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One thousand neonatal calves, allocated in a factorial design into four groups, were vaccinated subcutaneously with two doses each of either killed Escherichia coli (0111:B4) J5 bacterin or a UC Davis modified live, genetically altered (aro-) Salmonella dublin vaccine, or both, or with a placebo. In this prospective double-blind study to determine the immunogenicity and protective effects of both vaccines on bovine neonates in field conditions, calves were observed daily until 2 months of age, and serum samples from selected study calves were obtained at five different time points. No clinical adverse vaccine reactions were observed. Overall mortality was 7.5% (75 of 1000), E. coli and S. dublin infection being the most commonly associated aetiological agents of deaths. Both J5 (p < 0.01) and Salmonella (p = 0.05) vaccines were significantly effective in reducing the mortality rate but without an additive effect. The role of passive transfer was important in calf survival. The E. coli J5 and (aro-) S. dublin vaccination schedule employed significantly (p < 0.001) elevated J5 and Salmonella-specific serum ELISA antibody titres, respectively, by the sixth week of age.

Genetically altered stable nonreverting aromatic-dependent (aro-) Salmonella dublin, strain SL5631, was administered orally to healthy colostrum-fed calves as vaccine. Twenty-six calves were allotted to 4 groups. There were 2 experiments, each with a vaccinated and nonvaccinated control group. Skin testing with 0.1 ml of sonicated S dublin was performed 3 days prior to challenge exposure. The IgG and IgM titers to S dublin lipopolysaccharide (LPS) antigen were determined by ELISA on sera before initial vaccination and at 1.5 to 2 weeks after each vaccination. In experiment 1, six calves received a dose of 1.7 x 10(10) colony-forming units (CFU) of aro-S dublin SL5631 orally at 2 and 4 weeks of age. After the first vaccination, 2 of 6 calves developed fever, but all 6 calves continued to have normal appetite and mental attitude. Adverse changes were not observed after the second vaccination. At the time of challenge exposure at 6 weeks of age, all 12 calves were seronegative for IgG and IgM LPS-specific antibodies, and the difference in percentage increase in skin test reaction at 48 hours was not significant. At 6 weeks of age, the 6 vaccinates and 6 controls were orally challenge-exposed with 1.5 x 10(11) CFU of virulent S dublin T2340. Protection from challenge was not evident, as 3 of 6 controls and 5 of 6 vaccinates died after challenge exposure. In experiment 2, eight calves received a dose of 5 x 10(11) CFU of aro-S dublin SL5631 orally at 2, 3.5, and 5 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)

A commercially available Salmonella bacterin was administered to Holstein calves starting at 1 to 19 weeks of age. Serum samples were obtained before administering bacterin and at 2-week intervals thereafter. An ELISA with Salmonella dublin lipopolysaccharide (LPS) or S dublin whole cells as antigen, was used to measure specific IgG and IgM responses. Antibody responses to LPS were not detected from calves < 12 weeks old inoculated with killed bacterin. Immunoglobulin responses to whole-cell antigen were detected from all age groups of calves inoculated with the same killed Salmonella bacterin. Calves < 11 weeks old are able to produce immunoglobulins to some whole-cell antigens, but are unable to produce anti-LPS immunoglobulins when inoculated with killed Salmonella bacterin. This age-related response to killed Salmonella antigens may account, in part, for increased susceptibility to salmonellosis in calves < 12 weeks old. In comparison to the response for killed antigen, 8 calves given modified-live aromatic-dependent S dublin bacterin at 1 to 3 weeks of age had detectable anti-LPS immunoglobulins after immunization, although the response was not as rapid and was of a lesser magnitude than that of older calves given killed Salmonella bacterin.

