S. suis was identified, 21 accessions (7.6%) involving 37 pigs were cases in which 46 different isolates of S. suis were identified. The distribution of these isolates is shown in Tables 1 and 2. Because of the small sample size, statistical analyses were not performed. Although there was a slight increase in recovery of S. suis in the fall and winter months for all serotypes, S. suis was readily isolated throughout the year. In this study, there was a slight increase (approximately 5%) in the prevalence of serotypes 3, 7, and 8 and a decrease (approximately 10%) in the prevalence of serotype 2 from those reported previously. 12

The potential use of poly(methacrylic acid) hydrogels for oral administration of drugs and vaccines to ruminants

Abstract Poly(methacrylic acid) hydrogels were investigated for the delivery of a model antigen to the lower gastrointestinal tract of sheep. Hydrogels were tested by incorporating a radiopaque material, administering them orally to a sheep, and then radiographing the sheep. The potential for loading high molecular weight proteins into hydrogels was determined by absorbing hydrogels with culture supematants of the bacterium Pasteurella haemolytica . The hydrogels were dried, hydrated, and the culture supematants eluted. The eluents were assayed for the presence of the 102 kDa proteinaceous exotoxin. The hydrogels readily bypassed the first stomach and swelled releasing a radiopaque dye into the lower gastrointestinal tract. Chromium-loaded hydrogels were then administered to a sheep and intestinal contents were collected for 5 days. Chromium was detected in the intestinal contents of the sheep for 96 h with peak levels detected at 12–15 h after administration. Eluents of the hydrogels loaded with culture supematants contained readily detectable amounts of the proteinaceous exotoxin. PMA hydrogels were then absorbed with a vaccine consisting of culture supematants of a pulmonary bacterium P. haemolytica . Hydrogels containing vaccine were administered orally to calves. Calves were challenged by an intrabronchial dose of bacteria. The length of time each calf survived was noted. All surviving calves were killed 3 days post-challenge. A post-mortem examination was performed to evaluate the severity of the pneumonic lesions. Vaccinated calves had less pneumonia and lived longer than control calves. Results of this study indicate that poly (methacrylic acid) hydrogels could be used to administer drugs and proteinaceous vaccines orally to ruminants.

Streptococcus Suis Infection in Swine: A Retrospective Study of 256 Cases. Part I. Epidemiologic Factors and Antibiotic Susceptibility Patterns

A retrospective study of 256 cases of naturally acquired Streptococcus suis infections in swine submitted to the Indiana Animal Disease Diagnostic Laboratory from 1985 to 1989 was performed to determine the epidemiologic factors and antibiotic susceptibility patterns associated with S. suis serotypes 1–8 and 1/2. A standardized computer form was used to record the history, signalment, and clinical signs obtained from the records of selected cases and the microscopic lesions identified after review of the histopathology slides for each case. A computer statistics package (SAP) was used to evaluate the data. Although the number of recovered S. suis isolates increased in the fall and winter months, most serotypes were readily isolated throughout the year; only serotypes 1, 4, 7, and 1/2 increased in frequency of isolation in the fall, winter, and spring months. The majority (6 1.1%) of infected pigs in this study were < 12 weeks of age. More than 75% of pigs infected with serotypes 1, 6, 7, and 1/2 were < 12 weeks of age. There was extensive overlap in the age distributions for pigs with each serotype, and statistically significant differences for most serotypes were not observed. Fifty percent of pigs infected with S. suis serotypes 1 and 1/2 were 3–10 weeks of age, 50% of pigs infected with serotype 2 were 6–14 weeks of age, and 50% of pigs infected with serotypes 3, 4, 5, 7, and 8 were 2–16 weeks of age. Isolates of S. suis were not uniformly susceptible to penicillin, and a large percentage of isolates were resistant to many antibiotics in common usage. The results of this study indicated that the various serotypes of S. suis could not be readily separated based on antibiograms, epidemiologic factors (herd size, breed, etc.), or geographic location.

Prevention of Bacteremia in Dogs Undergoing Dental Scaling by Prior Administration of Oral Clindamycin or Chlorhexidine Oral Rinse

Dogs with periodontitis were used to determine the efficacy of an oral regimen of clindamycin versus chlorhexidine acetate oral rinse in reducing the total number of bacteria and the incidence of bacteremia before and after dental scaling. Aerobic and anaerobic bacteria, isolated from blood and gingival swab cultures, were identified to genus using an automated system.

Systemic and pulmonary immune response to intrabronchial administration of ovalbumin in calves

Local immunization of the respiratory tract may be the best way to achieve protection against respiratory pathogens. In order to do so successfully, it is important to fully understand how the immune response to antigen administered via the respiratory route develops. We studied the respiratory and systemic immune response after subcutaneous (SC) and intrabronchial (IB) inoculation of calves with ovalbumin (OVA). Eight calves received two SC inoculations of OVA and eight other calves received two SC and three additional IB inoculations of OVA. The occurrence of OVA-specific antibodies and antibody-secreting cells (ASC) was measured over time using isotype-specific enzyme linked immunosorbent assay (ELISA) and ELISPOT. SC immunization of calves did not result in OVA-specific IgA in bronchoalveolar lavage (BAL) fluid. Subcutaneous priming followed by intrabronchial challenge caused an initial IgG1 response in the bronchoalveolar lavage fluid, followed by a large IgA response. The presence of IgG1-ASCs indicated that the IgG1 was at least partially locally produced. Most of the OVA-specific IgA in the BAL fluid was secreted by pulmonary ASCs as indicated by the large number of IgA-ASCs in BAL samples and the low serum level of OVA-specific IgA. Antigen-specific IgG1 ASCs were detectable among peripheral mononuclear cells after culture with OVA.