Influence of temperature, social, and dietary stress on development and stability of protective microflora in chickens against S. typhimurium.

In the day-old chick, physiological stress in the form of high or low temperatures or feed and water deprivation either interfered with colonization or altered the protection provided by normal intestinal microflora against subsequent challenge with Salmonella typhimurium. The response was more obvious after individual exposure of chicks to salmonella than after exposure to infected seeders. Physiological and dietary stress at 2 weeks of age did not appreciably alter the S. typhimurium excretion pattern in treated chicks. The protective microflora appears to be quite stable under these stress conditions.

Therapeutic trials with native intestinal microflora for Salmonella typhimurium infections in chickens.

Chicks infected with Salmonella typhimurium at 1 day of age were treated with normal intestinal flora starting 3 or 9 days later. Chicks were reared on the floor in isolated pens with wood shavings for litter. Cloacal-swab cultures were the main criteria for determining S. typhimurium infection. A single treatment reduced the infection period, but multiple treatments hastened the process considerably: after 6-8 weeks, birds given multiple treatments yielded sporadic or no isolations of S. typhimurium. It appears that litter from birds colonized by a protective microflora may serve as a vehicle for transmitting the flora to succeeding groups of birds.

Influence of Mycoplasma gallisepticum, infectious bronchitis, and cyclophosphamide on chickens protected by native intestinal microflora against Salmonella typhimurium or Escherichia coli.

Chickens that have considerable resistance to Salmonella typhimurium or Escherichia coli infection by early development of a native intestinal microflora shed these bacteria following aerosol exposure to Mycoplasma gallisepticum and/or infectious bronchitis virus. Administration of cyclophosphamide to similarly treated chickens induced slight shedding of these bacteria, and the combination of cyclophosphamide and respiratory agents magnified the shedding rate. These agents also influenced the isolation rate of E. coli and S. typhimurium from the trachea and air sacs.

Reciprocal competitive exclusion of salmonella and Escherichia coli by native intestinal microflora of the chicken and turkey.

Both the native intestinal microflora of chickens that protected chicks against salmonellae and Escherichia coli and native turkey intestinal microflora were evaluated for their reciprocal protective capacity in both species against Salmonella typhimurium and a pathogenic strain of E. coli. Nalidixic-acid-resistant forms of the S. typhimurium and E. coli strains were used in seeder-bird and individual-bird challenge tests. Reciprocal protection was provided by native chicken and turkey intestinal microflora in chicks and poults against S. typhimurium and the pathogenic strain of E. coli. The chicken and turkey microflora appeared to be equally effective in protecting the two species from S. typhimurium, but protection against E. coli was somewhat greater in the chicken than in the turkey.

The influence of lactobacilli on the competitive exclusion of paratyphoid salmonellae in chickens.

The competitive exclusion of salmonellae by native gut microflora was investigated by treating chicks with various avian lactobacilli. The evaluation of protection was based on the number of salmonellae adhering to the mucosa of the crop and the cecum, enumeration of salmonellae in fecal droppings, and enrichment of cloacal swabs and fecal droppings using both individual and seeder bird tests. Lactobacilli reduced the number of salmonellae adhering to the crop mucosa by 1 to 2 logs. Treatment with lactobacilli did not lower the number of chickens shedding salmonellae or reduce the number of salmonellae adhering to the mucosa of the cecum. Lactobacilli as a single bacterial treatment played a minor role in protecting the crop, but no protection of the cecum was demonstrated.

Competitive exclusion of intestinal colonization of Escherichia coli in chicks.

A native gut microflora of chickens that protected chicks against Salmonella was evaluated against six pathogenic strains of Escherichia coli using similar methodology. Nalidixic-acid-resistant forms of the E. coli strains were derived for use in seeder-bird and individual-bird challenge tests. A substantial degree of exclusion of intestinal colonization of each of the six pathogenic strains of E. coli was secured in chicks that had been treated with native gut microflora. E. coli appeared to colonize chiefly the crop and cecum. Native gut microflora limited to a much lesser degree the "normal" E. coli present in the intestinal tract of test chicks. The mechanism of protection appears to be similar to that for Salmonella: native gut microflora and E. coli competed for sites of attachment.

Studies on the use of 9R strain of Salmonella gallinarum as a vaccine in chickens.

The 9R strain of Salmonella gallinarum produced hepatitis and splenic lesions without mortality in meat-type and brown-egg-producing strains of chicks, but not in Leghorns. It was not recovered from Leghorns for as long following vaccination as from the other strains of chicks. The infectivity of the 9R strain was determined by the genetic susceptibility and age of the host. Subcutaneous vaccination of 9R produced partial immunity to S. gallinarum in Leghorns as well as in meat-type and brown-egg-producing strains of chickens. Addition of an oil adjuvant appeared to interfere with protection and gave even less protection than did a vaccine prepared from an inactivated oil-adjuvanted smooth strain. Use of the 9R vaccine did not protect against intestinal colonization by S. typhimurium or S. infantis. Potential egg transmission of 9R following vaccination and of a pathogenic strain following challenge of vaccinated birds was indicated by ovarian infection with each strain and by isolation of the pathogenic strain from one egg. All chickens vaccinated subcutaneously with the 9R strain developed antibodies detectable by the microantiglobulin test, but only a few birds developed antibody levels detected by the whole-blood, microagglutination, and tube tests. The inactivated vaccine prepared from a smooth S. gallinarum strain produced the highest and most uniform antibody response. Antibody levels were not related to protection, which is probably dependent on cellular immunity.

The influence of native gut microflora on the colonization and infection of Salmonella gallinarum in chickens.

