Survival of parasite eggs upon storage in sludge

Destruction rates of parasite eggs in stored sludge were examined to help understand the fate of these agents of enteric diseases in sludge lagoons. Eggs from the roundworms, Ascaris spp., Toxocara spp., Trichuris spp., and the tapeworm, Hymenolepis spp., were treated with domestic sludges by aerobic or anaerobic processes. Sludge samples seeded with eggs were stored at 4 or 25 degrees C or in a container inserted into the ground to simulate lagoon conditions. The number of eggs recovered from the samples decreased with storage time. The viability and infectivity of eggs recovered were related to the storage temperature; i.e., the eggs stored at 4 degrees C remained viable longer than those stored at 25 degrees C. After 25 months at 4 degrees C, the Toxocara eggs and some Ascaris eggs remained both viable and infective, whereas most of these eggs stored at 25 degrees C were rendered nonviable after 10 to 16 months of storage in sludge. Although storage temperature was found to be the most important factor affecting the destruction and viability of these eggs, other factors, such as the type of sludge digestion, whether or not the eggs were digested along with the sludge or added later, storage in the soil versus sludge, pH, and egg species also exhibited some minor effects. These controlled laboratory studies suggest that lagooning of sludge can be an effective method for the elimination of parasite eggs, particularly in warmer geographical locations.     

Survival of Salmonellas and Ascaris Suum Eggs in a Thermophilic Biogas Plant

In a continuous biogas plant, receiving manure from 200 dairy cows and 400 calves and young stock, survival of salmonellas and Ascaris suum eggs was studied. The bacteria and parasite eggs were kept in filter sacs in the manure that had a temperature of 55°C. No viable salmonellas or Ascaris suum eggs could be found after 24h in the digester. Survival of salmonellas and Ascaris suum eggs was also studied in the manure pit where the manure was stored after digestion. The temperature in the manure pit varied between 22–27°C. Salmonellas survived 35 but not 42 days. On day 56, when the experiments had to be stopped, 60% of the Ascaris eggs were viable.     

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.     

Survival rates of parasite eggs in sludge during aerobic and anaerobic digestion.

The effects of mesothermic anaerobic or aerobic sludge digestion on survival of eggs from the roundworms Ascaris suum, toxocara canis, Trichuris vulpis, and Trichuris suis and from the rat tapeworm Hymenolepis diminuta were studied. Destruction of eggs throughout a 15-day treatment period, as well as their viabilities after reisolation, was analyzed. The laboratory model digesters used in this study were maintained at a 15-day retention schedule, partially simulating a continuously operating system. Ascaris eggs were destroyed in the anaerobic (23%) or aerobic (38%) digesters, and 11% Trichuris eggs were destroyed in the aerobic digesters. Trichuris eggs in anaerobic digesters and Toxocara eggs in either anaerobic or aerobic digesters were not destroyed. Destruction of eggs in digesters was correlated with the state of the eggs before subjection to the treatment processes; i.e., some Ascaris and Trichuris eggs were already embryonated in host intestinal contents or feces and hence past their most resistant stage. The viabilities of Ascaris and Toxocara eggs that survived the digestion processes were greater in anaerobically treated than in aerobically treated material. Eggs from Hymenolepis were nonviable before use in the experiments. However, they were more effectively destroyed in aerobic digesters than in anaerobic digesters.     

Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti

The inoculation of legumes with rhizobia is used to maximise nitrogen fixation and enhance the plant yield without using N fertilisers. For this reason many inoculant types were developed and optimised. In our study, the effects of the growth medium, the carrier, the temperature and the storage period were determined on the survival of Sinorhizobium meloliti. Secondary sludge from Communauté Urbaine de Quebec wastewater treatment plant and standard medium (YMB) were used for rhizobial growth. Dehydrated sludge from Jonquière wastewater treatment plant, peat and a mixture of peat and sludge were used as carrier materials. Results showed that the wastewater sludge offered better protection for rhizobia survival during freezing and thawing at -20 degrees C than the standard medium. In general, results also showed the suitability of using sludge as a carrier because it had the same or a higher potential than peat to support survival of S. meliloti. In the case of YMB-grown rhizobia, peat- and sludge-based carriers appeared to be similar in terms of survival rate during the storage at 4 and 25 degrees C. For secondary sludge-grown rhizobia, the survival was better in sludge than in peat based carrier. Generally, the cell count remained higher than 10(8) cells/g for up to 80 days at 4 and 25 degrees C in both carriers (sludge and peat). However, for the secondary sludge-grown cells stored in peat-based carrier at 4 degrees C, the viable cells decreased under 10(8) cells/g at the 81st day of storage but remained acceptable compared to the standard (10(7) cells/g of carrier).     

