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Cyathostomes in horses in Canada resistant to pyrantel salts and effectively removed by moxidectin.

Clinical trials using fecal egg count reduction tests and coproculture were conducted with yearlings and mares on a farm in 1997. Fecal samples were taken from each horse to estimate the number of strongyle eggs/g feces with Cornell-Wisconsin centrifugal flotation and Cornell-McMaster dilution techniques. Eleven of 15 yearlings, which had been on a daily feeding of grain with pyrantel tartrate for 66 d were found with strongyle eggs in feces. This was the first time the in-feed medication had been used on the farm. Nine yearlings were randomised into three groups; continuation of daily pyrantel tartrate or one treatment with pyrantel pamoate or moxidectin. Two of three yearlings given pyrantel tartrate or pamoate had no reduction in the eggs/g feces. These six yearlings were then given moxidectin and in all yearlings the eggs/g feces was reduced to zero. The 66 d of pyrantel tartrate use was an inadequate time for development of resistant cyathostomes and a hypothesis was the resistance was due to extensive use on the farm over many years of pyrantel pamoate at twice the label dose for control of tapeworms. That hypothesis was tested with 12 mares with strongyle eggs in the feces randomised into two treatment groups: pyrantel pamoate at label dose or moxidectin. Five of six mares given pyrantel had <80% reduction in egg/g feces. These mares were then given moxidectin and in all mares the eggs/g feces was reduced to zero. Only cyathostomes were found on culture and apparently there was side resistance among the pyrantel salts.

Animals↗

Control of the taeniosis/cysticercosis complex: future developments.

Cysticercosis is due to the establishment of the larval stage of the zoonotic cestode parasite Taenia solium. The infection causes substantial human morbidity and mortality, particularly in several Latin American countries and parts of Africa and Asia, as well as economic losses in pig husban dry due to condemnation of infected pork meat. The life cycle of T. solium includes human beings as definitive hosts and pigs as intermediate hosts. Cysticercosis is acquired by the ingestion of eggs released by human tapeworm carriers, who become infected after ingesting pork meat contaminated with cysticerci. Taenia solium transmission has been associated with poverty, lack of sanitary services and practices of rearing backyard pigs with free access to the areas that villagers use as toilets, as well as cultural behaviour. Nonetheless, due to the recent increase of migration and tourism, industrial countries are also reporting cases of human cysticercosis. There are many epidemiological studies that have been conducted mainly in Latin American countries that have evaluated intervention measures for control of cysticercosis including the development and testing of vaccines. Furthermore, the involvement of international agencies and institutions, such as the World Health Organization, the Food and Agriculture Organization and the International Livestock Research Institute, as well as the commitment of policymakers, scientists and field workers, are key means for the sustainable control and, hopefully, eradication of T. solium infections.

Animal Husbandry↗

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode.

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T. solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis.

Amino Acid Sequence↗

Risk factors associated with porcine cysticercosis in selected districts of Eastern and Southern provinces of Zambia.

To determine the risk factors associated with Taenia solium transmission in humans and pigs in the rural areas of Eastern and Southern provinces of Zambia, a questionnaire was administered in 788 households from 155 villages. Pigs were examined from 800 households. Tongue examination and enzyme-linked immunosorbent assay (Ag-ELISA) for the detection of circulating antigens of T. solium cysticerci were used to measure infection in pigs. A snowballing technique was utilised to select households with pigs. Prevalence of households with pigs infected with T. solium on tongue examination by district ranged from 12.7% to 32.1% with Ag-ELISA having a range of 30.0-51.7%. Of the total number of households visited, 18.8% and 37.6% had at least one pig positive for porcine cysticercosis on tongue examination and Ag-ELISA, respectively. Risk factors associated with T. solium infection were lack of pork inspection at slaughter (96.7%), consumption of pork with cysts (20.1%), selling of pork infected with T. solium cysticerci (18.3%), free-range husbandry system (83.2%) and absence of latrines (58.0). Free-range husbandry system (OR=1.68; 95% CI=1.36-2.07) was a significant risk factor for porcine cysticercosis in the surveyed areas. The result that pigs were mostly kept on free-range and semi-intensive husbandry systems may have permitted them to have access to eating human faeces that could be contaminated with tapeworm eggs. This study has shown that T. solium infection poses a high public health risk in the study areas and urban areas as well. We recommend that a human survey be conducted to verify the human exposure to taeniasis and/or cysticercosis in Zambia.

Abattoirs↗

Using electronic technology for Taenia solium education: educating the educators.

