Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis)

In previous studies we observed that human umbilical cord blood (HUCB) could have a protective effect on the onset of disease and time of death in MRL Lpr/Lpr mice which have an autoimmune disease that may be considered similar to human lupus. We believed a temporary xenograph may have occurred in these animals with the disease process delayed and the life span markedly increased. When HUCB is stored at 4 degrees C in gas permeable bags, there is a decrease of the cell reaction in mixed lymphocyte cultures. The blood, however, maintains a significant number of cells capable of producing replatable colonies. This study attempted to determine the effect of HUCB on SOD1 mice (transgenic B6SJL-TgN(SOD1-G93A)1GUR), which have a mutation of the human transgene, (CuZn superoxide dismutase gene SOD1) that has been associated with amyotrophic lateral sclerosis. We previously developed evidence that the survival of lethally irradiated mice was related to the number of human mononuclear cells administered. In the present study, we decided to investigate the effect of a relatively large dose of human mononuclear cord blood cells on SOD1 mice subjected to a sublethal dose of irradiation preceded by antikiller sera (rabbit anti-asialo). The SOD1 mice show evidence of paralysis at 4 to 5 months. The average expected lifetime of these mice is reported to be 130 days (Jackson Laboratory). In this experiment, there were 23 mice. Two mice died before the onset of paralysis. The remainder were divided into three groups: group I: control group of 4 untreated mice; group II: an experimental group of 6 mice treated with antikiller sera, 800 cGy irradiation plus 5 x 10(6) congenic bone marrow mononuclear cells; group III: another experimental group of 11 mice treated with antikiller sera, 800 cGy irradiation plus 34.2-35.6 x 10(6) HUCB mononuclear cells, previously stored for 17-20 days at 4 degrees C in gas permeable bags. The results were as follows: the average age at death was: (I) 127 days for the untreated control group, (II) 138 days for the group that received 800 cGy of irradiation and congenic bone marrow (BM) and (III) 148 days for the group that received irradiation and HUCB. (P < 0.001 HUCB vs control, p < 0.01 HUCB vs BM). The longest surviving mouse in each group was 131, 153, and 182 days old respectively. In summary, large doses of HUCB mononuclear cells produced considerable delay in the onset of symptoms and death of SOD1 mice. These preliminary results may not only indicate that amyotrophic lateral sclerosis is an autoimmune disease, but may also indicate a possible treatment for a devastating disease and possibly others.     

Nociceptive responses to altered GABAergic activity at the spinal cord

GABA agonists and antagonists were injected intrathecally at the spinal cord, to determine their effect on nociceptive thresholds. Tactile stimulation, applied against the flank by a medium diameter von Frey fiber (5.5 g force), elicited distress vocalizations after, but not before injection of the GABA antagonists, bicuculline MI or picrotoxin (0.25 and 1 microgram dosages). Vocalization threshold to tail shock was significantly reduced by bicuculline MI or picrotoxin. Tail flick withdrawal latency from radiant heat was not altered by GABA antagonists. The GABA agonist, muscimol, significantly elevated vocalization threshold to tail shock at a 5 micrograms dose. At a lower dose level (1 microgram), muscimol significantly reduced vocalization threshold to tail shock. Tail flick latency was significantly prolonged by the 5 micrograms dose of muscimol; however, flaccid paralysis of the hind limbs was also evident. Nociceptive thresholds were not altered by GABA or saline injection. These findings indicate that GABAergic activity contributes to the tonic modulation of nociception at the spinal cord.     

No lengthening of life span in mice continuously exposed to gamma rays at very low dose rates

Late effects of continuous exposure to ionizing radiation are potential hazards to workers in radiation facilities as well as to the general public. Recently, low-dose-rate and low-dose effects have become a serious concern. Using a total of 4000 mice, we studied the late biological effects of chronic exposure to low-dose-rate radiation as assayed by life span. Two thousand male and 2000 female 8-week-old specific-pathogen-free (SPF) B6C3F1 mice were randomly divided into four groups (one nonirradiated control and three irradiated). Irradiation was carried out for approximately 400 days using (137)Cs gamma rays at dose rates of 21 mGy day(-1), 1.1 mGy day(-1) and 0.05 mGy day(-1) with total doses equivalent to 8000 mGy, 400 mGy and 20 mGy, respectively. All mice were kept under SPF conditions until they died spontaneously. Statistical analyses showed that the life spans of mice of both sexes irradiated with 21 mGy day(-1) (P < 0.0001) and of females irradiated with 1.1 mGy day(-1) (P < 0.05) were significantly shorter than those of the control group. Our results show no evidence of lengthened life span in mice continuously exposed to very low dose rates of gamma rays.     

