Immunity to Escherichia coli in pigs: Serum Gamma Globulin Levels, Indirect Hemagglutinating Antibody Titres and Bactericidal Activity Against E. coli in pigs up to five Weeks of Age

Serum gamma globulin levels, indirect hemagglutinating antibody titres and bactericidal activity against the 0149:K91;K88ac:H10 Serotype of Escherichia coli were determined in pigs up to five weeks of age from vaccinated and non-vaccinated sows.

Gamma globulin levels at two days of age were approximately twice adult levels, by three weeks of age they were one quarter of adult levels and remained so until five weeks of age. Indirect hemagglutinating antibody activity was highest at two days of age, fell until three weeks of age and then rose. Little or no indirect hemagglutinating antibody activity was detected in sera taken at two days of age from pigs from non-vaccinated sows. Only three of 26 two day old pigs had demonstrable bactericidal activity; by three weeks of age 16 of 26 had bactericidal activity. Serum from piglets of vaccinated sows had no more bactericidal activity than did sera from non-vaccinated sows.

     

Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when the pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracellularis exposed pigs with or without current proliferative enteropathy.     

Response of Gnotobiotic Pigs to Escherichia coli

In a study of the response of gnotobiotic pigs to coliform infections, 45 one-week-old germfree pigs were divided into five groups and each group was inoculated orally with a different strain of Escherichia coli. Three of these were enteropathogenic swine strains, P307[08:K87(B), K88 a,b (L):H19]; P570 [0138:K81]; P568[0141:K85a,b(B), K88a,b(L):H4], one was a virulent human strain, H224, [026:K60(B6)], and one was a non-enteropathogenic swine strain, P581[OX13:K68]. It was attempted to protect a portion of the pigs with orally administered specific antisera and sera from non-immunized specific pathogenfree (SPF) pigs. Observations were made on the clinical response, bacterial counts of feces and intestinal contents, gross pathological changes, distribution of the organisms in organs and serum hemagglutinin titers.

Infection with E. coli P307 resulted in diarrhea, dehydration and death, unless the pig was protected with specific antiserum. The pigs infected with E. coli P570 had a transient diarrhea but retained their appetites and recovered. Those infected with the other three strains remained healthy throughout. No circulating hemagglutinating antibody against the test strains of E. coli could be detected in any of the pigs seven days or earlier post-inoculation.

Relationship could not be established between the numbers of viable E. coli in the feces and the presence of clinical colibacillosis. Orally administered specific antiserum afforded protection against strain P307, but did not reduce the number of E. coli in the gut or alter their distribution in the internal organs. This suggested that the protective effect of specific antibody in the intestine was due to its action on a metabolite (enterotoxin) produced by E. coli P307 rather than the organism itself.

     

Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody.

In this study, an immunochromatographic strip (ICS) was developed for the detection of antibody against Streptococcus suis serotype 2 (SS2). Colloidal gold particles labeled with staphylococcal protein A (SPA), which can bind to the F(C) fragment of mammalian immunoglobulin, were used as the detector reagent. The capsular polysaccharide (CPS) of SS2 and affinity-purified IgG from a healthy naive pig were immobilized on test and control regions of a nitrocellulose membrane, respectively. The ICS was used to 1) detect anti-CPS antibody in 14 sera taken from 4 SS2-infected pigs, 24 sera from pigs hyperimmunized with SS2, and 68 sera from pigs inoculated or infected with bacteria other than SS2; 2) determine anti-CPS antibody titers of 20 positive sera for comparison with enzyme-linked immunosorbent assay (ELISA); and 3) detect anti-CPS antibody in 226 clinical sera taken from diseased pigs also for comparison with ELISA. An ELISA used as a reference test determined the specificity and sensitivity of the ICS to be 97.1% and 86.3%, respectively. There was excellent agreement between the results obtained by ELISA and the ICS (kappa = 0.843). Additionally, there was strong agreement between the results of bacterial isolation from pig tonsils and ICS test (kappa = 0.658). Because it is rapid and easy to use, the test is suitable for the serological surveillance of SS2 at farms.     

