Australian parasitological research: now and in 2000.
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Diagnosis of parasitic infections in small animals is challenging, interesting, and fun, and involves the recognition of parasite stages based on size, morphology, color, and movement. Size and morphology are the major diagnostic parameters, and a calibrated microscope is the essential tool in the diagnostic laboratory. The fecal flotation technique is used routinely for the diagnosis of most parasites that reside within the gastrointestinal tract. The specific gravity and type of solution used often will influence the results obtained. A sugar flotation solution with a specific gravity of 1.27 is one that will float a majority of parasite stages with minimal distortion. Other techniques for diagnosis of specific gastrointestinal parasites include the direct smear and the merthiolate-iodine-formalin preservative method for diagnosis of Giardia sp, the Baermann technique for diagnosis of lungworm and other live larvae, the direct sedimentation technique for diagnosis of trematode eggs, the ether-formalin-sedimentation technique for diagnosis of trematode eggs and concentrating protozoan cysts from feces with high fat content, and the McMaster technique, a dilution modification of the fecal flotation technique. Techniques for evaluation of parasites in blood include the Knott's test, hematocrit method, and direct blood smear for diagnosis of microfilariae and thick and thin stained blood smears for diagnosis of Babesia sp, Haemobartonella sp, Cytauxzoon felis, Hepatozoon canis, and Trypanosoma cruzi. Tissue impression smears are used commonly for the diagnosis of Leishmania sp in dogs and biopsy specimens or aspirates are used for the diagnosis of Pneumocystis carinii. Diagnosis of acute toxoplasmosis can be accomplished with peritoneal and thoracic fluids, and organisms occasionally are detected in blood or spinal fluid. Serological tests for many parasitic diseases have been developed and often are used as supportive diagnostic tests.
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An epizootiological survey of leishmaniosis, coccidiosis and parasitic helminths in 67 foxes (Vulpes vulpes) was conducted in Guadalajara (central Spain). Examination for parasitic protozoa revealed prevalences of 74% Leishmania (determined by molecular methods) and 2.9% coccidia oocysts (fecal flotation). Survey of parasitic helminths (fecal flotation/necropsy) demonstrated the presence of nine species, including six nematodes, two cestodes and one trematode. Nematodes were the most common parasites of foxes, followed by cestodes and trematodes. Greater levels of nematodes like Uncinaria, with a free-living stage in its life-cycle, were found in foxes in areas where moist soils were likely to exist, in contrast to areas of semiarid characteristics, where Toxascaris leonina or Trichuris vulpis were predominant. With regard to helminths of importance as human pathogens, trichinoscopy revealed the presence of a relatively high number of foxes (8.9%) infected with Trichinella spiralis. Finally, Toxocara canis infection was less frequent (4.4%) than trichinellosis.
The course of Trypanosoma evansi infection in coatis (Carnivora, Procionidae) was followed for 262 days. Parasites were detected in all infected animals from day 2 post infection until the end of the study. No correlation between temperature and parasitemia was observed. Animals of the infected group demonstrated depression, weakness, lethargy and pale mucous membranes. Indirect fluorescent antibody tests detected anti-T. evansi antibodies within 7 to 14 days post infection and showed high levels until the end of the experimental period. The persistent parasitemia in coati and their relative tolerance to clinical signs suggested that this species develops a chronic disease and plays an important role in the epidemiology of trypanosomosis due to T. evansi in enzootic regions.
Over the past 15 years, there has been a dramatic evolution in molecular approaches to study parasites and parasitic diseases. Many of these advancements have been brought about through the development of new applications of the polymerase chain reaction (PCR). Enhancements in sensitivity that can be achieved using PCR now permit scientists to investigate changes at the level of a single cell, far below what is often needed for parasite-derived applications. PCR has had a substantial impact on advances made in the areas of parasite systematics and epidemiology, immunology and host-parasite interactions, recombinant DNA vaccine development and most recently, the analysis of whole genomes either through directly sequencing the DNA, the analysis of expressed sequence tags (ESTs) or through the rapidly growing field of functional genomics. This paper, however, focuses on the application of PCR methodology to parasite detection and differentiation, and the diagnosis of disease. Specific attention is given to advances provided by multiplex PCR, fluorescence-based "real-time" PCR, and the utilization of PCR as a quantitative technique.
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The reasons for the wide range of parasites occurring in South Africa are mentioned. An account is given of the establishment of veterinary research and training in South Africa. Some major breakthroughs and advances in the study or the control of ecto- and endoparasites as well as protozoal diseases are listed.
