Hidden diversity of cestodes in wild African carnivores: I. Non-taeniid cyclophyllideans.

Our knowledge of parasites in wildlife remains limited, primarily due to restricted access to samples, especially of parasites from protected species. This present study contributes to the comprehension of the enigmatic world of helminths of African wild mammals and cestode biodiversity by combining both molecular and morphological analysis. Cestode samples were opportunistically collected from 77 individual definitive hosts in South Africa, Namibia and Ethiopia, encompassing 15 different species of wild African carnivores and additionally domestic cats. The analysis revealed 32 different cyclophyllidean species of which 21 (65.6 %) represent previously unknown genetic entities. They belong to the families Mesocestoididae, Hymenolepididae, Dipylidiidae and Taeniidae. Here we cover the non-taeniid cestodes, while the taeniids will be addressed in a separate publication. Three of the non-taeniid species uncovered in this study could be assigned to the genus Mesocestoides and were isolated from servals and domestic cats. The white-tailed mongoose was found to be a suitable host for a species belonging to the Hymenolepididae, which was identified as Pseudandrya cf. mkuzii. Both feline and canine genotypes of Dipylidium caninum were detected in domestic cats, the canine genotype also in an African wolf. In addition to these, a novel species of Dipylidium was discovered in an aardwolf. Lastly, four distinct species of Joyeuxiella were found in this study, revealing a cryptic species complex and emphasizing the need for a taxonomic reassessment of this genus. Despite the limited scope of our study in terms of geography and sample size, the results highlight that biodiversity of cestodes in African wild mammals is grossly under-researched and follow-up studies are urgently required, in particular linking morphology to gene sequences.

When wildlife comes to town: interaction of sylvatic and domestic host animals in transmission of Echinococcus spp. in Namibia.

The present study was conducted in the isolated desert town of Oranjemund in the far south of Namibia. It is an extremely arid region where no livestock husbandry is practiced and only animals adapted to the desert can be found. However, in and around the city, artificial irrigation maintains lush green patches of grass that attract wild animals, in particular oryx antelopes (Oryx gazella). In 2015 four oryx antelopes were euthanised due to poor conditions and a post-mortem examination was conducted. Two were found positive for cystic echinococcosis and 16 cysts were collected for molecular analyses. In addition, faecal samples from black-backed jackals (n=5) and domestic dogs (n=9), which were regularly observed to feed on oryx carcasses, were collected and taeniid eggs isolated. Parasite species identification of the cysts and eggs was done by amplifying and sequencing the mitochondrial nad1 gene. Both oryx antelopes were found infected with E. ortleppi and one co-infected with E. canadensis G6/7. Both Echinococcus species were able to develop fertile cysts in oryx, making oryx antelopes competent hosts for these parasites. Therefore, the analysis of faecal samples was of high interest and although the numbers were quite small, taeniid eggs were found in three out of five faecal samples of jackals and in all nine dog samples. However, species determination was only successful with two jackal and one dog sample. All three were positive for E. canadensis G6/7. The absence of E. ortleppi may be due to the low number of faecal samples examined. In our small study, we discovered a rather unique lifecycle of Echinococcus spp. between jackals and domestic dogs as definitive hosts and oryx antelopes as intermediate hosts. Here, the presence of E. canadensis G6/7 is of particular concern, as it is the second most important causative agent of CE in humans.

Genetic characterization of Echinococcus species in eastern Ethiopia.

Cystic echinococcosis (CE) is a neglected zoonotic disease with considerable economic and public health burden worldwide, particularly affecting developing countries like Ethiopia. To initiate effective prevention and control of CE, comprehensive data on the local lifecycles of the various species/genotypes of Echinococcus are needed. In the present study, conducted in eastern Ethiopia, a total of 1106 livestock animals were examined at three slaughterhouses, which resulted in combined prevalence of morphologically and molecularly confirmed CE of 8.4% (75/891) in cattle, 1.1% (1/95) in sheep, 0.0% (0/95) in goats and 12.0% (3/25) in camels. All cystic lesions recovered during post mortem examination were assessed for cyst condition and underwent molecular characterization by PCR and sequencing of a 1081 bp fragment of the mitochondrial cox1 gene. A total of 175 cysts belonged to E. granulosus s.s. (n = 165), E. ortleppi (n = 6) and E. canadensis G6/7 (n = 4). Of all examined cysts, only 14 were fertile and contained protoscoleces, all from the lungs of cattle: 5 were E. granulosus s.s., 6 E. ortleppi and 3 E. canadensis G6/7. In sheep, only one sterile liver cyst of E. granulosus s.s. was found, while in camels seven sterile or caseated/calcified cysts of E. granulosus s.s. and E. canadensis G6/7 were found in liver and lungs. In conclusion, the prevalence of CE was rather low compared to other regions of Ethiopia, and, based on the number of fertile cysts, three Echinococcus spp. contributed almost equally to transmission. Cattle seem to be, epidemiologically, the most important livestock species. Our data provide a substantial basis for more detailed investigations of the transmission dynamics of CE in the study area.

