Immunity to Salmonella gallinarum during ontogeny of the chicken: III. Bactericidal antibody

The bactericidal activity of chicken serum before and after injection of 1–35-day-old chickens with Salmonella gallinarum has been studied. The bactericidal action of immune serum is temperature-dependent. Treatment of both normal and immune serum with mercaptoethanol, by heating to 56°, produced a ten-fold reduction in bactericidal activity. The ingested yolk contained bactericidal factors at 1 day of age. Bactericidal activity of normal serum declined very slowly during the first 5 weeks of development. Bactericidal activity released by injection of S. gallinarum, showed some specificity for the somatic-O antigens and a high degree of species specificity. Injection of S. gallinarum into 1-day-old chicks resulted in the rapid release of a high level of bactericidal activity into the circulation. The bactericidal antibody response to an injection of S. gallinarum increased from 4 to 35 days of development. In chickens of 3 weeks of age, or older, induced bactericidal antibody remained at a high level for several weeks.     

Immunity to Salmonella gallinarum during ontogeny of the chicken: IV. Immunocyto-adherence and a cytophilic antibody

Enhanced numbers of immunocyto-adherent (ICA) cells were found in spleens only after two injections of Salmonella gallinarum and then only if the chicken were at least 4 weeks old at the time of the second injection. There was a steady increase in the proportion of ICA cells elicited by a second injection of S. gallinarum during 6–14 weeks of age. As young chickens are quite resistant to infection with this organism by 5 weeks of age, ICA does not appear to be a protective immunological reaction. Increase in spleen cell populations exhibiting ICA were not always correlated with high titres of cytophilic antibody in the circulation of such birds. In fact, a single injection of S. gallinarum elicited high titres of cytophilic antibody in adult serum, when there was no increase in the level of ICA cells. Titres of cytophilic antibody were closely similar to those of agglutinins; however, these antibodies may be separate entities.     

Immunity to Salmonella gallinarum during ontogeny of the chicken: II. Induction of tolerance or priming by single doses of live or killed bacteria

Partial tolerance was induced in cells forming agglutinins by live or killed, virulent or avirulent strains of Salmonella gallinarum in chick embryos, 1-day and 1-week-old chicks. Tolerance was induced by a single injection of only 100–200 live virulent organisms in 1-day-old and 1-week-old chicks. However, 10⁹ avirulent organisms were required to induce a similar degree of tolerance in 1-week-old chicks. The primary agglutinin response paralleled the relative increase in spleen weight during early development and increased only slightly after 5 weeks of age. Priming to live bacteria occurred at 2 weeks of age; the magnitude of the secondary response showed little further increase with age. The induction of partial tolerance in cells producing agglutinins did not increase susceptibility to infection and further emphasizes that agglutinin production is not a protective immune mechanism for S. gallinarum infection.     

Evaluation of Salmonella enteritidis-immune lymphokines on host resistance to Salmonella enterica ser. gallinarum infection in broiler chicks

The prophylactic treatment of neonatal broiler chicks with lymphokines derived from S. enteritidis-immurazed chickens (SE-ILK) was evaluated for its effect on the birds' resistance to an experimental infection S. enterica ser. gallinarum (SG). On the day of hatch, chicks were injected intraperitoneally with either SE-ILK, control non-immune lymphokines (NILK), or were left untreated. Thirty minutes later, all chicks were orally gavaged with either 10(4) colony forming units (CFU) or 10(6) CFU SG. The chicks were observed twice daily for 10 days for morbidity and mortality. Chicks that died during the experiment had their livers cultured for SG. The survivors were killed and their livers, spleens and caecal tonsils cultured for SG. The prophylactic treatment of chickens with SE-ILK induced significant protection against extraintestinal SG infection when compared to the NILK-treated or non-treated controls as evidenced by: (1) a significant reduction (P< 0.005) in the mortality of chicks challenged with either 10(4) and 10(6) CFU SG; (2) an increased average weight gains of chicks challenged with either 10(4) and 10(6) CFU SG; and (3) a significant (P< 0.001) reduction in the number of chicks with organs culture-positive for SG. The results suggest that the prophylactic administration of SE-ILK can confer non-specific protection to chicks against a pathogenic species of Salmonella resulting in reduced morbidity, mortality, and organ infectivity caused by SG infections of broiler chicks, while enhancing performance during the first 10 days of Ufe.     

