Growth of salmonella in chickens' yolk sacs and its relationship to pathogenicity

Newly hatched chicks were inoculated in the yolk sacs with standardized suspensions of Salmonella anatum, S. heidelberg, or S. infantis. At intervals between 3 and 48 hr postinoculation, chicks from each group were sacrificed, the average number of viable cells per yolk sac was determined, and liver tissue from each chick was examined for Salmonella. Growth patterns of the three species were almost identical when each chick was inoculated with about 3.5 million cells, but S. heidelberg was recovered more frequently from the liver, and caused a much higher percentage of mortality than did either S. anatum or S. infantis. When 100-fold dilutions of the suspension of S. heidelberg were used, mortality and recovery rates of the bacterium from the liver were directly related to the number of cells injected. The logarithmic growth phase was lengthened as the number of cells in the inocula was decreased; consequently, there was little difference in the average number of S. heidelberg cells per yolk sac at 36 or 48 hr postinoculation regardless of number of cells injected. Results of these trials indicated that factors other than rate of multiplication in the yolk sac are responsible for observed differences between Salmonella species in degree of pathogenicity for baby chicks.     

Effects of antihelminthic treatment on cell-mediated immunity in Gentoo penguin chicks

Intestinal parasites suppose a cost to hosts as they compete directly for nutritional resources. Therefore, hosts must defend themselves against intestinal parasites by mounting an immune response. Many penguin species acquire parasites through their diet and transfer these parasites to their chicks when feeding them. High parasite loads in penguin chicks could have effects on their growth and body condition, and ultimately on their survival. Here, we evaluated the effect of parasites on the cell-mediated immune system in Gentoo penguin (Pygoscelis papua) chicks at Stranger Point (25 de Mayo/King George Island, South Shetland Islands). To this end, 12 chicks were experimentally deparasitized with a mixture of anthelminthic drugs (albendazole and praziquantel), whereas 10 others were kept as control. We measured cutaneous cell-mediated immunity in response to immunization with phytohemagglutinin (PHA). We also analyzed the leukocyte profile in both treated and control groups before and after the treatment. After the treatment, deparasitized birds showed larger foot-web swelling in response to PHA injection than control birds. Deparasitized penguins also showed lower eosinophil and monocyte counts than controls, whereas heterophils, lymphocytes, and total white blood cell counts did not differ between groups. Our results suggest that Gentoo penguin chicks parasitized with intestinal parasites suffer a cost in terms of reduced cell-mediated immune responses that could ultimately affect their survival.     

玳瑁和绿海龟幼体外周血细胞的观察与比较

对玳瑁(Eretmochelys imbricata)和绿海龟(Chelonia mydas)外周血细胞形态特征及其数量进行了观察、测定与比较.结果表明,在2种海龟外周血都观察到7种血细胞:红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞和血栓细胞,除了绿海龟观察到大、小2种嗜酸性粒细胞外,另外几种血细胞的形态结构与其他爬行动物相似.白细胞分类计数表明,2种海龟白细胞中以嗜中性粒细胞数量最多,其次是淋巴细胞和单核细胞,嗜酸性粒细胞仅有少数,嗜碱性粒细胞极少,并且此类细胞在玳瑁的白细胞分类计数中为零.玳瑁红细胞数量为(346.7±68.4)×10~3个/μl,比绿海龟红细胞含量少,绿海龟为(403.3±170.6)×10~3/μl;玳瑁白细胞及血栓细胞数分别为(7.7±1.9)×10~3个/μl和(9.6±2.2)×10~3个/μl,绿海龟分别为(7.3±2.8)×10~3个/μl和(7.5±3.7) ×10~3个/μl.     