Twenty-six clinically normal colostrum-fed dairy calves were allotted to 5 groups. Calves of groups 1 and 2 served as nonvaccinated controls and were challenge-exposed with variable numbers of organisms. Group-3 calves were vaccinated SC with a modified Salmonella typhimurium bacterin. The bacterin was composed of killed acid-hydrolyzed S typhimurium G30/C21 (Re-mutant) whole cells coated with alkali-hydrolyzed S typhimurium LT-2 lipopolysaccharide, as antigen, and monophosphoryl lipid A, as adjuvant. Calves of groups 4 and 5 were vaccinated with a 2% mineral oil-in-water emulsion containing lipopolysaccharide as antigen and monophosphoryl lipid A and trehalose 6-6'-dimycolate as adjuvants. Calves of groups 3-5 were vaccinated at 2 weeks of age and again at 4 or 6 weeks of age. Adverse reactions were not observed after vaccination. Calves were challenge-exposed orally at 6 or 8 weeks of age with 1.5 X 10(11) (groups 1 and 4), or 3.0 X 10(11) (groups 2, 3, and 5) colony-forming units of S typhimurium UCD 108-11. Mortality after challenge exposure was 2 of 5 group-1 calves; 4 of 5 group-2 calves; 5 of 6 group-3 calves; 1 of 5 group-4 calves; and 4 of 5 group-5 calves. Statistical difference between calves of similarly challenge-exposed groups was not evident, indicating failure of either vaccine to protect calves of this age from oral challenge exposure with virulent S typhimurium.

Ten Holstein calves were divided into 2 groups. Five calves served as nonvaccinated controls, and 5 calves were vaccinated IM at 2 and 3 weeks of age with 10(9) aromatic-dependent (aro-) Salmonella typhimurium strain SL1479 containing O antigens 1, 4, 12. Serious adverse reactions to vaccination were not observed in the calves. Mean maximum rectal temperature increase in the vaccinated calves was 1.5 C. One calf had diarrhea and depressed appetite for 1 day after vaccination. At 5 weeks of age, all calves were challenge exposed orally with 1.5 X 10(11) virulent S dublin strain SL1367 (O antigens 9,12). After challenge-exposure inoculum was given, 1 of 5 vaccinated calves died and 4 of the 5 nonvaccinated calves died (P less than 0.05). Thus, some cross serotype protection against S dublin was induced by parenteral vaccination of calves with aro- S typhimurium strain SL1479, although protection was not complete.

Chemiluminescence of bovine alveolar macrophages was used to study the development of opsonins in calves vaccinated parenterally with live aromatic-dependent strains of either S. dublin or S. typhimurium. These calves responded by producing Salmonella-specific opsonins detected by increased chemiluminescent responses, and were able to survive oral challenge with live virulent organisms of either serotype. Non-vaccinated calves of the same age lacked Salmonella-specific opsonins and were not able to survive challenge. Thus it was concluded that the ability to produce opsonins is among the immunological responses that are associated with protection against salmonellosis in calves. Antigenic similarities between S. dublin and S. typhimurium were shown by the ability of either organism to absorb significant amounts of opsonic capacity from the sera of calves vaccinated with either of the two vaccines. These antigenic similarities are thought to explain in part the ability of either vaccine to protect against challenge with either the homologous or heterologous Salmonella serotype.

A virulent Salmonella dublin isolate was made histidine-requiring (his-) to allow recognition. The his- derivative, SL1367 (still calf-virulent), was then given by transduction and mutation, a transposon-generated non-reverting aromatic biosynthesis (aro) defect; this defect caused loss of virulence for the mouse. The his- aro- derivative strain, SL1438, was effective as a live vaccine in mice. Twenty male Holstein calves were divided into 4 groups. Groups I, II, and III were vaccinated IM at 2 weeks and at 3 weeks of age with aromatic-dependent (aro-) S dublin strain SL1438. Groups I and III received freshly prepared vaccine and group II received lyophilized vaccine. Serious adverse reactions to the vaccination were not seen. After vaccination, the mean maximum increase in rectal temperature was 1.8 C in group I and III calves and 0.6 C in group II calves. Fewer group II calves developed diarrhea (1 of 5) or positive blood cultures (0 of 5) after vaccination compared with group I and III calves (6 of 10 and 5 of 10, respectively). Postvaccination diarrhea was mild and of short duration. Group IV was comprised of 5 nonvaccinated calves. At 5 weeks of age, all calves were challenge exposed orally. Group I, II, and IV calves were challenge exposed with 10(11) virulent S dublin SL1367. Group III was challenge exposed with 10(11) virulent S typhimurium UCD 108-11. Subsequently, fever and diarrhea (lasting 1 to 3 days), but no deaths, were observed in the vaccinated calves. Four of the 5 nonvaccinated (group IV) calves died (P less than 0.001) within 8 days after challenge exposure. Aro- S dublin SL 1438 did not cause serious adverse effects and provided protection against oral challenge exposure with either virulent S dublin or S typhimurium.

