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Abstract

Objective

To determine whether Mycobacterium paratuberculosis could survive in colostrum after pasteurization. Additionally, this study investigated the effect pasteurization had on IgG concentration in colostrum.

Animals

Colostrum samples were collected from cattle (beef and dairy) owned by the state of Ohio.

Procedure

Colostrum was divided into aliquots and inoculated with variable concentrations of M paratuberculosis (ATCC No. 19698: 104, 103, and 102 colony-forming units/ml). Half the samples at each concentration were subjected to pasteurization temperatures (63 C) for 30 minutes and the remainder were kept at approximately 20 to 23 C. All samples were incubated (Herrold's egg yolk medium with and without mycobactin J) and observed for growth during the next 16 weeks. Additionally, the IgG concentration of colostrum was determined by radioimmunoassay before and after pasteurization. Samples that coagulated at pasteurization temperatures were mechanically resuspended before measurement of IgG concentration.

Results

Growth of M paratuberculosis was retarded but not eliminated by pasteurization. Growth was observed in all unpasteurized samples incubated on Herrold's egg yolk medium with mycobactin J but in only 2 of 18 pasteurized samples similarly cultured. Growth from pasteurized samples appeared 5 to 9 weeks after growth was observed from nonpasteurized samples.

Mean colostral IgG concentration was 44.4 g/L in nonpasteurized samples and 37.2 g/L in pasteurized sample, a decrease of 12.3%. High-quality colostrum (> 48 g of IgG/L) had a significantly greater loss of IgG concentration than did colostrum of lesser quality (P = 0.002).

Conclusions

Pasteurization lessened, but did not eliminate, growth of M paratuberculosis from experimentally inoculated colostrum samples. Pasteurization resulted in a significant decrease in colostral IgG concentration but not to an unmanageable level that would preclude the colostrum's use for passive transfer of immunity.

Clinical Relevance

Colostrum is macrophage rich and may serve as a source of M paratuberculosis infection to calves. Pasteurization of colostrum may lessen the risk of infection, but will not totally eliminate M paratuberculosis. (Am J Vet Res 1996;57:1580–1585)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate polymerase chain reaction (PCR) for detection of Lawsonia intracellularis DNA in feces and an indirect fluorescent antibody test (IFAT) for detecting serum IgG antibodies in pigs exposed to L intracellularis.

Animals

15 seven-week-old pigs and 42 three-week-old pigs.

Procedure

During 3 experiments, 23 pigs were inoculated with a pure culture of L intracellularis, 31 pigs served as noninoculated controls, and 3 pigs were used as sentinels. Fecal shedding of L intracellularis was monitored by use of PCR analysis at 7-day intervals. At euthanasia, the ileum was obtained for PCR and histologic analyses. Serum was obtained at 7-day intervals for use in the IFAT.

Results

Polymerase chain reaction analysis detected L intracellularis DNA in the feces of 39% of the inoculated pigs; by postinoculation days 21 to 28, 90% of inoculated pigs developed IgG antibodies detected by IFAT. Neither L intracellularis DNA nor IgG antibodies were detected in any of the noninoculated control pigs at euthanasia. Sera from pigs inoculated with enteric pathogens other than L intracellularis did not contain detectable antibodies that reacted with L intracellularis by use of the IFAT.

Conclusion

The IFAT for L intracellularis IgG antibody detection appeared to be a more sensitive antemortem test for detecting pigs experimentally infected with L intracellularis than was a PCR method for direct detection of the organism in the feces.

Clinical Relevance

Not all animals that are infected with L intracellularis shed the organism in feces at detectable amounts. (Am J Vet Res 1998;59:722-726)