Effect of Brilliant Green on the Motility of Salmonellae and Other Selected Microorganisms

The effect of Brilliant Green on motility was studied with Salmonella anatum, S. derby, S. tennessee, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa. Semisolid tryptic soy-agars containing 0, 20, or 40 mg of Brilliant Green per liter were used as the motility media. Both concentrations of Brilliant Green inhibited the growth of the non-Salmonella species through the semisolid agar. For 24 hr, the Brilliant Green appeared to limit the growth of the salmonellae; however, by 48 hr the salmonellae were able to grow through the semisolid agars. The presence of Brilliant Green in the motility media aided in the detection of Salmonella when mixed cultures were used.     

Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils

Heterophils are the predominant polymorphonuclear leukocytes (PMNs) in poultry. The oxidative burst of activated heterophils, which generates reactive oxygen species (ROS), is one of the first line cellular defenses against invading microorganisms. In this report, the oxidative response of heterophils from neonatal chicks to in vitro stimulation by various inflammatory agonists was investigated using a fluorescence microplate assay. Both non-opsonized formalin-killed Salmonella enteritidis and Staphylococcus aureus were able to stimulate heterophil oxidative burst. The phorbol myristate acetate (PMA) was the most potent stimulant for the chicken heterophil oxidative response, whereas, the bacterial cell surface components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were less effective. Protein kinase C (PKC) is an essential signaling component regulating heterophil oxidative response to stimulation by PMA, LPS, LTA and S. enteritidis. However, inhibition of PKC did not affect the oxidative response to stimulation by S. aureus, suggesting differential signaling pathway responsible for the activation of oxidative burst by Gram-negative S. enteritidis and Gram-positive S. aureus. Inhibition of mitogen activated protein (MAP) kinase p38 and extracellular response kinase (ERK) by SB 203580 and PD 098059, respectively, did not inhibit activated oxidative burst.     

Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.     

Growth and Destruction of Salmonella typhimurium in Egg White Foam Products Cooked by Microwaves

Currently, in both home and institutional food preparation, attempts are being made to produce high quality foods with a minimum of time and effort. Research is being carried out to develop equipment capable of cooking foods in a fraction of the time required by conventional methods; as a result, the problem arises as to the bacteriological safety of these products. We investigated the microbiological aspects of lemon and chocolate foam pies before and after cooking by microwaves for less than 2 min. Pies prepared with sterile equipment under sanitary conditions were inoculated with washed cells from a 24-hr broth culture of Salmonella typhimurium and were incubated for 24, 48, and 72 hr at 33 C. The same procedures were followed in model systems to determine the effects of various sugar and pH levels on the survival of S. typhimurium. No S. typhimurium was detected in inoculated cooked or uncooked lemon pies by the plating method; with the Lactose Broth pre-enrichment method, survivors were detected in lemon pies immediately after preparation. After electronic cooking, no survivors were detected in lemon pies by plate counts, whereas cells were recovered from chocolate pies by the Lactose Broth method. Both chocolate and lemon pies had lower counts throughout the 72-hr incubation period than the model systems compared to them. With the model systems, at pH 7.3, media containing sugar inhibited the growth of S. typhimurium but did not cause a significant reduction in counts during the incubation times studied. At pH 3.7, media without sugar yielded no cells with the Lactose Broth pre-enrichment method after 48 hr of incubation, whereas media with sugar were not sterile until after 72 hr of incubation. Apparently, the presence of sugar in the medium had a protective influence which made the lethal effect of the low pH less severe.     

Replication of Poliovirus in Phytohemagglutinin-stimulated Human Lymphocytes

Poliovirus replication has been studied in human lymphocytes during the course of blastogenesis under phytohemagglutinin (PHA) stimulation. Enhancement of virus replication in PHA-stimulated leukocyte cultures was due to an increase in number of virus-producing cells. Virus yield was approximately 10 plaque-forming units (PFU) per producing cell, both in stimulated and in nonstimulated cultures. Adsorption and penetration studies showed that freshly drawn lymphocytes (unlike other leukocytes) were resistant to virus infection, but they became susceptible to the virus during PHA stimulation. Also, the eclipse of the virus after penetration was enhanced during blastogenesis of the lymphocytes. Our findings suggested that the monocytes in the leukocyte cultures were infected initially. In PHA-stimulated cultures, the virus then spread to lymphocytes which became susceptible to virus infection during blastogenesis. Polymorphonuclear cells died within 24 to 48 hr after initiation of the cultures and apparently could not support poliovirus replication.     