A block in the common aromatic biosynthesis (aro) pathway makes E. coli, Salmonella, etc., exacting for aromatic metabolites; two of these, paraaminobenzoic acid and dihydroxybenzoic acid, are not mammalian metabolites. Bacteria needing substances not available in host tissues should be unable to grow there and so be non-virulent. Transposon-generated deletion and deletion-inversion mutations at aroA caused loss of mouse virulence in Salmonella typhimurium; e.g., LD50, intraperitoneal route, increased from less than 10 to greater than 10(6), and LD50 by feeding increased from ca. 10(5) to greater than 10(8). Furthermore, a single injection of 10(5) live aro- bacteria protected mice of a Salmonella-susceptible line, BALB/c, against challenge, 4 weeks later, with 5 X 10(5) virulent S. typhimurium (i.e., greater than 10(4) LD50); a single dose of the live-vaccine strain given i.p. also protected against challenge one month later by feeding 2 X 10(7) of a virulent strain. Mice vaccinated by feeding 2 X 10(8) live aro- bacteria were unaffected by feeding, one month later, 2 X 10(7) of a virulent S. typhimurium strain ( = ca 100 LD50). Intravenous injection of microparticulate silica did not reduce the mouse i.p. LD50 of an aro- strain, and 3 i.p. injections of cyclophosphamide, 4 mgm, caused only a small reduction. Thus the reduced virulence of the aro- strain does much depend on the integrity of host cellular defense mechanisms. Non-reverting aro- derivatives made from proven calf-virulent S. typhimurium and S. dublin strains are being tested at the School of Veterinary Medicine, University of California at Davis. None of 26 calves given aro- live-vaccine bacteria intramuscularly (usually 10(9)) and none of 8 calves fed ca. 10(11) live aro- bacteria, died or became seriously ill. Vaccinated and control calves were challenged by feeding 10(11) bacteria of a virulent strain, S. typhimurium or S. dublin. This challenge caused death of 14 of 16 non-vaccinated calves. The results, to date, in vaccinated animals indicate that one aro- strain of each of the two Salmonella species is effective if given as a live vaccine in two intramuscular doses.

The purpose of this investigation was to relate cutaneous delayed hypersensitivity reactions to the degree of immunity induced in calves given a live virulent or a modified-live strain of Salmonella typhimurium. Calves were placed into 1 of 5 groups on the basis of the vaccinal strain given and route of the vaccination: (I) nonvaccinated controls, (II) vaccinated twice by IM inoculation with small doses of the live virulent strain, (III) vaccinated (IM) twice with the modified-live strain, (IV) vaccinated (orally) twice with the modified-live strains, and (V) vaccinated (IM, and then orally) twice with the modified-live strain. Skin testing was performed by intradermal injection of fragmented S typhimurium as antigen. Double skin fold thickness and visual assessments were recorded at 3, 6, 24, 48, and 72 hours after antigen was administered. Biopsy samples for histopathologic evaluation were obtained. After vaccination and skin testing were complete, calves were orally challenge exposed with 1.5 X 10(11) virulent S typhimurium. Cutaneous delayed-type hypersensitivity reactions (48 hours) were observed in all except controls (group I) and 2 of 3 calves vaccinated orally with the modified-live vaccine strain (group IV). Significant correlations between positive skin tests and protection from challenge exposure were observed except in group V calves, in which positive skin test results were seen, but adequacy of the immunity (or survival after challenge exposure) was inconsistent.

