Immunity to Escherichia coli in pigs: Antibody Secretion by the Mammary Gland after Intramammary or Intramuscular Vaccination with an E. coli Vaccine

Indirect hemagglutinating antibody titres in individual gland samples of colostrum and milk from 13 sows were measured. Five of the sows were vaccinated via a mammary gland and five by the intramuscular route with a live formalinised Escherichia coli vaccine and three remained as non-vaccinated controls.

Antibody titres were higher in colostral and milk whey from the vaccinated sows than from non-vaccinated groups. The inoculated gland in the group of sows given vaccine by the intramammary route secreted milk containing markedly more antibodies to the vaccine E. coli strain than did the non-vaccinated glands. Milk from the vaccinated gland did not contain higher titres to heterologous E. coli O antigens than milk from non-vaccinated glands. Serum titres were the same or higher than the titres in colostrum from non-vaccinated glands.

     

Role of glycoconjugates in adherence of Salmonella pullorum to the intestinal epithelium of chicks

1. The distribution of glycoconjugates on the surface of Salmonella pullorum and the ileal epithelium of chicks was demonstrated by lectin cytochemistry. The role of glycoconjugates in adherence of S. pullorum to the ileal epithelium was determined by a sugar inhibition assay using the scanning electron microscope.

2. S. pullorum exhibited binding to Concanavalin A (Con A) and wheat germ agglutinin (WGA) but not to soyabean agglutinin (SBA), Ricinus communis agglutinin (RCA) and Ulex europaeus agglutinin (UEA).

3. The ileal epithelium of chicks bound WGA, Con A and SBA. The binding sites for WGA were on the brush border and cytoplasm of columnar enterocytes as well as on goblet cells. The binding of Con A was confined to the cytoplasm of columnar enterocytes, while SBA bound, in a limited way, to the brush border of columnar enterocytes.

4. After an oral dose of S. pullorum, adherence to the ileal epithelium was inhibited by methyl α‐D‐mannopyranoside.     

Synergism between the endogenous viral loci ev6 and ev9 in inducing immunological tolerance to avian leukosis virus

1. The course of infection by exogenous avian leukosis virus was followed in a commercial strain of White Leghorn domestic fowls by measuring viral antigen in feather pulp and egg albumin. Ten days after hatching, 2 out of 360 birds tested positive and at 286 days of age about 60% of the birds had been antigen positive at least once.

2. Among the antigen positive birds, two groups could be distinguished: those which permanently and those which transiently expressed viral antigen. Permanent antigen expression was associated with low antibody titres, while transient antigen expression was associated with high antibody titres.

3. The strain segregated for the two endogenous viral genes ev6 and ev9, both of which express endogenous viral envelope protein, and have been implicated in affecting immune‐responsiveness. The antibody titre in individuals positive for both ev6 and ev9, was significantly lower than in those which had none or only one of the two ev‐genes. In addition, individuals positive for both ev‐genes occurred more frequently in the group permanently positive for viral antigen than in the group transiently antigen positive.

4. The results indicate that there was a strong synergism between ev6 and ev9 in reducing the antibody response to exogenous avian leukosis virus infection, perhaps by inducing immune tolerance or interfering with antibody formation.     

Differentiation Among Strains of Goat Mycoplasma by Incident Light Immunofluorescence

Antigenic differentiation between strains of goat mycoplasma was studied by direct fluorescent antibody reactions employing incident (vertical) ultraviolet light. Agar colonies of the mycoplasma grown in petri dishes were fixed by alcohol in situ, and stained with conjugated globulin before examination with ultraviolet light.

The fluorescent antibody (FA) conjugate against Vom strain of Mycoplasma mycoides var. capri was Vom strain-specific, no cross reaction with Mexico, Connecticut, or Maryland strains. Similarly, the Mexico strain conjugate was specific for colonies of Mexico, and did not cross with the Vom, strain. Additionally, the conjugate of the PG-2 strain of Mycoplasma agalactiae, which was specific for the colonies of PG-2 was refractory for the strain #99 of M. agalactiae.