Antimetrazol action of two potential anticonvulsants, CM 40907 and SR 41378, in immature rats.

The action of two potential anticonvulsants, CM 40907 (10-50 mg/kg i.p.) and SR 41378 (1.25-20 mg/kg i.p.) against metrazol-induced seizures was studied in rats 7, 12, 18 and 25 days old. Two types of motor seizures--minimal, clonic and major, generalized tonic-clonic--were elicited by a 100-mg/kg dose of metrazol (s.c.) and their incidence and latency were evaluated. The severity of seizures was expressed as a score on a 5-point scale. Dimethylsulfoxide, an organic solvent, exhibited anticonvulsant action only in doses far exceeding those used for dissolving the two anticonvulsants. Both drugs suppressed minimal as well as major seizures in all age groups studied in a dose-dependent manner, SR 41378 being approximately four times more potent than CM 40907. The latencies could be measured only in animals given low doses of anticonvulsants. CM 40907 did not change the latencies whereas SR 41378 prolonged them. The severity of seizures was decreased again in a dose-dependent manner. There were only minor changes in the efficacy of CM 40907 among the four age groups. On the contrary, SR 41378 exhibited an extreme efficacy in 7-day-old rat pups, where even the 1.25 mg/kg dose significantly decreased the incidence and severity of seizures. The efficacy in the remaining three age groups was approximately at the same level as in adult rats.     

Induced sterility of Glossina pallidipes Austen males after irradiation in a nitrogen atmosphere

The sterile insect technique relies on sterilization of males using ionizing radiation. Life cycle stage, and the environmental conditions under which irradiation is carried out are crucial to the provision of good‐quality insects. To identify an optimal radiation strategy for Glossina pallidipes Austen, 1903, 13‐day‐old males were irradiated at different doses in a nitrogen atmosphere. The following day the males were mated with 8‐day‐old virgin females. Pupal production of mated females was monitored for 6 weeks, and induced sterility was determined by probit analysis. Survival of the males that mated was also monitored. At least 95% sterility of irradiated males was achieved with a 158 Gy dose in nitrogen and a 125 Gy in air. Irradiation significantly lowered the probability of survival between 30 and 100 days of age (especially flies irradiated in air), but probabilities of survival were similar outside this period for irradiated and unirradiated flies. Exposure of 2‐ or 13‐day‐old males to sterilizing radiation induced similar levels of sterility in both air and nitrogen.     

Effect of negative air ions on morphine-induced changes in the latency of the tail-flick reflex

Adult male Long-Evans rats were exposed to negatively charged air ions at high concentrations (7 x 10(5)/cm3) for six days. Sham-exposed rats were treated identically except that the source of ions was not activated. At the end of the exposure, the latency of the tail-flick reflex was measured in each rat before and 30 and 60 min after an injection of morphine sulphate. The tail-flick reflex was initiated by thermal stimulation. Two heat settings were employed, the lower considered to impart a submaximal and the higher a maximal thermal stimulus. Three morphine doses were tested: 0.5, 1.0, and 1.5 mg/kg. Statistically significant differences between ion-exposed and sham-exposed rats were observed in tail-flick latencies 30 min after the administration of the two lower doses, but not after the highest dose of morphine sulphate. These differences were found at both intensities of thermal stimulation. Tail-flick latencies measured in each group prior to morphine injection were not affected by negative-ion exposure. The data indicate that exposure of rats to negative air ions tends to inhibit the action of morphine on the latency of the tail-flick reflex at morphine doses below 1.0 mg/kg.     

