Influence of Temperature on Bacterial Infection of the Hen's Egg

Temperature of incubation had a marked effect on infection of eggs in which the air cells had been inoculated with a washed suspension of Serratia marcescens. There was no evidence of bacterial multiplication or spoilage in eggs held at 10 C for 42 days. Multiplication occurred in the shell membranes of eggs held at 30 or at 37 C when the yolk made contact with these membranes, and continued in the contents of the egg, at which time the first signs of spoilage appeared. In a few eggs, very large populations were present in the shell membranes and in the albumen. In eggs inoculated with Pseudomonas fluorescens and held at 10 C, bacterial multiplication occurred in the shell membranes in the first 7 days of incubation. These populations did not appear to change in size in the 7- to 14-day period of incubation. Renewed multiplication and concomitant spoilage of the contents was observed in many of the eggs thereafter.     

The Growth of Gram-Negative Bacteria in the Hen's Egg

Summary: Bacteriological and chemical methods were used to follow the course of infection in eggs, incubated at 27°, the air cells of which had been inoculated with a suspension of washed bacteria. In the 3–4 days following inoculation, limited bacterial multiplication occurred in the inner membrane of the air cell but very few organisms entered the albumen. These populations then remained static or decreased slightly until renewed multiplication occurred 12–30 days after inoculation. This was induced by contact of the yolk and the shell membranes: it occurred on the 12–20th day in eggs in which the yolk moved towards the site of inoculation, but later when the yolk moved in the opposite direction. At this time there was a general infection of the egg contents and significant changes occurred in the pH and glucose concentration in the albumen. In eggs that had been inoculated with chromogenic and/or proteolytic bacteria, the first macroscopic changes of the contents were seen at this time. The rate and extent of the initial multiplication was influenced by the composition of the fluid used to suspend the washed bacteria and, in all instances, the fastest multiplication occurred when iron was added to the inoculum. Moreover, renewed multiplication occurred when iron was added to the albumen of eggs in which the bacteria were in the stationary phase.     

Action of Pseudomonas fragi on the Proteins of Pig Muscle

Considerable salt-soluble protein degradation was observed in pork muscle inoculated with Pseudomonas fragi. During a 20-day incubation period at 10 C, the samples proceeded to rank spoilage or putrefaction. There was a large decrease in the salt-soluble protein fraction and a corresponding increase in nonprotein nitrogen. Disc gel electrophoretic patterns showed that breakdown of the salt-soluble proteins had occurred after incubation for 20 days. During incubation for 10 days at 10 C, P. fragi produced large amounts of extracellular proteolytic activity in ground pork. Most of the proteolytic activity appeared immediately after spoilage occurred. However, a significant increase in the ability to hydrolyze casein and a slight increase in the ability to hydrolyze denatured hemoglobin occurred prior to spoilage.     

THE PREVENTION OF MICROBIAL SPOILAGE IN WHOLE SHELL EGGS BY HEAT TREATMENT METHODS

SUMMARY: The prevention of microbial spoilage in whole shell eggs by heat treatment methods has been investigated. After shell eggs had been immersed for 3 min in water at 144·5 ± 0·5°F the incidence of microbial rotting was reduced by over 90%. Consistent reductions in spoilage losses were obtained with farm steeped eggs, eggs produced under commercial conditions, and eggs held in cold storage for 6 months. Heat treatment efficiently controlled green, sour and black rots in stored eggs, but although reductions were noted in the number of mould rots, it cannot be guaranteed that this type of spoilage can be controlled with similar certainty.
Internal quality in heat treated eggs was satisfactory. During protracted holding in cold storage, heat treatment appeared to result in a better internal egg quality and a more stable keeping quality, than when untreated eggs were held under similar conditions.     