Free access
in American Journal of Veterinary Research

Summary

Effect of estrogen (E2) and progesterone (P4) on uterine antibacterial activity and immunoglobulin concentrations in mares was studied. In 2 in vitro experiments, 6 mixed-breed mares were ovariectomized, and uterine fluid and blood serum were analyzed. Antibacterial assay methods were used to determine inhibitory effects on Streptococcus zooepidemicus of uterine fluid samples collected on days 3, 5, and 8, and serum obtained on day 8 of treatment. Single radial immunodiffusion methods were used to quantify amounts of IgA and IgG in uterine fluid and serum on days 3, 5, 8, and 14 of treatment. Neither E2 nor P4 increased activity of serum and uterine fluid against S zooepidemicus. Numbers of colony-forming units per milliliter of bacteria were significantly (P < 0.01) lower in control Hanks’ balanced salt solution with 1.0% gelatin (hbssg) than in uterine fluids. Bacterial numbers were significantly (50%) greater in uterine fluids and serum than in hbssg controls for both treatments. Both fluids, especially serum, supported significantly (P < 0.01) more growth of S zooepidemicus than did hbssg when incubated for 0, 2, and 4 hours. These findings are in contrast to previous reports of antibacterial activity in the uterus of sexually intact mares undergoing an estrous cycle: great reduction of bacterial count in uterine fluid from mares in diestrus, and significant increases in bacterial numbers in uterine fluid or serum from mares in estrus. Treatment comparisons between serum and uterine fluid IgA and IgG concentrations were not significantly different, although overall IgA concentration in the uterus was higher than concentration in serum. The IgG concentration in uterine fluid was higher in P4- than E2-treated mares. However, IgG concentration was significantly (P < 0.01) higher in uterine fluid on day 8 in P4-treated mares than on day 3 or 5. Results of this study indicate that neither immunoglobulin concentration nor hormone treatment has a direct effect on streptocidal activity.

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To elucidate kinetics of Bartonella henselae bacteremia and IgG response, evaluate antibiotic therapy, and investigate challenge exposure in cats.

Animals

Specific-pathogen-free cats.

Procedure

Cats were inoculated with B henselae or B quintana and monitored. Convalescent cats were challenge exposed with B henselae. Amoxicillin, enrofloxacin, erythromycin, and tetracycline HCI were evaluated for effect on B henselae bacteremia.

Results

Cats developed B henselae bacteremia within 1 week; bacteremia persisted for longer than 2 months before subsiding spontaneously. IgG antibody titer developed shortly after onset of bacteremia; antibody coexisted with bacteremia for several weeks and remained detectable after bacteremia subsided. Cats inoculated with B quintana remained abacteremic. On challenge exposure to B henselae, cats previously infected with B henselae remained abacteremic; cats previously inoculated with B quintana supported B henselae infection. Tetracycline HCI and erythromycin depressed B henselae bacteremia; however, duration of bacteremia remained similar to that in untreated cats. Obvious signs of illness were not observed.

Conclusions

Long-duration, high-titer B henselae infections were highly reproducible in cats. Convalescent cats were immune to reinfection. B quintana-inoculated cats did not have evidence of infection and were susceptible to B henselae challenge exposure. Antibiotic therapy was incompletely efficacious in terminating cat bacteremia.

Clinical Relevance

A cat with an inapparent B henselae infection must provisionally be regarded as a possible reservoir for infection for a minimum of 2 to 3 months. Convalescent cats are resistant to reinfection. Usual antibiotic therapy was not completely efficacious. Measurement of IgG antibody can be used to detect past or current infection. (Am J Vet Res 1996;57:1714–1719)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine systemic and mucosal antibody responses in calves to Pasteurella haemolytica 1:A and to 2 major outer membrane proteins (OMP) and 1 major iron-regulated OMP of P haemolytica 1:A.

Animals

23 crossbred calves.

Procedure

2 experiments were performed. In the first experiment, 6 calves were vaccinated and challenge exposed intranasally with an aerosol of P haemolytica 1:A and 6 calves were only challenge exposed. In the second experiment, 8 calves were vaccinated in the area of the tracheal bifurcation with an aerosol of P haemolytica 1:A and 3 calves were used as controls. Serum, nasal secretions, and bronchoalveolar lavage (BAL) samples were collected, and IgG1, IgG2, IgA, and IgM titers were determined. Nasal secretions and BAL samples were also submitted for bacterial culture.

Results

Serum antibody responses in the 2 groups were similar. Antibody titers in nasal secretions and BAL samples increased in calves vaccinated intranasally. In calves vaccinated in the area of the tracheal bifurcation, antibody titers increased in BAL samples but not in nasal secretions. Antibody responses did not correlate with results of bacterial culture.

Conclusions

Results indicated that intranasal administration of P haemolytica 1:A may be a better method for stimulating protective immune responses in the upper portion of the respiratory tract than lung administration. The single dilution ELISA provided a reliable and economical method for determining antibody titers. (Am J Vet Res 1998;59:727-732)

Free access
in American Journal of Veterinary Research

SUMMARY

Enzyme-linked immunosorbent assays were established to detect Breda virus antigen in feces and homologous antibodies of the IgGl, IgM, and IgA isotypes in serum. With the aid of solid-phase immune-electron microscopy, torovirions in fecal material were observed. The course of natural infection was studied in 10 sentinel calves that had been obtained from different farms, and housed together at 1 week of age. They were separated from other cattle until the age of 10 months.

