Tissue sterility in uneviscerated carcasses.

Sheep muscle tissue removed aseptically from control carcasses, from uneviscerated carcasses held at 20 degrees C for 24 h, and from carcasses of sheep subjected to stress before slaughter was examined for the presence of bacteria. All samples from a total of 68 carcasses were sterile. Whole-body autoradiography of mouse carcasses showed that 14C-labeled fixed bacteria injected after death remained in the lumen of the intestine. Live bacteria did not penetrate the mucosal surface until the tissue structure had been disrupted by proteolytic enzymes. Bacteria were unable to penetrate sections of intestine longitudinally until considerable structural breakdown had occurred, indicating that blood and lymph vessels do not normally offer a pathway for microbial invasion from the intestine. Clostridia, which have been reported to be responsible for deep spoilage of meat, reached maximum numbers 24 to 28 h after death in the intestines of guinea pig carcasses stored at 20 degrees C, but did not invade carcass tissues until the stomach ruptured as a result of proteolysis between 2 and 3 days after death.     

Survival of bacteria in carcasses.

Bacteria injected into the bloodstream of guinea pigs shortly before death decreased in number in carcass tissues for about 1 h after death. If initial bacterial numbers were sufficiently low, all bacteria were eliminated, and carcass tissues were sterile 24 h after death. Carcass tissue sterility was maintained with an initial density of Clostridium perfringens or Salmonella typhimurium of 20 cells per g or with an initial density of the other species examined of several hundred cells per gram. With larger numbers of strict and facultative anaerobes, growth commenced after 3 h in carcasses incubated at 30 degrees C. Spores of C. perfringens were killed over the same period as vegetative cells, but growth did not commence until 8 h after death. Bactericidal activity in carcass tissues must therefore be taken into account in evaluating the significance of reports of deep-tissue contamination of carcasses from meat animals.     

Survival of bacteria in carcasses.

Bacteria injected into the bloodstream of guinea pigs shortly before death decreased in number in carcass tissues for about 1 h after death. If initial bacterial numbers were sufficiently low, all bacteria were eliminated, and carcass tissues were sterile 24 h after death. Carcass tissue sterility was maintained with an initial density of Clostridium perfringens or Salmonella typhimurium of 20 cells per g or with an initial density of the other species examined of several hundred cells per gram. With larger numbers of strict and facultative anaerobes, growth commenced after 3 h in carcasses incubated at 30 degrees C. Spores of C. perfringens were killed over the same period as vegetative cells, but growth did not commence until 8 h after death. Bactericidal activity in carcass tissues must therefore be taken into account in evaluating the significance of reports of deep-tissue contamination of carcasses from meat animals.     

Effect of delayed evisceration on the microbial quality of meat.

The postomortem invasion of muscle and other tissues by bacteria from the intestinal tract was studied with the use of radioactive tracers. The injection of 14C-labeled bacteria or spores into the intestines of guinea pig carcasses within 24 h of death resulted in the rapid spread of 14C throughout carcasses. When live bacteria were injected along with the labeled cells, it was not possible to isolate viable organisms from the body tissues if the living animal had been exposed to the bacteria. It appears that animals are immune to their normal intestinal flora and that this immunity persists after death; thus passage of these bacteria into the lymphatic system does not necessarily result in the presence of live bacteria in carcass tissues. It therefore seems that a delay of up to 24 h before evisceration would not lead to deep tissue contamination of the carcass by organisms usually present in the intestines. Further evidence for this hypothesis was obtained by showing that muscle and lymph nodes from uneviscerated lamb carcasses hung for 24 h at 20 C remained sterile.     

Effect of delayed evisceration on the microbial quality of meat.