Low-Temperature Production of Urocanic Acid by Spoilage Bacteria Isolated from Mahimahi (Coryphaena hippurus)

Urocanic acid production was studied in 166 bacterial cultures isolated from mahimahi which had been stored for 14 days on ice in seawater. After 4 days of incubation at 10°C in histidine-supplemented Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), urocanic acid was measured by thin-layer chromatography. Fifty-eight of the cultures were positive for urocanic acid, with the eight most active isolates producing ca. 10 mg/100 ml. These observations, coupled with a report that urocanic acid may be the predominant histidine metabolite in fish held at low temperatures, all suggest that urocanic acid may be a useful alternative to histamine as a spoilage index in scombroid and other fish that are rich in endogenous histidine.     

Effect of Temperature of Liquid Nitrogen on Radiation Resistance of Spores of Clostridium botulinum

An apparatus consisting of a Dewar flask and a relay system controlling the flow of liquid nitrogen permitted the irradiation of samples in tin cans or Pyrex tubes at temperatures ranging from 0 ± 1.5 C to -194 ± 2 C. An inoculated pack comprising 320 cans of ground beef containing 5 × 10⁴ spores of Clostridium botulinum 33A per can (10 cans per radiation dose) was irradiated with Co⁶⁰ at 0 and -196 C. Incubation was carried out at 30 C for 6 months. Approximately 0.9 Mrad more radiation was required to inactivate the spores at -196 C than at 0 C. Cans irradiated at -196 C showed partial spoilage at 3.6 Mrad and no spoilage at 3.9 Mrad; the corresponding spoilage-no spoilage doses at 0 C were 2.7 and 3.0, respectively. The majority of positive cans swelled in 2 to 14 days; occasional swelling occurred as late as 20 days. At progressively higher doses, swelling was delayed proportionally to the radiation dose received. The remaining nonswollen cans had no toxin after 6 months of storage, although occasional cans contained very low numbers of viable spores comprising on the average 0.1% of the original spore inoculum. The D₁₀ values in phosphate buffer were 0.290 Mrad for 0 C and 0.396 Mrad for -196 C; in ground beef, the corresponding D₁₀ values were 0.463 Mrad and 0.680 Mrad, respectively. These D₁₀ values indicate that the lethal effect of γ rays decreased at -196 C as compared with 0 C by 13.5% in phosphate buffer, and by 47% in ground beef.     

Cultivation of the cercaria of Himasthla quissetensis (Trematoda) on the chick chorioallantois

Cercariae of Himasthla quissetensis were cultivated on the chick chorioallantois maintained at 38 ± 1°C and a relative humidity of 70–75%. Of 68, 6-day-old eggs, each inoculated with 100 cercariae, 28 (41%) were examined 1–9 days post-inoculation. Of the 28 eggs, 23 (82%) were infected with a total of 224 live worms plus 10 encysted metacercariae. Worms contained blood or hematin-like material in their intestinal caeca and showed considerable development of reproductive structures. A total of 58, 6- or 7-day-old chorioallantoic-worms were serially transferred to new 6-day-old embryos, but only a single live worm was recovered after 13 days on two membranes. Based on fixed and stained specimens, 7-day-old chorioallantoic-worms increased their body area about 4× compared to cercariae and the 13-day-old worm increased its body area about 10×.     

Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate.     

USE OF BLUE LAKE AS AN INDICATOR OF BACTERIAL PENETRATION INTO EGGS

Blue Lake, an insoluble dye, was evaluated as an indicator of potential bacterial penetration into eggs. Various groups of eggs (fresh-laid; commercial; water-washed) were dipped in 0.25% (w/v) Blue Lake in 0.1% Triton X-100 solution for 2 min and incubated at room temperature up to 1 ± 24 h. Penetration was detected by counting the blue dots on shell membranes after breaking the eggs. Commercial eggs allowed the easiest penetration. All commercial eggs showed blue dots even at 2 min incubation and the average count reached 111/egg at 1 h. Water-washed eggs allowed much less penetration than commercial eggs and the counts of blue dots on those eggs were 10/egg at 2 min and 22 at 1 h. Fresh-laid eggs did not allow any penetration up to 24 h. Above results corresponded very well with the penetration study with Salmonella enteritidis and also with the morphological study of eggshell surfaces using electron microscopy where fresh-laid eggs had intact cuticle layers, but commercial eggs did not. Thus, Blue Lake dye might be used as a rapid indicator of bacterial penetration through eggshells.     