Up to the age of 4 months, all calves regularly excreted Breda virus in the feces. Irrespective of the existence of IgG1 isotype maternal antibodies, all calves had early IgM responses in serum, but lack of IgA seroconversion. In 7 calves, antibody titer decreased below detection, whereas 3 calves had an isotype switch, resulting in persistent IgG1 titer. After introduction into the dairy herd at 10 months of age, all calves had diarrhea, and shedding of Breda virus was observed in 8 of them. Seroconversion for all antibody isotypes was observed, indicating lack of mucosal memory. In contrast, coronavirus infection in the presence of maternal antibodies led to isotype switch in all calves but one, and a memory response was observed after introduction into the dairy herd.

Free access
in American Journal of Veterinary Research

Summary

Aerosol vaccination is used effectively to immunize poultry against Newcastle disease, but to the authors’ knowledge, this vaccination procedure is not well studied in other species. The efficacy of im and aerosol vaccination of pigs against Mycoplasma hyopneumoniae infection was evaluated. Twenty-one pigs from a Mycoplasma-free herd were randomly allotted by litter and body weight into 3 groups. One group was given aerosolized phosphate-buffered saline solution (pbss) by inhalation. The second group (aero) was given aerosolized M hyopneumoniae vaccine by inhalation. The third group (im) was given the same vaccine by im injection. Vaccination by im administration was repeated once, and aerosol vaccination was repeated twice at 2-week intervals. Two weeks after the last vaccination, all pigs were intra-tracheally challenge-exposed with 3 ml of broth culture containing 107 color-changing units (ccu) of a low-passage strain of virulent M hyopneumoniae. Pigs were observed daily for coughing. Four weeks after challenge exposure, all pigs were necropsied. Percentage of lung affected by gross pneumonia was measured, bronchioalveolar lavage fluid (balf) cells were counted, and quantitative culture for mycoplasmas was performed on lung sections. Additionally, M hyopneumoniae-specific antibodies were measured in prevaccination, postvaccination, and postchallenge-exposure serum and balf by use of indirect elisa. Mean prevalence of persistent coughing in pigs of the aero group (4.6 d/pig) was not different from that in pigs of the pbss group (3.7 d/pig). Prevalence of coughing in im vaccinated pigs (1.0 d/ pig) was lower (P < 0.05) than that in pigs of the pbss group. Mean gross lung lesion scores and balf cell counts were not different between the aero (15% pneumonia, 5,233 cells/μl) and pbss (11% pneumonia, 3,022 cells/μ1) groups, but were lower (P < 0.05) in the im group (1.5% pneumonia, 400 cells/μl) than in the pbss group. Mean lung mycoplasmal counts were not significantly (P < 0.05) different among the pbss (105.6 CCU/g), aero (105.3 CCU/g), and im (103.3 CCU/g) groups. Postvaccination M hyopneumoniae-specific IgG or IgA was not detectable in balf after either vaccination procedure. Postvaccination M hyopneumoniae-specific serum IgG concentration was not different among the 3 groups. Postchallenge exposure M hyopneumoniae-specific IgG and IgA were detectable in balf of all pigs, but were not different among the 3 treatment groups. Postchallenge exposure-specific serum IgG concentration was not different between the pbss (mean OD, 0.739) and aero (mean OD, 0.672) groups, but was higher (P < 0.05) in the im group (mean OD, 1.185) than in the pbss group. Aerosol vaccination failed to induce local and systemic antibody responses detectable by elisa, and failed to protect pigs against mycoplasmal pneumonia. Intramuscular vaccination failed to induce local and systemic antibody responses detectable by elisa, but substantially reduced the clinical signs and lesions caused by challenge exposure to virulent M hyopneumoniae.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate in vitro antigenic relations, in vivo cross-protection, and isotype antibody responses to a winter dysentery (WD) and calf diarrhea strain of bovine coronavirus (BCV).

Design and Animals

Gnotobiotic and colostrum-deprived calves were inoculated oronasally with a WD (DBA) or a calf diarrhea (DB2) BCV, and were challenge exposed with the heterologous BCV.