Targeted therapy of spinal cord glioma with a genetically modified Salmonella typhimurium

Objective:  Spinal cord tumours are highly malignant and often lead to paralysis and death due to their infiltrative nature, high recurrence rate and limited treatment options. In this study, we measured antitumour efficacy of the Salmonella typhimurium A1-R tumour-targeting bacterium strain, administered systemically or intrathecally, to spinal cord cancer in orthotopic mouse models.
Materials and methods:  Tumour fragments of U87-RFP were implanted by surgical orthotopic implantation into the dorsal site of the spinal cord. Five and 10 days after transplantation, eight mice in each group were treated with A1-R (2 × 10⁷ CFU/200 μL i.v. injection or 2 × 10⁶ CFU/10 μL intrathecal injection).
Results:  Untreated mice showed progressive paralysis beginning at day 6 after tumour transplantation and developed complete paralysis between 18 and 25 days. Mice treated i.v. with A1-R had onset of paralysis at approximately 11 days and at 30 days; five mice developed complete paralysis, while the other three mice had partial paralysis. Mice treated by intrathecal injection of A1-R had onset of paralysis at approximately 18 days and one mouse was still not paralysed at day 30. Only one mouse developed complete paralysis at day 30 in this group. Intrathecally treated animals had a significantly better survival than the i.v. treated group as well as over the control group.
Conclusions:  These results suggest that S. typhimurium A1-R monotherapy can effectively treat spinal cord glioma.     

Modification of radiation myelopathy by the transplantation of neural stem cells in the rat

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2-C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of (60)Co gamma rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 microl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 microl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.     

Sex-and age-dependent effects of prenatal exposure to caffeine on open-field behavior, emergence latency and adrenal weights in rats

Pregnant rats were provided with drinking water containing 0, 0.23 or 0.3 mg/ml of caffeine throughout gestation. These concentrations gave rise to daily doses of 0, 28 and 36 mg/kg. Open-field behavior and latencies to emerge from a darkened chamber were observed in offspring at regular intervals from 1 to 8 months after birth. The main results revealed increases in open-field locomotor and rearing activity with 28 but not 36 mg/kg/day. The opposite pattern characterized emergence latency. These changes were more typical of male rats particularly when older. Combining the present results with those of an earlier study by the authors strengthened the curvilinear trends observed and led to the conclusion that, low doses of prenatal caffeine increase activity and decrease emotionality. Higher doses may have the opposite effects to the point that the significant differences from control subjects reported earlier can occur. When 8 months old, female but not male rats prenatally exposed to 36 mg/kg/day of caffeine had significantly heavier adrenal glands than controls.     

Time-dependent neurosphere-forming ability of adult rat spinal cord after irradiation

To determine whether there was evidence for long-term time-dependent changes in neurosphere-forming ability of rat spinal cord after irradiation, a 15-mm length of spinal cord (C2-T2) of 10-week-old female rats was irradiated with a single dose of 2, 5, 10 or 19 Gy. Cells were isolated from the central 10-mm segment of the irradiated spinal cord immediately or at 0.5, 1, 2 or 5 months to form neurospheres. The number and sizes of neurospheres were determined at day 10, 12, 14 and 16 in vitro. The multipotential properties of neurosphere cells were assessed by immunocytochemistry using lineage-specific markers for neurons and glia. In nonirradiated controls, the number and size of the neurospheres decreased with increasing age of the animals. Regardless of the time after irradiation, there was a dose-dependent decrease in the number and size of neurospheres obtained from the irradiated cord compared to age-matched controls. Using three-way ANOVA, the number of neurospheres was dependent on radiation dose (P < 0.0001), time after irradiation (P < 0.0001), and day of counting in vitro (P < 0.0001). Compared to cells cultured immediately after irradiation, there was an increase in the relative plating efficiency of neurospheres cultured 1 month after irradiation. However, no further increase was apparent up to 5 months after irradiation. The multipotential properties of neurosphere cells in vitro remained unchanged with increasing time after irradiation. These results may suggest a time-dependent recovery of radiation damage using neurosphere-forming ability as the end point and agree with data that show time-dependent recovery of radiation damage in spinal cord using histological or functional end points.     

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.     

Age at reproductive maturity and effect of age and time of day on sex attraction in the potato psyllid Bactericera cockerelli

Abstract The potato psyllid, Bactericera (= Paratrioza) cockerelli (?ulc) (Hemiptera: Triozidae), is a major pest of potato. Studies were conducted to determine the age at which both males and females reach reproductive maturity and the effect of age and time of day on sex attraction. Adult B. cockerelli reach reproductive maturity within 48 h post‐eclosion, with females being mature on the day of eclosion and males at 1 day post‐eclosion. Oviposition generally began 2 days after mating but was delayed when females mated within 2 days post‐eclosion. In laboratory olfactometer assays, the age of females used as odor sources and the age of males assayed to these odors did not affect sex attraction, with both younger (1–4‐day‐old) and older (8–10‐day‐old) males being attracted to females, regardless of female age (1–4‐day‐old or 8–10‐day‐old). Males assayed to live females at different times during the photophase (between 8:00 and 20:00 hours) were attracted to females between 11:00 and 17:00 hours, showing a temporal periodicity in sex attraction with B. cockerelli at least during the photophase.     