It was therefore possible to utilize an immunofluorescent technique (incident ultraviolet light) to demonstrate differences among strains of M. mycoides var. capri and M. agalactiae.

     

Some Observations on the Diagnosis and Epidemiology of Leptospirosis in Swine

The results of combined epidemiological, clinical, serological, bacteriological and histopathological studies following an outbreak of disease caused by L. pomona on a farm stocked with cattle, sheep, pigs, goats and horses maintained for experimental purposes, are reported.

The incidence of infection was high in horses, cattle and pigs. A few low titres were seen in sheep. The goats were not infected. Apart from a single bovine abortion all the clinical symptoms observed occurred in pregnant sows. Seven of these aborted or gave birth to stillborn pigs within a six week period.

Fifteen species of wildlife were trapped or shot on the farm during the year following the outbreak. L. pomona was isolated from four skunks and a porcupine. Epidemiological studies indicated that wildlife reservoir hosts were the primary source of infection for the domestic livestock.

Leptospiruria and the serological response were studied in a group of eight infected sows. Microscopic agglutination titres of 10² or less could not be associated with leptospiruria and the duration of leptospiruria was found to range from a few weeks to over two years in individual sows. Direct dark-field examination of urine proved superior to guinea-pig inoculation as a method of detecting leptospiruria and it is suggested that the former technique could be adopted with advantage as a routine aid to diagnosis.

     

Induction of maggot antimicrobial peptides and treatment effect inSalmonella pullorum-infected chickens

Antimicrobial peptides (AMP) are important components of the host innate immune response, as they exert broad-spectrum antimicrobial activities against pathogenic microbes. The AMP allow housefly larva (maggots) to live in harsh environments filled with pathogenic bacteria. In this study, maggot AMP were induced by incubation with inactivatedSalmonella pullorum and crudely extracted. The concentration and antimicrobial activity of the maggot AMP were then measured. In bird experiments, chickens were artificially infected withS. pullorum, and the maggot AMP extracts were used to treat the infected chickens. The expression level of AMP was significantly enhanced byS. pullorum stimulation, and the antibiotic activity of theS. pullorum-induced AMP was significantly stronger than that of the noninduced AMP, especially againstS. pullorum. In the bird experiments, based on survival rate, blood indicators, and intestinal bacterial changes, maggot AMP and antibiotics were successful in treating theS. pullorum-infected chickens. In conclusion, AMP have the potential for further development as a convenient, alternative antibiotic strategy to reduce the use of antibiotics and disease resistance.     

A Preliminary Study of the Development of 19s and 7s Immunoglobulins in Sera of Two Sheep Vaccinated With Brucella Abortus, Strain 19

The pattern of antibody response to vaccination with Brucella abortus, strain 19, was studied in two sheep. Agglutinative activity was detected by the third and fifth days and complement - fixing activity by the fifth and seventh days post-vaccination.

Density gradient centrifugation and DEAE cellulose column chromatography showed the 19S antibody developed first, followed soon after by 7S antibody. The former had disappeared by the 25th day but the latter persisted longer in both sheep. A small amount of 19S antibody was detected in sheep 1 following a booster dose of vaccine but 7S antibody constituted the major secondary response.

The standard tube agglutination test was found to be more efficient than the complement-fixation test for titration of 19S antibody. An increase in the salt concentration to 10% in tube agglutination test rendered it more sensitive in demonstrating 7S antibody.

     

Symptoms and Pathology Produced By Toxic Microcystis Aeruginosa NRC-1 In Laboratory and Domestic Animals

Toxicity tests with lyophilized M. aeruginosa NRC-1 cells have been conducted using mice, guinea pigs, rabbits, chickens, ducks, two calves and one lamb as the test animals.

The symptoms and pathological changes are described. On an equivalent weight basis it required three to five times the oral dosage to kill the large animals and birds as it did to kill the laboratory animals. The symptoms were less pronounced and the survival times were longer in the more resistant animals. Enlargement and congestion of the liver with necrosis of the hepatic cells were constant and pathognomonic. These findings are in general agreement with the observations of other workers who have examined the toxicity of naturally occurring Microcystis waterblooms.