Thermal inactivation of eight Salmonella serotypes on dry corn flour.

Dry heat was used to inactivate Salmonella newington, Salmonella typhimurium, Salmonella anatum, Salmonella kentucky, Salmonella cubana, Salmonella seftenberg, Salmonella thompson, and Salmonella tennessee in corn flour at 10 and 15% moisture. The flour was spray inoculated at 10(5) Salmonella cells per g and then stored at 49 degrees C (120 degrees F); viable Salmonella cells were counted on Trypticase (BBL Microbiology Systems) soy agar plates every 30 min for the first 4 h and then at 4-h intervals for 20 additional h of storage. After 24 h, 99.9% of all Salmonella cells were killed. S. thompson and S. tennessee were more resistant to heat inactivation than the other serotypes. Naturally occurring contamination by Salmonella spp. in dry food products could be significantly reduced with this treatment.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Rapid Determination of Salmonella in Samples of Egg Noodles, Cake Mixes, and Candies

A glass apparatus system was compared with a standard enrichment broth-selective agar method to test samples of egg noodles, cake mixes, and candy for the presence or absence of salmonellae. The glass apparatus system used fermentation of mannitol, production of H(2)S, or motility, in conjunction with a serological test of flagellar antigens, to detect salmonellae. No salmonellae were detected in 173 samples of food products. Of these samples, 171 were found to be Salmonella-negative after 48 hr with the glass apparatus system. After 72 hr, the standard Salmonella procedure yielded 38 samples which produced Salmonella false-positive results on selective agars. Inoculation of samples with cultures of Salmonella showed that approximately one inoculated cell could be detected after 48 hr of incubation with the glass apparatus. The standard Salmonella test requires a minimum of 72 hr for completion. Compared with the standard Salmonella test, the glass apparatus system is a more rapid and simple system that can be used to determine the presence or absence of Salmonella in these food products.     

Cellular immunological responses of pheasant during endogenous development of Eimeria colchici

We examined the time course and histological localisation of the developmental stages of Eimeria colchici. The prepatent period in the caeca of pheasants was 6 days. The patent period began on day 7 post-infection (p.i.) and ended on day 11 p.i. with peak production of oocysts on days 8-9. The peripheral blood lymphocytes of pheasant chicks showed a significant increase in proliferation to E. colchici antigen from day 5 p.i., with peak on day 14 p.i. The metabolic activity (respiratory burst) of heterophils increased on days 3, 4 and 14 p.i. The total number of peripheral blood leukocytes and lymphocytes in the infected pheasant chicks had increased by day 2 p.i. and reached a maximum on day 4 of the experiment. Days 5 and 6 p.i. were characterised by a drop in the number of these cells.     

Changes in nucleotide DNA structure and adhesive properties of blood leukocytes in early period after irradiation of animals]

Postirradiation changes of adhesive properties and supercoiled nucleoid DNA structure of blood leukocytes were studied in Macaca nemestrina and in rats. The dynamics of membrane changes demonstrated a temporary increase of the leukocyte adherence 24 h after nonlethal irradiation of rats, followed by a decrease of this parameter to normal levels on h 48. After lethal irradiation of both animal species the increase in adhesive leukocytes fraction was shown already on hr 3. This higher index was observed until the end of experiments (5 days). Early (on h 3-6) temporary loosing of supercoiled DNA structure was demonstrated in the total blood leukocytes of nonlethally irradiated animals, this seems to be connected with lymphocyte changes. This process was not dependent on altered adhesive properties of leukocyte membranes. The membrane changes of leukocytes preceded decondensation of supercoiled DNA after lethal irradiation of animals. Loosing of supercoiled DNA progressively increased on hour 24 and in later postirradiation period.     

Accessory replicons of species of Salmonella and Shigella.