The effect of colchicine on the transport of axonal protein in the chicken

1. Small doses (1-10mug) of colchicine injected into the ventral horn of the spinal cord of the chicken caused paralysis in the legs. 2. Colchicine had no effect on the incorporation of leucine into proteins of the spinal cord but markedly decreased the total amount of protein flowing into the axons of the sciatic nerve. 3. This axonal flow of protein proceeded at two rates: a high rate (300mm/day) and a low rate (2mm/day). Although both groups of proteins were affected, the slow transport of protein was more profoundly blocked by colchicine. 4. The results suggest that axonal flow is dependent on the neurotubular system in the axon.     

Further observations on the pathogenesis of rabies in guinea-pigs after experimental infection with the Flury strain

Summary Following inoculation of guinea-pigs with the Flury strain of rabies virus in the muscles of the foreleg, the virus could be recovered from the cervico-thoracic cord on the 4th day. From the 6th day on the virus spread in both directions, to the brain and to the lumbosacral cord. There was a close parallelism between the virus distribution and the clinical symptoms: the first signs of paralysis were observed in the inoculated leg. Intravenous inoculation into the veins of the penis resulted in a mortality rate at least as high as that induced by intramuscular injection. The symptoms and the virus distribution demonstrated that the virus first invaded the anterior part of the central nervous system. From these experiments and others previously reported, we may conclude that after intramuscular inoculation the first segment of the central nervous system, invaded by the virus, corresponds with the site of inoculation, whereas after intravenous inoculation no such correlation exists.     

Cocaine as a cause of congenital malformations of vascular origin: experimental evidence in the rat

Cocaine hydrochloride was administered to pregnant Sprague-Dawley rats as a single intraperitoneal dose or as two doses 1-4 hours apart. A single dose administered on day 16 of gestation was teratogenic in a dose-dependent manner, with 40 mg/kg being a no-effect dose and 50 mg/kg the lowest teratogenic dose; 80 mg/kg was lethal to the dam. Forty-eight hours after exposure to a teratogenic dose on day 16 of pregnancy, the fetuses showed severe hemorrhage and edema in the their extremities, particularly the footplates, tail, genital tubercle, and upper lip/nose. When the fetuses were examined on day 21 of gestation, the main externally visible malformations were reduction deformities of the limbs and tail. When two doses of cocaine were administered 1-4 hours apart, the incidence of affected fetuses increased as the time interval between the two doses decreased. Two doses of cocaine administered 2 hours apart were not teratogenic on day 9, 10, 11, 12, 13, or 14 of gestation but did induce reduction deformities on days 15, 16, 17, 18, or 19. The same dose administered 1 hour apart was teratogenic on days 14-19. In general, cocaine administration on gestational days 14, 15, or 16 induced more severe and more widespread hemorrhage and edema than administration on days 17, 18, or 19. In the latter cases, damage was restricted to the distal parts of the hindlimb digits and the tail. The results show that in the rat cocaine is only teratogenic during the late organogenic or postorganogenic period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of recombinant human interleukin 11 on thrombocytopenia and neutropenia in irradiated rhesus monkeys

The effects of recombinant human interleukin 11 (rhIL11) on thrombocytopenia and neutropenia in irradiated rhesus monkeys were evaluated after administration different doses at different times. Twenty-three rhesus monkeys were exposed to a total-body irradiation (TBI) with a single dose of 3 Gy 60Co gamma rays. Either placebo, rhIL11 at a dose of 30, 60 or 120 microg/kg day(-1) on days 0-13, or rhIL11 at a dose of 60 microg/kg day(-1) on days 13-26 after TBI was administered to the animals. The results showed that the immediate treatment with rhIL11 but not treatment on days 13-26 resulted in much higher platelet nadirs than in the placebo-treated group. The accelerated recovery of platelets to normal levels after TBI was demonstrated in all groups treated with rhIL11, but the effects of rhIL11 were independent of dose. However, rhIL11 treatment could also accelerate the recovery of leukocytes to normal levels. The numbers of colony-forming bone marrow cells (CFU-E, CFU-Mix, CFU-MK and CFU-GM) in all groups treated with rhIL11 were increased 4- to 14-fold relative to those of the placebo group on day 30. We conclude that rhIL11 may directly promote megakaryocyte development and ameliorate myelosuppression in irradiated monkeys.     

