Efficacy of an injectable combination anthelmintic (nitroxynil+clorsulon+ivermectin) against early immature Fasciola hepatica compared to triclabendazole combination flukicides given orally or topically to cattle.

The objective was to compare the efficacy against artificially induced 2- and 4-week old early immature triclabendazole-susceptible liver flukes (Fasciola hepatica) of an injectable combination of nitroxynil, clorsulon and ivermectin with oral and pour-on combination formulations containing triclabendazole. Groups of yearling Angus or Angus cross cattle were confirmed fluke free before being artificially infected with 500 Sunny Corner strain triclabendazole-susceptible liver fluke metacercariae. Two or four weeks after infection, cattle were treated with the test combination Nitromec (10.2mg/kg nitroxynil, 2.0mg/kg clorsulon, 0.2mg/kg ivermectin), or oral Flukazole C+Se (triclabendazole/oxfendazole/Selenium), oral Fasimec C (triclabendazole/ivermectin) or Genesis Ultra Pour-On (triclabendazole/abamectin). At intervals cattle were weighed, faecal sampled for liver fluke egg counts and blood sampled for liver serum enzyme analysis. Cattle were slaughtered 14 weeks after infection for recovery of adult flukes; fluke egg counts and liver pathology assessment. All cattle increased in body weight by 0.4-0.8kg/day but there were no significant differences between control and treated groups or between the treatment groups. Geometric mean 14-week fluke egg counts and total fluke counts for all treatments, were significantly less (p<0.05) than the control group, except for the group treated with Genesis Ultra Pour-On, 2 weeks after infection. Nitromec treatment of 2-week old flukes was 83% and 95% effective as assessed by 14-week egg and fluke counts, respectively, compared to Flukazole C; 96% and 99%, Fasimec C; 70% and 46%, and Genesis Pour-On, which was ineffective, with egg and fluke count reductions of 0% and 8%, respectively. Against 4-week old flukes, Nitromec treatment was 88% and 99% effective when assessed by 14-week egg and fluke counts, respectively, with Flukazole C; 98% and 99%, Genesis Pour-On; 98% and 82% and Fasimec C; 91% and 61% effective, respectively. Group mean levels of the bile duct-associated enzyme gamma glutamyl transpeptidase (GGT) and the parenchymal associated enzymes, aspartate amino-transferase (AST) and glutamate dehydrogenase (GLDH) increased above the normal range 8 and 11 weeks after infection in the untreated control animals and the group treated 2 weeks after infection with Genesis Pour-On. The groups treated with Fasimec at 2 or 4 weeks after infection, also had elevated enzyme levels. The use of liver-associated enzyme assay is supported as supplementary indicators of fluke-induced pathology.

The prevalence, organ distribution and fertility of cystic echinoccosis in feral pigs in tropical North Queensland, Australia.

An investigation was carried out to study the prevalence of Echinococcus granulosus hydatidosis in feral pigs (Sus domesticus) in the Charters Towers region of tropical North Queensland. Data were collected from a total of 238 carcasses, which were hunted and shot in the Burdekin River catchment area. Organs of the abdominal, thoracic, and pelvic cavities were examined for the presence of hydatid cysts. In the laboratory, cysts and hydatid cyst fluids were examined under a stereoscopic binocular microscope and a compound microscope. An overall prevalence of E. granulosus hydatid cysts in feral pigs was found to be 31.1%. There was no significant difference in either sex or age between infected and non-infected feral pigs. The predilection sites of cysts were livers (23%) and lungs (62%), with more cysts in lungs (252) than livers (48). The ratio of livers to lungs infected with fertile cysts was 1:4 compared to 1:8 sterile cysts. The overall fertility of cysts was 70.1%. The percentage of fertile cysts in liver and lung was 79.2% and 68.7%, respectively. The diameter of fertile cysts ranged from 15 to over 60 mm. There was no significant difference in size between fertile and non-fertile cysts in lungs. The high prevalence rate and fertility of cysts in feral pigs confirm that feral pigs can take part in the sylvatic cycle of the parasite in the region. The public health significance of this observation is potentially very important.