Procedure

Nasal swab and feces specimens and blood samples were collected. Fecal and nasal specimens were assayed for BCV shedding by antigen-capture ELISA or immune electron microscopy. Bovine coronavirus antigens were detected in nasal epithelial cells by immunofluorescence. Antibody titers to BCV in serum were assayed by virus neutralization (VN), and BCV antibody isotype titers in feces and sera were quantitated by ELISA.

Results

All calves developed diarrhea and shed BCV nasally and in feces, then recovered and were protected from BCV-associated diarrhea after challenge exposure with the heterologous BCV. After challenge exposure with either strain, fecal shedding of DBA was detected in 1 of 4 calves and nasal shedding of DB2 was detected in 2 of 4 calves. Immunoglobulin M was the principal coproantibody to BCV early, followed predominantly by IgA. Immunoglobulin G1 coproantibody titers to BCV were low, but increased after challenge exposure. Immunoglobulin G1 antibodies were predominant in serum. After challenge exposure, all serum antibody isotype titers increased except IgG2. The VN antibody responses paralleled serum IgG1, antibody responses.

Conclusions and Clinical Relevance

Immunoglobulin A coproantibodies at challenge exposure were associated with protection against diarrhea. Nasal shedding of BCV after challenge exposure confirmed field data documenting reinfection of the respiratory tract of cattle, suggesting that, in closed herds, respiratory tract infections constitute a source of BCV transmission to cows (WD) or young calves. (Am J Vet Res 1996;57:48-53)

Free access
in American Journal of Veterinary Research

Summary

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 × 1010 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 × 1011 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 × 1011 cfu of aro-S dublin SL5631 orally at 2, 3.5, and 5 weeks of age. The vaccine dose and volume (300 ml) were 30 times that of experiment 1. After each vaccination, some calves (7, 6, and 2 calves for first, second, and third doses, respectively) developed fever, but all calves continued to have normal appetite and attitude. At 7 weeks of age, the 8 vaccinates and 6 controls were orally challenge-exposed with 1.5 × 1011 cfu of virulent S dublin T2340 (same dose as experiment 1). At the time of challenge exposure, all 8 vaccinated calves had elisa titers to IgG and IgM lps-specific antibodies significantly above those of nonvaccinated calves (P < 0.01 and P < 0.05, respectively), 5 of 8 had a strongly posisitive skin test reaction to lps, and the group mean percentage increase in skin thickness 48 hours after intradermal injection was 135% (P = 0.01). The 6 control calves had negative elisa results and mean increase in skin thickness of 34%. Protection from challenge exposure was evident as vaccinates remained blood culture-negative, whereas 5 of 6 controls were blood culture-positive; vaccinates did not develop diarrhea, whereas all controls developed diarrhea. All vaccinates survived, but 3 of 6 controls died after challenge exposure (P = 0.05).

Failure of orally administered vaccine to protect calves in experiment 1 appeared attributable to insufficient antigenic stimulation when 1.7 × 1010 cfu of aroS dublin SL5631 was administered. In experiment 2, a larger number of vaccinal organisms given orally was able to induce a measurable systemic immune response and protection, but the vaccine volume makes it unlikely to be practical for field use.

Free access
in American Journal of Veterinary Research

Summary

A blocking elisa was developed to detect antibodies directed against porcine epidemic diarrhea virus (pedv). The pedv antigen was first incubated with dilutions of test sera. Any antigen that was not blocked by antibodies in the serum was assayed in a double-antibody sandwich elisa, using 2 monoclonal antibodies directed against different antigenic sites on pedv as capture and detecting antibodies, respectively. The blocking elisa was compared with a fixed-cell elisa that used monolayers of Vero cells infected with pedv prototype strain CV777 as a solid phase and a conjugate of an IgG-specific monoclonal antibody for antibody detection. Pigs were inoculated with pedv strain CV777 or 1 of 2 field isolates, and antibody responses were measured by use of the 2 tests. Antibodies were detected by the blocking elisa as early as postinoculation day 7 and, by the fixed-cell elisa, as early as postinoculation day 14. From day 14 on, antibody titers for both tests correlated highly. Titers for the fixed-cell elisa were 5.4 times higher than those for the blocking elisa. The latter technique is easier to perform and discriminates well between infected and noninfected pigs, which makes this test useful for routine diagnosis and serologic surveys of porcine epidemic diarrhea.

Free access
in American Journal of Veterinary Research