In Taenia solium endemic areas, intervention programs include a variety of actions aimed to improve sanitary infrastructure and environment, pig husbandry conditions, detection and treatment of tapeworm carries and cysticercosis cases, among others. For a successful long-term effect, these actions must be associated with community participation and health education programs. Prior to designing/conducting health education programs tailored to specific populations, individuals involved in delivering education, as well as other professionals, students, and community leaders, must be well prepared, their knowledge increased, updated or strengthened. Educators must first be well educated to convey clear and accurate messages. Current electronic technology provides invaluable resources for teaching and learning. Learners using electronic materials benefit from a dynamic, non-linear and flexible interaction that can render long lasting effects compared to traditional passive methods. Electronic resources also provide the capability of producing one standardized template, versatile enough to be adapted, translated, modified, and updated. In this presentation, we introduce our preliminary work on the first interactive media presentation on T. solium which will hopefully, soon, become an interactive tutorial that could be utilized in health education in both developing and developed countries.

Adult↗

The emergence of Taenia solium cysticercosis in Eastern and Southern Africa as a serious agricultural problem and public health risk.

Pig production has increased significantly in the Eastern and Southern Africa (ESA) region during the past decade, especially in rural, resource-poor, smallholder communities. Concurrent with the increase in smallholder pig keeping and pork consumption, there have been increasing reports of porcine cysticercosis in the ESA region. This article reviews the findings concerning the presence and impact of porcine cysticercosis in seven of the ESA countries. Most of the reported findings are based on surveys utilising lingual palpation and post-mortem examination, however, some also used serological assays. In Tanzania, community-based studies on porcine cysticercosis indicate a prevalence of 17.4% in the northern highlands district of Mbulu and a prevalence range of 5.1-16.9% in the southern highlands. In Kenya recent surveys in the southwestern part of the country where smallholder pig keeping is popular indicate that 10-14% of pigs are positive for cysticercosis by lingual examination. Uganda has the most pigs in Eastern Africa, most of which are kept under smallholder conditions. Preliminary surveys in 1998 and 1999 at slaughterhouses in Kampala indicated a prevalence of porcine cysticercosis between 0.12 and 1.2%, however, a rural survey in northern Uganda in 1999 indicated 34-45% of pigs slaughtered in selected villages were infected. Additionally, a new survey of 297 pigs slaughtered in Kampala in 2002 indicated that pigs from the central region of the country were negative for cysticercosis while 33.7% of the pigs coming from the rural Lira district in the north were positive. Interestingly 8 piglet foetuses removed from an infected slaughtered sow coming from Lira district were all found to harbour cysts of T. solium providing evidence of congenital transmission of porcine cysticercosis. In Mozambique, abattoir records indicate that porcine cysticercosis is present in all provinces of the country. A serological survey on pigs in rural Tete Province found 15% of pigs positive. In Zimbabwe, a retrospective study in official abattoirs around the country from 1994 to 2001 reported a mean prevalence of 0.34% which is in contrast to a post-mortem survey in 1999, which showed that the prevalence of porcine cysticercosis in rural west Zimbabwe where smallholder pig keeping is popular was 28.6%. In Zambia, abattoir records reported porcine cysticercosis in six of the nine provinces. Routine meat inspection of 1316 pigs at a slaughter slab in Lusaka showed that 20.6% of the pigs had cysticercosis whereas serological testing of 874 pigs at the same abattoir indicated that 56.6% were found to have circulating antigens of Taenia solium. Field surveys based on lingual palpation in Southern and Eastern Provinces of Zambia revealed prevalences of 8.2-28.4 and 5.2%, respectively. South Africa has the largest number of pigs in Southern Africa and cysticercosis has been recognised as a problem in the country for many decades. There is strong evidence supporting the high prevalence of neurocysticercosis infecting humans from resource-poor areas of the country where pigs are being raised under smallholder conditions. In spite of this community-based surveys on porcine cysticercosis have never been conducted in South Africa and the last slaughterhouse survey was conducted nearly 40 years ago. The prevalences of porcine cysticercosis found in these ESA countries rank among the highest in the world and the disease is emerging as an important constraint for the nutritional and economic well being of resource-poor smallholder farming communities. The current findings suggest the widespread presence of human tapeworm carriers and thus a high risk of human cysticercosis in both rural areas and urban centres in the ESA region. More research is required in the region to assess the extent and public health and economic impact of T. solium infection in order to determine whether and what prevention and control efforts are needed.

Abattoirs↗

The current status of neurocysticercosis in Eastern and Southern Africa.