Helper T cell and antibody responses to infection of CBA mice with Babesia microti.

Helper T cell cytokine and antibody responses were investigated in mice after infection with Babesia microti (King strain). Infection of CBA mice with 106 parasitized erythrocytes resulted in the development of a transitory high parasitaemia which peaked 14 days post infection (DPI), and was resolved at 24 DPI. Th1 responses were activated predominately during the acute phase (6-18 DPI) whereas Th2 responses predominated during the recovery phase (14-28 DPI) as detected by the reverse transcriptase polymerase chain reaction. Increased expression of Th1 cytokines was first detected at 6 DPI (IL-2) and 8 DPI (IFN-gamma) and their peak levels were reached at 12 DPI. After the peak levels were reached, they progressively declined and fell to baseline levels (22 DPI). Increased expression of Th2 cytokines (IL-4 and IL-10) first appeared at 14 DPI, peaked at 20 DPI and Th2 cytokine levels were elevated till the end of the study (28 DPI). Levels of serum IFN-gamma detected by a sandwich ELISA correlated well with IFN-gamma gene expression and were detectable at 8-18 DPI. IgM against B. microti was first detected in serum by ELISA at 4 DPI, and peaked at 10 DPI. The levels of IgM subsequently declined but remained positive at low titre till the end of study. IgG against B. microti was first detected at 8 DPI and peak levels were reached at 24 DPI and remained at that level until the end of study. The results of the present study show that Th1 cytokines predominated in the early inflammatory response and might be involved in control of levels of acute parasitaemia whereas the Th2-associated responses, including expression of IL-4 and IL-10 and the production of parasite-specific IgG, might be the functional means for the reduction and clearance of the parasite from the body. It was concluded that an effective vaccine against Babesia spp. should be designed to induce Th1 responses to maintain the parasitaemia at unfulminating levels and also maintain Th2 responses to clear the parasite from the body.

Inhibition of growth of cultured Babesia microti by serum and macrophages in the presence or absence of T cells.

Serum and macrophages from the acute-phase (days 12-14 p.i.) and recovery-phase (days 23-25 p.i.) of infection of mice with Babesia microti were analyzed for their ability to inhibit the in vitro growth of B. microti in the presence or absence of T cells. Recovery-phase serum was inhibitory to the growth of B. microti, whereas, acute-phase serum had no inhibitory effects. Both acute- and recovery-phase macrophages inhibited B. microti growth. The co-culture of acute- but not recovery-phase T cells with macrophages from uninfected control mice was inhibitory to the growth of B. microti. Growth of B. microti was also inhibited in cultures containing macrophages from uninfected control mice plus culture supernatant fluid from acute-phase but not recovery-phase T cells. The supernatant fluid from B. microti cultures with acute-phase T cells contained IFN-gamma detected by a sandwich ELISA, whereas cultures with control T cells or recovery phase T cells did not. Results of the present study suggest the likelihood of a protective role against B. microti in mice for antibody which appeared in recovery-phase serum and for macrophages activated by IFN-gamma from acute-phase T cells.

In vitro culture of equine strongylidae to the fourth larval stage in a cell-free medium.