The toxicities and structures of microcystin and of six other biologically active cyclic polypeptides are summarized. The pathological effects produced by microcystin in laboratory and domestic animals resemble those produced in man but differ from those produced in animals by the toxic peptides of Amanita phalloides.

     

Incident Light Immunofluorescence of Alcohol-fixed Colonies of Ruminant Mycoplasma

Two species of ruminant mycoplasma colonies had to be fixed in ethyl alcohol so that incident immunofluorescence method could be applied. In addition, the stain reaction had to be kept for 90 minutes at 37°C.

This fluorescent antibody (FA) method was developed to identify colonies of Vom strain of Mycoplasma mycoides var. capri, V-5 strain of M. mycoides var. mycoides, and PG-2 strain of M. agalactiaeon agar, using fluorescent ultraviolet light. Fluorescence was not demonstrated when heterologous conjugates or normal rabbit serum conjugate were applied but the reaction appeared to be specific for each strain of mycoplasma.

The FA method was able to differentiate specific mycloplasma colonies in mixed cultures.

     

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.

     

Feeding of Ferrets with the Raw Meat and Liver of Chickens Chronically Poisoned with Toxic Groundnut Meal

Chickens were fed a ration containing 30 per cent of toxic groundnut meal for up to six weeks. The concentration of aflatoxin (toxic metabolites of Aspergillus flavus) in the above ration was 3.06 p.p.m. At the end of 2nd, 4th or 6th week the birds were killed. The meat was removed from the bones and put through a meat grinder. The livers of three groups were pooled together. Three control groups of birds kept on commercial pellets were treated similarly.

Female ferrets, two years of age, were used in the present study. They were divided into four groups. The first three groups were given for one month meat from chickens fed the toxic ration for 2, 4, and 6 weeks, respectively. Each of these three groups contained one control ferret that was fed with the meat of chickens fed a commercial ration for a similar period of time. One half of the 4th group was fed pooled liver from intoxicated birds and one half was fed liver from control birds.

No significant changes in the ferret tissues were observed as a consequence of feeding them with the meat or liver from the chickens chronically poisoned with toxic groundnut meal.

     

Entérite nécrotique chez le poulet de gril II. Caractères des souches de Clostridium perfringens isolées

A Gram positive bacillus, strictly anaerobic, was isolated from the viscera of all diseased birds showing lesions of necrotic enteritis. Its morphology and biochemical reactions, the presence of alpha and thêta hemolysins and the production of a lecithinase-C in vitro, all these characteristics indicated a similarity to those belonging to the group of Clostridium perfringens.

The two hemolysins were neutralized in vitro only by the antitoxin A. Broiler chickens injected I.V. with a Viande-Foie (VF) broth culture of Clostridium perfringens together with the antitoxin A survived, whereas those receiving antitoxin C died. These results seem to indicate that this organism belongs to the type A. This bacillus was sensitive to a great variety of antibiotics, except neomycin.

     

Some observations on the use of the morantel sustained‐release bolus in first season‐grazing calves on a Belgian dairy cattle farm

Summary

The efficacy of the morantel sustained‐release bolus (MSRB) in controlling gastrointestinal parasites in first‐season grazing calves was evaluated on a dairy cattle farm in Belgium. The calves grazed a pasture which had been used by bolus‐treated animals in the three previous years. The effect of bolus administration was determined with respect to live weight gain, faecal egg shedding, herbage larval counts, serum pepsinogen levels and ELISA antibody titres.

In spite of an incomplete reduction of faecal egg shedding during the first months of the grazing season, bolus administration resulted in the prevention of parasitic gastro‐enteritis in the calves. A weight gain advantage of 35,2 kg of the bolus‐treated animals over the controls was noted already at two months after turnout. This weight gain advantage was maintained until housing.

The usefulness of serum pepsinogen values and ELISA antibody titres as parameters in prevention experiments is stressed. Both serological parameters gave more information concerning infection level than did the faecal egg output and the herbage larval counts.     

