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.

Derivation of Pasteurella multocida-free rabbit litters by enrofloxacin treatment.

Pasteurella multocida is an important bacterial pathogen of rabbits that is easily transmitted from infected does to their kits prior to weaning. Enrofloxacin, a flouroquinolone antibiotic, is effective at limiting nasal carriage of P. multocida in rabbits. To determine if enrofloxacin treatment of pregnant does infected with P. multocida can be used to produce P. multocida-free litters, groups of 3 rabbits were inoculated intranasally on day 10 of gestation with 1.0 x 10(6) P. multocida CFUs. Beginning on day 14, one group received enrofloxacin IM (5 mg kg-1, BID), and a second group received enrofloxacin in the drinking water (200 mgl-1). IM treatment continued until kindling, while PO treatment continued 1 week after kindling. A third group was infected but received only IM saline, and a fourth group was infected but not treated. In addition, a fifth group was neither infected nor treated. Culture of nasal lavage samples and tissues from does and kits showed that both routes of enrofloxacin treatment failed to completely eliminate P. multocida from does, but all kits from enrofloxacin-treated does were free from P. multocida. These results suggest that treatment does with enrofloxacin during the periparturient period may interrupt transmission of P. multocida from infected does to their kits and that this treatment may be useful for deriving Pasteurella-free rabbits from infected does.

Induction of systemic and mucosal immune response in cattle by intranasal administration of pig serum albumin in alginate microparticles.

Biodegradable microparticles are an efficient mucosal delivery system that protect antigens from the harsh mucosal environment and facilitate their uptake by M cells at the epithelium of mucosal-associated lymphoid tissue. In this study, we determined the systemic and mucosal immune response in calves following intranasal and oral immunization with pig serum albumin (PSA) encapsulated in alginate microparticles. The size of the particles ranged from 1 to 50 microm in diameter, with 95% of the particles being smaller than 5 microm. High levels of anti-PSA IgG1 antibodies were found in the serum, nasal secretions, and to a less extent in saliva of calves vaccinated intranasally, but not orally, with PSA-microparticles. There was no significant increase of PSA-specific IgA. A weak lymphocyte proliferative immune response was observed in peripheral blood mononuclear cells (PBMCs), and few anti-PSA antibody-secreting cells (ASC) were detected in the blood of calves immunized intranasally. The combined systemic and mucosal response observed in intranasally immunized animals may be attributed to the wide variation in the size of the alginate microparticles, with smaller particles translocating to regional lymph nodes and inducing a systemic immune response, and larger particles being retained in the NALT and inducing a mucosal immune response. The procedure presented here may be useful as an intranasal vaccine against respiratory diseases in cattle.

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.

Use of ELISA to detect toxigenic Pasteurella multocida in atrophic rhinitis in swine.

The use of an enzyme-linked immunosorbent assay (ELISA) as a means of detecting dermonecrotoxin-producing strains of Pasteurella multocida was investigated. The assay was evaluated as a means to identify toxigenic P. multocida isolates recovered from nasal secretions of swine with atrophic rhinitis. The sensitivity and specificity of the ELISA for detecting dermonecrotoxin-producing P. multocida strains were compared to those of mouse-inoculation and cytotoxicity assays. The ELISA was highly sensitive and more specific than animal inoculation or tissue culture assay and is thus a more effective method for screening swine herds for the presence of toxigenic strains of P. multocida. The ELISA is a rapid, effective, economical way to identify toxigenic P. multocida isolates.

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 (SAS) 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 (61.1%) 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Streptococcus 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 (R2 = -0.19, P = 0.003), as were respiratory signs and gross lesions in the CNS (R2 = -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.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsphere uptake by the intestine of White Leghorn chickens.

A study was conducted to determine the effective size for latex microsphere uptake in the intestine of white leghorn chickens. Three trials were conducted in which ligated intestinal segments of anesthetized 8-wk-old chickens were injected with 0.2-, 0.5-, 2-, 6-, 10-, or 20-mu diameter fluoresceinated latex microspheres. Microspheres were counted in brush border, epithelium, and lamina propria of each intestinal segment, liver, and spleen. After 1 hr, the 0.2-, 0.5-, and 2-mu microspheres were oriented along the brush border of epithelial cells and microsphere uptake into the epithelium and lamina propria was observed in the duodenum, ileum, cecum, cecal tonsil, and colon. Uptake of microspheres of 6, 10, and 20 mu diameter into epithelium and lamina propria was not observed in any intestinal segment. Also, no microspheres of any diameter were observed in sections of liver and spleen to suggest that there was no appreciable entry of microspheres into the bloodstream within 1 hr after administration. The results indicated that uptake of microspheres by the chicken intestine is a size-dependent process with microspheres < or = 2 mu being taken up to an equal extent by most segments of intestine.

