Experimental Salmonella enteritidis phage type 4 infection and egg transmission in Japanese quails

Japanese quails were inoculated orally with 1 x 10(10) colony-forming units of Salmonella enteritidis phage type 4 isolated from chicks to obtain information concerning S. enteritidis infection and egg transmission. The inoculation resulted in a bacteraemic infection with seeding of the liver, spleen, intestine, peritoneum, ovule, ovary and oviduct. Some infected birds showed diarrhoea, ruffled feathers, depression, loss of appetite and death. However, most infected birds remained clinically normal with normal egg production. Salmonella enteritidis was isolated from the albumen of 7, yolk of 15, shell of 13, and shell membrane of 15 of 164 eggs. The experiment suggests that S. enteritidis phage type 4 is invasive for Japanese quails and the infected eggs laid by S. enteritidis infected quails are probably the result of transovarian infection.     

Pathologico-anatomic, bacteriologic and serologic findings in laying hens from small neighborhood flocks with Salmonella enteritidis phage type 4 infection

Two small free range flocks with 32 hens, whose Salmonella enteritidis contaminated eggs caused salmonellosis in man, were investigated for localisation of the agent. Salmonella enteritidis phage type 4 was isolated from 12 of 32 hens (37.5%). Eight hens (25%) harbored the bacterium in the ovary and/or the oviduct. The rapid slide agglutination test with Salmonella pullorum-antigen revealed 21 positive hens (66%). All eight hens with Salmonella enteritidis phage type 4 in the reproductive tract were seropositive. The significance of these findings for the control of Salmonella enteritidis phage type 4-infections in poultry is discussed.     

The effect of killed Salmonella enteritidis vaccine prior to induced molting on the shedding of s. enteritidis in laying hens

Effects of administering killed Salmonella enterica serovar enteritidis (SE) vaccines to laying hens prior to induced molting on egg production and on shedding of SE were investigated. Forty hens were vaccinated with one of two SE vaccines available commercially in the United States and Japan. Twenty-five days after vaccination, feed was withdrawn for 2 wk from 20 vaccinated plus 10 unvaccinated hens to induce molt. Four days after molt induction, all hens were challenged with a dose of 2.4 X 10(9) of SE. For the 25 days following administration of the SE bacterins, egg production in vaccinated hens showed approximately a 15% decrease. After molt induction, egg production in molted hens ceased and then returned to normal levels 8 or 9 wk postvaccination. Through the 3-mo experimental period, the decreases in numbers of eggs laid in the unvaccinated/molted group and two vaccinated/molted groups were 225 (26.2%), 245 (28.4%), and 274 (31.9%), respectively, compared with 860 in the unvaccinated/unmolted group. There was no significant difference in egg lay at the P < 0.05 level among the former three groups. Hens in the vaccinated/molted groups shed about two logs less SE than hens in the unvaccinated/molted group 3 14 days postchallenge (P < 0.05 or 0.01). These results indicate that vaccination prior to induced molting might be effective in preventing the exacerbation of SE problems within flocks in which the potential for SE contamination may exist.     

Salmonella enteritidis clearance and immune responses in chickens following Salmonella vaccination and challenge

Our previous work showed that the cell-mediated immunity (CMI) was enhanced by live Salmonella vaccine (LV). The objective of this study was to evaluate the impact of live and killed Salmonella vaccines on Salmonella enteritidis (SE) clearance and to determine if the clearance was mediated by cell-mediated and/or humoral immunity. Chickens were first immunized at 2 weeks of age followed by a booster dose at 4 weeks, challenged with live SE 2 weeks later (6-week-old) and tested for CMI, antibody response and SE clearance 1-week post SE-challenge (7-week-old). Spleen cell proliferation induced by SE-flagella and Concanavalin A (Con A) were significantly higher and SE shedding was significantly lower in the LV group. The splenic CD3 population was significantly lower and B cells were higher in the control group compared to all the SE-challenged groups (with and without vaccination). Serum antibody to SE-flagella and envelope were significantly higher in the KV group compared to all the other groups. These results suggest that LV protects against SE infection, probably by enhancing the CMI.     

Use of pilocarpine-induced alimentary secretions to measure intestinal shedding of Salmonella enteritidis in chickens.

A technique has been developed that uses the parasympathomimetic drug pilocarpine to induce alimentary secretions in chickens for measuring local immune responses to Salmonella enteritidis strain SE6. A study was conducted to determine if these secretions could also be used to detect intestinal SE6 shedding. White leghorn chickens infected with 1 x 10(9) SE6 were samples weekly using cloacal swabs, and the isolation rates from these samples were compared with alimentary secretions induced by oral administration of phosphate-buffered saline followed 45 minutes later with an intraperitoneal injection of 5% pilocarpine. At 9 days postinfection, isolation rates from the alimentary secretions were significantly higher than isolation rates from the swabs, and by day 16 they were double those from the swabs. In separate small experiments, alimentary secretions induced by pilocarpine alone also had significantly more SE6 isolations than did cloacal swabs on two of three sampling times examined. Direct culture of feces resulted in numerically but not significantly greater SE6 isolations than did cloacal swabs on two of three sampling times. These results indicate that induced intestinal material is a better sample source than cloacal swabs for detecting S. enteritidis intestinal infections in chickens and could have many applications in intestinal pathogenesis research.     