Studies of Escherichia Coli in Gnotobiotic Pigs: IV. Comparison of Enteropathogenic and Nonenteropathogenic Strains

Four gnotobiotic pigs were infected with an enteropathogenic strain of Escherichia coli, and 4 were infected with a nonenteropathogenic strain of E. coli. Pigs killed in pairs at 6, 12, 24, and 48 hours PI. Four pigs were maintained as germfree controls. The discussions were based on the results of 1) clinical observations, 2) necropsy observations, 3) counts of viable E. coli in segments of the small intestine, 4) attempts to isolate E. coli from the heart, liver, and bile, 5) microscopic examination of fixed intestinal sections to determine the location of E. coli and morphologic evidence of the host response, and (6) determinations of the pH of the contents of the various portions of the gastrointestinal tract.

No diarrhea, fluid accumulation, or impairment of the digestive capacity were noted in the pigs infected with the nonenteropathogenic strain of E. coli. The number of viable E. coli detected in the respective segments of the homogenized small intestine was similar in pigs infected with either strain.

Diarrhea occurred continuously starting 18 hours PI in the pigs infected with the enteropathogenic strain and killed 24 or 48 hours PI. The pH of the contents of the cecum and colon became markedly more alkaline simultaneously with the increase in the heterogeneity and fluid content of the cecum and colon and thus appeared to correlate well with the onset of the clinical diarrhea. No enteritis was detected grossly or microscopically.

The characteristics that determine the enteropathogenicity of a strain of E. coli could not be defined from the results, but it was noted that the host response appeared to be quite similar to that of infant rabbits experimentally infected with Vibrio cholera.

     

In ovo oral vaccination with cmpylobacter jejuni establishes early development of intestinal immunity in chickens

1. Chick embryos were orally immunised at day 16 of incubation by injection of heat‐killed Campylobacter jejuni organisms into the amniotic fluid. The response to vaccination was observed at 5 d after hatching or, in some birds which received a postnatal oral booster vaccination, at 7 d after hatching, and the response was observed at 14 d of age.

2. The titres of antibody in serum, bile and intestinal scrapings, the distribution of immunoglobulin‐containing cells in the spleen, duodenum and ileum and the expression on peripheral blood leukocytes (PBL) of the T cell surface markers CD3, CD4 and CD8 were determined.

3. Whereas low titres of anti‐flagellin antibody were detected in serum, bile and intestinal scrapings of unimmunised birds, high titres were observed in immunised birds.

4. An increase in antibody of all isotypes was detectable in serum but the elevation in IgA antibody in intestinal scrapings and bile was particularly striking. This response was reflected in a dramatic increase in immunoglobulin‐containing cells, detected by fluorescent histology, particularly diose associated with IgA and IgM isotypes in the spleen and intestine of immunised birds.

5. Secondary oral boosting after hatching resulted in a depression in serum anti‐flagellin antibody in immunised birds compared to pre‐boosting titres (although still significandy higher than in non‐immunised controls) but an increase in IgA antibody in intestinal scrapings and bile. The number of immunoglobulin‐containing cells was also increased after boosting.

6. Neither immunisation regimen caused a significant change in the numbers of circulating CD3, CD4 or CD8 T cells.

7. These results indicate that in ovo oral immunisation with C. jejuni antigens stimulates the precocious development of immunity in chicks.     

Contribution à l'étude quantitative de la flore bactérienne du gros intestin des porcs dysentériques

The bacterial flora and the pH of the large intestine of dysenteric swine during acute subacute and chronic phases have been submitted to quantitative and qualitative studies. The methods used are based on primary isolation and differentiation of the bacteria by the use of selective media and the subsequent differentiation using the replica plating technique. The most characteristic changes are the following:

1. A significant increase of the pH of the chyme in the large intestine during acute dysentery

2. A significant increase of Vibrio, Escherichia coli and Staphylococcus in the colon and cecum during acute dysentery.

3. A significant increase of Shigella in the colon and cecum during subacute dysentery.

4. The almost total disappearance of Aeromonas and of the yeasts in the large intestine during acute, subacute and chronic dysentery.