Some information has been documented on the epidemiology of neurocysticercosis in Eastern and Southern Africa through the monitoring of hospital-based patients with neurocysticercosis, community-based serological surveys of particular socio-economic groups of people and surveys of porcine cysticercosis. Studies have revealed that non-pork eaters have as great a chance of infection as a pork eater, the Xhosa-speaking people of the Eastern Cape Province have the highest prevalence of cysticercosis/taeniosis in South Africa probably due to the common practice of free-range pig farming and the lack of sanitation in these areas. Several studies have revealed high prevalence rates in children and interestingly, patients with active cysts suffering from epilepsy. A startling mode of transmission is where self-trained healers use Taenia segments either for benevolent (e.g. in the treatment of severe intestinal tapeworm infections) or malevolent (evil) purposes (e.g. women "poisoning" an unfaithful husband or lover by adding the contents of Taenia solium segments to beer).

Africa, Eastern↗

Prevalence of taeniasis and cysticercosis in a population of urban residence in Honduras.

To determine the prevalence of antibodies to Taenia solium and taeniasis in a population of urban residence, an epidemiological study was carried out in a military institution located in Tegucigalpa, the capital city of Honduras. A total of 404 persons were interviewed to collect epidemiological data, investigate antibodies to T. solium cysticercus by the enzyme-linked immunoelectrotransfer blot (EITB) assay and taeniasis by Ritchie's concentration technique. A total of 41 persons that were living at the headquarters and were traveling frequently to their rural hometowns were regarded as rural residents, the remaining 363 persons were considered urban residents. The seroprevalence of antibodies was 22% (9/41) in the rural residents and 15% (54/363) in the urban residents. The overall seroprevalence was 15.6% (63/404). Analyses of risk factors for seropositivity in the urban residents showed that seropositivity was statistically associated with poor household conditions such as raising pigs (odds ratio (OR), 5.39; 95% confidence interval (CI), 1.42-19.50), lack of potable water (OR, 3.66; CI, 1.25-9.94), lack of sanitary toilet (OR, 2.92; CI, 1.35-6.05) and earthen floor (OR, 2.48; CI, 1.28-4.73). Also seropositivity was associated with low academic education (OR, 2.5O; CI, 1.33-4.69) and lack of knowledge about the parasite (OR, 2.39; CI, 1.26-4.49). Out of 328 persons examined for intestinal parasites, two soldiers (0.6%) coming from rural areas were identified as Taenia sp. carriers. T. solium was identified in one case. Although T. solium infections originate and are more prevalent in rural villages, the high seroprevalence found in this study indicates that they can also be found at important levels in urban communities. Migration of tapeworm carriers from rural areas to the city grants the transmission of cysticercosis when poor environmental and social conditions are present.

Adolescent↗

Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism.

Echinococcosis, a disease caused by infection with the larval stage of a tapeworm parasite of the genus Echinococcus, is of major socio-economic importance, and studying genetic variability within and between Echinococcus populations has important implications for disease control and epidemiology. Various DNA approaches have been used to study Echinococcus genetics, but most methods do not allow the accurate display or definition of mutational/allelic variation. To overcome this limitation, we established a mutation scanning approach. Single-strand conformation polymorphism (SSCP) of two different enzymatically amplified mitochondrial (mt) DNA regions was evaluated using seven different genotypes of Echinococcus (defined as G1, G4, G6, G8, O, V and M2). The NADH dehydrogenase 1 gene (ND1) or the cytochrome c oxidase subunit 1 (CO1) were amplified by polymerase chain reaction from parasite DNA, denatured and directly subjected to electrophoresis in a non-denaturing gel matrix. Each of the seven genotypes examined could be delineated from one another based on characteristic and reproducible banding patterns. The results demonstrate the usefulness of SSCP for the direct visual display of sequence variation in mtDNA of Echinococcus without the need for DNA sequencing or restriction analyses, and indicate its potential for studying allelic variability in a range of other genes.

Animals↗

Interaction of the 5'-phosphates of the anti-HIV agents, 3'-azido-3'-deoxythymidine and 3'-azido-2',3'-dideoxyuridine, with thymidylate synthase.

A study has been made of the interaction of 3'-azido-3'-deoxythymidine 5'-phosphate (AZTMP) and 3'-azido-2',3'-dideoxy-uridine 5'-phosphate (AZdUMP) with thymidylate synthase. With the enzyme from L1210 cells and the tapeworm Hymenolepis diminuta, AZTMP was a weak inhibitor competitive with respect to dUMP (Ki = 6.3 mM and 0.5 mM); hence cytotoxicity of AZT, in cells in which accumulation of AZTMP is not high, is not due to inhibition of cellular thymidylate synthase. AZdUMP, with the L1210 enzyme, was a weak substrate (competition with dUMP described by apparent Ki = 4.7 mM), excluding conversion of AZdUMP to AZTMP as a source of toxicity of 3'-azido-2',3'-dideoxyuridine. An efficient procedure is described for enzymatic phosphorylation on a preparative scale of dideoxynucleosides.

