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[Fluctuation Of The Egg Production Amounts According To Worm Burden And Length Of Ascaris Lumbricoides]

This study was undertaken to observe the fluctuation pattern of E.P.G. (eggs per gram of feces) according to the number of infected worms and developmental status of Ascaris lumbricoides. The cases were selected from 2,109 inhabitants of Hwasung Gun, Kyunggi Do, Korea, who had received pyrantel treatment in dose of 10 mg/kg during April 1977-May 1980 and who were cooperative enough to collect whole-day stool specimens for 2 days. Among them, 602 cases who had been infected with 1-5 worms were subjected to analysis. Egg examination was undertaken by means of cellophane thick smear technique and the amount of produced eggs was checked by Stoll's egg counting technique. The Ascaris worms were collected from stool specimens, counted in number in each case, and each worm was measured by length. The results could be summarized as follows: 1. According to the increase of worm burden per case from l to 5, the egg detection rate increased from 33.4 % to 95.7 %. And the same pattern was observed when the only burden was considered. 2. There were ample variations in the individual E.P.G. counts even among the cases with same burden, although the mean E.P.G. counts among the cases were quite proportionately increased from 842 to 3,467 according to the number of female worms, 1-4. 3. However, from the distribution pattern of the number of each grade of E.P.G. count, it was revealed that the lower the number of females the more cases were with low grade of E.P.G. 4. It was observed that the eggs could be detected after the female worm grew longer than 12.6 cm and the amount of eggs increased until the worm became about 25.0 cm, where a plateau was made in E.P.G. 1,300~1,400, and decreased significantly after 27.5 cm. And yet, wide variations in E.P.G. counts were noted even among the worms of similar growth. Accordingly, the E.P.G. value had better be used as a useful index to estimate the intensity of Ascaris infection and to compare the intensity between two communities, rather than be used to estimate the individual worm burden.

Journal Article↗

Polychaete worms--a vector for white spot syndrome virus (WSSV).

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.

Animals↗

Injection of recombinant interleukin 3 hastens worm expulsion in mice infected with Trichinella spiralis.

The effects of interleukin 3 (IL-3) on worm expulsion were studied in mice infected with Trichinella spiralis. C3H/He mice were treated with a total of 10(4) U IL-3 or saline by daily peritoneal injection from day-5 to day-1. Muscle larvae were given orally to both groups of mice on day 0. The muscle worn burden in infected mice was assessed on day 28. The worm burden in mice treated with recombinant IL-3 (rIL-3) was significantly suppressed as compared with that in control mice. A reduction in the worm burden was observed in mice treated with rIL-3 from day-5 to day-1 but not in those treated day 16 to day 20. This suggests that IL-3 could up-regulate the host defense response to intestinal worms but not to parenteral stage worms. When various doses of rIL-3 were given to mice and the intestinal worm burden was assessed on day 5, protection was observed only in mice treated with a total of 10(4) U rIL-3 but not in those given either 3.5 x 10(3) or 10(3) U. A kinetics study on the recovery of intestinal adult worms showed that rIL-3 treatment hastened worm expulsion. The mucosal mast-cell response observed in the small intestine of rIL-3-treated mice was induced earlier and was greater than that seen in the control. The host defense response induced by rIL-3 could not be inhibited by treatment with anti-IL-4 or anti-IL-5 monoclonal antibody. Under such an experimental condition in this study, at least, the numbers of mast cells per villus crypt unit observed in mice treated with rIL-3 and anti-IL-4 antibody were slightly lower than those seen in mice treated with rIL-3, but the difference was not significantly different. These results suggest that IL-3 can induce the expulsion of T. spiralis worms without the cooperation of IL-4 or IL-5 in mice.

Animals↗

Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida.

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.

Animals↗

Worm kinetics and serum IgE in hooded lister rats infected with the acanthocephalan Moniliformis moniliformis and the nematode Nippostrongylus brasiliensis.