Immunity to bacterial infection in the chicken

Bacterial infections remain important to the poultry industry both in terms of animal and public health, the latter due to the importance of poultry as a source of foodborne bacterial zoonoses such as Salmonella and Campylobacter. As such, much focus of research to the immune response to bacterial infection has been to Salmonella. In this review we will focus on how research on avian salmonellosis has developed our understanding of immunity to bacteria in the chicken from understanding the role of TLRs in recognition of bacterial pathogens, through the role of heterophils, macrophages and γδ lymphocytes in innate immunity and activation of adaptive responses to the role of cellular and humoral immunity in immune clearance and protection. What is known of the immune response to other bacterial infections and in particular infections that have emerged recently as major problems in poultry production including Campylobacter jejuni, Avian Pathogenic Escherichia coli, Ornithobacterium rhinotracheale and Clostridium perfringens are discussed.     

Studies on Immunity and Pathogenesis of Salmonellosis: I. Antigen—Antibody Reactions on Circulating Leucocytes of Chickens Infected with Salmonella gallinarum

A technique is described for the withdrawal of circulating leucocytes at intervals from chickens infected with Salmonella gallinarum, and for testing these cells for the presence of cytophilic antibody and bacterial polysaccharide by a cytopathic test.

The results of these experiments have shown that during the early stages of infection, circulating leucocytes develop a marked susceptibility to cytophilic antibodies in serum and later to bacterial polysaccharide. It has also been shown that humoral antibodies, demonstrable by the antiglobulin haemagglutination test, have cytophilic properties.

These results are discussed in relation to the pathogenesis of Salm. gallinarum infection in chickens, and it is suggested that during acute infection, cellular antigen—antibody reactions occur which may result in the development of a hypersensitive reaction.

     

Measurement of antigen—antibody complexes in Salmonella gallinarum infection

Following oral infection of a 12-week-old chicken with an overnight broth culture of Salm. gallinarum, serum was removed on the 9th day and tested for the presence of haemagglutinating antibody using erythrocytes modified with a Westphal lipopolysaccharide extract of Salm. gallinarum. The serum was found to have a high titre and on `Sephadex' fractionation and ultracentrifugal preparation showed a marked IgM response.

Spectrophotometric measurements were made of interactions between the O-antigen extract and the whole serum or its component IgM and IgG fractions. Hapten—antibody complexes were measurable with the whole serum and the IgM fraction, but no complexes were detected with the IgG fraction under the conditions of the experiment.

     

An immunological basis for the anaemia of acute Salmonella gallinarum infection of chicken. I. Haematological changes and their association with in vivo modification of the erythrocytes

The haematological changes occurring during the course of acute Salmonella gallinarum infection of chicken have been investigated. A severe, acute anaemia, together with reticulocytosis and hepato-splenomegaly, were regularly observed. The findings indicated that these changes were due to increased extravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity.

Coincidentally with the development of the anaemia, erythrocytes became regularly modified serologically in vivo, suggesting a possible relationship between these two phenomena. In contrast, in chickens which died of the per-acute form, where normal haematologic values were found, in vivo erythrocyte modification was not detected.

As the anaemia increased in severity, a change in the type and in vivo erythrocyte modification was also frequently noted.

It is considered that in vivo erythrocyte modification initiates the severe haematological changes observed and it is postulated that the underlying mechanism of increased erythrocyte destruction is immunological.

     

Immunity to experimental fowl typhoid in chickens induced by a virulence plasmid-cured derivative of Salmonella gallinarum.

Chickens were immunized by two intramuscular inoculations at 1 and 14 days of age with virulence plasmid-cured derivatives of Salmonella gallinarum and were challenged 14 days later by oral inoculation of ca. 50 50% lethal doses (LD50) of fully virulent S. gallinarum 9. Mortality in the nonimmunized and immunized groups were 36 and 3%, respectively. This difference was highly significant (P less than 0.01). A significant reduction in mortality was also produced following oral challenge with 5,000 LD50 doses. The LD50 values by intramuscular inoculation of the challenge organism into nonimmunized and immunized chickens were log10 (0.13 +/- 1.57) and (9.74 +/- 2.72), respectively. Immunization was effective whether chickens were immunized at 1 and 14 days of age or at 21 and 35 days of age. Serum agglutinins were present in immunized chickens. Immunization with plasmid-cured Salmonella pullorum gave less protection, and immunization with Escherichia coli K-12 possessing the virulence plasmid of S. gallinarum gave none. The plasmid-cured S. gallinarum was made both rough by virulent bacteriophage activity and nalidixic acid resistant (Nalr) to produce a strain designated 9VP-phi rNalr. It was compared with a Nalr mutant of the rough 9R vaccine strain designated 9 Nalr for virulence and immunogenicity. 9VP-phi rNalr was slightly less protective and less virulent than was the 9R vaccine strain.     