Effects of gamma irradiation on the life stages of yellow flower thrips, Frankliniella schultzei (Trybom) (Thysanoptera: Thripidae)

Irradiation at a minimum absorbed dose of 250 Grays (Gy) has been approved by the USDA as a quarantine treatment for certain fruits in Hawaii to control four species of tephritid fruit flies. Subsequent research must determine whether this dose is sufficient to control other quarantine pests, such as mealybugs, thrips, mites, beetles, moths, and scale insects, on other commodities with export potential that are approved for irradiation treatment for fruit flies. This study demonstrated that irradiation at 250 Gy caused non‐emergence of eggs and pupae, failure of larval development, and sterility of adults of yellow flower thrips, Frankliniella schultzei (Trybom). Adults were the most resistant stage tested, with 100% mortality at 57, 36 and 30 days post‐treatment for the 250, 350 and 400 Gy treatments, respectively. Untreated adults survived up to 66 days. After receiving an irradiation dose of 250 Gy, no one‐ to two‐day old eggs hatched successfully, while 3–4‐day old eggs hatched but did not develop beyond the larval stage. Of the controls, 96.0% of 1–2‐day old eggs and 75.9% of the 3–4‐day old eggs hatched and survived through pupation. No first or second instar larvae treated with a target dose of 250 Gy were able to pupate. When pupae were irradiated at 250 Gy, 37% emerged as adults and all were sterile compared to 88.3% emergence of controls.     

The effect of FSH on LH induced testosterone secretion in the immature hypophysectomized male rat

The effect of gonadotropin pretreatment of hypophysectomized male rats on LH stimulated serum testosterone concentrations was studied. A 5 day pretreatment period began 2 days after hypophysectomy at 21 or 24 days of age. On the day following the pretreatment period the animals received an intraperitoneal injection of saline or LH 60 min before blood collection. Animals pretreated with NIH-FSH-B1, or with doses of LH approximating the amount present in the NIH-FSH, had increased testosterone concentrations after LH stimulation compared to similarly stimulated saline pretreated animals. Pretreatment with more highly purified FSH Ex 199C at a lower dose than the minimum effective dose of NIH-FSH was also effective. There was no synergistic or additive effect when FSH Ex 199C and LH pretreatments were combined. FSH Ex 199C is more potent and contains appreciably less LH contamination than NIH-FSH-B1. The results obtained using FSH Ex 199C indicate that FSH, independent of LH contamination, can increase testes response to LH stimulation.     

Demyelination Occurred as the Secondary Damage Following Diffuse Axonal Loss in a Rat Model of Radiation Myelopathy

The main purpose of the present study was to examine the time and dose-dependent course of demyelination in the rat radiation myelopathy model in the first 180 days after irradiation of the spinal cord. An irradiated cervical spinal cord rat model (C2-T2 segment) was generated using a ⁶⁰Co irradiator to deliver 50 Gy and 100 Gy, respectively. The behavioral dysfunction was observed by the forelimb paralysis scoring system. The histological damage in the irradiated spinal cord was examined by hematoxylin/eosin staining, luxol fast blue staining, immunohistochemical analysis, methylene blue/Azure II staining, and uranyl/lead salts staining. The gene expression of oligodendrocyte-related markers were also determined by quantitative real-time PCR. The complete loss of forelimb motor function in all animals was observed at 180 days 50 Gy post-irradiation and at 120 days 100 Gy post-irradiation. We demonstrated that a 50 and 100-Gy single-dose irradiation of the C2-T2 spinal cord segment resulted in diffuse axonal loss and elicited secondary demyelination damage in the spinal cord. We further observed that 100-Gy irradiation reduced the gene expression of myelin oligodendrocyte glycoprotein in irradiated spinal cord. Taken together, our data not only define diffuse axonal loss as the main histological damage but also provide the first evidence that demyelination occurred as the secondary damage in irradiated spinal cord.     

Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.