Samples of Echinococcus granulosus from seven pigs from Mexico were compared with isolates of the parasite from pigs in Poland and representative strains and species of Echinococcus. Isolates from pigs in Mexico were found to be genetically identical to E. granulosus from Polish pigs and distinct from other major genotypes by sequencing part of the mitochondrial cytochrome c oxidase I (COI) mtDNA locus, restriction fragment length polymorphism (RFLP) of the polymerase chain reaction (PCR) amplified rDNA internal transcribed spacer (ITS) 1 using five different enzymes, and random amplified polymorphic DNA (RAPD) analysis. These results were complemented by data on hook morphology and together strengthen the view that Echinococcus maintained in a cycle involving pigs and dogs is a distinct strain that is conserved genetically in different geographical areas. The present study supports the close relationship of the cervid, camel and pig strains and raises the question of their taxonomic status.

To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). The mean index of association (IA=0.92) significantly deviated from zero, and the average number of multilocus genotypes (G/N ratio) was 0.21. Twenty-four multilocus genotypes were defined from the combination of alleles at the three loci. The Kenyan sub-populations showed Fst=0.28 and analysis of molecular variance showed significant divergence (22.7%) between the Laikipia, Kulal and Galana regions. The regional and host distribution of multi-locus genotypes significant population differentiation and high Nei's genetic distances suggest existence of genetic sub-structuring within T. evansi stocks while the few multi-locus genotypes and deviation of association index from zero indicate the lack of recombination. In conclusion, this study reveals that some genetic sub-structuring does occur within T. evansi, which has a clonal population structure.

Many issues concerning the taxonomy of Echinococcus have been resolved in recent years with the application of molecular tools. However, the status of Echinococcus maintained in transmission cycles involving cervid intermediate hosts remains to be determined. The recent characterization of the parasite from cervids in Finland has highlighted the paucity of data available, particularly that from North America. In this study, we have characterized a large number of Echinococcus isolates from cervids from Western Canada on the basis of morphology and molecular genetic techniques. Our results support earlier studies suggesting that Echinococcus of cervid origin is phenotypically and genetically distinct to Echinococcus maintained in domestic host assemblages, and also confirms that Echinococcus of cervid origin does not constitute a genetically homogeneous group. However, our data do not support the existence of 2 distinct genotypes (strains/subspecies) with separate geographical distributions. Our data appear to support the existence of only 1 species in cervids, but additional isolates from cervids and wolves in other endemic regions should be characterized before a final decision is made on the taxonomic status of Echinococcus in cervids.

There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei , T. evansi, T. b. rhodesiense and T. b. gambiense ) gave a constant product of approximately 480 bp; T. congolense , savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp , T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon , T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi . The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.

Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (κ = 0.12), PCR (κ = 0.11) and MHCT (κ = 0.05). However, there was a higher agreement between farmers’ classification of disease with the PCR test (κ = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 ± 0.5) and from infected calves (19.5 ± 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable “pen-side” diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.

Molecular tools are increasingly being used to address questions about parasite epidemiology. Parasites represent a diverse group and they might not fit traditional population genetic models. Testing hypotheses depends equally on correct sampling, appropriate tool and/or marker choice, appropriate analysis and careful interpretation. All methods of analysis make assumptions which, if violated, make the results invalid. Some guidelines to avoid common pitfalls are offered here.