Shigella and Salmonella strains isolated from clinical samples were examined. Out of 42 Shigella strains tested, 17 (40%) were found to be colicinogenic and another 3 were lysogenic. All three lysogens yielded a phage antigenically homologous to coliphage P2. Out of 30 strains tested, only 1 was found to be resistant to both neomycin and sulfamethoxazole. Out of 48 strains of Salmonella tested for drug resistance, only 2 showed multiple drug resistance. In contrast to Shigella isolates, the Salmonella isolates were infrequently (approximately 5%) bacteriocinogenic. The frequency of lysogeny in Salmonella strains was found to be 6% when tested on Salmonella typhimurium LT2, but by using a set of five indicators belonging to species Salmonella potsdam, Salmonella mbadanka, Salmonella dublin, Salmonella london, and Salmonella wandsworth, 50% of the strains were shown to be lysogenic. Salmonella phages related to P22 were recoverable from Salmonella saintpaul, Salmonella indiana, and Salmonella heidelberg. Some isolates of S. typhimurium yielded a temperature-sensitive and P22-heterologous phage which was found to be a more efficient transducer of bacterial genetic markers than P22. EcoRI-generated fragments of the DNA of some phages permitted the establishment of a clonal descent for some of the wild-type lysogenic bacterial strains. This last observation points out the potential usefulness of prophages as epidemiological markers.     

Lymphocyte and granulocyte migration across the endothelial layer of bovine pulmonary artery intimal explants towards lymphocyte conditioned medium

An intravenous infusion of endotoxin into sheep results in accumulation of equal numbers of lymphocytes and granulocytes in the pulmonary microcirculation. The role of the sequestered lymphocytes in acute lung injury is not known. The present study examines whether lymphocyte migration through pulmonary endothelium contributes to endothelial damage and also examines the effect of lymphokines on granulocyte migration. Bovine pulmonary artery intimal explants were mounted in Boyden chambers and conditioned media, prepared from bovine peripheral blood lymphocytes, was used as the chemoattractant. The rate of 51Cr labelled bovine granulocyte lymphocyte migration into intimal explants was determined over a 3 hr incubation period. Permeability changes were assessed by adding trace amounts of 14C-sucrose and 3H-water to the upper well and following their rate of equilibration with the lower well. 6-Keto-PGF1 alpha was measured in the upper well. Lymphocyte conditioned media was found to be chemotactic for both lymphocytes and granulocytes (lymphocyte migration at 60 min: lymphocyte conditioned media = 18.5 +/- 2.3%, mean +/- s.e. RPMI control = 12.5 +/- 1.5; granulocyte migration at 120 min: conditioned media = 36.1 +/- 5.7, RPMI control = 18.2 +/- 3.0). Ultrastructural examination revealed leukocyte migration followed an orderly sequence during which the leukocytes maintained close contact with the adjacent endothelial cells. No structural evidence of endothelial cell damage was seen at any time examined. Granulocyte migration was associated with an increased rate of 14C-sucrose equilibration after 2 hr of incubation (lower well counts/upper well counts at 2 hr, RPMI control = 0.18 +/- 0.02; lymphocyte conditioned medium = 0.30 +/- 0.04) indicating alteration in the endothelial barrier function. Leukocyte migration, particularly lymphocyte migration, was accompanied by a marked increase in prostacyclin accumulation (3 hr: no leukocytes, 188 +/- 17 ng/ml; lymphocytes, 560 +/- 104). These in vitro findings suggest that lymphocytes and lymphokines may be involved in acute lung injury and also that permeability changes associated with granulocyte migration may depend on the chemoattractant.     