An efficient and reliable method is described for the culture of equine strongyles from the third (L3) to the fourth (L4) larval stage. Medium consists of 50% fetal calf serum and 50% NCTC with additions of L-glutamine, NaHCO3, yeast extract, bactopeptone, and dextrose. The gas phase used is of prime importance; it is a mixture of 10% CO2, 5% O2, and 85% N2. Strongylus vulgaris, Strongylus edentatus, Strongylus equinus, Triodontophorus brevicauda, Triodontophorus serratus, Triodontophorus tenuicollis, Oesophagodontus robustus, Cylicocyclus insigne, and mixed species of cyathostomes were cultured to the L4 stage. Oesophagodontus robustus was cultured to the fifth larval stage. Depending on species, 44-95% of Strongylinae L3 inoculated into this system molted to L4. Although some development of the Cyathostominae L3 occurred, only a small portion (1%) completed ecdysis to L4. Viability in cultures of all species remained high (> 60-70% larvae surviving) for at least 4 wk (cyathostomes) and as long as 6 mo (S. edentatus). The addition of equine hemin to cultures of S. vulgaris and O. robustus L4 enhanced development and prolonged viability of these larvae. Hemin had no effect on cultures of S. edentatus or S. equinus, and it was not tested in cultures of other species.

Clinical and pathological findings of Babesia infection in dogs.

The clinical and pathological findings of Babesia infection in 32 dogs in northern Australia are presented. Eleven different breed types were represented from 6 localities in north Queensland and one locality in northern Western Australia. Twenty three (72%) were males. Babesia-infected dogs were grouped by the degree of haematological disturbance and clinical severity: Acute babesiosis (25/32), all pups with severe haemolytic anaemia; subclinical carriers (5/32) with non-specific malaise, characterised haematologically by a normal erythrogram but marked leucopenia; chronic anaemia, observed in 2 adult dogs. Pups were azotaemic (serum urea greater than 6.6 mmol/l) and had elevated serum bilirubin levels (20.8 to 48.5 mmol/l). Total serum protein was usually within the normal range. Pups that died were also hypoglycaemic and severely hyperkalaemic (K+ greater than 10 mmol/l). Low parasitaemias in routine blood smears complicated diagnosis but smears made from ear or toe capillaries, or after haematocrit concentration, greatly enhanced finding parasitised cells. At necropsy, pallor and jaundice were the most consistent observations. Haemoglobinuric nephrosis, an active reticulo-endothelial system and capillaries packed with large numbers of infected erythrocytes were the main histopathological findings. A combination of imidocarb dipropionate at 5 mg/kg body weight, given intramuscularly, with fluid therapy and blood transfusion was the most successful treatment.

Prevalence and abundance of equine strongyles (Nematoda: Strongyloidea) in tropical Australia.

A postmortem survey of 57 horses in tropical northern Queensland revealed 41 (89%) infected with intestinal strongyles. Thirty-five strongyle species (8 large strongyles and 27 small strongyles [Cyathostominae]) were recorded of which 9 species are reported from Australia for the first time. The 14 most prevalent small strongyles were Cyathostomum catinatum (in 76% of horses), Cyathostomum coronatum (65%), Cyathostomum pateratum (33%), Cyathostomum labiatum (30%), Cylicostephanus calicatus (70%), Cylicostephanus longibursatus (67%), Cylicostephanus goldi (43%), Cylicostephanus minutus (26%), Cylicocylus nassatus (67%), Cylicocyclus leptostomus (41%), Cylicocylus insigne (41%), Cylicocyclus radiatus (33%), Cylicocyclus brevicapsulates (22%), and Poteriostomum imperidentum (24%). The remaining cyathostomes were each found in less than 15% of horses. The 4 most common large strongyles were Triodontophorus serratus (30%), Strongylus vulgaris (28%), Strongylus equinus, and Strongylus edentatus (both 22%). The number of species of small strongyles per horse showed a marked variation (mean 10.3, range 2-21) but bore no relationship to either the total number of strongyles per horse, age, sex, and breed of horse, or season. Total number of strongyles per horse (mean 15,890, range 20-165,000) was less than in recent surveys in Europe and the U.S.A. Most horses had low worm burdens, whereas a very small number were heavily infected. Ninety-seven per cent of the total strongyle counts were small strongyles. Strongylus species contributed just over 1%. Small numbers of large strongyles per horse were usual with T. serratus (mean 570), S. vulgaris (mean 330), and S. equinus (mean 330) the most numerous.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal translation of equine strongyle infective larvae to herbage in tropical Australia.