Airborne infection of laying hens with Salmonella enteritidis phage type 4.

Hens were exposed to small-particle aerosols containing different concentrations of Salmonella enteritidis phage type 4. They developed a systemic infection and some birds were still excreting the organism in the faeces when killed 28 days after infection. S enteritidis was present for a similar period in a wide range of alimentary tract issues and in the ovary and oviduct.     

The relationship between the duration of fecal shedding and the production of contaminated eggs by laying hens infected with strains of Salmonella enteritidis and Salmonella Heidelberg

Egg contamination by Salmonella Enteritidis has remained a significant public health problem for nearly two decades, and Salmonella Heidelberg has also been recently implicated in egg-transmitted human illness. Colonization of the intestinal tract is a necessary precursor to the invasion of reproductive organs and subsequent deposition inside eggs laid by infected hens, but the relationship between the persistence of Salmonella in the intestinal tract and the likelihood of egg contamination has been uncertain. In this study, groups of laying hens were inoculated with large oral doses of strains of Salmonella Enteritidis and Salmonella Heidelberg, including variants of the original parent strains that had been reisolated from eggs laid by infected hens in a prior study. The shedding of Salmonella in voided feces was monitored for 6 wk postinoculation, and all eggs laid by infected hens between 5 and 22 days postinoculation were cultured for Salmonella in their contents. The mean duration of fecal shedding was significantly longer for the previously passaged Salmonella strains (26.7 days) than for the original parent strains (17.5 days), and the passaged strains caused a significantly higher frequency of egg contamination (6.4%) than did the parent strains (3.3%). However, the duration of fecal shedding and the frequency of egg contamination were not correlated for any of the Salmonella Enteritidis or Salmonella Heidelberg strains.     

Enzyme-linked immunosorbent assay for the detection of Salmonella enteritidis infection in chickens

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.     

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology

Age-related susceptibility patterns of turkeys, broilers, and specific pathogen-free (SPF) White Leghorn chickens to experimentally induced infection with turkey or chicken rotavirus isolates were compared. The following determinants were evaluated: clinical signs, onset and duration of virus production, viral titers, involvement of intestinal villi in the replication of the virus, and the development of antibodies against the virus. Older turkeys and chickens were more susceptible than were their younger counterparts, turkeys were more susceptible than were broiler and White Leghorn chickens (regardless of age), and broiler chickens were slightly more susceptible than were age-matched White Leghorn chickens. Turkeys developed diarrhea, accompanied by high viral titers within 1 day after inoculation with virus. Viral antigen was found in the epithelial cells of the intestinal villi throughout the intestinal tract and some cells of the cecal tonsils. Antibodies could be detected as early as 4 to 5 days after inoculation. These findings were more pronounced in turkeys inoculated at 112 days of age than in birds inoculated at a younger age. Age-related susceptibility patterns were similar in White Leghorn and broiler chickens. Infection was subclinical in birds less than 56 days old, whereas older birds developed soft feces. Egg production in the White Leghorn chickens decreased after being inoculated with virus at 350 days of age.     

Microbiological and histopathological effects of an induced-molt fasting procedure on a Salmonella enteritidis infection in chickens.

A study was undertaken to determine if a 2-week feed-removal protocol, as is used by industry to induce a molt in aging hens, would affect the course of a Salmonella enteritidis infection. White leghorn hens aged 69-84 weeks were deprived of feed to induce a molt, and on day 4 of the fast, the birds were orally infected with 5 x 10(6) S. enteritidis. S. enteritidis organisms were enumerated in the spleen on day 6 and from the alimentary tract on days 7, 14, 21, 28, and 35. Little difference was detected in numbers of S. enteritidis from spleens of molted and unmolted hens. Significantly more molted hens shed detectable intestinal S. enteritidis than unmolted hens on day 14 (one of two trials) and day 21 (one of two trials). Intestinal levels of S. enteritidis were increased 100- to 1000-fold in the molted birds on day 7 (one of two trials) and day 14 (two of two trials), and many of the hens exhibited bloody alimentary secretions. Histological examination of the intestinal tract of S. enteritidis-infected molted hens showed increased inflammation in the epithelium and lamina propria of colons and ceca, compared with unmolted infected hens.     