Animals↗

Biochemistry and clinical chemistry of vitamin B12 transport and the related diseases.

This review focusses on research performed by the author and coworkers. The absorption, turnover and excretion of cobalamin and the pathogenesis of cobalamin deficiency states are described and the laboratory tests used to diagnose these states are discussed. Topics dealt with in detail include: overall turnover, daily need, enterohepatic circulation and excretion of cobalamin and other corrins . The soluble proteins mediating cobalamin transport and their cellular receptors are described and their nomenclature, isolation, structure and mode of action, the role of calcium in the membrane transport, the evolution of these systems and the analogies with transport systems for other substrates are discussed together with deficiency states, especially fish tapeworm anemia and familial selective vitamin B12 malabsorption with proteinuria. Folate deficiency is a relatively rare cause of megaloblastic anemia in Scandinavia but common in North America and explanations for this difference are suggested. The methods of assaying cobalamin in serum and plasma and the performance of radiovitamin B12 absorption tests are critically evaluated. The measurement of intrinsic factor in gastric juice, serum, amniotic fluid and urine is described.

Anemia, Megaloblastic↗

A new diagnostic tool for neurocysticercosis is a member of a cestode specific hydrophobic ligand binding protein family.

A protein of unknown function has been identified as a key serological tool for diagnosis of human tapeworm neurocysticercosis, a major worldwide neurological disease. Our own sequence analysis predicts that this protein is a member of a newly identified cestode specific oligomeric hydrophobic ligand binding protein family. In this report, using a rat cestode model, we confirm that homologues of this protein can bind fatty acids and their derivatives, and thus suggest a biological function for this key diagnostic tool.

Acyl Coenzyme A↗

On the phylogenetic positions of the Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eucestoda) inferred from 18S rRNA.

A phylogenetic analysis of tapeworms (Eucestoda) based on complete sequences of the 18S rRNA genes of 43 taxa (including new sequences of 12 species) was carried out, with the emphasis on the groups parasitising teleost fish and reptiles. Spathebothriidea and Trypanorhyncha (the latter group being paraphyletic) appeared as basal groups of the Eucestoda but their position was not stable. The tetrafossate orders (Litobothriidea, Lecanicephalidea, Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea) were well separated from the remaining groups. Results supported polyphyly of the Pseudophyllidea formed by two distinct clades: one with diphyllobothriids (Diphyllobothrium, Schistocephalus, Spirometra and Duthiersia) and another including Abothrium, Probothriocephalus, Eubothrium and Bothriocephalus. The former pseudophyllidean clade formed a separate branch with the Caryophyllidea (Khawia and Hunterella) and Haplobothriidea (Haplobothrium), the latter taxon being closely related to either caryophyllideans or diphyllobothriids in different analyses. Proteocephalideans formed a monophyletic group in all analyses and constituted a clade within the Tetraphyllidea thus rendered paraphyletic. Within the Proteocephalidea, the Acanthotaeniinae (Acanthotaenia from reptiles in Africa) and Gangesiinae (Gangesia and Silurotaenia from silurid fish in the Palearctic Region) were separated from parasites of freshwater fish and mammals. The family Proteocephalidae was found to be paraphyletic due to the placement of a monticelliid species, Monticellia sp., in a clade within the former family. The genus Proteocephalus appeared as an artificial assemblage of unrelated taxa which is congruent with previous molecular analyses.

Animals↗

Phylogenetic analysis of European species of Proteocephalus (Cestoda: Proteocephalidea): compatibility of molecular and morphological data, and parasite-host coevolution.

The phylogeny of European species of the tapeworm genus Proteocephalus was studied, based on partial 18S rDNA and morphological data. The group was found to be monophyletic. The analysis showed a low informative value of available morphological characters in comparison with molecular data. The morphological matrix resulted in a poorly resolved tree which is, however, compatible with the topology (Proteocephalus osculatus (Proteocephalus torulosus (Proteocephalus macrocephalus, Proteocephalus filicollis) (Proteocephalus tetrastomus, Proteocephalus percae, Proteocephalus longicollis))) based on the 18S rDNA data. A comparison performed by the program TreeMap showed a lack of significant congruency between parasite and host phylogenies. Therefore, the distribution of species in their hosts appears to be independent of the phylogeny and it is likely to be a result of host-switching, rather than co-speciation events.