After infection with the intestinal helminths Moniliformis moniliformis or Nippostrongylus brasiliensis, worm-specific IgE first appeared in the serum rats between days 10 and 24 p.i., varying with host age, worm species and worm dose used. The rate of increase in specific IgE was comparable regardless of the worm species, infection dose or host age and a peak response was observed about 1 month after the sera turned positive. In the M. moniliformis infections, these events took place long before the beginning of worm expulsion on day 63 in high-dose (50 worms) infections, and potentiation of heterologous IgE was not observed. In contrast, IgE stimulation by N. brasiliensis infections was detected as potentiation of anti-ovalbumin IgE, anti-M. moniliformis IgE and total IgE. Most of the total IgE in the serum of M. moniliformis-infected rats was likely to be the worm-specific IgE. Anthelminthic removal of M. moniliformis revealed that the presence of residual worms was necessary to maintain worm-specific IgE production.

Aging↗

The effect of gamma irradiation on Nematospiroides dubius. Factors affecting the survival of worms in a primary infection in mice.

The infective larvae of Nematospiroides dubius were exposed to various levels (0-30 krad) of gamma irradiation by means of a Cobalt 60 source. Groups of mice were infected with these larvae and autopsied 5 weeks later for worm counts. It was found that male worms were more susceptible to irradiation than female worms. In both instances, however, the survival curve on a semi logarithmic plot was characterised by a shoulder at low doses and an exponential component at the higher levels of exposure. No male worms were recovered from mice infected with larvae given more than 12 krad but some female worms were capable of surviving 20 krad. The fecundity of female worms was reduced by 61% at 4 krad and totally ablated at 8 krad. Further experiments demonstrated that the survival of irradiated N. dubius in vivo was related to the extent of the damage caused at the time of irradiation and was not dependent on additional host parameters. Thus neither the number of irradiated worms inoculated nor the sex of the host radically altered the sex ratio or proportion of the worms lost as a result of irradiating the larvae. Furthermore, treatment with cortisone or sublethal irradiation of the host did not increase the proportion of surviving worms. It was, therefore, concluded that a host immune response was not involved.

Animals↗

Schistosoma mansoni: changes in elemental composition in relation to the age and sexual status of the worms.

Changes during development in the elemental contents (Ca, P, K, Mg and S) of male and female Schistosoma mansoni from both mixed and single sex infections have been measured for the first time. In mixed infections, there is a significant difference between the calcium content of male and female worms; female worms having between 2 and 3 times more calcium. In mature female worms from mixed infections, after an initial rise in the calcium content corresponding to the time when vitelline development occurs, the amount of calcium ranged from 0.36 to 0.65 mg/g dry wt, whereas the amounts in similarly aged male worms ranged between 0.19 and 0.32 mg/g dry wt. While the type of infection does not affect the calcium content of male worms, a significant difference in the calcium levels in female worms from mixed and single sex infections was found. The amount of calcium in females from single sex infections ranged from 0.21 to 0.39 mg/g dry wt and this is considerably less than the levels recorded in females from mixed infections. This difference in calcium content between mixed and single sex infections can be related to the differences in degree of development of the vitelline gland in the two types of female worm. The amounts of phosphorus, potassium, magnesium and sulphur in both male and female worms from both single and mixed infections are similar, although females from mixed infections contained slightly more phosphorus than either females from single sex infections or either type of male worm.

Age Factors↗

The mode of action of suramin on the filarial worm Brugia pahangi.