Radiation Sterilization of Bacon for Military Feeding

Sliced, cured bacon, packed in cans and seeded with 6 × 10⁵ spores per can of Clostridium botulinum strains 33A or 41B, or with 3 × 10⁶ spores per can of strains 36A, 12885A, 9B, or 53B, was irradiated to various dose levels with γ radiation. Evidence provided by swelling, toxicity, and recoverable C. botulinum with 2,200 inoculated, irradiated cans demonstrated that: (i) 4.5 Mrad were more than adequate as a sterilization dose; (ii) the experimental minimal sterilizing dose was 2.0 Mrad, and the theoretical 12-log reduction dose was 2.65 or 2.87 Mrad depending on the method of calculation; (iii) some spoilage occurred at dose levels below 2.0 Mrad; (iv) all visible spoilage of irradiated bacon was due to strains 33A and 12885A only, whose D values were, respectively, 0.141 and 0.177 Mrad based on spoilage data, and 0.221 and 0.188 Mrad, respectively, when based on recovery data; (v) toxic cans did not always result in swelling, nor did swollen cans always produce toxic spoilage; and (vi) viable C. botulinum can exist for at least 8 months in storage at 30 C without producing visible or toxic spoilage at doses below 2.0 Mrad.     

Fertility and hatchability of eggs laid in the pullet-to-breeder transition period and in the initial production period

The initial eggs produced by broiler breeder hens are relatively small compared with later in the production cycle. An evaluation of indices related to hatchability is required when these eggs are to be used for the production of broiler chicks. Two experiments were conducted to evaluate characteristics related to the hatchability of eggs from pullet-to-breeder transition phase, at 25 and 27 weeks of age, and from the peak of production period and five weeks later, at 32 and 37 weeks of age. Eggs from birds 25 weeks had a lesser fertility in Experiment 1. Mortality occurred unevenly in early (1–5 days), middle (6–17 days) and late (18–21 days) incubation, and greater mortality was observed after the internal membrane was ruptured. The younger the hen, the lighter the egg, chick, and shell, and the longer the time required to complete the hatching process. In Experiment 2, greater mortalities were observed at the early period (1–5 days) and after “pipping” of the internal and external membranes. Embryos from heavy eggs of breeder hens 37 weeks of age took less time to complete the hatching process. Results indicated the larger the egg, the heavier the chick and shell, and the lesser the shell percentage. As breeder age advanced, characteristics related to egg fertility and hatchability improved.     

Survival and persistence of nonspore-forming biothreat agents in water

Aims: To determine whether nonspore‐forming biothreat agents can survive and persist in potable water that does not contain a disinfectant. Methods and Results: Autoclaved, de‐chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml^?1). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature. Conclusion: Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore‐forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C. Significance and Impact of the Study: The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent.     

Incubation of eggs of tuatara, Sphenodon punctatus

Eggs of the tuatara, Sphenodon punctatus , were incubated either buried or half buried in vermiculite at constant temperatures of 15, 18, 20, 22 and 25 °C and constant water potentials between —90 and —400 kPa. Many clutches failed completely, possibly because they had been taken from females prior to proper shell development. Failed eggs were significantly smaller than successful eggs. Incubation is unsuccessful at 15 °C. Hatching success is high between 18 and 22 °C but low at 25 °C, but equally successful between 18 and 22°C. Incubation is strongly influenced by temperature, with mean incubation periods of 328 days at 18 °C, 259 days at 20 °C, 169 days at 22 °C and 150 days at 25 °C. Water potential generally has little influence on incubation time at a given temperature. Buried eggs hatch sooner than partially buried eggs at 20 °C but the large range makes significance dubious.
Eggs on the driest substrata at 18 and 20 °C lose water initially but then gain water through the rest of incubation. Eggs in all other conditions gain water throughout incubation, with the rate of i water absorption being maintained or increasing late in incubation. The suggestion that increasing rate of water absorption late in incubation facilitates explosive hatching is not supported. Egg mass at the time of hatching varies from 132 to 398% of initial values, depending on incubation conditions. Final egg mass is not affected significantly by incubation temperature. Hence, rates of absorption increase with temperature.
Water potential has no influence on hatchling size. However, hatchlings from buried eggs generally are significantly larger than those from partially buried eggs.     