Morphological changes of Ascaris spp. eggs during their development outside the host

Information on the infective stage of Ascaris lumbricoides and the pathology caused by the parasite is widely available in the literature. However, information about early embryonic development of A. lumbricoides and its life cycle outside the host is limited. The purpose of this study was to describe the morphological changes within the developing embryo during incubation in vitro at 28 C, as well as to explore differences in egg viability during incubation. Ascaris suum eggs (4,000 eggs/ml), used as a model for A. lumbricoides , were placed for incubation in 0.1N H(2)SO(4) at 28 C in the dark for 21 days. Every day, sub-samples of approximately 100 A. suum eggs were taken from the incubation solution for microscopic evaluation. Development, morphological changes, and viability of the first 40 eggs were observed and documented with photos. During this study, 12 stages were identified in the developing embryo by standard microscopy, 2 of which had not been previously reported. By the end of the first wk, most developing embryos observed were in the late-morula stage (72.5%). On day 14 of incubation, 90% had developed to larva-1 stage, and by day 21, 100% had developed to larva-2 stage. No significant differences were found in the viability recorded in a continuum from day 5 to day 21 of incubation (chi-square, P > 0.05). The result of this study complements and expands the stages of development of Ascaris spp. outside the host previously reported in the literature. It also suggests the potential use of early stages of development of the nematode to determine viability and safety of sewage sludge, wastewater, or compost after treatment recommended by USEPA.     

Tolypocladium cylindrosporum (Deuteromycotina:Moniliales), a fungal pathogen of the mosquito Aedes australis

A laboratory fermenter was used to produce up to 12 l of infective Tolypocladium cylindrosporum blastoconidia in Sabouraud dextrose broth. Two media derived from coconuts were also demonstrated as suitable alternative systems for the production of viable blastoconidia. T. cylindrosporum conidia when dried at 37 degrees C and stored at 4 degrees C retained their viability for 10 months, but, when stored at 25 degrees C, the conidia lost viability after 2 months and blastoconidia did not survive the drying process. Distilled water suspensions were a simple, economic technique for the long-term storage of spores at both 4 and 25 degrees C. The adsorption of conidia onto silica gel crystals was a very suitable technique for the storage of stock culture material at 4 degrees C. The virulence, production and storage capabilities of both spore types were examined.     

Decontamination by anaerobic stabilisation of the environment contaminated with enteronematode eggs Toxocara canis and Ascaris suum

Investigations were carried out under operating conditions of Field Composting Factory in Brezno (Slovak Republic) to determine the effect of anaerobic stabilization of organic wastes from public areas on the survival of model helminth Toxocara canis and Ascaris suum eggs. Due to anaerobic conditions, low temperature, low C:N ratio and changes in physical and chemical properties of organic waste, less than 64% of A. suum eggs remained viable after 150 days of stabilisation. The anaerobic stabilisation had a greater effect on the viability of T. canis eggs than on A. suum eggs. The infectivity of T. canis eggs was confirmed by a follow-up experiment in laboratory mice. A small number of T. canis larvae were found in their brain and muscles on day 28 after infection. The results refer to the risks of dissemination, survival and potential spread of endoparasitic developmental stages in the environment through organic wastes subjected to low temperature stabilisation.     

Preservation of rainbow trout (Oncorhynchus mykiss) eyed eggs using a perfluorochemical as an oxygen carrier

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.     

Prevalence and viability of eggs of Toxocara spp. and Toxascaris leonina in public parks in eastern Spain

To demonstrate the prevalence of Toxocara spp. and Toxascaris leonina eggs in parks in Murcia city, eastern Spain, a total 644 soil samples were examined from nine parks. More than 67% of parks and 1.24% of soil samples were contaminated and the mean egg density per sample was eggs per 100 g of soil. Over 97% of eggs identified were viable. Only one sample was positive for Toxascaris leonina. The present findings suggest that shady conditions are important for the occurrence and viability of Toxocara spp. and Toxascaris leonina eggs in soil as there were significantly more positive samples in shaded and moist areas compared with open and dry habitats.     