The mode of action of suramin upon Brugia pahangi has been investigated in vivo and in vitro. The drug was without effect on the glycolytic activity of worms in vitro at 2 X 10(-4) M. The lack of effect was correlated with the failure of [14C]suramin to penetrate the worms in vitro. Suramin bound to the surface of worms in vitro presumably by virtue of its polyanionic nature. B. pahangi adults ingested [14C]suramin in vivo but no reduction in the rate of lactate production, of glucose utilization or in the rates of uptake of [14C]glucose, [14C]leucine or [14C]adenosine was observed in worms recovered from jirds between weeks 1 and 5 following 4 daily doses of suramin at 50 mg/kg given intraperitoneally. Worm death occurred between weeks 5 and 7 but this delayed drug effect was not the result of a progressive accumulation of suramin in the worms. Ultrastructural changes were observed in the intestinal epithelium of worms from suramin-treated jirds and parallel observations on worms exposed to Trypan blue in vivo suggest that both polyanionic compounds are restricted to the intestinal lumen of the worms. The evidence presented is consistent with the concept that, in B. pahangi, suramin acts at the surface of the intestinal epithelium and not by primarily inhibiting glucose catabolism or inhibiting phagosome and lysosome fusion as previously demonstrated for bloodstream trypanosomes and mammalian macrophages, respectively.

Adenosine↗

Relationship between faecal egg count and worm burden of Opisthorchis viverrini in human autopsy cases.

The relationship between faecal examination for egg output and worm burden of Opisthorchis viverrini in man of 181 autopsy cases from Northeast Thailand is described. Diagnosis of the parasite infection by stool examination for the presence of eggs was less sensitive than the worm recovery technique. Using Stoll's dilution and formalin-ether technique, no eggs were detected in the faeces of 20 cases harbouring low worm burdens (less than 20 worms). The quantitative faecal egg count by Stoll's dilution technique showed a strikingly close positive correlation with the number of worms recovered (r = 0.96, P less than 0.001) indicating a strong linear association between eggs per gram of faeces (epg) and worm burden. The number of epg per worm was inversely correlated to the worm burden (P less than 0.001), suggesting that density-dependent constraints on fecundity could operate to restrict egg output in heavy infections. The accuracy of egg counts for estimating worm burden and its relevance to parasite epidemiological research are discussed.

Adolescent↗

Immunoglobulin E and mast cell responses are related to worm biomass but not expulsion of Hymenolepis diminuta during low dose infection in rats.

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.

Animals↗

De-worming school children and hygiene intervention.

Helminths or worm infestations refer to worms that live as parasites in the human body and are a fundamental cause of disease associated with health and nutrition problems beyond gastrointestinal tract disturbances. Globally, over 3.5 billion people are infected with intestinal worms, of which 1.47 billion are with roundworm, 1.3 billion people with hookworm and 1.05 billion with whipworm. School children aged 5 - 15 years suffer the highest infection rate and worm burden that attributes to poor sanitation and hygiene. About 400 million school-age children are infected with roundworm, whipworm and hookworm worldwide, a large proportion of whom are found in the East Asia region (Cambodia, China, Lao PDR, Thailand and Vietnam). These parasites consume nutrients from children they infect, thus retarding their physical development. They destroy tissues and organs, cause abdominal pain, diarrhoea, intestinal obstruction, anaemia, ulcers and other health problems. All of these consequences of infection can slow cognitive development and thus impair learning. De-worming school children by anthelmintic drug treatment is a curative approach for expelling the heavy worm load. However, drug therapy alone is only a short-term measure of reducing worm infection and re-infection is frequent. Control measures through improved sanitation, hygiene and de-worming are needed to prevent infection and re-infection. UNICEF has supported many governments in this (and other) regions to assist in the provision of water supply and sanitary facilities and intensive hygiene education in many schools through the Water, Environment and Sanitation (WES) programme. The UNICEF supported school sanitation and hygiene education (SSHE) programme, and other programmes, could effectively enhance behaviour change in children to break the routes of worm transmission and other waterborne diseases.

Adolescent↗

The role of adult worms in suppressing functional protective immunity to Heligmosomoides polygyrus bakeri challenge infections.