Strains of Salmonella sp with complete nonreverting aromatic biosynthesis (aro) defects are expected to be nonvirulent, in respect to invasive infection, because they need the aromatic metabolites paraaminobenzoate (for making folate) and dihydroxybenzoate (for making enterochelin) which are not available in host tissues. Derivatives with transposon-generated complete nonreverting aro-defects were prepared from 3 mouse-virulent strains of S typhimurium, namely, FIRN, WRAY, and UCD. The latter 2 parent strains originally were isolated from calves and are known to be calf-virulent. The resultant aromatic-dependent (aro-) strains were used to vaccinate 27 calves (2 to 3 weeks old), usually giving 2 doses by the IM route (10(9) bacteria) or orally (1.5 X 10(11)). Vaccination did not cause severe ill effects in any calf. Thus aro- defects cause loss of virulence for calves, as previously shown for mice. Vaccinated and control calves were challenge exposed, usually at 5 weeks of age, by feeding 1.5 X 10(11) cells of 1 of 2 calf-virulent S typhimurium strains, either UCD 108-11 or SL1323. Of the 16 challenge-exposed control calves, all became anorectic and depressed (CNS), and 15 had diarrhea. Fourteen of the 16 died; all tested tissues were bacteriologically culture-positive for Salmonella at necropsy. Vaccination with the live UCD aro- vaccine strain, SL1479 by either of 2 schedules (IM or orally) appeared effective.(ABSTRACT TRUNCATED AT 250 WORDS)

A diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA) was used to record immunoglobulin development of colostrum-fed calves vaccinated with aromatic dependent (aro-) Salmonella and challenged with either the homologous as a heterologous serotype. IgG was detected by using a peroxidase conjugated rabbit antibovine IgG, whereas IgM and IgA were measured using a double sandwich technique. Although IgG levels to Salmonella endotoxins increased after exposure to Salmonella, they were found to be high in many calves prior to vaccination. However, IgM antibody levels were consistently low prior to vaccination, and their increase was a more reliable indicator of the vaccination and immune status of the calves. IgA levels were generally low and of less predictive value.

Migration of bovine macrophages under agarose was used to assess cellular immunity in 7 nonvaccinated calves and 9 calves vaccinated with Salmonella typhimurium. The 9 vaccinated calves were allotted to 4 groups. Group I calves were vaccinated twice orally with small doses of virulent S typhimurium; group II calves were vaccinated twice orally with genetically altered aromatic-dependent (aro-) S typhimurium SL3261; group III calves were vaccinated twice IM with small doses of virulent S typhimurium; and group IV calves were vaccinated twice IM with aro- S typhimurium SL1479. Samples of blood were obtained from these calves at 2 weeks after the 2nd vaccinal dose was given, and lymphocytes were harvested, using lymphocyte separation medium. Lymphocytes in serum-free medium were then incubated with S typhimurim antigen for 48 hours. Lymphocytes were then transferred to antigen-free medium and incubated for 48 hours, and the supernatant was assayed for the migration-inhibition factor (MIF). Lymphocyte supernatant was assayed for MIF by incubating it for 48 hours with 2.0 X 10(4) alveolar macrophages in agar wells. The macrophage migration distance was measured and compared with control values. Macrophage migration was inhibited in the presence of supernatant of lymphocytes from vaccinated calves that had been incubated with antigen, indicating the presence of the MIF in the supernatant. Migration distances, as a percentage of control, were 33% for group I calves (oral vaccination, virulent vaccinal organism), 60% for group II calves (oral vaccination, aro- vaccinal organism), 41% for group III (IM vaccination, virulent organism), and 25% for group IV (IM vaccination, aro- vaccinal organism).(ABSTRACT TRUNCATED AT 250 WORDS)