Polyerositis and Arthritis Due to Escherichia coli in Gnotobiotic Pigs

Forty gnotobiotic pigs from six litters were exposed orally to Escherichia coli 083:K·:NM at 69 to 148 hours of age, while 17 pigs from the same litters served as unexposed controls. Clinical signs of infection included fever, anorexia, diarrhea, lameness, and reluctance to move.

Eighty-four percent of the exposed pigs in four litters died, while only 13% in two litters died. Gross and microscopic lesions included serofibrinous to fibrinopurulent polyserositis in 96% of the exposed pigs in four litters and 33% of the exposed pigs in two litters. A few pigs had gross and/or microscopic lesions of arthritis. Escherichia coli was routinely isolated from the serous and synovial cavities of infected pigs.

Anti-hog cholera serum administered orally as a colostrum substitute gave partial protection against E. coli infection.

     

Isolation of Leptospira interrogans serovar bratislava from stillborn and weak pigs in Iowa

Leptospira interrogans serovar bratislava was isolated from a herd of swine in Iowa with a history of stillborn and weak neonatal pigs. Placentas, kidneys, and lungs of stillborn and weak pigs from 3 litters were processed to detect leptospires by use of bacteriologic culture and fluorescent antibody testing. Sera from stillborn and weak pigs were tested to detect agglutinating antibody against leptospires. A low antibody titer against L interrogans serovar bratislava was detected in the sera of stillborn and weak pigs. Small numbers of leptospires were sometimes detected in tissues by use of the fluorescent antibody test. Serovar bratislava was isolated from placentas, stillborn pigs or weak pigs from each of the 3 litters.     

A serological survey to determine the prevalence of infection with Treponema hyodysenteriae in Western Australia

A serological survey to detect antibody titres against Treponema hyodysenteriae was conducted on pigs from 106 herds in Western Australia. Titres indicating a positive result in the tests were determined by examining 400 sera from 4 herds known to be free of swine dysentery, and sera from immunised or experimentally infected pigs. Samples of serum from 40 bacon-weight pigs from each of the 106 herds were then collected at 2 abattoirs. Each serum was tested in enzyme-linked immunosorbent assays (ELISA) against the lipopolysaccharide of T hyodysenteriae of serogroups A, B and E, respectively. To assist in evaluating the test, 19 herds were resampled and retested, and faecal samples from 17 herds were cultured for T hyodysenteriae. Thirty-five of the 106 herds (33%) had serological evidence of infection when only one batch of sera from each herd was tested. The ELISA to detect T hyodysenteriae infection in herds using 40 sera was estimated as having a sensitivity of 77.3% and a specificity of 81.8% based on the owners' opinion of their herds disease status. Prevalence of infection within herds ranged from 2.5% to 47.5%, with a mean of 18%.     

Enzootic Pneumonia of Pigs: Complement-Fixation Tests for the Detection of Mycoplasma Antibodies in the Serum of Immunized Rabbits and Infected Swine

The direct, the modified direct and the indirect complement-fixation tests were investigated as methods for the detection of antibodies for the enzootic pneumonia mycoplasma and for Mycoplasma hyorhinis in the serum of infected pigs and of immunized rabbits.

Only the modified direct complement-fixation test in which the guinea-pig complement is supplemented with fresh, normal unheated calf serum was suitable for the detection of mycoplasma antibodies in sera of infected swine. Based on the close correlation between the production of typical lung lesions in experimentally infected pigs and the appearance of significant serum antibody titres, the modified direct complement-fixation test provides for the first time a sensitive, specific in vitro method for the detection of enzootic pneumonia in the live pig. This test also permitted the in vitro differentiation of the mycoplasma causing enzootic pneumonia from M. hyorhinis which causes polyserositis.

Antibodies in the sera of rabbits were demonstrable by the ordinary direct complement-fixation test. However, in contast to the observation made with swine sera, only a slight quantitative antigenic difference between the enzootic pneumonia mycoplasma and M. hyorhinis was seen when the tests were performed with rabbit serum antibodiies.

     

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of infectious bovine rhinotracheitis viral antibody in cattle.