A native intestinal microflora of chickens that is protective against paratyphoid salmonellae appeared to be partially protective against Salmonella gallinarum and abbreviated the excretion time of S. gallinarum in White Leghorn chicks. Treated birds had a lower incidence of systemic infection measured by liver and spleen culture, and fewer birds developed infection in the intestinal tract. Most of the chickens positive by cloacal swabs excreted S. gallinarum throughout the test period. The low level of protection provided by competitive exclusion suggested that the practice would not prove useful in the practical control of fowl typhoid.

Further studies on competitive exclusion for controlling Salmonellae in chickens.

A native intestinal microflora of chickens which is protective against salmonella readily transferred to penmates and apparently to birds in adjacent pens. The microflora not only minimized infection resulting from exposure following colonization of the gut with microflora, but significantly abbreviated the period of infection when introduced after a salmonella infection was established in chicks. A microflora with undiminished protective activity, sensitive to only a few commonly used antibacterials, was established in a SPF-Cofal/Marek-negative population. Intestinal microflora from mourning doves was at least partially effective in protecting chicks against a naladixic-acid-resistant strain of Salmonella infantis. In limited tests with 2 of 3 sources of protective microflora, the growth rate of chicks in the absence of salmonellae was significantly improved. A hypothesis involving specificity of attachment between the glycocalyces of the protective microflora and of the intestinal mucosa is offered as the likely mechanism of protection.

A supplemental test system to measure competitive exclusion of Salmonellae by native microflora in the chicken gut.

An improved individual challenge test system is described for evaluation of competititve exclusion of salmonellae in day-old chicks by native gut microflora. Evaluation of salmonella protection is based on culture of cloacal and fecal samples by enrichment methods plus enumeration of salmonellae in fresh droppings collected from test groups. The use of a nalidixic-acid-resistant strain of salmonella allowed for ease of measurement. Comparative data are included that were developed by parallel tests by the seeder bird and this individual challenge test system on salmonella protection provided by microflora from a specific-pathogen-free population and from intestinal microflora propagated anaerobically in several media. The individual-bird challenge test system appeared to yield a more precise measurement of protection than the seeder-bird system and indicated that trypticase soy broth is as effective as VL broth for anaerobic culture of the protective microflora. Fresh fecal suspensions were somewhat more dependable than anaerobic cultures in providing a high level of protection against subsequent salmonella infection.

Evaluation of organic acids and other compounds as Salmonella antagonists in meat and bone meal.

Twelve compounds were evaluated as salmonella antagonists in commercial meat and bone meal. The test organism was a nalidixic acid-resistant strain of Salmonella infantis. Adequate moisture to support salmonella growth was assured by the addition of 40% water to each 50 g test sample. Compounds were tested at levels permitted for food additives except formalin which has not been approved. Salmonella counts were determined every few days by spread plates and the pH was recorded from the initial 1:10 dilution. Although the salmonella population initially declined in the presence of several additives, none of the compounds were effective in preventing salmonella multiplication in the MBM except formalin at levels greater than .1%.

Evaluation of several methods of detecting salmonellae in groups of chickens.

Chickens exposed experimentally to Salmonella agona, S. blockley, S. enteritidis, S. infantis, and S. typhimurium were tested serologically by microagglutination and microantiglobulin procedures and culturally by cloacal swabs and environmental samples. Chicks infected at one day old yielded the highest level of positive cloacal swab cultures, whereas exposure of older birds gave maximal serologic titers. Recovery of salmonella from environmental samples is dependent on a number of factors, including excretion rate by the population and survival rate of salmonellae in the environment. Serologic titers persisted after salmonellae could no longer be isolated from cloacal swabs or environmental samples. The possibility of false-positive serologic reactions was explored in two groups of chickens, one exposed to other Enterobacteriaceae, and one composed of eight specific-pathogen-free flocks. Occasional serologic microantiglobulin titers were observed only in the latter group. Extensive culturing of the environment from these flocks had negative results.

Protecting chicks and poults from Salmonellae by oral administration of "normal" gut microflora.

Resistance of young chicks and poults to salmonella exposure was substantially increased by early oral administration of intestinal contents or feces from selected adult chickens. Protection was secured also by administering anaerobic broth cultures of intestinal microflora from selected donor birds. Protection, was substantial for 63 days, the longest period tested, although it could be overcome by severe exposure. The protective mechanism appears to be a consequence of competitive exclusion of salmonella by "normal" microflora of the gastrointestinal tract.

Examination of poultry litter for Salmonellae by direct culture and fluorescent antibody technique.

Direct culture and fluorescent antibody (FA) procedures were used for examining approximately 6200 samples of poultry floor or nest litter for salmonellae. Each sample was cultured directly in tetrathionate brilliant-green (TBG) broth, incubated at 42 C for 48 hours, and plated on brilliant green (BG) agar. The FA procedure was conducted from pools of 4-8 samples prepared by transferring 1 ml supernatant from TBG at 24 hr into 50 ml of either gram-negative (GN) broth, selenite cystine, or selenite brilliant-green sulfapyridine. After 6 hr of incubation at 42 C, BG agar plates were streaked from each pool, and smears were prepared for FA. Salmonella was isolated by plating from 268 of 854 pooled units. Recovery was 97% by direct culture, 86% from GN broth, and 57% from the selenite secondary enrichments. In the FA procedure, GN broth was superior to either of the selenites as the secondary medium from which to prepare smears, giving more FA-positive pools, a higher percentage of confirmed positives, and a lower percentage of false negative pools. FA examination of litter samples can be expedited by using sample pools. A major deficiency of the FA technique was the high percentage of false-positive reactions as judged by inability to isolate salmonellae. From litter cultured directly in TBG, salmonellae were isolated from 97% of the total number of pools culturally positive by combined culture methods.