Effect of spectinomycin on Escherichia coli infection in 1-day-old ducklings.

Two challenge trials and one confirmation trial were conducted to evaluate the efficacy of spectinomycin in the treatment of 1-day-old ducklings infected with Escherichia coli. In the challenge trials, ducklings were injected in the right posterior thoracic air sac with 0.2 cm3 of broth containing 10(8) colony-forming units E. coli (strain O78, E38)/ml. Spectinomycin at dosage levels of 2.5 mg, 5.0 mg, and 10.0 mg of activity was injected subcutaneously 6 hours following infection. The confirmation trial was conducted to confirm the challenge trials; procedures were similar to those used in the challenge trials, except that only the 5.0 mg of activity dosage of spectinomycin was used. In both types of trials, spectinomycin-treated ducklings had significantly lower mortality and higher average weight gain, average daily gain, and feed consumption than infected unmedicated controls. These results indicate that spectinomycin is effective in treating ducks for experimentally induced colibacillosis caused by E. coli (strain O78, E38).

Intranasal vaccination of New Zealand white rabbits against pasteurellosis, using alginate-encapsulated Pasteurella multocida toxin and potassium thiocyanate extract.

OBJECTIVE: We evaluated the efficacy of intranasal administration of Pasteurella multocida toxin (PMT) and a potassium thiocyanate extract of P. multocida (CN) encapsulated in alginate microspheres, compared with unencapsulated PMT and CN antigens, in protection of rabbits against pasteurellosis. METHODS: New Zealand male rabbits (n=24) were allotted randomly into four intranasally administered vaccine groups: 1, PMT/CN; 2, microencapsulated PMT/CN with or; 3, without subcutaneous priming; and 4, empty microspheres (control). Blood samples and nasal wash specimens were collected before vaccination and one week after each vaccination (days 7, 21, 35, and 49). Rabbits were primed subcutaneously with either unencapsulated PMT/CN or aluminum hydroxide (control) (day 0), vaccinated intranasally (days 14, 28, and 42), challenged intranasally with live P. multocida (day 56), and necropsied (day 60). RESULTS: Compared with controls, PMT/CN-immunized rabbits had significantly higher concentrations of serum IgG and IgM, nasal IgG, and bronchoalveolar lavage fluid IgA and IgG against CN. Immunized rabbits had 100% survival rate and low numbers of bacteria in liver and lungs; the control group had 50% survival rate and higher numbers of bacteria (> 4x) per gram of tissue in liver and lungs. CONCLUSION: The PMT/CN microspheres stimulated systemic and mucosal immune responses similar in effectiveness (protection) to those in response to unencapsulated PMT/CN administration.

Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves.

The role of neutrophils in the development of peracute lung lesions of bovine pneumonic pasteurellosis was investigated. Eight calves were divided into two groups of four calves each. Group I was treated with intravenous phosphate-buffered saline and served as the neutrophil sufficient calves. Group II was treated with intravenous hydroxyurea which produced a state of neutropenia. When peripheral blood neutrophil numbers dropped below 300 cells/microL in group II, all calves were challenged with an intrabronchial bolus of Pasteurella haemolytica in the log phase of growth. An acute inflammatory process occurred in both groups of calves indicated by a rise in body temperature. While pulmonary lesions occurred in both groups by six hours postinoculation, they varied in pathological characteristics. Pulmonary lesions in the neutrophil sufficient calves consisted of fibrinopurulent alveolitis-bronchiolitis with associated alveolar septal necrosis, interlobular edema, and intravascular thrombi. The neutrophil deficient calves had extensive intra-alveolar edema, interlobular edema, intraalveolar hemorrhage, atelectasis, and focal areas of alveolar septal necrosis. These results show that P. haemolytica can induce severe pulmonary tissue damage through both neutrophil dependent and neutrophil independent mechanisms.