Adult Heligmosomoides polygyrus bakeri, radiolabelled with [35S]-methionine were successfully transferred to naive NIH mice by oral gavage. Adult worms and radiolabel could be detected up to 45 days post-infection. Adult worms gavaged into immune NIH mice, immunized with a drug abbreviated larval infection, were rejected within 45 days. These adults worms were unable to ablate the development of a functional protective response to a larval challenge infection in the NIH strain. In fact 50 adult worms were sufficient to significantly immunize NIH mice against a larval challenge infection. However, adult worms were able to suppress the development of a functional protective response in an outbred CFLP strain. Although a protective immune response could not be elicited to a challenge infection in CBA mice, the presence of gavaged adult worms was shown to increase the susceptibility of mice to a challenge infection. For all mouse strains, no significant difference in levels of L4 antigen-specific serum IgG, IgG1, IgG2a, and IgA existed between immune mice and groups of mice immunosuppressed by adult worms. Levels of L4 antigen-specific serum IgG1 were significantly lower in the poorly immunizable CBA strain compared to CFLP and NIH strains. No correlation was found across mouse strains between the intensity of the antibody response and the mean worm burdens per animal group. In addition, no correlation was found between levels of L4 antigen-specific antibody within each mouse and the loss of worms by individual mice.

Animals↗

Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces fecundity in Strongyloides venezuelensis-infected mice.

We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR(-/-)) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR(-/-) and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR(-/-) mice. Although PAFR(-/-) infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR(-/-) mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR(-/-) mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR(-/-) mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR(-/-) mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.

Animals↗

Gordius worm found in a three year old girl's vomitus.

Since the Gordius worm is a parasite of crickets and several arthropods, cases of humans infected with this worm have been rare and accidental. A Gordius worm was obtained from a three-year-old girl who consulted a local clinic in Gwangju, Kyunggi-do, Korea. She lived in a rural area, and had eaten an insect that looked like a cricket. She expelled the worm in vomitus 15 minutes later; in fact, she expelled two worms, but one was discarded. The worm had a grayish white color and an intact outer surface. It was 16 cm in length and 0.6 cm wide. The posterior end of the worm was spirally enrolled and furcated into two caudal lobes, which were nearly cylindrical but showed a somewhat concave medio- ventral surface. The cloacal aperture was round and situated anterior to the point of bifurcation of the lobes. The cloacal aperture was encircled by a dark ring, which was a little removed from the aperture. The crescent fold was reddish brown, and no hairs were noticed over the entire body surface. The worm had the morphological features of a male Gordius. Accidental human cases involving the Gordius worm are rare and this is the first such case in Korea.

Administration, Oral↗

Increased human IgE induced by killing Schistosoma mansoni in vivo is associated with pretreatment Th2 cytokine responsiveness to worm antigens.

In schistosomiasis endemic areas, children are very susceptible to postchemotherapy reinfection, whereas adults are relatively resistant. Different studies have reported that schistosome-specific IL-4 and IL-5 responses, or posttreatment worm-IgE levels, correlate with subsequent low reinfection. Chemotherapy kills i.v. worms providing an in vivo Ag challenge. We measured anti-worm (soluble worm Ag (SWA) and recombinant tegumental Ag (rSm22.6)) and anti-egg (soluble egg Ag) Ab levels in 177 Ugandans (aged 7-50) in a high Schistosoma mansoni transmission area, both before and 7 wk posttreatment, and analyzed these data in relation to whole blood in vitro cytokine responses at the same time points. Soluble egg Ag-Ig levels were unaffected by treatment but worm-IgG1 and -IgG4 increased, whereas worm-IgE increased in many but not all individuals. An increase in worm-IgE was mainly seen in >15-year-olds and, unlike in children, was inversely correlated to pretreatment infection intensities, suggesting this response was associated both with resistance to pretreatment infection, as well as posttreatment reinfection. The increases in SWA-IgE and rSm22.6-IgE positively correlated with pretreatment Th2 cytokines, but not IFN-gamma, induced by SWA. These relationships remained significant after allowing for the confounding effects of pretreatment infection intensity, age, and pretreatment IgE levels, indicating a link between SWA-specific Th2 cytokine responsiveness and subsequent increases in worm-IgE. An exceptionally strong relationship between IL-5 and posttreatment worm-IgE levels in < 15-year-olds suggested that the failure of younger children to respond to in vivo Ag stimulation with increased levels of IgE, is related to their lack of pretreatment SWA Th2 cytokine responsiveness.

