Ferron L. Andersen

Effect of temperature on survival of the free-living stages of Trichostrongylus colubriformis.

Desiccation of the 3rd stage larvae of Trichostrongylus colubriformis was beneficial to their survival at temperatures below freezing and at 35 to 50 C, but was of indifferent effect at intermediate temperatures of 20 and 25 C. After 128 days, half the clean larvae which had been desiccated and stored at -95 C were still alive, as compared with none of those that had not been desiccated; however only 7% of the larvae which had been desiccated in fecal pellets were still alive. After 128 days at -20 C, 5% of the larvae that had been desiccated in fecal pellets were alive, and at -10 C, 12%. At the optimum survival temperature (4 C), the best survival for any treatment occurred in the control larvae kept in water; 92% were still alive after 128 days, as compared with 60% in moist fecal pellets, 65% of the larvae desiccated within fecal pellets, and 17% of the clean desiccated larvae. At 20 C, survival was not as good as at 4 C, and at 25 C it was still worse; desiccation was generally neither beneficial nor harmful at these temperatures. At 35 C, 10% of the larvae desiccated in pellets were still alive after 128 days, and less than 1% of the undesiccated ones in pellets. At 45 and 50 C, larvae desiccated in fecal pellets survived as long as 64 days as compared with 12 days in moist fecal pellets. Larvae which had been stored in water for approximately 1 month or longer at 4 C were not as resistant to desiccation as younger larvae. Eggs in the advanced stage of embryonation were approximately as resistant to desiccation as infective larvae. Unembryonated eggs were only slightly resistant to desiccation. First and 2nd stage larvae removed from the fecal pellets were highly susceptible to drying, but small numbers survived at least 8 days in dried fecal pellets at 30 C and 65 to 75% RH. It appears that there is generally sufficient moisture within fecal pellets deposited on pasture for the eggs to develop to the resistant embryonated egg stage before the moisture evaporates. Additional external moisture is then probably required before development to the next resistant (3rd larval) stage can occur. After the infective larvae migrate from the pellets onto the herbage, they probably desiccate again and remain so until rehydrated with rain or heavy dew, or until eaten by grazing hosts. Parasites which have free-living stages as part of their normal life cycles are frequently subjected to environmental conditions far from optimum for their survival. Although we do not understand how all parasites withstand such deleterious conditions as freezing or hot dry weather, we do have considerable information on how certain groups survive, at least in small numbers. Some parasites, for example, form stages highly resistant to environmental extremes, such as cysts, spores, or metacercariae. Certain parasitic arthropods overwinter in the egg stage, even though the adults are all killed by freezing weather. Some helminth parasites may be acquired by transport hosts, such as earthworms or arthropods, and yet remain infective for their respective definitive Received for publication 12 August 1967. * This investigation was supported by research grants AI-06197 from the NIAID, NIH, and CC-00037 from the Communicable Disease Center, U. S. Public Health Service, Department of Health, Education, and Welfare. t Present address: Department of Zoology and Entomology, Brigham Young University, Provo, Utah 84601. hosts if the transport hosts are subsequently ingested. Existence within the transport host, while not obligatory, nevertheless provides a favorable temporary environment for the parasite and also prevents death from desiccation as long as the transport host continues to live. The life cycles of most trichostrongylid nematodes which parasitize sheep and cattle are similar in that the eggs of the adult worms are passed to the outside with the feces. Under suitable conditions of temperature and moisture these eggs develop to 1st and 2nd stage larvae and finally to 3rd stage (infective) larvae. All stages occur within the hosts feces after deposition on the ground, or on the vegetation near the ground surface. Many investigators (Monnig, 1930; Stewart and Douglas, 1938; Crofton, 1948; Gordon, 1948; Silverman and Campbell, 1959; Prasad, 1959; Rose, 1963; Kates, 1965; Andersen, Wang, and Levine, 1966; Williams and Mayhew, 1967) have found that embryonated eggs and infective larvae are the stages most resistant to the deleterious temperature extremes that would probably occur on pasture at different times of the year. It is not clear why these stages

Frequency distribution of Echinococcus granulosus hydatid cysts in sheep populations in the Xinjiang Uygur Autonomous Region, China

Age-prevalence and age-intensity data of Echinococcus granulosus hydatid cysts in sheep populations were collected in an abattoir in the Xinjiang Uygur Autonomous Region, Peoples Republic of China. The frequency distribution of the larval cysts per sheep was empirically described by the negative binomial model, with parameter k being 0.5273. A mathematical model for the life cycle of E. granulosus was applied to the collected data and the results show that the infection pressure on sheep was 0.4362 (female) or 0.4119 (male) infections per year, the mean number of cysts increased linearly by 0.8824 (female) or 0.9971 (male) cysts every year and acquired immunity was too low to depress this rate of increase. According to certain definitions of steady states for taeniid populations, it was concluded that at least in some parts of Xinjiang, the life cycle of E. granulosus was and may still be in an endemic steady state. Consequently, the regular dog-dosing program would readily drive the infection from an endemic state towards extinction.