Induction of pulmonary antibodies to Pasteurella haemolytica following intraduodenal stimulation of the gut-associated lymphatic tissue in cattle.

The induction of pulmonary antibodies to a bacterial antigen following intraduodenal (D) stimulation of the gut-associated lymphatic tissue (GALT) was investigated. Six calves were divided into two groups of three calves each. The GALT-primed calves received an ID dose of live Pasteurella haemolytica A1 followed by a subcutaneous (SC) dose of killed P. haemolytica. The sham-primed calves received an ID dose of phosphate-buffered saline solution (PBSS) followed by a SC dose of killed bacteria. Serum and pulmonary lavage fluids were collected weekly from each calf and assayed for titers of leukotoxin neutralizing antibodies (LNA), as well as IgG and IgA (lavage fluids only) to P. haemolytica. The GALT-primed calves responded to the ID stimulation by bacteria with increased serum IgG. The sham-primed calves had no change in antibody titers following ID stimulation. The GALT-primed calves had increased serum IgG, lavage IgG and IgA and increased LNA titers in both lavage fluids and serum following the SC dose of killed bacteria. The sham-primed calves demonstrated only an increase in serum IgG following the SC inoculation. A challenge study to evaluate if antibodies induced by GALT stimulation could reduce pulmonary lesions was performed using six calves divided into two groups. One group received an ID dose of P. haemolytica followed two weeks later by a SC dose of killed P. haemolytica. The sham vaccinated calves received an ID dose of PBSS followed in two weeks by a SC dose of killed bacterin. Calves were challenged by an intrapulmonary dose of live P. haemolytica A1 eleven days after the SC inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary immunity in calves following stimulation of the gut-associated lymphatic tissue by bacterial exotoxin.

Antibodies in serum and pulmonary lavage fluids were measured in calves following stimulation of the gut-associated lymphatic tissue (GALT) by inoculation of crude leukotoxin of Pasteurella haemolytica into the duodenum through a surgically placed catheter. Nine calves free of P. haemolytica were divided into two groups. Group 1 received an intraduodenal (ID) inoculation of leukotoxin and group 2 received an ID inoculation of phosphate buffered saline. Serum and pulmonary lavage fluids were collected weekly and assayed for antibodies specific to P. haemolytica including immunoglobulin (Ig)G, leukotoxin neutralizing antibodies (LNA), and IgA (lavage fluids only). The multiplicative increase (over baseline) in each class of antibody titer following ID inoculation of leukotoxin, the composite geometric mean increase of all antibodies together, and the composite number of the five antibody titers which increased at least fourfold were computed. Results showed that the geometric mean of each antibody titer and the two composite indices was higher in the GALT-primed groups than in the sham-primed group. The differences were statistically significant (p less than 0.05) for serum IgG and for the two composite indices. This experiment demonstrates for the first time that GALT stimulation by bacterial exotoxins results in increased pulmonary antibody levels in calves.

Hematological changes in calves exposed to a mixture of lipopolysaccharide and crude leukotoxin of Pasteurella haemolytica.

The purpose of this investigation was to determine if culture supernatants of Pasteurella haemolytica containing crude leukotoxin and lipopolysaccharide (CLCL) causes disseminated intravascular coagulopathy (DIC) when injected into calves. The effect of intraduodenal (ID) exposure followed by a subsequent subcutaneous (SC) inoculation of either heat-treated or untreated CLCL was evaluated. The relative contribution of the crude leukotoxin and lipopolysaccharide (LPS) to the virulence of P. haemolytica was evaluated. One group of calves received an ID inoculation of CLCL followed two weeks later by a SC inoculation of CLCL; one group received an ID inoculation of tissue culture medium followed two weeks later by a SC inoculation of CLCL; and a third group received an ID inoculation of CLCL followed two weeks later by a SC inoculation of heat-treated CLCL. Hematological parameters used to evaluate DIC included white cell count, platelet count, neutrophil number, fibrinogen, fibrin degradation products, one stage prothrombin time (OSPT), activated partial thromboplastin time, body temperature and clinical signs. Each parameter was measured in calves at 0, 2, 4, 6, 12 and 24 h following the SC inoculation of CLCL. Each group had significant changes over time in all parameters except body temperature. Calves that received a SC inoculation of heat-treated CLCL had smaller changes in all parameters except OSPT compared to the other groups. Results suggest that the LPS and leukotoxin of P. haemolytica exert additive effects on the coagulation cascade and number of peripheral leukocytes, and that the ID inoculation of CLCL does not affect the response of calves to a SC inoculation of toxin.