Animals↗

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig-human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.

Animals↗

Partial characterisation of carbohydrate-rich Echinococcus granulosus coproantigens.

Coproantigen ELISA based tests for diagnosis of canine echinococcosis provide high specificity and sensitivity. However, the antigenic molecules present in faeces from infected dogs have not yet been characterised. While initial attempts to determine the molecular weights of Echinococcus granulosus coproantigens by SDS-PAGE and Western blotting with coproantigen reactive capture antibodies were equivocal, they suggested presence of a significant carbohydrate component. Periodate treatment of coproantigen positive faecal supernatants resulted in a significant reduction (53%) in ELISA activity, suggesting that carbohydrates are involved in the antigenic structure of E. granulosus coproantigens. Protease treatment of antigenic molecules resulted in an 11% reduction in absorbance in ELISA, indicating that protein components were also present which affected by enzyme activity. Lectin-binding ELISA assays indicated strong affinity of E. granulosus coproantigens to concanavalin agglutinin and Lens culinaris agglutinin, and moderate binding to wheat-germ agglutinin and peanut agglutinin. No binding was detectable to Ulex europaensis agglutinin-I, Bandeiraea simplicifolia or Dolichos biflorus agglutinin. These data indicate that E. granulosus coproantigens from infected dog faeces possibly contained alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues. To verify the role of carbohydrate moieties in coproantigens, faecal samples were treated with exoglycosidase and tested in the coproantigen ELISA. Treatment with beta-galactosidase or N-acetyl-beta-glucosamine reduced ELISA activity by 44 and 30%, respectively. Incubation with a panel of other specific exoglycosidases including alpha-galactosidase as well as alpha-L-fucosidase, alpha-mannosidase, beta-mannosidase, alpha-glucosidase, beta-glucosidase, beta- fructosidase, or neuraminidase, did not alter coproantigen detection in ELISA. The results indicate that coproantigens present in faeces from E. granulosus naturally infected dogs were highly glycosylated and contain beta- galactose and N-acetyl-beta-glucosamine. The putative relationship of antigenic molecules with the tapeworm glycocalyx is discussed.

Acetylglucosamine↗

Investigations on the protonephridial system of post-larval Gyrocotyle urna and Amphilina foliacea (Cestoda).

The gross morphology and ultrastructure of the different parts of the protonephridial system of the monozoic tapeworms Gyrocotyle urna and Amphilina foliacea are described. The terminal cell in both species has numerous cilia which are interconnected and extend into the lumen of the first canal cell. The filtration area is built up from projections of two cells, the terminal cell and the first canal cell. The first canal cell forms a solid hollow cylinder without a cell gap and a desmosome as found in Neodermata other than cestodes and Udonella. In Gyroctyle the nucleus of the first canal cell is located in the wall cytoplasma whereas more distally located ductules of both species have subepithelial cell bodies containing the nuclei. In both taxa the protonephridial canal system is reticulate. In Amphilina the distal canals lack non-terminal ciliary flames, such ciliary tufts can be found in the larger capillaries of Gyrocotyle. The capillary cilia have rootlets and the ultrastructure of the duct wall cytoplasm containing large numbers of vesicles indicates highly active transport processes. The morphology of the protonephridial systems is discussed with regard to the evolution of Neodermata (especially of the Cestoda) and the function of the protonephridial system in cestodes as a probable organ of nutrient distribution.

Animals↗

Transmission of hydatid disease to sheep from wild dogs in Victoria, Australia.

Adult tapeworms and metacestodes of Echinococcus granulosus were found commonly in wildlife on Crown Land (Alpine National Park or State Forest) in the area around Mansfield, Victoria. A total of 756 sheep from 32 farms in the areas were examined at slaughter. Seventeen of the farms (Group A) adjoined Crown Land and wild dogs commonly entered these farms and killed sheep. A high prevalence of hydatid infection was found in sheep from 15 of these farms. Serology and arecoline purging failed to identify infection with E. granulosus in any domestic dogs on these farms, despite some farmers sometimes feeding their dogs with offal of sheep and/or kangaroos. The remaining 15 farms (Group B) were in an area where wild dogs did not occur. Sheep infected with hydatid cysts were present on 2 farms in Group B, but E. granulosus infection was not evident in wildlife species examined from this area. The only sheepdog to have detectable serum antibodies against E. granulosus was a dog from a farm separating the 2 Group B farms where infected sheep were found. Echinococcus granulosus were not found when this dog was purged. The implications of the results of this study are discussed in terms of transmission and control.

Animals↗