Molecular identification of zoonotic hookworms in dogs from four counties of Kenya.

All canine hookworms are known to be zoonotic, causing infections ranging from transient skin irritations to prolonged 'creeping eruptions', eosinophilic enteritis and even patent intestinal infections. There is little information on canine hookworm species and their public health significance in sub-Saharan Africa. This study determined the prevalence and species of hookworms in dogs from different climatic zones of Kenya. Dog faecal samples were collected from the environment, and hookworm eggs were isolated by zinc chloride flotation and subjected to DNA extraction. Polymerase chain reaction (PCR) assays targeting the internal transcribed spacer (ITS) 1 and 2, 5.8S and 28S ribosomal RNA of Ancylostoma spp. and Uncinaria stenocephala were performed, and hookworm species were identified by PCR-restriction fragment length polymorphism (RFLP) or DNA sequencing. Hookworm eggs were detected by microscopy in 490/1621 (30.23%, 95% CI 28.01-32.54) faecal samples. Estimates of faecal prevalence were high in counties receiving higher rainfall (Narok 46.80%, Meru 44.88%) and low in those with a more arid climate (Isiolo 19.73%, Turkana 11.83%). In a subset of 70 faecal samples, Ancylostoma caninum (n = 59) was the most common species, followed by A. braziliense (n = 10) and A. cf. duodenale (n = 1). This study reports for the first time the detection of A. cf. duodenale in dog faeces and zoonotic hookworm species in Kenyan dogs. These findings emphasize the need for control measures such as enforcing laws for restraining stray dogs, regular deworming of dogs, and public health awareness programmes aimed at informing communities on outdoor use of footwear.

Ecology and Life Cycle Patterns of Echinococcus Species.

The genus Echinococcus is composed of eight generally recognized species and one genotypic cluster (Echinococcus canadensis cluster) that may in future be resolved into one to three species. For each species, we review existing information on transmission routes and life cycles in different geographical contexts and - where available - include basic biological information of parasites and hosts (e.g., susceptibility of host species). While some Echinococcus spp. are transmitted in life cycles that involve predominantly domestic animals (e.g., dog - livestock cycles), others are wildlife parasites that do or do not interact with domestic transmission. In many cases, life cycle patterns of the same parasite species differ according to geography. Simple life cycles contrast with transmission patterns that are highly complex, involving multihost systems that may include both domestic and wild mammals. Wildlife transmission may be primary or secondary, i.e., resulting from spillovers from domestic animals. For most of the species and regions, existing information does not yet permit a conclusive description of transmission systems. Such data, however, would be highly relevant, e.g., for anticipation of geographical changes of the presence and frequency of these parasites in a warming world, or for initiating evidence-based control strategies.

Genetic polymorphism and population structure of Echinococcus ortleppi.

The zoonotic cestode Echinococcus ortleppi (Lopez-Neyra and Soler Planas, 1943) is mainly transmitted between dogs and cattle. It occurs worldwide but is only found sporadically in most regions, with the notable exception of parts of southern Africa and South America. Its epidemiology is little understood and the extent of intraspecific variability is unknown. We have analysed in the present study the genetic diversity among 178 E. ortleppi isolates from sub-Saharan Africa, Europe and South America using the complete mitochondrial cox1 (1608 bp) and nad1 (894 bp) DNA sequences. Genetic polymorphism within the loci revealed 15 cox1 and six nad1 haplotypes, respectively, and 20 haplotypes of the concatenated genes. Presence of most haplotypes was correlated to geographical regions, and only one haplotype had a wider spread in both eastern and southern Africa. Intraspecific microvariance was low in comparison with Echinococcus granulosus sensu stricto, despite the wide geographic range of examined isolates. In addition, the various sub-populations showed only subtle deviation from neutrality and were mostly genetically differentiated. This is the first insight into the population genetics of the enigmatic cattle adapted Echinococcus ortleppi. It, therefore, provides baseline data for biogeographical comparison among E. ortleppi endemic regions and for tracing its translocation paths.

Microdiversity of Echinococcus granulosus sensu stricto in Australia.

Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato.