The postomortem invasion of muscle and other tissues by bacteria from the intestinal tract was studied with the use of radioactive tracers. The injection of 14C-labeled bacteria or spores into the intestines of guinea pig carcasses within 24 h of death resulted in the rapid spread of 14C throughout carcasses. When live bacteria were injected along with the labeled cells, it was not possible to isolate viable organisms from the body tissues if the living animal had been exposed to the bacteria. It appears that animals are immune to their normal intestinal flora and that this immunity persists after death; thus passage of these bacteria into the lymphatic system does not necessarily result in the presence of live bacteria in carcass tissues. It therefore seems that a delay of up to 24 h before evisceration would not lead to deep tissue contamination of the carcass by organisms usually present in the intestines. Further evidence for this hypothesis was obtained by showing that muscle and lymph nodes from uneviscerated lamb carcasses hung for 24 h at 20 C remained sterile.     

The Effect of Lactic Acid Sprays on Campylobacter jejuni Inoculated onto Poultry Carcasses

Spraying poultry carcasses with 1 % lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4°C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1 % or 2 % lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4°C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident Campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2 % lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

The biology of Diadromus collaris (Hymenoptera: Ichneumonidae), a pupal parasitoid of Plutella xylostella (Lepidoptera: Plutellidae), and its interactions with Oomyzus sokolowskii (Hymenoptera: Eulophidae)

The ichneumonid Diadromus collaris (Gravenhorst) has been recorded in many parts of the world as an important parasitoid of the diamondback moth, Plutella xylostella (Linnaeus), a serious pest of brassica vegetable crops worldwide. Some aspects of its biology and its interactions with Oomyzus sokolowskii (Kurdjumov), another major parasitoid of the same pest, were studied in the laboratory. At 25 degrees C, female wasps did not have mature eggs in their ovaries until about 12 h after emergence. Both males and females mated successfully 24-48 h after emergence, and females started to oviposit one to two days after emergence. Unmated females produced male progeny only; mated females produced progeny of both sexes. The development rate of the parasitoid increased linearly with temperature from 15 to 30 degrees C, with an estimated low temperature threshold of 7.4 degrees C and a thermal constant of 225.1 day-degrees for development from egg to adulthood. Rates of survival from larva to adulthood were about 90% between 20 and 28 degrees C and decreased as temperature decreased or increased. No immatures survived to adulthood at 35 degrees C. When provided with honey solution, the females lived on average 8.3, 11.5 and 7.0 days, and parasitized 26, 44 and 46 host pupae at 20, 25 and 30 degrees C, respectively. Female wasps could be stored at 15 degrees C for up to four weeks without detrimental effects on reproduction. Females of D. collaris attacked host pupae already parasitized by O. sokolowskii, inserting their ovipositor into the hosts at a similar frequency as into unparasitized host pupae, but they did not lay eggs inside the hosts.     

UV-C irradiation reduces microbial populations and deterioration in Cucurbita pepo fruit tissue

Tissue slices of zucchini squash (Cucurbita pepo L., cv. Tigress) fruit were exposed to ultraviolet-C (UV-C) radiation from germicidal lamps for 1, 10 or 20 min; however, only 10 and 20 min UV-C exposure significantly reduced microbial activity and deterioration during subsequent storage at 5 or 10 degrees C. UV-C treated slices had higher respiration rates than controls; however, the ethylene production of the slices was not affected by UV-C treatments. Slight UV-C irradiation damage (reddish brown discoloration) was detected on the surface of 10 and 20 min-treated slices after 12 days of storage at 10 degrees C. Slices stored at 5 degrees C did not show UV-C damage. Chilling injury was not observed until after 20 days of storage at 5 degrees C. The symptoms of chilling injury appeared as dried sunken brown spots on the surface of cortex tissue. UV-C treatments did not affect the degree of chilling injury during storage at 5 degrees C. UV-C treatment also had no consistent effect on sugar or malic acid concentrations. The most pronounced effect of UV-C irradiation was to retard microbial growth thereby providing a basis for the frequently observed delay in senescence and subsequent deterioration in fruit tissues.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Methods for decreasing interstitial immunoglobulin in tissue slices and cryostat sections