5. A significant decrease of Klebsiella, in the cecum, during acute dysentery and of the fungi during subacute dysentery.

6. Decrease of Streptococcus in the colon during acute dysentery.

7. The total quantitative flora of the large intestine do not change very much.

     

Studies of Escherichia coli in Gnotobiotic Pigs VI. Effects of Feeding Bacteria-Free Filtrates of Broth Cultures

Nine gnotobiotic pigs derived from one gilt were fed bacteria-free filtrates prepared from: 1) cultures of an enteropathogenic strain of Escherichia coli 09:K·:NM (Strain 340), 2) cultures of a nonenteropathogenic strain of E. coli 08.K·.H16 (Strain CDC-1466-56), and 3) uninoculated culture medium.

Diarrhea was observed initially two to four hours after feeding the filtrate prepared from the enteropathogenic E. coli. The duration of diarrhea was five to ten hours. No diarrhea was observed after feeding filtrate prepared from uninoculated medium or cultures of nonenteropathogenic E. coli.

The pH values of the feces increased with the onset of diarrhea and decreased to normal after diarrhea stopped.

No histopathological lesions were found.

     

Serologic, Pathologic, and Symptomatic Aspects of Mycoplasmosis of Turkeys

Adult male turkeys were exposed to Mycoplasma gallisepticum isolate 1010 by either intratracheal inoculation, intrasinus inoculation, intranasal inoculation or by contact with inoculated turkeys. The symptomatic, serologic, and pathologic responses to the different types of exposure were compared. Tracheitis occurred only in birds exposed intratracheally or by contact, and sinusitis occurred only in birds exposed via the sinuses. Antibody titers, determined by hemagglutination-inhibition and tube agglutination tests, were initially higher in turkeys exposed by intratracheal and intrasinus inoculation than in those exposed by other means. There were no appreciable differences among the four groups in incidence or severity of air sac lesions.

In addition, the effects of intrasinus exposure to isolate 1010 were compared with those produced by similar exposure to a different isolate, M. gallisepticum isolate 1150. Isolate 1010 caused a slightly higher incidence of sinusitis, but a much lower incidence of tracheitis than isolate 1150. Air sac lesions did not differ in incidence or severity. The differences observed indicate differences in tissue predilection of the isolates.

     

The Effect of Chlortetracycline Feed Additive on Experimental Salmonella Infection of Swine and Antibiotic Resistance Transfer

Four Salmonella strains were used. With two, S. canada and S. enteritidis, establishment of infection in pigs was poor. Although improved establishment was achieved with two strains of S. typhimurium, no infections were extensive. No significant difference was detected between excretion of infecting strains by pigs receiving chlortetracycline at levels of ten gm per ton of feed and that by control counterparts. Comparable establishment of S. typhimurium S192 was achieved in pigs receiving 20 or 40 gm of chlortetracycline per ton.

Resistant variants were isolated from some animals in all experiments using S. typhimurium S192. Almost without exception such variants were isolated only after specimen enrichment. The numbers of specimens from test animals yielding resistant variants were more than twice those of control counterparts. Numbers of such variants in material from the former were much greater.

In vitro transfer of resistance to S. typhimurium S192 was demonstrated both in waters from drinking troughs and during specimen enrichment. The former, indicating the risk of transfer in pigs' environments and packing houses, has disturbing implications for public health. The latter emphasizes the difficulties of determining antibiotic sensitivity of pathogens as they occur in the animal body.

     

The Supracloacal Chromolipoid Body of the Black Vulture (Coragyps atratus): Anatomical, Histological and Histochemical Considerations

During histological and physiological investigations of black vultures (Coragyps atratus) dissection revealed the presence of an “organ”, the supracloacal chromolipoid body, which has no counterpart among other warm blooded animals.

The organ occurs in both sexes and in birds of different ages. It is located in the median sagittal plane dorsal to the cloaca. It is enveloped by a smooth muscle-connective tissue capsule and has a rich blood supply.

The supracloacal body is yellow-brown in color being composed chiefly of islands of pigment cells.

Histochemically, the pigment is a chromolipoid as defined by Ciaccio.