Resistance to fecal shedding of salmonellae in pigs and chickens vaccinated with an aromatic-dependent mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the effectiveness of an aromatic-dependent mutant of Salmonella typhimurium as a parenteral vaccine for prevention of fecal shedding of Salmonella spp. Pigs and chickens were vaccinated IM, with 1 x 10(9) and 1 x 10(8) organisms, respectively, followed by a second identical vaccination 2 weeks later. Salmonella organisms were not detected by analysis of fecal or cloacal swab specimens from any animal after vaccination. Deleterious side effects were not noticed after vaccination. Pigs were challenge-inoculated PO with 1 x 10(12) virulent S typhimurium 1 week after the second vaccination. Chickens were challenge-inoculated PO with 3 x 10(8) organisms of either S enteritidis or the virulent parent strain of S typhimurium 3 weeks after the second vaccination. Vaccinated pigs shed Salmonella spp significantly less frequently than did nonvaccinated pigs. Vaccinated chickens challenge-inoculated with either S enteritidis or S typhimurium also shed Salmonella less frequently than the corresponding nonvaccinated control birds; however, the difference was not significant.     

Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment.

Seven Canadian layer flocks with Salmonella enteritidis in their environment were investigated to determine the numbers of hens infected with S. enteritidis, the localization of S. enteritidis in organs of infected hens and the numbers of S. enteritidis-infected eggs produced by two affected flocks. By a microagglutination test (MAT) using S. pullorum antigens, these flocks had more seropositive hens (mean 51.9 +/- 16.9%) than two Salmonella-free flocks (mean 13.0 +/- 4.2%). Culture of tissues of 580 hens (433 seropositive) from the seven flocks detected 26 (4.5%) S. enteritidis-infected hens from two flocks. In one flock, 2/150 hens were infected with S. enteritidis phage type (PT) 8, which was confined to the ceca, and no Salmonella spp. were isolated from 2520 eggs (one day's lay). In the second flock, where 24/150 hens were infected with S. enteritidis PT13, extraintestinal infection was found in nine hens and involved the ovaries and/or oviduct in two hens. Salmonella enteritidis PT13 was isolated from one sample of egg contents and from one sample of cracked shells from among 14,040 eggs (one day's lay) from this flock. The overall prevalence of S. enteritidis-contaminated eggs from the two flocks with infected hens was less than 0.06%. Other Salmonella spp. isolated were S. heidelberg from 58 hens (10%), and S. hadar, S. mbandaka and S. typhimurium from one hen (0.2%) each. The MAT with antigens of S. pullorum had a sensitivity of 81% and a specificity of 24% for detecting S. enteritidis-infected hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Environmental contamination and detection of Salmonella enterica serovar enteritidis in laying flocks.

Faecal, dust and other environmental samples were collected from the floors, droppings belts, egg-collection systems and other areas of 14 cage-layer flocks, 10 barn egg production flocks and seven free-range flocks, and cultured for Salmonella species. The distribution of the organism varied with its prevalence and with the vaccination status of the birds. No one sample type was found to be suitable for identifying all contaminated houses. Salmonella was also frequently found on egg-packing equipment and in samples from rodents and wild birds.     

Growth of Salmonella typhimurium and Salmonella enteritidis in egg yolks from highly immunized hens.

This experiment was conducted to ascertain whether the growth of Salmonella Enteritidis (SE) or Salmonella Typhimurium (ST) would be suppressed in the presence of antibodies contained in egg yolks. Specific pathogen-free chickens (102 days of age) were subcutaneously immunized with oil-adjuvanted bacterin of SE or ST, twice within a four-week interval. During 160 to 170 days of age, eggs were collected, the yolks were removed and mixed with an equal volume of physiological buffered saline, inoculated with ten colony forming units (CFU) of SE or ST, and incubated at 37 degrees C or 20 degrees C for 23 hr. The growth of organisms in each yolk solution was examined. The egg yolk derived from non-immunized hens was examined in the same manner as the controls. There was no difference in the growth titer between the antibody-positive yolk and the negative yolk. The result suggests that the antibodies in the yolk do not influence the growth of each organism, even if the hens are highly immunized.     

Salmonella enteritidis infection in two species of psittaciformes.

In 1990, Salmonella enteritidis phage type 4 was recovered from two young (less than 20-week-old) lilac-crowned Amazon parrots (Amazona finschi Schlater), one in Tennessee and one in Kansas. The parrot from Tennessee was treated for a plugged naris and anorexia before the S. enteritidis infection was discovered. The parrot from Kansas exhibited signs of septicemia and died within 24 hours of examination. An apparently healthy green-cheeked conure (Pyrrhura molinae) on the same premises as the parrot from Tennessee was positive for S. enteritidis phage type 4 on a cloacal swab. These are the first reported cases of avian infection with S. enteritidis phage type 4 in the United States. Because several infectious agents were present simultaneously in the Amazon parrots, it was difficult to determine the precise role of S. enteritidis phage type 4 in the clinical presentations.     