To determine whether lymphoid antigens and cellular morphology can be preserved after long-distance transport in buffer or cell culture medium, we stained cryostat sections prepared from human tonsil samples that had been kept at 4 degrees C or 20 degrees C for 24, 48 or 72 h. B-Cell antigens, T-cell antigens, and Ia antigens were well preserved after storage up to 72 h in buffer or medium at 4 degrees C. Interstitial immunoglobulin (Ig) was decreased following all incubation procedures. We then investigated methods to diminish interstitial Ig in cryostat sections, since it would be inconvenient to keep 2-3 mm tissue slices in buffer or medium prior to freezing and subsequent Ig staining. Cryostat sections were air dried or briefly fixed in acetone prior to washing in buffer or medium at 4 degrees C, 20 degrees C or 37 degrees C for 1, 2 or 24 h. Then sections were air dried or washed prior to acetone fixation and immunostaining. A method for washing cryostat sections was developed which diminished interstitial Ig without compromising the quality of immunostaining or cellular detail. These methods are especially useful for studying samples of lymphoid tissue in which the presence of large quantities of interstitial Ig obscures the detection of monotypic Ig staining patterns.     

Cold-hardiness in Pelecitus fulicaeatrae (Nematoda: Filarioidea), a parasite of the ankles of Fulica americana (Aves).

The filarioid nematode Pelecitus fulicaeatrae (Diesing, 1861) is considered cold-hardy. Adults and microfilariae became motile when placed in saline at 22 C after having been removed from thawed carcasses of their host, the American coot (Fulica americana Gmelin) (Aves: Gruiformes). Adult nematodes from 5 of 12 carcasses became active as did microfilariae from 4 of 5 carcasses. Carcasses had been frozen at an undetermined temperature below 0 C for an initial 14 days and then at -21 to -24 for 100-159 days.     

Initiation of fatty acid synthesis in rat mammary glands.

The rate of fatty acid synthesis from [6-14C]glucose in mammary tissue remained low until parturition at 22 days of gestation and increased 10-fold at 1 day post partum. Administration of progesterone on days 20 and 21 or removal of pups at parturition abolished this increase. In the latter case, administration of prolactin, corticosterone or oxytocin had no stimulatory effect; tissue from suckled glands in which the ducts had been ligated at parturition also showed no increased rate in 24 h. Foetoplacentectomy on day 18 did not stimulate fatty acid synthesis but subsequent suckling by foster pups did. Whereas lactose synthesis is initiated by withdrawal of progesterone from the circulation, a further stimulus related to removal of milk by suckling is required to initiate fatty acid synthesis.     

Vivo clearance of enteric bacteria from the hemolymph of the hard clam and the American oyster.

American oysters, Crassostrea virginica, and hard clams, Mercenaria mercenaria, were experimentally contaminated with Escherichia coli, Salmonella typhimurium, and Shigella flexneri either by intracardial injection or via the natural route of ingestion. Bacterial inactivation in the hemolymph was monitored for 72 h after exposure to these enteric pathogens at 20 and 6 degrees C. At 6 degrees C, both mean bacterial uptake by ingestion and subsequent clearance was singificantly lower that at 20 degrees C. However, substantial bacterial clearance from the hemolymph occurred for both shellfish at each temperature. At 20 degrees C, viable bacteria were no longer detectable after 24 h in hemolymph of either clams or oysters after exposure to contaminated water containing 4 x 10(3) bacteria per ml.     

Vivo clearance of enteric bacteria from the hemolymph of the hard clam and the American oyster.