Frequency distribution of Echinococcus granulosis in dog populations in the Xinjiang Uygur Autonomous region, China

Age-prevalence and age-intensity data of Echinococcus granulosus in dog populations were collected in four counties in the Xinjiang Uygur Autonomous Region, Peoples Republic of China. The frequency distribution of the parasite per dog was adequately described by the negative binomial model, with parameter k being 0.0571. The mathematical model of the life cycle of E. granulosus constructed by Roberts et al. (1986--Parasitology, Vol. 92, pages 621-641) was used to estimate the epidemiologic parameters of E. granulosus in Xinjiang. This model showed that the prevailing infection pressure on dogs was 0.4560 infectious insults year-1 and the mean length of an infection was 1.4975 years. The effect of acquired immunity on the prevalence of E. granulosus in dogs was considered to be insignificant.

Effect of praziquantel on adultEchinococcus granulosus in vitro: Scanning electron microscopy

The effect of praziquantel in vitro at concentrations of 5, 50 and 500 ppm for 1 h resulted in the progressive breakdown of the tegument and in morphologic distortion of adultEchinococcus granulosus when compared to controls. Scanning electron microscopy of all specimens treated in the various concentrations of praziquantel showed loss of most, if not all, of the rostellar hooks and changes in the structure of the suckers. Many of the tapeworms immediately detached from the hosts gut upon being placed in the drug, and all treated cestodes exhibited contraction or swelling, particularly in the penultimate proglottid. Intense contraction was apparent in the worms exposed to the higher drug concentrations. Characteristic conical microtriches on the terminal proglottid, as observed in the control specimens, became fused and matted when exposed to 5 ppm of praziquantel. At a drug concentration of 50 ppm, the tegumental surface developed grooves or furrows between clumps of fused microtriches, while 500 ppm caused production of holes within the denuded tegument of the parasite. Ovoid bodies, presumed to be eggs, were observed on the outer surfaces and just below the tegument of tapeworms treated with concentrations of 50 ppm. These structures also appeared to adhere to the outer surfaces of specimens exposed to 500 ppm. In view of the foregoing, special care should be taken in handling and disposing of feces from infected or suspect dogs after praziquantel treatment, since the breakdown in the tegumental surface ofE. granulosus presumably results in the release of potentially infective eggs.

In vivo efficacy and ultrastructural effects of mitomycin C against experimental alveolar hydatid disease

The in vivo efficacy and ultrastructural effects of mitomycin C were determined against alveolar hydatid disease in experimentally infected animals and compared to mebendazole treatment. Mitomycin C inhibited the mean cyst mass of treated versus control animals by 84.1% which was statistically significant at the alpha = 0.01 level. Mebendazole given daily inhibited the mean cyst mass by 80.1%, while mebendazole administration on the same treatment schedule as that used for mitomycin C inhibited the mean cyst mass by 70.4%. Ultrastructurally, mitomycin C was not observed to affect the tegumental microtriches (microvilli) or the microtubular system. However, an increase in the number and accumulation of round to oval electrondense vesicles was observed within the subtegument. These inclusion bodies became vacuolated, subsequently degenerated, and formed myelin-like figures. Mitomycin C, like mebendazole, was only cystistatic in its effects on the cyst stage of Echinococcus multilocularis as evidenced by the growth of treated cyst material following inoculation into helminth-free animals.

FINE STRUCTURE AND FREEZE-ETCH STUDY OF THE PROTOSCOLEX TEGUMENT OF ECHINOCOCCUS MULTILOCULARIS (CESTODA)

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.

FREEZE-ETCH CHARACTERIZATION OF THE TEGUMENTS OF THREE METACESTODES: ECHINOCOCCUS GRANULOSUS, TAENIA CRASSICEPS, AND TAENIA TAENIAEFORMIS

The objective of this study was to characterize the teguments of metacestodes of Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis using the freeze-etch technique. Metacestodes of E. granulosus (19 mo old), T. crassiceps (28 days old), and T. taeniaeformis (34 days old) from gerbils, mice and rats, respectively, were fixed for 2 hr in 3% glutaraldehyde and then prepared for freeze-etching and thin sectioning by standard techniques. Freeze-etch replicas of the teguments of all three species displayed morphologic characteristics that were generally in agreement with previous ultrastructural work, although some new features and interpretations arose from use of this technique. For each species there was a concentric ring structure within the microthrix base, and cytoplasmic extensions of the perikarya into the distal tegument were membrane-bound rather than confluent bridges; these extensions frequently branched within the tegument. In addition, channels running from the proximal tegumental membrane to, and opening at the distal surface of, the tegument were seen in thin sections.

Prevalence of Trichinella spiralis in wild animals on two Illinois swine farms.