Colonization of reproductive organs and internal contamination of eggs after experimental infection of laying hens with Salmonella heidelberg and Salmonella enteritidis

Internal contamination of eggs laid by hens infected with Salmonella enteritidis has been a prominent international public health issue since the mid-1980s. Considerable resources have been committed to detecting and controlling S. enteritidis infections in commercial laying flocks. Recently, the Centers for Disease Control and Prevention also reported a significant association between eggs or egg-containing foods and S. heidelberg infections in humans. The present study sought to determine whether several S. heidelberg isolates obtained from egg-associated human disease outbreaks were able to colonize reproductive tissues and be deposited inside eggs laid by experimentally infected hens in a manner similar to the previously documented behavior of S. enteritidis. In two trials, groups of laying hens were orally inoculated with large doses of four S. heidelberg strains and an S. enteritidis strain that consistently caused egg contamination in previous studies. All five Salmonella strains (of both serotypes) colonized the intestinal tracts and invaded the livers, spleens, ovaries, and oviducts of inoculated hens, with no significant differences observed between the strains for any of these parameters. All four S. heidelberg strains were recovered from the interior liquid contents of eggs laid by infected hens, although at lower frequencies (between 1.1% and 4.5%) than the S. enteritidis strain (7.0%).     

Effect of daily feed intake in laying period on laying performance, egg quality and egg composition of genetically fat and lean lines of chickens

1. The objectives of this study were to evaluate the effects of feed intake on laying performance, egg quality and egg composition in a Fat line and a Lean line during the laying period (34 to 54 weeks of age). 2. The experiment was a 2 × 2 factorial design with two dietary intake levels (nutrition recommendation and 75% of recommendation) and two broiler genotypes (Fat line and Lean line). Hens (384 of each line) were randomly divided at 23 weeks of age into 4 treatments, with each treatment represented by 12 replicates of 16 birds each. The experiment started when the rate of lay reached 5% and continued until 54 weeks of age. 3. The results indicated that there was a significant interaction between daily feed intake and genotype on egg production, egg weight, percentage yolk, yolk/albumen ratio and yolk cholesterol content. Fat line hens produced significantly more eggs and had a lower incidence of cracked eggs than the Lean line hens. The reduction in feed intake decreased egg weight and increased egg production, egg-shape index and cholesterol content of yolk significantly.     

Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep

Two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes. The seroprevalence of C. burnetii infection was studied by using an ELISA and the immunofluorescence (IF) assay was applied to the contents of vaginal swabs. In addition, a PCR assay, with primers based on a transposon-like repetitive region of the C. burnetii genome (trans-PcR), was used for the highly sensitive and specific detection of C. burnetii in vaginal swabs, milk and faeces. Of the 34 animals tested at parturition, eight (24 per cent) were positive by ELISA, 11 (32 per cent) were positive by IF, and 15 (44 per cent) were positive when the vaginal swab extract was subjected to the trans-PCR assay. C. burnetii was therefore detected by PCR in the vaginal swabs of seven seronegative ewes. However, five weeks after lambing, 16 (47 per cent) of the animals tested were ELISA positive but only two animals (6 per cent) were positive by PCR. Among the ELISA- and PCR-positive animals, eight (25 per cent) shed coxiella in their milk and six (18 per cent) did so in their faeces.     

The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens,followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

Three groups of 100 individually marked salmonella-free chickens were followed for a period of 53 wk. The chickens were infected as day olds by crop instillation of 10(8) colony-forming units: one group with Salmonella enteritidis and a second group with Salmonella typhimurium. A third group was kept uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 8 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yolk samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods in surveillance programs, in particular to detect flocks in early stages of infection before a measurable serologic response has been raised.     

Avian leukosis virus infection and congenital transmission in lines of chickens resisting selection for reduced shedding

Two lines (D and E) of three breeder lines of chickens that had resisted selection for reduced avian leukosis virus (ALV) congenital transmission on the breeder's premises did not resist the same selection procedures (tests for gs-antigen in albumen) under laboratory conditions. The incidence of ALV congenital transmission in the remaining third line (F) was spontaneously reduced from 13% to 0.9%. Environmental ALV exposure of uninfected chicks after hatching induced 7-10% of the progeny from lines E and F to become congenital transmitters but had negligible effects on line D. Neither errors in identifying dams nor horizontal transmission leading to congenital transmission were great enough to explain the lack of improvements in the three lines on the breeder's premises. Conditions of environmental exposure on the breeder's farm seem most likely to account for the resistance to reduced shedding. These findings suggest that the effectiveness of testing and selection procedures used to reduce ALV may be greatly influenced by the environment.