An enzyme-linked immunosorbent assay was developed to detect bovine serum antibody to infectious bovine rhinotracheitis virus. The specificity of this assay in 304 bovine sera, collected from an infectious bovine rhinotracheitis virus-free herd, was 100%; in sera from 62 cattle inoculated with an intranasal vaccine, its diagnostic sensitivity was 27.4% at one month and 100% at six months, postvaccination. In 303 bovine sera with standard serum neutralizing antibody titers of greater than or equal to 1:2 it showed 100% sensitivity; and in 463 random diagnostic samples, comparative tests indicated that enzyme-linked immunosorbent assay detected more seropositive animals (61.6%) than the standard serum neutralizing test (49.9%). The enzyme-linked immunosorbent assay method was considered to be technically superior as a routine diagnostic test for the detection of infectious bovine rhinotracheitis viral antibody in bovine sera.     

<Emphasis Type="Italic">Ungulate copiparvovirus 2</Emphasis> in healthy and postweaning multisystemic wasting syndrome-affected pigs

A SYBR Green-based real-time polymerase chain reaction (qPCR) was designed to detect Ungulate copiparvovirus 2, also known as porcine parvovirus 4 (PPV4). The test was applied to search for PPV4 DNAemia in sera from 1- to 4-month-old pigs displaying signs of postweaning multisystemic wasting syndrome (PMWS), as well as in sera from healthy swine at equivalent age and in sera from older healthy animals (>6 months old). High levels of PPV4 DNA were detected in PMWS-affected pigs. The mean viral DNA load in PMWS-affected pigs was 5.2 × 10⁷ copies/mL, whereas in young healthy pigs it was 1.4 × 10⁵ copies/mL (P ≤ 0.001). Although the copy numbers were lower in younger PMWS-affected individuals, this result sheds some light on the possible association between PPV4 viral load detection in this group and the immune impairment caused by PMWS.     

Vibriosis: Demonstration of Vibrio fetus and Vibrio bubulus Organisms in Preputial Fluid by Immunofluorescence and Cultural Techniques

Fluorescent conjugates were prepared from the sera of calves immunized with four Vibrio fetus strains and one Vibrio bubulus strain. The fluorescent antibody technique (FAT) was then used to detect vibrio organisms in preputial fluid collected from 67 bulls belonging to a Canadian artificial insemination (AI) unit. The V. fetus conjugates reacted with both V. fetus var venerealis and V. fetus var intestinalis. V. fetus was found in 20 animals (29.9%), 13 of which also harboured V. bubulus. In two cases, the FAT failed to detect V. fetus which was isolated by concurrent bacteriological examinations.

It was concluded that the FAT can be a rapid method of detecting some carrier bulls but more reliable results are obtained when a combination of FAT and bacteriological methods is employed. It was found that a single sample giving negative results is inconclusive and additional tests are required before making a final diagnosis. The FAT can also be used to differentiate V. fetus isolates from V. bubulus.

     

Experimental Atrophic Rhinitis in Gnotobiotic Pigs

Twenty-nine caesarian derived colostrum deprived germfree pigs were reared in isolators in groups of three to four per isolator. At seven days of age each group was inoculated intranasally with one of four strains of Bordetella bronchiseptica (designated B, J, L and 55B), or Pseudomonas aeruginosa or a mucoid strain of Escherichia coli, all previously isolated from nasal mucus of pigs affected with clinical atrophic rhinitis. Another group was inoculated simultaneously with B. bronchiseptica B and Pasteurella multocida. The animals were observed for clinical signs of atrophic rhinitis and monitored bacteriologically at weekly intervals for seven weeks. Then they were bled for serology and killed and their respiratory organs examined for gross and histopathological lesions.

All of the pigs inoculated with the Bordetellae had inflammation of the nasal mucosa and developed positive serum antibody titers against all four of the Bordetella strains used in this study. Strain J caused sneezing and turbinate atrophy in three of four pigs. One of the three pigs inoculated with strain L died in ten days from bronchopneumonia and pericarditis and had turbinate atrophy. Strains B and B55 caused no turbinate atrophy, but two out of three pigs inoculated with both B. bronchiseptica B and P. multocida had turbinate atrophy. No nasal lesions were observed in the pigs inoculated with E. coli or P. aeruginosa or in the noninoculated germfree controls.