Comparison of the intracellular bacterial killing activity of leukocytes in musculocutaneous and random-pattern flaps

The in vivo physiologic response to gram-negative bacterial inoculation within wound cylinder spaces enclosed by the deep surface of paired musculocutaneous and random-pattern flaps was studied in the canine model. Leukocyte function was assessed by calculating the following values: leukocyte counts, bacterial counts, phagocytic indices, and intracellular bacterial killing ratios. The following results were observed in the wound cylinder spaces after bacterial inoculation with 5 X 10(7) of Pseudomonas aeruginosa bacteria: (1) the numbers of mobilized leukocytes within each wound cylinder space flap were not statistically different, (2) the bacterial counts were significantly lower in the musculocutaneous flap wound cylinder space at both 24 and 48 hours, (3) the phagocytic activity of the leukocytes within musculocutaneous flap wound cylinder space was 1.5 times greater than the leukocytes in the random-pattern flap wound cylinder space, and (4) the intracellular bacterial killing ratio of the musculocutaneous flap leukocyte was 83 percent versus 26 percent in the random-pattern flap leukocyte, a significant difference.     

Effect of low and high doses ofSalmonella enteritidis PT4 on experimentally infected chicks

Chicks (1-d-old, three groups, each containing 50 chicks) were inoculated with 2×10² and 2×10⁸ CFU ofSalmonella enteritidis; the third group were kept as uninoculated control. Five birds from each group were euthanized at intervals from 6 h to 4 weeks post-inoculation (pi). In the lowdose groupS. enteritidis was isolated from 60% cecal samples at 18 h pi. and from 20% of livers at 3 d pi. Individual variation in the frequency ofS. enteritidis recovery was observed in this group. The clearance of salmonella from the organs was faster in the low-dose group, and salmonella was not isolated from the liver and cecum at 21 and at 27 d, pi, respectively. However, in the high-dose group,S. enteritidis was isolated from all ceca and 80% of liver 6 h pi, and salmonella was detected in the cecum and liver throughout the experiment. Serous typhlitis and unabsorbed yolk sac were the most prevalent lesions in both groups. Granulomatous nodules in the cecum were found occasionally in some cases in both inoculated groups, which can play a role as reservoirs in carrier chicks.     

濒危鱼类稀有白甲鱼外周血细胞特征

为了研究濒危鱼类稀有白甲鱼（Onychostoma rara）外周血细胞的特征,以采自长江中游沅江水系清水江共计21尾稀有白甲鱼的血液为材料,采用常规方法对稀有白甲鱼外周血细胞的组成、形态、大小和数量进行了观测。结果显示,稀有白甲鱼红细胞数量为（1.75±0.44）×106 个/ L,白细胞数量为（4.91±1.95）×105 个/ L。在血涂片上共计观察到了5种白细胞,包括淋巴细胞、血栓细胞、单核细胞、嗜中性粒细胞和嗜酸性粒细胞,没有发现嗜碱性粒细胞。其5种白细胞数量比例差异较大,其数量比例关系为：淋巴细胞>血栓细胞>嗜中性粒细胞>单核细胞>嗜酸性粒细胞。这5种白细胞的大小也有所不同,其大小关系为：单核细胞>嗜中性粒细胞>嗜酸性粒细胞>淋巴细胞>血栓细胞。与已报道的鱼类相比,稀有白甲鱼白细胞的数量明显较高,红细胞数量较多、体积相对较小,可能与其适应流水生活相关。     

Paw oedema test for detection of Salmonella enterotoxin : modification and standardization

Paw oedema test (POT) was standardized with modifications for the detection of Salmonella enterotoxin. Instead of measuring the weight of the inoculated paws after amputating the limbs at 48 hr post-inoculation, percent relative thickness of the order of 121 +/- 3.8% at 24-48 hr was found to be a better index. This test yielded parallel results to rabbit ligated ileal loop (RLIL) technique. The test was positive with enterotoxic crude cell lysates (CL) and cell free-culture-supernatants (CFCS) of S. newport and S. typhimurium, partially purified and purified enterotoxin of S. newport and purified cholera toxin. The test was found to be specific in that non-enterotoxic CFCS did not cause significant increase in the thickness. Minimum detection level of purified S. newport enterotoxin was estimated to be as low as 20 micrograms. Thus, the modified POT was considered to be an effective and economical bioassay model for the detection of Salmonella enterotoxin.