American oysters, Crassostrea virginica, and hard clams, Mercenaria mercenaria, were experimentally contaminated with Escherichia coli, Salmonella typhimurium, and Shigella flexneri either by intracardial injection or via the natural route of ingestion. Bacterial inactivation in the hemolymph was monitored for 72 h after exposure to these enteric pathogens at 20 and 6 degrees C. At 6 degrees C, both mean bacterial uptake by ingestion and subsequent clearance was singificantly lower that at 20 degrees C. However, substantial bacterial clearance from the hemolymph occurred for both shellfish at each temperature. At 20 degrees C, viable bacteria were no longer detectable after 24 h in hemolymph of either clams or oysters after exposure to contaminated water containing 4 x 10(3) bacteria per ml.     

Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem

Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.     

Morphological and ultrastructural analysis of sheep primordial follicles preserved in 0.9% saline solution and TCM 199

The objective was to determine the morphological and ultrastructural features of sheep primordial follicles preserved in either 0.9% saline solution or TCM 199 at different temperatures. Soon after death, the ovarian pair of each ewe (n = 5) was divided into 25 fragments. One fragment was immediately fixed for morphological evaluation (control). The other 24 fragments were randomly distributed in tubes containing 2 ml of 0.9% saline solution or TCM 199 and maintained at 4, 20 or 39 degrees C for 2, 4, 12, or 24h. Based on histological assessment, storage of ovarian fragments in 0.9% saline solution at 20 degrees C for up to 24h and in both solutions at 39 degrees C for 4, 12 or 24h increased (P < 0.01) the percentage of degenerate primordial follicles compared with controls. In contrast, preservation at 4 degrees C in both solutions, kept the percentage of morphologically normal primordial follicles similar to control values. Although histological integrity of primordial follicles was maintained in fragments stored at 20 degrees C for up to 24h in TCM 199, these results were not confirmed by ultrastructural analysis. Based on transmission electron microscopy, only primordial follicles stored at 4 degrees C for up to 24h, at 20 degrees C for up to 12h and at 39 degrees C for up to 2h in both solutions were ultrastructurally normal. In conclusion, sheep primordial follicles were successfully preserved at 4 degrees C for up to 24h, at 20 degrees C for up to 12h and at 39 degrees C for 2h in 0.9% saline solution or TCM 199.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine.

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469--3473]. This activity is attributed to an enzyme provisionally named 'macromolecular heparin depolymerase'. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.     

Cryopreservation of mouse ovarian tissue following prolonged exposure to an Ischemic environment.

In cases in which ovarian tissue is to be cryopreserved for tissue or gene banking it is important to maintain its integrity and viability. This study examined how delays between the death of an animal and the collection/cryopreservation of its ovarian tissue influenced follicle viability. Mouse ovaries were placed in PBS+antibiotic (in vitro) or left within the body (in situ) at room temperature for 0, 3, 6, 12, or 24 h following the death of the donor. These ovaries were cryopreserved at 1 degrees C/min on dry ice or in a -84 degrees C freezer using a passive cooling device or by conventional slow cooling (0.3 degrees C/min). The ovaries were grafted under the kidney capsule of ovariectomized recipient mice and collected 2 weeks later, and the size and number of follicles were determined. Cryopreserved ovarian tissue grafted immediately after the death of the donor contained numerous viable and healthy follicles independent of the cooling procedure (dry ice, 134 +/- 32; -84 degrees C, 165 +/- 54; slow, 214 +/- 55 follicles per half ovary). Tissues stored in vitro before cryopreservation retained viable follicles up to 12 h after death (dry ice, 30 +/- 15; -84 degrees C, 86 +/- 45; slow, 93 +/- 33), whereas tissue left in situ had significantly reduced follicle numbers within 3 h of death (dry ice, 36 +/- 12; -84 degrees C, 19 +/- 6; slow, 28 +/- 7). No significant difference was found between the cooling rates tested, indicating that a passive cooling container which cools at 1 degrees C/min is a suitable alternative to conventional slow cooling. We conclude that ovarian tissues for cryobanking should be cryopreserved as soon as possible after collection or death of the animal to ensure maximal follicular survival.