The effects of low temperature on fertilized rabbit ova in vitro,and the normal development of ova kept at low temperature for several days

Fertilized rabbit ova at the 2-blastomere stage kept in rabbit serum were stored at low temperatures for various lengths of time. They were then cultured at 38 degrees C. for about 24 hours to determine their viability. A number of the viable ova were finally transplanted into recipient does. It was found that rapid cooling of ova to 5 degrees or to 0 degrees C. was more harmful to the subsequent viability of ova than slow cooling. Rapid cooling was not more lethal to the ova than slow cooling, but did prevent their future normal cleavage. There was no difference between those ova cooled rapidly or slowly to 10 degrees C. It was concluded that temperature shock has an adverse effect on ova, especially at the lower temperatures, though temperature shock can be remedied by acclimatization (slow cooling). Thus, the physiological significance of temperature shock would seem to be broadened. The optimal temperature for the storage of ova was investigated. It was found that 10 degrees C. was the best temperature; at this temperature viable ova were obtained after storage for 144 to 168 hours. At 0 degrees , 5 degrees , or 15 degrees C. the ova were viable for 96 to 120 hours, while at 22-24 degrees C., only for 24 to 48 hours. The percentage of dead ova was low at a favorable temperature, increasing only at the end of the storage period. At an unfavorable temperature, however, the rate of death increased steadily from beginning to end of storage. The percentage of abnormally cleaved ova (arrested cleavage and fragmentation) remained at a low level at first at a favorable temperature, but then increased just before or during death of the ova. A critical time for the viability, the abnormal cleavage, and the death of ova was characteristic of each temperature. About 24 to 28 per cent of the viable ova remaining after being stored at 0-15 degrees C. for 2 to 4 days and cultured at 38 degrees C. for 24 hours were capable of development into normal young. The compatibility of serum and ova, the absence of a correlation between the viability of the ova and the source of the fertilizing spermatozoa, and the fertilization of superovulated ova (i.e., the percentage of fertile does in follicular phase and in luteal phase, the percentage of unfertilized ova and of fertilized ova at different stages, the percentage of does that had produced a normal number of ova or had produced a large number of ova, etc.), are reported. The possibility of a more efficient utilization of the germ cells of valuable animals by means of the present techniques, and the possibility of a new approach to the experimental investigation of mammalian genetics and development, have been mentioned.     

Lethal effects of freezing Echinococcus multilocularis eggs at ultralow temperatures

Methods for killing Echinococcus multilocularis eggs within stool or intestinal samples, without damaging the diagnostic value of the sample, would significantly reduce the risk of animal health providers acquiring alveolar hydatid disease. The first objective of this study was to determine whether E. multilocularis eggs located in fox intestines can survive storage at -70 C for at least 4 days. Results showed that none of 72,000 E. multilocularis eggs remained infectious to defined strains of mice under these conditions, yet, similar eggs recovered from nonfrozen carcasses stored at 4 C for the same time period were viable. The structural identities of adult worms and eggs were not significantly altered by the freezing and thawing processes. These results indicate that ultracold temperatures can be used to kill or inactivate E. multilocularis eggs, making them safe to handle when diagnosing this parasite in definitive hosts. The second objective of this study was to determine whether E. multilocularis eggs could survive freezing to -70 C if commonly used cryopreservation protocols were used. The use of the cryoprotectant solution, 5% dimethyl sulfoxide-35% saline-60% lamb serum, with a -1 C/min freezing rate was unable to prevent the eggs from being killed by freezing to -70 C. Rapid cooling by plunge freezing into liquid nitrogen was also lethal to E. multilocularis eggs. Only a few of the many potential cryopreservation protocols were tested in this study, so it is not yet possible to completely rule out the possibility of preserving these eggs at ultralow temperatures, but it does indicate that temperatures below -70 C are lethal to eggs even under favorable storage conditions.     

Inactivation of viable Ascaris eggs by reagents during enumeration.

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

Long-term storage and viability of Ochlerotatus albifasciatus (Diptera: Culicidae)

The viability of Ochlerotatus albifasciatus (Macquart) eggs stored at room temperature and at 5 degrees C was studied over 31 months. After 12, 18 and 31 months of storage, eggs were acclimatized at 22 degrees C for ten days, and then inundated twice every seven days. The effect of the storage period on the percentage of hatching was analyzed by one way ANOVA. Differences on the hatching response between the first and second flooding were analyzed by paired t-test. Differences on the hatching response between the two storage conditions were analyzed by Mann-Whitney rank test. Results showed that (1) Oc. albifasciatus eggs were able to survive and hatch over 31 months; (2) the percent hatching of eggs stored at 5 degrees C was higher than that of eggs stored at room temperature; and (3) low temperatures and long periods without water favor installment hatching.     

Survival of Brucella abortus strain RB51 lyophilized and as liquid vaccine under different storage conditions.

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (-25, 4 and 25 degrees C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5 x 10(8), 1 x 10(9), or 5 x 10(9) cfu/ml) and two storage temperatures (4 or 25 degrees C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0.05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0.05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25 degrees C had a more rapid decline in viability (P< 0.05) when compared to vaccine stored at -25 or 4 degrees C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at -25 degrees C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25 degrees C (P< 0.05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0.05) viability during storage at 4 or 25 degrees C. When compared to liquid SRB51 vaccine stored at 25 degrees C, storage at 4 degrees C was associated with a slower decline in viability (P< 0.05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.     

Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport

BACKGROUND: The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. METHODS: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2-8 degrees C), room temperature (18-24 degrees C) and 37 degrees C for up to 72 h. Samples were analyzed for viable CD34+ cells/microL at 0, 24, 48 and 72 h. RESULTS: The mean viable CD34+ yield prior to storage was 7.7 x 10(6)/kg (range 0.7-30.3). The mean loss of viable CD34+ cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34+ cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34+ cells remained after storage at 37 degrees C for 24 h. Only PBSC products and not BM showed temperature-related loss of CD34 viability. Greater loss of viable CD34+ cells was observed for allogeneic PBSC compared with autologous PBSC. DISCUSSION: These results demonstrate that the optimum temperature for maintaining the viability of CD34+ cells, during overnight storage and transport of freshly harvested HPC, is 2-8 degrees C. These findings will allow the development of standard guidelines for HPC storage and transport.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Effect of temperature on long-term storage of codling moth granulovirus formulations

Codling moth, Cydia pomonella (L.), is the major pest of apple (Malus spp.) in the western United States and many other regions of the world. The codling moth granulovirus (CpGV) provides a selective and safe means of its control. We assessed the long-term stability and storage potential of two commercial formulations of CpGV, Cyd-X, and Virosoft. All assays were performed with individual C. pomonella neonate larvae in 2-ml vials on 1 ml of artificial larval diet that was surface inoculated with 10 microl of the test virus suspension. Baseline quantitative assays for the two formulations revealed that the LC50 and LC95 values (occlusion bodies per vial) did not differ significantly between the formulations. For year-long studies on Cyd-X stability, the product was stored at -20, 2, 25, and 35 degrees C, and quantitative bioassays were conducted after 0, 3, 6, and 12 mo of storage. Cyd-X retained good larvicidal activity from -20 to 25 degrees C, and it was the least negatively affected at the lowest temperature. Storage of Cyd-X at 35 degrees C was detrimental to its larvicidal activity within 3 mo of storage. For longer term storage studies, Cyd-X and Virosoft formulations were stored at 2, 25, and 35 degrees C, and assayed for larvicidal activity over a 3-yr period. For recently produced product, a 10-microl sample of a 10(-5) dilution of both formulations resulted in 95-100% mortality in neonate larvae. Larvicidal activity for the Cyd-X formulation remained essentially unaffected for 156 wk when stored at 2 and 25 degrees C, but it began to decline significantly after 20 wk of storage at 35 degrees C. The Virosoft formulation stored at 2 degrees C also remained active throughout the 3-yr study, but it began to decline in larvicidal activity after 144 wk at 25 degrees C and 40 wk at 35 degrees C. The information reported in this study should be useful to growers and commercial suppliers for avoiding decreases in CpGV potency due to improper storage conditions.     

Insecticidal properties and microbial contaminants in a Spodoptera exigua multiple nucleopolyhedrovirus (Baculoviridae) formulation stored at different temperatures

The Spodoptera exigua (Hübner) multiple nucleopolyhedrovirus (SeMNPV) is currently being tested as a biological insecticide for use in greenhouse crops in southern Spain. We performed a study in which semipurified SeMNPV occlusion bodies (OBs) were formulated in phosphate-buffered saline, pH 6.5, with 5% (vol:vol) glycerol and 0.15% (wt:vol) sorbic acid, and they were stored at -20, 4, or 25 degrees C during 18 mo. Initial aerobic counts (+/-SE) averaged 1.4 (+/-0.17) x 10(7) colony-forming units/ml after 17-h incubation at 37 degrees C. Aerobic counts of microorganisms that contaminated OB formulations stored at 250C decreased markedly over the period of the study, whereas only small decreases were observed in counts from OBs stored at 4 or -20 degrees C. The principal microbial contaminants of OB suspensions were Enterococcus spp., Enterobacteriaceae, and yeasts. Potential human pathogens (Salmonella, Shigella, and Vibrio species) were not detected, and populations of Staphylococcus aureus and Bacillus cereus were extremely low. Compared with newly formulated OBs, the estimated LD50 values of OBs stored at 25 degrees C increased by >16,666-fold over the 18 mo of storage, whereas LD50 values were not greatly affected by storage at 4 or -20 degrees C. Significant changes over time in OB concentrations were only observed in the 25 degrees C treatment. Complete degradation of viral DNA was observed at 25 degrees C but not in refrigerated or frozen OBs. We conclude that OB formulation with bacteriostatic or antioxidant additives, together with storage and distribution in refrigerated conditions, will likely result in an SeMNPV biopesticide shelf life that exceeds 18 mo.