Longevity in faeces, migration to and survival on herbage of mixed strongyle infective larvae (approximately 70% cyathostomes: 30% large strongyles) from experimentally deposited horse faeces was studied in the dry tropical region of North Queensland for up to 2 years. Larvae were recovered from faeces deposited during hot dry weather for a maximum of 12 weeks, up to 32 weeks in cool conditions, but less than 8 weeks in hot wet summer. Translation to herbage was mainly limited to the hot wet season (December-March), except when unseasonal winter rainfall of 40-50 mm per month in July and August allowed some additional migration. Survival on pasture was estimated at 2-4 weeks in the summer wet season and 8-12 weeks in the autumn-winter dry season (April-August). Hot dry spring weather (pre-wet season) was the most unfavourable for larval development, migration and survival. Peak counts of up to 60,000 larvae kg-1 dry herbage were recorded. The seasonal nature of pasture contamination allowed the development of rational anthelmintic control programs based on larval ecology.

Protection of yearling ponies against Strongylus vulgaris by foalhood vaccination.

The long-term efficacy of an irradiation attenuated larval (L3) vaccine against Strongylus vulgaris was tested in ponies which were reared on pasture. Prior to foaling, mares were divided into two groups. One group of mares and foals received regular (eight weekly) treatment with ivermectin and the second group remained untreated. Half the foals in each pasture group were vaccinated at eight to ten weeks of age. Foals were weaned at three to four months of age and maintained on separate pastures. At eight to ten months of age, ponies were placed in box stalls and half of each treatment group were challenged with S. vulgaris (5 x 1000 L3). Clinical signs and lesions typical of acute verminous arteritis were found at necropsy in the ivermectin treated non-vaccinated challenged yearlings. Ivermectin treated vaccinated challenged yearlings did not show these clinical signs, had markedly reduced to absent arterial lesions and showed an 89 per cent reduction in arterial larval burdens post mortem. Significant differences in clinical signs, arterial lesions or arterial larval burdens were not seen between vaccinated and non-vaccinated foals reared without benefit of ivermectin treatment.

Prevalence and intensity of non-strongyle intestinal parasites of horses in northern Queensland.

A quantitative post-mortem study of 57 horses from northern Queensland was done to determine the prevalence and intensity of non-strongyle intestinal parasites. The following species (% prevalence) were found: Draschia megastoma (39%); Habronema muscae (43%); Gasterophilus intestinalis (34%), G. nasalis (30%); Parascaris equorum (15%); Strongyloides westeri (6%); Probstmayria vivipara (2%); Oxyuris equi (26%); Anoplocephala magna (2%); A. perfoliata (32%). Mean parasite numbers of individual species ranged from 10 to 1310. Prevalence and intensity data were compared to recent studies in Western Australia and in the United States of America. Differences between stabled and paddocked horses were noted, particularly for botfly larvae and spiruroids. Climatic and seasonal changes in prevalence were restricted to H. muscae, G. nasalis and P. equorum with highest prevalence in the wet season or in horses from wet coastal areas. Only P. equorum showed any age effect being restricted to horses less than 5 years old. Breed and sex of horses was not important. The likelihood of changing parasite population dynamics with improved anthelmintic regimen is discussed.

Abundance and survival of infective larvae of the cattle nematodes Cooperia punctata, Haemonchus placei and Oesophagostomum radiatum from faecal pats in a wet tropical climate.

Observations were made on the abundance and survival of Haemonchus placei, Cooperia punctata and Oesophagostomum radiatum infective larvae from cattle faecal pats exposed at various times of the year in north Queensland wet tropics. Pats exposed in the hot, wet season yielded abundant larvae on herbage. In the dry season, although low numbers of infective larvae were usual, considerable numbers were produced under conditions of heavy dews on dense herbage. Irrespective of season of deposition of pats, the resulting larvae persisted generally for not longer than 10 to 12 weeks, and in large numbers for only 2 to 6 weeks. The findings suggest that prevention of contamination in the wet season, and in the dry season when light rainfalls are accompanied by heavy dews on dense herbage, will result in low levels of larval infestation on herbage. Rotational grazing in the area is suggested as a means of worm control.