The results indicate a variation in the ability of different strains of B. bronchiseptica to cause turbinate atrophy in pigs and demonstrate that nasal infections by these organisms stimulate serum antibody response. Presence of P. multocida appears to increase the severity of the lesions. As the E. coli and Pseudomonas failed to produce atrophic rhinitis, they are probably of no significance as primary etiological agents in the atrophic rhinitis syndrome in swine.

     

Swine Enzootic Pneumonia: Age Susceptibility and Treatment Schemata

Pigs were found to be susceptible to Mycoplasma hyopneumoniae-induced swine enzootic pneumonia (SEP) when four hours old. Chlortetracycline was incapable of preventing transmission of SEP from infected pigs on the drug to susceptible, untreated pigs. When continuous medication was started at one or two weeks postinoculation, chlortetracycline partially or completely inhibited formation of SEP lesions but did not clear M. hyopneumoniae from inoculated pigs. Chlortetracycline administered orally was capable of completely suppressing the formation of SEP lesions in inoculated pigs if given prophylactically and if milk was withheld for several hours after each treatment; lesion suppression was incomplete if milk was given ad libitum. In either case treated animals remained infected with M. hyopneumoniae.     

Transfer of maternal antibody against group A rotavirus from sows to piglets and serological responses following natural infection

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of antirotaviral antibody in sera and faeces from pigs and used to study the dynamics of antirotaviral antibody responses in three cohorts of pigs. Piglets acquired antirotaviral antibody by sucking their dams soon after birth. Antirotaviral antibodies of IgA and IgG classes were detected in both colostrum and milk of all sows tested but IgM class antibodies were not. The antibody levels in colostrum were eight to 32 times higher than those in milk which was collected 18 days post partum. The levels of antibody in piglets' sera were comparable to those in colostrum but declined quickly to low levels by one month old. Maternal antibody was also detected in the faeces of piglets up to 18 days old. Natural rotavirus infection occurred in each of these cohorts when the geometric mean ELISA titres of maternal antibody in their sera declined to 1/1600 (by days 21, 25 and 30 for cohorts 1, 2 and 3, respectively). However, a positive correlation was not obtained between the levels of antirotaviral antibody and protection in individual litters within each of the cohort groups. In each of the cohorts, rotavirus infection usually occurred in one or two piglets first and then spread to other piglets in the same cohort. It is therefore suggested that maternally derived antibody is protective against rotavirus infection in piglets only for the first one or two weeks. Following natural infection with rotavirus, increases in serum antibodies were detected in two of the three cohorts by 20 to 30 days after the average time of onset of faecal shedding of virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fractionation on Cross-Linked Dextran, Sephadex G-25, of Sera from Sheep Infected with Johne's Bacilli. Preliminary Report

Ten sera collected during the winter months from six sheep infected with Johne's bacilli were fractionated on Sephadex G-25 columns, and all fractions tested for complement-fixing antibody, anticomplementary properties and for supplementing and inhibitory activities when added to complement-fixation tests of a heterologous antigen-antibody system: bovine or ovine antiserum with Brucella abortus antigen. Serum from a normal sheep was similarly fractionated and examined.

The complement-fixing activity with a carbohydrate fraction of Johne's bacilli was confined principally to supernatant fractions of the earlier eluates containing the greater part of the serum proteins. Some of the unheated earlier and later fractions displayed a limited degree of supplementing effect. Inhibitory activity was demonstrated by certain of the earlier and later eluates after they had been heated, particularly those of antisera with initially low complement-fixing antibody titer.

     

Humoral Antibody Responses of Swine Infected Experimentally with Group E Streptococcus II. Antistreptokinase and Antistreptodornase Responses

Swine infected experimentally with group E Streptococcus (GES) produced significant antibody titers against streptokinase (streptococcal fibrinolysin, SK) and streptodornase (streptococcal deoxyribonuclease, SD). The antibodies directed against SK (antistreptokinase, ASK) appeared two to nine weeks postexposure and persisted for the duration of the experiment (nearly six months). The ASK inhibited SK produced by GES antigenic types III, IV, and V, by GES devoid of type specific antigen, and by a group P Streptococcus. Selected strains of GES serotypes I and II and group U Streptococcus did not produce detectable SK.