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex^®) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta , left untreated (U), treated with 10 μg ml⁻¹ nisin (N), Fibrimex^® (F) or Fibrimex^® containing 10 μg ml⁻¹ nisin (FN), held aerobically at 4 °C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 °C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.     

Earlier hatching time predisposes Cobb broiler chickens to tibial dyschondroplasia

Fertile eggs from Cobb 500 broiler breeder hens were incubated to provide low starting egg shell temperatures (EST; 36.9°C to 37.3°C) which were gradually increased to 37.8°C during the first 7 to 15 days of incubation compared with eggs incubated with a constant EST of 37.8°C (standard conditions) over the first 18 days of incubation. Time of individual chick hatching (measured at 6 h intervals from 468 h of incubation), chick weight, chick length and yolk weight were measured at take-off and BW was measured at 7, 14, 28, 34 and 42 days of age. Male birds at 34 and 42 days of age were assessed for their ability to remain standing in a latency-to-lie test. At 34 and 42 days, male birds were examined for leg symmetry, foot pad dermatitis, hock bruising and scored (scale 0 to 4, where 0=no lesion and 4=lesions extending completely across the tibial growth plate) for tibial dyschondroplasia (TD) lesions. The lower EST profiles caused chicks to hatch later than those incubated under the standard EST profile. Chicks which hatched at ⩽498 h incubation grew faster over the first 7 days than those that hatched later. There were significantly more birds (only males were studied) that hatched from the lower EST profiles with TD scores of 0 and 1 and fewer with score 4 at 34 days than those hatched under the standard profile. Male birds at 34 days with TD lesions ⩾3 stood for significantly shorter times than males with TD scores ⩽2. Moreover, male birds at 34 and 42 days with TD lesion scores of ⩾3 hatched significantly earlier and grew significantly faster over the first 2 weeks of age than did male birds with TD scores ⩽2. It appears possible to decrease the severity and prevalence of TD in the Cobb 500 broiler by ensuring that the birds do not hatch before 498 h of incubation.     

Biomass Partitioning in Tomato Plants Infected with Meloidogyne incognita

Tomato plants were inoculated with Meloidogyne incognita at initial populations (Pi) of 0, 1, 10, 50, 100, and 200 (x 1,000) eggs per plant and maintained in a growth chamber for 40 days. Total fresh biomass (roots + shoots) at harvest was unchanged by nematode inoculation with Pi of 1 x 10⁵ eggs or less. Reductions in fresh shoot weight with increasing Pi coincided with increases in root weight. Total fresh biomass declined with Pi above 1 x 10⁵ eggs, whereas total dry biomass declined at Pi above 1 x 10⁴ eggs. The greatest reduction percentages in fresh shoot biomass induced by root-knot nematodes occurred in the stem tissue, followed by the petiole + rachis; the least weight loss occurred in the leaflets. Although biomass varied among shoot tissues, the relationship between biomass of various shoot tissues and Pi was described by quadratic equations. The linear and quadratic coefficients of the equations (stem, petiole + rachis, or leaflets on Pi) did not differ among tissues when calculations were based on standardized values. Meloidogyne incognita-infected plants had thinner leaves (leaf area/leaf weight) than did uninfected plants. Reductions in leaf weight and leaf area with nematode inoculation occurred at nodes 5-15 and 4, 6-14, respectively. Losses in plant height and mass due to nematodes reflected shorter internodes with less plant mass at each node.     

Reduction of Olive Knot Disease by a Bacteriocin from Pseudomonas syringae pv. ciccaronei

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10⁵ and 10⁸ CFU ml⁻¹, respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml⁻¹ at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml⁻¹) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.     