Development of free-living stages of equine strongyles in faeces on pasture in a tropical environment.

The development of the free-living stages and yields of infective third stage strongyle larvae in faeces from a horse with a mixed natural infection deposited on pasture plots were studied over a 2-year period in a coastal area in tropical north Queensland. Two sets of faecal masses (one exposed to, and the other protected from the action of a natural population of dung beetles) were deposited monthly and after 7 days faecal samples were taken for larval recovery and counts. Hatching and development of the free-living stages occurred in faeces on pasture throughout the year. Development was rapid as infective stages were reached within a week of faecal deposition in all months. Yields of infective larvae were affected by the season and the action of dung beetles on the faecal masses. Highest yields were obtained from both beetle-exposed and protected faeces during winter (June to August) and lowest yields were in spring (September to November). High temperatures in spring and summer resulted in low yields of larvae, however, the dry conditions in spring made this season the most unfavourable period. In autumn and winter the temperatures were never low enough to stop or markedly slow down the rate of development, and allowed the development of large numbers of infective larvae. Dung beetle activity was observed throughout the year, and exposed faeces were usually completely dispersed within 24 h of deposition. This resulted in lower yields of infective larvae from these than from protected faeces. Though larval yields were lower, the actual numbers were still substantial so as to cast doubt on the usefulness of these beetles as biological control agents for equine strongylosis in the dry tropics.

In vitro drug screening in isolated male Onchocerca gibsoni using motility suppression.

A primary in vitro screen was developed to screen for drug activity against isolated Onchocerca gibsoni. The assay estimates variation in motility through the use of a motility meter. Of the seven compounds tested in the screen; ivermectin, CGP 6140, CGP20376, Mel W and furapyrimidone gave MI50 concentrations (the concentration at which the motility was reduced to 50% of the control value at 72 hours) below 10(-4) M, whereas suramin gave variable results depending on the varying susceptibility of individual worms and levamisole at 10(-4) M had no significant effect on the worms. The effects of these drugs were not reversible as removal of the worms into drug-free medium caused no increase in motility. Thus the reduction in motility is regarded as indicating significant metabolic damage. The results compared favourably with reported in vivo tertiary screens for activity against Onchocerca species. This is a quantitative, inexpensive and reproducible method for assessing the effectiveness of drugs against Onchocerca and could be included into the primary screens for activity against filarial worms.

Development and survival of free-living stages of equine strongyles under laboratory conditions.

In a series of laboratory studies the optimum conditions for the development and survival of the free-living stages of strongyle parasites occurring in horses in tropical north Queensland were determined. No differences in behaviour were noted between the strongyle species. Development to the infective stage occurred only between 10 and 35 degrees C. The rate was affected by temperature, taking 15-24 days and 3 days, respectively, at the lowest and highest temperatures for the developing stages to reach the infective third stage. Yields of infective larvae were very low outside the range 20-33 degrees C, and were highest at 28 degrees C. Survival of infective larvae was good between 20 and 33 degrees C, and large numbers were recovered after 3 months in faeces incubated at 20-28 degrees C. At 33 and 37 degrees C larval survival was affected by the moisture content of the faeces, with infective larvae surviving better in dry than in moist faeces; even a residual moisture level of 40% significantly reduced the number of larvae recovered from faeces incubated at 37 degrees C for 1 month. Moisture also affected larval development, especially at the higher temperatures of 25-39 degrees C. When faecal moisture content fell to less than or equal to 20% by 3 days, larvae which had not yet reached the infective stage were still pre-infective at 7 days, while all larvae in faeces with adequate moisture had reached the infective third stage. It was not possible to determine the critical faecal moisture level below which larval development ceased, however, 28 degrees C (range 25-33 degrees C) was found to be the optimum temperature. Larval development was very rapid and yields of infective larvae highest at this temperature.