The potential for the non-coding intergenic rDNA spacer (IGS) to DNA fingerprint Giardia duodenalis isolates was investigated. Conserved PCR primers, specific for the flanking large and small rDNA genes, were used to amplify the IGS from 52 in vitro-cultured Giardia isolates. Four distinct IGS-PCR size groups (1.35-1.6 kb) were observed, which correlated closely with the major genetic assemblages established previously for the same isolates using isoenzyme analysis. IGS-PCR size groups A (1.42 kb), C (1.4 kb) and D (1.35 kb) corresponded to isoenzyme assemblage A, and IGS-PCR group B (1.6 kb) to isoenzyme assemblage B. Amplified products from IGS-PCR size groups A and B, which contained 50/52 isolates, were subsequently digested with 8 different restriction enzymes and their profiles compared. Analysis separated isolates within each IGS-PCR size group into 2 distinct clusters which correlated almost exactly with the same genetic groups established previously using isoenzyme electrophoresis. Within each cluster, both methods exhibited a similar capacity to distinguish between Giardia genotypes although they established different genetic relationships between individual isolates. Much of the variability associated with the IGS was attributed to isolates harbouring multiple IGS-sequence types. Restriction analysis of IGS-PCR products amplified from cloned and parent lines of a human isolate BAH 39, which contains multiple IGS variants, showed that trophozoite populations are homogeneous with respect to the types of IGS-variants they maintain. Furthermore, in vitro culture of the cloned isolate BAH39c9 over a 6-year period also failed to reveal variation in IGS-PCR digestion profiles. These results suggest that IGS-PCR RFLP profiles are inherently stable. IGS-PCR analysis was successfully applied to 11 Giardia cyst samples highlighting the potential for this approach to genotype Giardia isolates without the need for in vitro culture.

The parasitological, clinical efficacy and tolerability of albendazole in the treatment for both giardiasis and hookworm infection in a remote Aboriginal population was investigated. Albendazole at a dose rate of 400 mg daily for 5 days was highly effective in reducing hookworm egg numbers and both Giardia antigen and cysts. The 36.6% prevalence of Giardia prior to treatment fell to 12% between days 6 and 9, 15% for days 10-17 and rose to 28% between days 18 and 30. Tolerability and clinical efficacy were excellent. The effect of albendazole on hookworm was longer lasting than that on Giardia, reducing percent infection from over 76-2% on days 6-9 and zero by day 18-30 despite conditions highly conducive to rapid re-infection. We conclude that albendazole is highly efficacious against both parasites when used as described but that long term community benefit may require additional education programmes to avoid re-infection with Giardia although treatment strategies would seem appropriate for hookworm.

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities, worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development.

In this chapter, the contribution of molecular tools in understanding the aetiology and ecology of infectious diseases is examined in the context of molecular epidemiology (ME). ME is seen as providing the ‘tools’, both laboratory and analytical, which have predictive significance in epidemiological investigations of the causation of disease. A diversity of questions can be addressed with these tools which can conveniently be viewed as particular regions of DNA and grouped according to the different hierarchical levels of specificity by which infectious agents can be characterized. These groupings and the applications of the different molecular tools are described, and consideration given to the most appropriate methods of analysing data from ME investigations.

EvolutionVolume 51, Issue 1 p. 289-294 Brief CommunicationFree Access SELF-FERTILIZATION WITHOUT GENOMIC OR POPULATION STRUCTURING IN A PARASITIC TAPEWORM A. J. Lymbery, A. J. Lymbery alanl@agbyl.agric.wa.gov.au Agriculture Western Australia, PO Box 1231, Bunbury, Western Australia, 6231 Australia School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this authorC. C. Constantine, C. C. Constantine School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this authorR. C. A. Thompson, R. C. A. Thompson School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this author A. J. Lymbery, A. J. Lymbery alanl@agbyl.agric.wa.gov.au Agriculture Western Australia, PO Box 1231, Bunbury, Western Australia, 6231 Australia School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this authorC. C. Constantine, C. C. Constantine School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this authorR. C. A. Thompson, R. C. A. Thompson School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 AustraliaSearch for more papers by this author First published: 31 May 2017 https://doi.org/10.1111/j.1558-5646.1997.tb02411.xCitations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume51, Issue1February 1997Pages 289-294 ReferencesRelatedInformation

Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.