An immunological basis for the anaemia of acute Salmonella gallinarum infection of chicken. II. The relationship of the immune response to the development of haemolytic anaemia

The ability of chickens to respond immunologically during acute Salmonella galinarum infection has been examined in relation to the underlying mechanism of the associated haemolytic anaemia.

In spite of the severity and acuteness of the experimental infection, the majority of the chickens showed a marked immune response to lipopolysaccharide (LPS) derived from the challenge organism. Peak antibody titres occurred 5–6 days after infection, coincidentally with in vivo erythrocyte modification and with maximum destruction of erythrocytes. However, those animals which died of the per-acute form of the disease (within 3 days of challenge) showed neither antibody response, nor in vivo erythrocyte modification, and did not develop anaemia.

It was also observed that erythrocytes which were positive to the direct Coombs test persisted in the circulation of surviving chickens for comparatively long periods (on average 4 days).

The evidence is consistent with the hypothesis that an immunologically-mediated mechanism may be responsible for the development of the anaemia.

     

Differences in resistance to Salmonella enterica serovar Gallinarum infection among indigenous local chicken ecotypes in Tanzania.

A study was conducted to evaluate the disease resistance potential in 105 chickens of six indigenous local chicken ecotypes in Tanzania by orally challenging 1-week-old chicks with 2.5 x 10(8) colony-forming units of virulent S. Gallinarum. For 14 days post infection, clinical signs, necropsy findings, antibody titres, packed cell volume, leukocyte population count, and viable bacterial cell counts in the liver and spleen were recorded. Clinical signs were recorded daily but other parameters were recorded on the day of infection, then on days 3, 6, 10 and 14 after infection. Clinical signs of fowl typhoid were evident in chickens from day 3 post infection and disappeared by day 9 post infection. Pathological lesions on sacrificed birds included enlargement of the liver and spleen with foci of necrosis on the liver, spleen and myocardium. The mean viable bacterial cell counts in the liver and spleen varied between ecotypes, although the differences were not statistically significant. There were significant differences in the leukocyte population in the peripheral blood, with one ecotype (Morogoro-medium) showing a consistent and significantly higher heterophil count compared with other ecotypes. It was concluded that there is a selectable resistance potential to S. Gallinarum among the local chicken ecotypes in Tanzania that may be attributable to non-specific host immune responses. Further studies are suggested.     

Innate resistance of mice to Salmonella typhi infection.

The basis for the natural resistance of mice to Salmonella typhi was examined. In contrast to Salmonella typhimurium, the virulence of S. typhi for mice was independent of the mouse strain and was not affected by inactivation of murine macrophages with silica. However, mice were more susceptible to S. typhi when given iron alone or iron and an iron chelator. The results suggest that the failure of S. typhi to undergo net growth in murine tissues reflects an inability of the bacterium to multiply rather than rapid killing by resident macrophages.     

The role of dendritic cells during Salmonella infection

One type of phagocytic antigen-presenting cell (APC) - the dendritic cell (DC) - may have specialized functions during infection with the bacterium Salmonella, including a possible role in transporting Salmonella across the intestinal barrier. In addition, changes in the number, localization and cytokine production of CD8alpha+, CD8alpha-CD4+ and CD8alpha-CD4- DC subsets occur during infection. DCs function in stimulating bacteria-specific T cells by direct presentation of Salmonella antigens and as bystander APCs. Studying the function of DCs during Salmonella infection provides insight into the capacity of these sophisticated APCs, which are a key link between innate and adaptive immunity, to initiate and modulate the immune response to a bacterial infection.     