Evaluation of an orally administered vaccine, using hydrogels containing bacterial exotoxins of Pasteurella haemolytica, in cattle.

Poly(methacrylic acid) hydrogels were tested for oral delivery of a vaccine against Pasteurella haemolytica infection in cattle. Culture supernatants of P haemolytica, the most common bacterium associated with pneumonia in cattle, were used as the antigens in the vaccine. Hydrogels containing culture supernatants were administered orally to calves. Calves were then challenge-exposed with virulent P haemolytica. Calves were euthanatized 3 days after challenge exposure. The lungs of each calf were scored for severity and size of pneumonic lesions. Results indicated that vaccinated calves had smaller, less severe pneumonic lesions and lived longer than nonvaccinated calves. These results indicated that hydrogels can be used to deliver vaccines orally to calves to enhance resistance to pneumonia caused by P haemolytica.

Enhancement of respiratory immunity to Pasteurella multocida by cholera toxin in rabbits.

Cholera toxin (CT) is a potent adjuvant for the mucosal immune system. The purpose of this study was to determine if coadministration of CT with a potassium thiocyanate extract of Pasteurella multocida (PTE) leads to enhanced anti-PTE antibody activity and increased protection of rabbits against infection with P. multocida and associated disease. Groups of rabbits were immunized intranasally on days 0, 7, and 14, with phosphate buffered saline (PBS), 200 micrograms of CT, 1.0 mg of PTE, or 1.0 mg PTE with 200 micrograms CT. Nasal lavage and serum samples were collected over 28 days after initial immunization and evaluated by ELISA for specific antibody directed against PTE. Marked increases in serum (IgG) and nasal lavage (IgA) anti-PTE antibody activity were found beginning after day 14 in rabbits immunized with PTE. Rabbits immunized with PTE and CT demonstrated further increases in this activity. Tracheobronchial lavage samples collected at the time of necropsy demonstrated a significant level of anti-PTE IgA activity in animals immunized with PTE, and coadministration with CT stimulated a further significant increase in this activity. Groups of similarly immunized rabbits were challenged 16 days after initial immunization with 5 x 10(7) CFUs of P. multocida. Nasal lavage samples were cultured for P. multocida over the next 10 days. Rabbits were euthanized within 10 days after challenge, tissues cultured for P. multocida, and histopathologic lesion severity graded using a numeric scale. Rabbits immunized with PTE survived longer, had less severe lesions of the lungs, pleura, and liver, and fewer P. multocida CFUs cultured from samples than PBS or CT controls. Coadministration of CT led to further reductions in lesion severity of those tissues and numbers of P. multocida CFUs cultured from samples. Increased nasal turbinate atrophy of rabbits immunized with PTE with or without CT was associated with increased mean survival time. In summary, coadministration of CT with PTE enhanced protective immunity to P. multocida disease and infection in rabbits.

Interaction of Mycoplasma hyopneumoniae and Pasteurella multocida infections in swine.

To investigate the interaction between Mycoplasma hyopneumoniae and Pasteurella multocida infection, 32 pigs were randomly assigned by litter, sex, and weight to 4 treatment groups. Group-1 pigs were inoculated with M hyopneumoniae and allowed to recover from M hyopneumoniae infection. Group-2 pigs were vaccinated against M hyopneumoniae and then inoculated with M hyopneumoniae. Group-3 pigs were inoculated with M hyopneumoniae and developed clinical signs of mycoplasmosis. Group-4 pigs had never been exposed to M hyopneumoniae. All pigs were initially seronegative for M hyopneumoniae. All pigs were subsequently inoculated with P multocida and euthanatized 2 weeks later. Pasteurella multocida was isolated only from the lungs of group-3 pigs, and these pigs had a significantly higher median percentage of lung surface area affected by pneumonia than did pigs in the other groups. For group-3 pigs, percentage of lung surface area affected by pneumonia was positively correlated with the number of P multocida colonies isolated. We concluded that P multocida is not a primary respiratory pathogen in pigs, but that M hyopneumoniae infection can render the lungs susceptible to P multocida colonization and infection. Pigs recovered from or vaccinated against infection with M hyopneumoniae were resistant to P multocida infection.

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.