Various wild animal species on two Illinois swine farms were studied for evidence of Trichinella spiralis infection. On one farm only grain and commercial supplement were utilized as swine feed. Of 239 wild animals examined from this farm, only a Norway rat (Rattus norvegicus) and a white-footed mouse (Peromyscus leucopus) were found infected with T. spiralis. This is believed to be the first report of natural T. spiralis infection in P. leucopus. Cooked garbage was fed to swine on the second farm. Of the 250 wild animals examined from this farm, only two Norway rats (R. norvegicus) were found infected with T. spiralis. Gould (1945), and Zimmermann and Hubbard (1963), have reviewed the literature on specific wildlife hosts of Trichinella spiralis, and Zimmermann and Hubbard (1963), found that 324 (3.9%) of 8,308 wildlife specimens collected in Iowa from 1953 to 1963 were infected with T. spiralis. However, the role of wildlife in swine infections and the effects of interacting wild animal populations on maintenance of the parasite remain unresolved. Previously, other workers (Kerr, 1942; Schwartz, 1952, 1960), and more recently Zimmermann and Brandly (1965), have demonstrated that the prevalence of T. spiralis infection in garbage-fed pigs is higher than rates in farm-raised swine as a group. One may hypothesize that wild animals living on a swine farm that utilizes garbage as feed would have a higher prevalence of T. spiralis Received for publication 11 October 1967. * Dr. Martin, at the time of this study, was assigned by the National Communicable Disease Center, Atlanta, Georgia, to the Illinois Department of Public Health, Springfield. He is currently regional public health veterinarian with the department. t Dr. Schnurrenberger is chief public health veterinarian, Illinois Department of Public Health, Springfield, and senior member of the Center for Zoonoses Research. + Dr. Andersen, at the time of this study, was assistant professor of veterinary pathology and hygiene, University of Illinois College of Veterinary Medicine, and associate member of the Center for Zoonoses Research. He is currently associate professor of zoology, Brigham Young University, Provo, Utah. ? Mr. Hsu is a research assistant in veterinary pathology and hygiene, University of Illinois College of Veterinary Medicine. infection than wild animals from a farm that does not use garbage for swine feed. This study was designed to determine the prevalence of trichinelliasis in wild animal species on two Illinois swine farms, one that feeds garbage to swine and one that does not. MATERIALS AND METHODS Farm S, in northeastern Illinois, is a square area of 1,100 acres, with a swine-breeding herd of approximately 40 sows. The farm is divided almost equally between cropland and pasture. The swine are fed locally grown corn with commercial supplement. Farm G, in east central Illinois, is a rectangular area of approximately 20 acres. Cooked garbage is fed to the offspring of 10 to 12 sows. All the farm area except for the dwelling and the hog lots was used until May 1966, as an open dump by the owner who hauls trash commercially. Samples of diaphragm from 20 swine raised on Farm G were collected and examined by the United States Department of Agriculture in 1964, as part of a nationwide survey of garbage-fed swine (Jefferies, 1966). Two of the 20 swine were found infected with T. spiralis larvae at that time. Wild animals were trapped on Farm S in August 1964 and 1965, and in March, May, and August 1966 (Table I). Animals were collected on Farm G in November 1965 and March, July, and November 1966 (Table II). The species, age, sex, and location of capture were recorded for each animal. The whole muscular part of the diaphragm was taken from each of the small mammals; approximately half of the diaphragm muscle was collected from each of the larger mammals. From birds, 5 to 10 g of breast muscle was collected. Small rodent identification was confirmed by Dr. Richard D. Andrews, Department of Zoology, Eastern Illinois University, Charleston, Illinois. All samples were examined under the trichinoscope or digested in 1% pepsin and 0.5% HC1 for 12 hr. Digested sediment was examined under the trichinoscope or microscope with 10 X magnification. Samples collected from Farm S in August

Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices.

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.

Survival of protoscolices of Echinococcus granulosus at constant temperatures.

A study was conducted to determine the effects of storage at constant temperatures upon the survival of protoscolices of Echinococcus granulosus from hydatid cysts removed from infected sheep. Parallel tests were conducted on intact cysts from both lung and liver, and on protoscolices stored within 1-ml samples of hydatid fluid. The longest survival times of any of the samples tested at each temperature were: -20 C, 1 hr; -10 C, 4 hr; 1 C, 16 days; 10 C, 16 days; 20 C, 8 days; 30 C, 4 days; 40 C, 2 days, and 50 C, 2 hr. In general, protoscolices survived considerably better when stored within intact cysts than when in 1 ml of hydatid fluid. At temperature ranges where putrefaction occurred, protoscolices in cysts from lung survived longer than those from liver. The ability of these protoscolices to survive extended periods of time after an infected sheep has died or been killed suggests that stringent preventive and control measures should be established in areas where hydatid disease is endemic. Animal pits at community dumping grounds where sheep carcasses might be discarded should be enclosed or covered, and all stray and roving dogs in those regions should be rigidly controlled.