Adolescent↗

In vitro effect of Mirazid on Schistosoma mansoni worms.

Measuring Mirazid ability for contracting the worm muscle and its effect on the worm surface ultra-structure can be used to monitor the in vitro effect of any drug. This study aims at investigating the actual effect of Mirazid (a new schistosomicide; purified oleo-resin extract of Myrrh, derived from Commiphora molmol plant) on S. mansoni worms by detecting its in vitro effect. Three groups of white albino mice (5 mice in each group) were infected by 100 cercariae for each mouse. The 3rd group served as a control group. Seven weeks post-infection the mice were sacrificed, perfused and worms were collected. Muscle tension of the worms collected from the first group of mice, was assayed in response to Mirazid in rising concentrations of 100, 200, 300 and 400 nM. The in vitro effect of Mirazid on the muscle tension of single S. mansoni worm was determined using a special device to determine percentage of change in worm length (% shortening). The drug elicited somatic muscle contraction and reached the highest response with 400 nM Mirazid. The maximal increase in the muscle tension (48% shortening) was induced by the highest concentration (400 nM) of the drug. The worms collected from the second group of mice were scanned by electron microscopy. The worms were exposed each to 10 ul of Mirazid in concentration of 10(-6) and collected after 10, 20 and 30 minutes of exposure. Ten minutes exposure caused disruption of the tegument and collapse of tubercles. After 20 minutes, the tegument appeared edematous with more disruption and more shrinkage of tubercles. After 30 minutes, the tegument appeared markedly edematous with extensive disruption of the inter-papillary areas and sensory bulbs. The spines covering the tubercles appeared to be lost.

Animals↗

Dracunculiasis: an approach to hasten worm expulsion.

Twenty patients with Guinea worm disease having a total of 29 emerging worms in a rural village of North West Frontier Province, were put on a 5-day course of antibiotics and antiinflammatory drugs and compared with age-matched control patients. The ulcer sites of emerging worms were kept continuously moist, and 18 worms were extracted from 14 patients during the above mentioned period. The average time taken for the entire worm to be expelled in these patients was considerably shorter (13.6 days) than in controls (89 days). Seven (39%) of these worms were expelled spontaneously whereas the remaining 11 (61%) were pulled out manually with relative ease. Fifteen (75%) patients in this study had a single emerging worm, three (15%) had two emerging worms and two (10%) patients had four or more emerging worms.

Ampicillin↗

Schistosoma mansoni: the effects of a subcurative dose of praziquantel on the ultrastructure of worms in vivo.

The in vivo effects of a subcutaneously-administered subcurative dose (200 mg/kg body weight of mouse) of praziquantel on the structure of Schistosoma mansoni was investigated. Worms were collected at varying times post-treatment and processed for both SEM and TEM examination. Praziquantel caused extensive structural changes to both male and female worms within 15 min of treatment although variations in the amount of drug-induced damaged was observed between male and female worms. In female worms although some tegumental vacuolation was observed within 15 min, the major structural change was an often extensive vacuolisation of the subtegumental tissues followed by varying degrees of structural disruption to the subtegumental and gastrodermal musculature. In male worms the initial effects were a vacuolisation of parts of the dorsal tegument and loss of tegumental cytoplasm due to the pinching off of evaginations of the outer surface. With increasing time post-treatment there was an increase in the amount of tegumental damage, particularly in male worms, with total disruption of parts of the outer surface occurring. Also in male worms there was an increase in the amounts of vacuolisation of the parenchymal tissues and in the degree of structural damage to the musculature. In those female worms where subtegumental damage was not extensive changes in the structure of the differentiating vitelline cells were noted.

Animals↗