Multilocus enzyme electrophoresis was used to examine the relatedness of 52 isolates of Clavibacter toxicus, the agent of annual ryegrass toxicity. These included 37 Western Australian (WA) field isolates sampled in 3 distinct locations over a 2-year period, and 15 isolates sampled from 6 different host plant species in 3 states in Australia over approximately 8 years. Seventeen reference strains for the related genera Curtobacterium, Rhodococcus and Arthrobacter were examined for comparison. The 69 isolates were divided into 29 electrophoretic types (ETs), separated by genetic distances of 0.06 to 0.81. The C. toxicus isolates fell into 12 ETs, 11 of which formed a tightly clustered group separated by a genetic distance of 0.23 or less. Thirty-one of the WA field isolates of C. toxicus fell into a single ET, and four into another ET. Clavibacter toxicus therefore formed a closely related group which was genetically distinct from the other plant pathogenic species, and a dominant widely disseminated strain of the species was identified in WA.

Hydatid disease (echinococcosis) is one of the most important parasitic zoonoses and remains a public health and economic problem of global proportions. There are currently four recognized species in the genus Echinococcus: E.granulosus, E.multilocularis, E.oligarthrus, and E.vogeli. Because of the extensive variation in Echinococcus, it is very important to characterize the aetiological agents in different endemic areas to determine transmission patterns, particularly where there is the possibility of interaction between cycles. The first step in a taxonomic revision of the genus Echinococcus is to establish an appropriate species concept. Therefore, the purpose should be to update what is known about the nature and extent of variation in Echinococcus to provide a sound basis for a phylogenetic analysis. The current classification within the genus Echinococcus is not compatible with historical relationships between taxa. Phylogenetic analysis of DNA sequence data provides no support for the concept that E.grunulosus is a monophyletic group and it cannot be considered an evolutionary species. There is an urgent need for the molecular characterization of strains of E. multilocularis as well as strains of Echinococcus in lions and cervids, so that this taxonomic revision can be completed.

Journal Article FORTRAN Programs for Analyzing Population Structure From Multilocus Genotypic Data Get access C. C. Constantine, C. C. Constantine School of Veterinary Studies, Murdoch UniversityMurdoch, Western Australia 6150 Search for other works by this author on: Oxford Academic PubMed Google Scholar R. P. Hobbs, R. P. Hobbs School of Veterinary Studies, Murdoch UniversityMurdoch, Western Australia 6150 Search for other works by this author on: Oxford Academic PubMed Google Scholar A. J. Lymbery A. J. Lymbery Department of AgricultureP.O. Box 1231, Bunbury, Western Australia 6230 Australia Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Heredity, Volume 85, Issue 4, July 1994, Pages 336–337, https://doi.org/10.1093/oxfordjournals.jhered.a111475 Published: 01 July 1994 Article history Received: 30 July 1993 Accepted: 04 February 1994 Published: 01 July 1994

Host induced changes in morphological characters of the rostellar hooks of Echinococcus granulosus were used to determine the origin of infection in definitive hosts in rural areas of southeastern Australia where wild and domestic cycles of transmission may interact. The morphological characters studied vary depending on the species of intermediate host (macropod marsupials, sheep) in which protoscoleces develop, and these characters are retained in adult worms. It was therefore possible to determine whether definitive hosts (dingoes and foxes) acquired infection by consuming protoscoleces of E. granulosus from macropods or from sheep. The results correlated well with the known distribution of intermediate hosts and illustrate the practical value of such morphological markers in epidemiological studies.

Hydatid disease (echinococcosis) remains a public health and economic problem of global proportion. Treatment usually requires major surgery and the prognosis for some forms of the disease is poor. The variable transmission patterns exhibited by the causative agents of this complex zoonosis, and inadequate support both internationally and nationally, have resulted in the establishment of control campaigns that are usually organized and funded at a local level. Here, Andrew Thompson, Ian Robertson, Robin Gasser and Clare Constantine describe a novel, targeted campaign of education and surveillance that has recently been initiated in Western Australia, where a totally artificial cycle of transmission for Fchinococcus granulosus has been established as a result of human activity.