Pathological Reaction of Crested Wheatgrass Cultivars to Four Meloidogyne chitwoodi Populations

Meloidogyne chitwoodi populations from Tulelake, California; Ft. Hall, Idaho; Beryl, Utah; and Prosser, Washington, significantly (P < 0.05) reduced dry shoot weights of crested wheatgrass (Agropyron cristatum L., Gaertn. and A. desertorum, Fisch. ex Link, Schult.) cultivars Hycrest, Fairway, and Nordan in experiments conducted in a greenhouse and growth chamber. Shoot growth depression, root galling, and nematode reproduction indices were greatest (P < 0.05) on plants inoculated with 5,000 eggs/plant. Nematode populations from Tulelake, Ft. Hall, and Beryl significantly (P < 0.05) reduced the growth of the three grass cultivars at 15, 20, 25, and 30 C; the greatest reductions occurred at 20 and 25 C. There were significant differences in the virulence of the nematode populations at high (30 C) and low (15 C) soil temperatures. At 15 C, plant growth was reduced more by the Beryl and Tulelake than by the Ft. Hall population; whereas at 30 C, the Ft. Hall population was more virulent than the Beryl and Tulelake populations. Root galling and nematode reproduction were greater on plants inoculated with Beryl and Tulelake populations at 15 C than on plants inoculated with the Ft. Hall population, while the Ft. Hall population had the most pronounced effects at 30 C.     

Culture of naked quail (Coturnix coturnix japonica) ova in vitro for avian transgenesis: Culture from the single-cell stage to hatching with pH-adjusted chicken thick albumen

We first examined the pH change of the albumen of quail (Coturnix coturnix japonica) eggs before and after they were laid, as well as that of laid eggs. The pH rose rapidly after laying and continued to increase gradually in storage. Incubation at 37.5°C accelerated the increase in the pH of infertile eggs, while that of fertile eggs remained low during incubation. Referring to these results, we obtained a protocol for producing quail hatchlings by culture in vitro from naked ova. The naked ovum was filled with chicken (Gallus domesticus) thick albumen, the pH of which had been adjusted to 7.2–7.4. The ovum was cultured at 41.5°C in 20% CO₂ in air for the first 24 h. Then the embryo was moved to a surrogate quail egg shell that had been filled with non-pH-adjusted chicken thin albumen and cultured for a further 48 h in 100% air. The embryo was transferred again to a surrogate chicken egg shell and cultured under the same conditions until hatching. The culture yielded quail chicks with a hatchability of 19.4%. The method proposed here should be applicable to the production of transgenic birds.     

Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives.

A strain of enterohemorrhagic Escherichia coli serotype O157:H7 isolated from a patient in an apple cider-related outbreak was used to study the fate of E. coli O157:H7 in six different lots of unpasteurized apple cider. In addition, the efficacy of two preservatives, 0.1% sodium benzoate and 0.1% potassium sorbate, used separately and in combination was evaluated for antimicrobial effects on the bacterium. Studies were done at 8 or 25 degrees C with ciders having pH values of 3.6 to 4.0. The results revealed that E. coli O157:H7 populations increased slightly (ca. 1 log10 CFU/ml) and then remained stable for approximately 12 days in lots inoculated with an initial population of 10(5) E. coli O157:H7 organisms per ml and held at 8 degrees C. The bacterium survived from 10 to 31 days or 2 to 3 days at 8 or 25 degrees C, respectively, depending on the lot. Potassium sorbate had minimal effect on E. coli O157:H7 populations, with survivors detected for 15 to 20 days or 1 to 3 days at 8 or 25 degrees C, respectively. In contrast, survivors in cider containing sodium benzoate were detected for only 2 to 10 days or less than 1 to 2 days at 8 or 25 degrees C, respectively. The highest rates of inactivation occurred in the presence of a combination of 0.1% sodium benzoate and 0.1% potassium sorbate. The use of 0.1% sodium benzoate, an approved preservative used by some cider processors, will substantially increase the safety of apple cider in terms of E. coli O157:H7, in addition to suppressing the growth of yeasts and molds.