The antibodies directed against SD (antistreptodornase, ASD) appeared two to three weeks postexposure, reached a peak about six weeks postexposure, and persisted at high levels for nearly six months (the duration of the experiment). The ASD inhibited SD produced by all known antigenic types of GES, by GES devoid of type specific antigen, and by strains of groups P and U Streptococcus. The antibodies failed to inhibit SD produced by group C Streptococcus.

The potential utilization of ASK and ASD titers as serological means of identifying swine infected with GES (carrier swine) is discussed.

     

Establishment of a Salmonella-Free Guinea Pig Colony

Salmonellosis due to Salmonella typhimurium was enzootic in a guinea pig breeding colony for over 25 years. A Salmonella-free auxiliary colony was established by removing weanlings from the infected colony to a clean area, and preventing infection. Examination of agglutinin titers and necropsy specimens indicated that the auxiliary colony was still free from Salmonella 18 months after its establishment while 24% of the guinea pigs dying in the infected colony yielded Salmonella typhimurium.     

Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis and pneumonia in swine.

A total of 163 pigs from nine farrow-to-finish herds representing various levels of atrophic rhinitis (AR) were selected for postslaughter examination of AR and pneumonia. Nasal swabs and lungs were cultured for detection of Bordetella bronchiseptica and Pasteurella multocida. Seventy-three pigs were examined at eight weeks of age and 90 contemporaries at six months of age. Mean AR scores were 1.21 and 1.11 for the eight week and six month old pigs, respectively (0 = normal, 3 = severe). In individual pigs increasing AR score was related to increasing pneumonia score in eight week old pigs but not in six month old hogs. In eight week old pigs, B. bronchiseptica and P. multocida were isolated more frequently from pigs with higher AR scores. From nasal swabs of six month old hogs, Bordetella was almost never recovered while Pasteurella was frequently isolated score. Toxigenic type DP. multocida was isolated from nasal cultures of only seven (4%) pigs and from lung cultures of only one pig. Pasteurella was never isolated from lungs of the eight week old pigs and Bordetella never from the six month old hogs. The isolation rate of P. multocida, predominantly type A, from lungs of six month old pigs increased from 11% in grossly normal lungs to 86% in lungs with severe pneumonia. Pigs from one herd free from lesions of AR and pneumonia were also examined; type AP. multocida was isolated from nasal cultures of one of six eight week old pigs. Somatic antigens of P. multocida were determined for 94 nasal and 20 lung isolates. Somatic serovar 3 was found in 93% of the nasal isolates and in all lung isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of antibodies to Lawsonia intracellularis in pig herds in Australia

Objective Proliferative enteropathy (PE) of pigs is caused by Lawsonia intracellularis. Clinical severity appears to depend, at least partly, on the infective dose and strain of L. intracellularis. Serological tests are able to detect subclinical disease. The Bioscreen ELISA for detecting L. intracellularis-specific antibodies is widely used to monitor the circulating antibody status of pigs in Australia, but its sensitivity and specificity have not been reported. The aim of the present study was to measure the seroprevalence of antibodies to L. intracellularis in growing pigs in Australia. Methods Test sera were sourced from 1817 serum samples collected from finisher pigs from 63 herds across Australia in 2001, selected from a larger sample of 180 herds to represent the contribution that each herd size makes to the number of pigs produced. The test sera were the most recent collection of pig sera from all states and samples had been stored at −80°C from 2001 until testing was conducted in 2008. Sera were tested using the BioScreen ELISA. Results All herds tested positive for L. intracellularis-specific antibodies. The mean percentage of positive samples within each herd was 84.2% (range 31.3–100%). Conclusions Lawsonia intracellularis is endemic in pig herds in Australia and cost-effective strategies to reduce reliance on antibiotics, such as vaccination and/or all-in/all-out pig flow coupled with cleaning and disinfection of pens, are warranted.