Analysis of 85 studies on gastrointestinal nematode infections in first-grazing season (FGS) calves is presented. The studies cover a 26-year period and were carried out in 13 countries in Western Europe. Both control and chemoprophylactic-treated (early in the season) FGS calf groups were included. In 53 of the 85 studies, clinical outbreaks of parasitic gastroenteritis (PGE) were observed during the FGS in the control groups. The mean initial age (and weight) of the control calf group was significantly associated with PGE outbreaks: 82% of control calf groups < or = 6 months of age had outbreaks of PGE, compared to only 33% of control calf groups > 6 months of age. In 92% of trials where the geometric mean faecal egg count (MFEC) was > or = 200 EPG on Day 56, PGE outbreaks were observed, but where it was < 200 EPG, only 29% had PGE. The use of these two factors in assessing the likelihood of PGE outbreaks in untreated calf groups in the future FGS is therefore, proposed. No chemoprophylactic-treated groups had PGE, but there was a highly significant negative relationship between maximum faecal egg counts in the chemoprophylactic-treated calves and the proportion of the trial covered by the different chemoprophylactic systems. Higher stocking densities were significantly associated with higher pasture contamination in both control and chemoprophylactic-treated calves. A highly significant positive relationship between the weight gained in the chemoprophylactic-treated groups and the estimated duration of the various chemoprophylactic systems was found, but there were large variations in weight gains (60-160 kg) between groups even with the same chemoprophylactic. Despite this and other highly significant associations, it was not possible to indicate what weight gains were obtained by the end of the FGS, from factors measured early in the FGS.
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The epidemiology of nematode infection was studied in fattening grazing cattle from weaning (April 1994) to market at the end of their second autumn (July 1995). Sixty Aberdeen Angus calves of seven months of age were randomly allocated by weight to two groups: GT, treated every three weeks with doramectin (200 mcg/kg); and GI, an infected group, only treated with fenbendazole (7.5 mg/kg) at weaning and on the 1st of October. The two groups were grazed together on contaminated lucerne pastures until July, on 'clean' oat pastures until October and again on contaminated lucerne until the end of the trial. Fecal egg counts (epg), herbage larvae (L3), serum pepsinogen (Pep) and blood eosinophils (Eo) were evaluated monthly. Eight steers were slaughtered for worm recovery, three in July 1994, three in December 1994 and two in July 1995. Grazing feed intake was estimated by fecal output (chromic oxide method)/l-diet digestibility and to measure non-specific response, Brucella antibodies were detected at 11 and 40 days post-vaccination in early winter. Fecal egg counts, Pep and Eo of GI increased (P<0.01) from April to July when there was a moderate-to-high level of infection. Ostertagia, Trichostrongylus and Cooperia were the predominant genera. By late winter, all parameters decreased on oat 'clean' pastures and increased again when cattle returned to moderately infected lucerne. During summer, the parameters measured reflected the negligible numbers of L3 on pastures until early autumn. At this time, increased numbers of L3 were followed by a moderate rise (P<0.01) of epg, Pep and Eo values. During winter, GT showed higher (P<0.04) Brucella antibody IgG titers while feed intake of GI was 24.9% depressed (P<0.02). There were total cumulative weight-gain (WG) differences (P<0.001) between groups (GT=263.1 kg; GI=214.3 kg). During the second autumn, the mean WG of GT steers was 16.6 kg greater (P<0.04) than that of GI. Vaccination titres against Brucella suggested non-specific depression of immunity, while higher Pep and Eo levels in second-year steers may have reflected hypersensitivity reactions.
Merino wether lambs were individually confined and fed a basal diet of oaten chaff containing essential minerals which was untreated or contained 3% urea. Within each dietary group animals were orally infected with either 200 H. contortus (H), 1000 T. colubriformis (T) or both species (H + T) thrice weekly or remained uninfected (C). Weight gain, wool production, and parasite burden were measured over a 19-week period. Sheep on the diet containing urea gained more weight, consumed more feed and grew more wool of higher fibre diameter than their counterparts given no urea. On both diets uninfected sheep consumed more feed than infected sheep and the sheep given no urea and infected with both H and T worm species consumed the least feed. Parasitised sheep gained less weight than uninfected control sheep. Sheep with urea in their diet had lower faecal egg counts when infected with H alone or with H and T but there was no effect of urea on egg count of sheep infected with T alone. In contrast, T numbers after slaughter were reduced in sheep fed diets containing urea whereas H numbers were not affected by diet. It was concluded that supplementation with urea can increase resilience to parasitism thereby improving production and also enhance resistance mechanisms against worms in young sheep on low quality roughage diets. These responses can be partly attributed to stimulation of feed intake, presumably due to enhanced ruminal digestion, but also to elevated rumen NH3-N levels which would be expected to have increased rumen microbial protein synthesis and availability to the intestines.
Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneider's medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.
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