Clinical pathology of experimental stephanuriasis in pigs.

Haematology and serum aspartate aminotransferase and alanine aminotransferase activities were studied during the migration phase of Stephanurus dentatus in the livers of experimentally infected pigs. There was no evidence of anaemia but total leucocyte counts were raised and peripheral eosinophilia began 2 to 3 weeks after infection. Peak eosinophilia occurred 6 to 7 weeks after infection and levels were still elevated at 20 weeks. Lymphocyte and neutrophil numbers remained constant. Reinfection did not stimulate a secondary eosinophil response. Only aspartate aminotransferase was temporarily elevated. The gross pathology resulting from the infections is described. Several clinicopathological differences in the response of pigs to invasion of the liver by S. dentatus were noted compared to those produced by Ascaris suum but none are pathognomonic.

The effect of praziquantel on calcium in Hymenolepis diminuta.

Praziquantel (PZ) at concentrations down to 5 x 10(-8) M induced a rapid contraction of Hymenolepis diminuta musculature. This effect was accompanied by a strong inhibition of 45Ca2+ incorporation which showed some dependence on Ca2+ concentration. Ca2+ efflux experiments showed that PZ markedly stimulated the release of Ca2+ from tapeworms preloaded with 45Ca2+, with the effluxed Ca2+ being derived from a small fast pool and a larger slow pool. This stimulatory effect appeared., like PZ-induced muscle contraction, to be independent of external Ca2+. By carrying out 45Ca2+ exchange experiments under near equilibrium conditions and atomic absorption spectroscopy it could be demonstrated that PZ resulted in a net excretion of endogenous Ca2+. In PZ-induced contracted worms adenylate nucleotide levels and the adenylate energy charge were not significantly different from those of untreated control worms. Also, PZ had no effect on Ca2+-stimulated ATPase activity of the tapeworm's tegumental brush border. Nor did the drug alter the activities of Ca2+-ATPases in whole homogenates of worms or mitochondria, microsomal or soluble fractions. Although the mechanism of PZ-induced changes in Ca2+ transport was not elucidated, it is suggested that the sustained release of endogenous Ca2+ may affect the sequence of excitation-contraction coupling and that such interference may cause the observed massive contraction of the tapeworm's musculature.

Anthelmintic treatment of prepatent stephanuriasis with flubendazole, levamisole and disophenol and the effects on liver-specific serum enzymes.

Haematological parameters and liver specific serum enzymes were examined in pigs during the first 12 weeks of liver migration of larvae following experimental infection with 1000 infective Stephanurus dentatus larvae. No significant changes in total red blood cell counts, packed cell volume, or haemoglobin content were observed. Total white blood cell counts and circulating eosinophils rose rapidly from days 5 and 19 after infection, respectively. Treatment with a mixture of levamisole (LEV) at 10 mg/kg and flubendazole (FLU) at 50 mg/kg in feed four weeks after infection halted the leucocyte response and returned values to normal in two weeks. Disophenol (DIP) at 15 mg/kg subcutaneously restricted the leucocyte response but it was only terminated following FLU treatment alone on day 61. No effects of S dentatus or either anthelmintic treatments on liver specific serum enzymes glutamate dehydrogenase, sorbitol dehydrogenase or gamma-glutamyl transpeptidase were found. Animals killed seven, 26 and 54 days after treatment showed significant resolution of fibrotic liver lesions after LEV + FLU but not after DIP. We conclude that LEV + FLU is an effective treatment for prepatent stephanuriasis but that liver damage is insufficiently traumatic to release sufficient enzymes into serum to be pathognomonic or to assess anthelmintic efficacy.