Antibodies in action: role of human opsonins in killing Salmonella enterica serovar Typhi

Although vaccines have been available for over a century, a correlate of protection for typhoid fever has yet to be identified. Antibodies are produced in response to typhoid infection and vaccination and are generally used as the gold standard for determining vaccine immunogenicity, even though their role in clearance of Salmonella enterica serovar Typhi infections is poorly defined. Here, we describe the first functional characterization of S. Typhi-specific antibodies following vaccination with a new vaccine, M01ZH09 (Ty2 ΔaroC ΔssaV). We determined that postvaccination sera increased the uptake of wild-type S. Typhi by human macrophages up to 2.3-fold relative to prevaccination (day 0) or placebo samples. These results were recapitulated using immunoglobulins purified from postvaccination serum, demonstrating that antibodies were largely responsible for increases in uptake. Imaging verified that macrophages internalized 2- to 9.5-fold more S. Typhi when the bacteria were opsonized with postvaccination sera than when the bacteria were opsonized with day 0 or placebo sera. Once inside macrophages, the survival of S. Typhi was reduced as much as 50% when opsonized with postvaccination sera relative to day 0 or placebo serum samples. Lastly, bactericidal assays indicated that antibodies generated postvaccination were recognized by complement factors and assisted in killing S. Typhi: mean postvaccination bactericidal antibody titers were higher at all time points than placebo and day 0 titers. These data clearly demonstrate that there are at least two mechanisms by which antibodies facilitate killing of S. Typhi. Future work could lead to improved immunogenicity tests associated with vaccine efficacy and the identification of correlates of protection against typhoid fever.     

Development of carbapenem resistance during therapy for non-typhoid Salmonella infection

Multidrug-resistant Salmonella infection is a global problem, and carbapenems may represent the last therapeutic choice. We report a case of infection caused by ceftriaxone-resistant and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium. A bla_CMY-2-containing Tn6092, located on a self-transferable IncI1 plasmid, was found in all isolates derived from the patient. During ertapenem treatment, the strain developed carbapenem resistance. Apart from the OmpD deficiency found in all isolates, the strain further developed OmpC deficiency through a single gene mutation, and became carbapenem-resistant. Salmonella appears to be very plastic in developing antimicrobial resistance. Care must be taken by physicians when treating multidrug-resistant Salmonella infection.     

The appearance and nature of opsonins for goat erythrocytes during the development of the chicken

The appearance of opsonins to goat erythrocytes during embryonic development of the chick has been examined with respect to increase in total macrophage population and increasing incorporation of maternal antibody. The decline in opsonizing power of serum shortly after hatching, probably due to the decline of maternal antibody, is offset by the active production of opsonins. Transfer of increasing concentrations of adult immune sera to embryos resulted in proportionate increases in clearance rates, showing that immune antibody was an efficient opsonin in vivo. Treatment of normal and immune adult sera before introduction into the chick embryo's circulation indicated that some of the natural opsonin was heat-labile at 56° and that reductively cleaved IgM antibody was not opsonic. IgG and IgM sera of similar titre had similar opsonizing power.     

Immunity to Trichinella spiralis. I. Transfer of resistance by two classes of lymphocytes.

Rats can be solidly immunized against Trichinella spiralis by a series of methyridine-terminated oral infections with T. spiralis larvae. Injections of thoracic duct lymphocytes (TDL) obtained from such animals can protect normal rats against a Trichinella challenge. The protective cells belong to two populations which differ with respect to their adherence to affinity columns prepared with rabbit antibody to rat F(ab')2. Immune lymphocytes in the column-adherent B cell fraction are inhibited by vinblastine, whereas those in the non-adherent, T cell fraction are resistant to this drug. The above observations suggest that acquired resistance to T. spiralis is mediated by two classes of lymphocytes: B cells which are delivered to the thoracic duct and hence to the blood while still in active cycle, and T cells which have a potentially long life-span and presumably belong to a pool of recirculating small lymphocytes.     

Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection.

The role of macrophages in the innate immunity of (CBA/N female X DBA/2N male)F1 female mice to Salmonella typhimurium was assessed with silica, an agent which has been reported to selectively inactivate macrophages. Silica, administered intravenously to mice, markedly decreased the phagocytic capacity of splenic macrophages but had no effect on splenic responsiveness to the B-cell mitogen lipopolysaccharidide or the T-cell mitogen phytohemagglutinin, nor did it affect the frequency of surface immunoglobulin-positive cells (B cells). Silica given to mice 1 day before intraperitoneal challenge decreased the 50% lethal dose of S. typhimurium 100-fold. The incidence of survival of mice given silica up to 14 days before infection with a sublethal dose of organisms was also decreased. This susceptibility could also be demonstrated when silica was given 10 days, but not 20 days, after S. typhimurium infection. Poly-2-vinylpyridine-N-oxide, a lysosomal stabilizing agent, abrogated the silica effect. Deaths among silica-treated mice followed uncontrolled multiplication of the organism in the spleen. These results provide direct evidence that macrophages play an essential role in natural immunity to murine typhoid and demonstrate the efficacy of silica as a tool to analyze macrophage function.