Echinococcus granulosus, formerly regarded as a single species with a high genotypic and phenotypic diversity, is now recognised as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. This diversity is reflected in the mitochondrial and nuclear genomes and has led to the construction of phylogenetic trees and hypotheses on the origin and geographic dispersal of various taxa. Based on phenotypic characters and gene sequences, E. granulosus (sensu lato) has by now been subdivided into E. granulosus sensu stricto (including the formerly identified genotypic variants G1-3), Echinococcus felidis (the former 'lion strain'), Echinococcus equinus (the 'horse strain', genotype G4), Echinococcus ortleppi (the 'cattle strain', genotype G5) and Echinococcus canadensis. The latter species, as recognised here, shows the highest diversity and is composed of the 'camel strain', genotype G6, the 'pig strain', genotype G7, and two 'cervid strains', genotypes G8 and G10. There is debate whether the closely related G6 and G7 should be placed in a separate species, but more morphological and biological data are needed to support or reject this view. In this classification, the application of rules for zoological nomenclature led to the resurrection of old species names, which had before been synonymised with E. granulosus. This nomenclatural subdivision of the agents of cystic echinococcosis (CE) may appear inconvenient for practical applications, especially because molecular tools are needed for identification of the cyst stage, and because retrospective data on 'E. granulosus' are now difficult to interpret without examination of voucher specimens. However, the increased awareness for the diversity of CE agents - now emphasised by species names rather than genotype numbers - has led to a large number of recent studies on this issue and a rapid increase of knowledge on geographical spread, host range and impact on human health of the various species. E. granulosus s.s., often transmitted by sheep, is now clearly identified as the principal CE agent affecting humans. Contrary to previous assumptions, genotypes G6/7 of E. canadensis readily infect humans, although CE incidences are rather low where E. canadensis predominates. Sub-Saharan Africa seems to be the region with the highest diversity of Echinococcus, and wild carnivores may play a more important role in the lifecycles of various species than previously assumed. Still, a number of issues remain unclear, e.g. possibly diverging parameters of diagnostic tests among the species, different responses to vaccines and, importantly, possibly required modifications of clinical management due to differences in pathogenicity.

Echinococcus spp. in central Kenya: a different story.

Research on cystic echinococcosis (CE) has a long history in Kenya, but has mainly concentrated on two discrete areas, Turkana and Maasailand, which are known to be foci of human CE in Africa. Here, we report on a survey for CE in livestock from central to northeastern Kenya, from where no previous data are available. A total of 7,831 livestock carcasses were surveyed. CE prevalence was 1.92% in cattle (n = 4,595), 6.94% in camels (n = 216), 0.37% in goats (n = 2,955) and 4.62% in sheep (n = 65). Identification of the parasite was done using an RFLP-PCR of the mitochondrial nad1 gene, which had been validated before against the various Echinococcus taxa currently recognized as distinct species. From a total of 284 recovered cysts, 258 could be identified as Echinococcus granulosus sensu stricto (n = 160), E. ortleppi (n = 51) and E. canadensis (n = 47) by RFLP-PCR of nad1. In cattle, fertile cysts occurred mostly in the lungs and belonged to E. ortleppi (31 of 54), while the vast majority were sterile or calcified cysts of E. granulosus s.s.. Most fertile cysts in camels belonged to E. canadensis (33 of 37); sterile or calcified cysts were rare. Goats harboured fertile cysts of E. ortleppi (n = 3)--which is the first record in that host species--and E. canadensis (n = 1), while all cysts of E. granulosus were sterile. Only sterile cysts were found in the three examined sheep. Typically, all cysts in animals with multiple infections belonged to the same species, while mixed infections were rare. Our data indicate that the epidemiological situation in central to northeastern Kenya is clearly different from the well-studied pastoral regions of Turkana and Maasailand, and the apparently low number of human CE cases correlates with the infrequent occurrence of E. granulosus s.s.

[The ecology of ticks, tick-borne diseases and biological tick control in Baden-Württemberg]. / Ökologie von Zecken als Überträger von Krankheitserregern in Baden-Württemberg und biologische Zeckenbekämpfung.

Ticks and tick-borne diseases are of great significance for the health of humans and animals. However, the factors influencing their distribution and dynamics are inadequately known. In a project financed by the Baden-Württemberg Ministry of the Environment, Climate and Energy Industry, as part of the program BWPLUS, interdisciplinary specialists work together to determine the influence of weather, (micro)climate, habitat, land use, human activities, and the population dynamics of host animals on the distribution and abundance of ticks and the diseases that they transmit in Baden-Württemberg. The project comprises four modules: the large-scale distribution of ticks in Baden-Württemberg (module 1), detailed studies of host-tick-pathogen interaction in relation to the microclimate (module 2), and the spatial occurrence of important tick-borne pathogens (module 3). The fourth module involves the comprehensive analysis and synthesis of all data in order to determine the relative importance of the factors studied and to develop a risk model. Recently, intensive investigations into tick control have been undertaken using various entomopathogenic fungi and nematodes as well as a parasitoid wasp. Our aim was to determine whether these natural enemies could be used to effectively reduce the number of free-living ticks.

A survey for Echinococcus spp. of carnivores in six wildlife conservation areas in Kenya.

To investigate the presence of Echinococcus spp. in wild mammals of Kenya, 832 faecal samples from wild carnivores (lions, leopards, spotted hyenas, wild dogs and silver-backed jackals) were collected in six different conservation areas of Kenya (Meru, Nairobi, Tsavo West and Tsavo East National Parks, Samburu and Maasai Mara National Reserves). Taeniid eggs were found in 120 samples (14.4%). In total, 1160 eggs were isolated and further analysed using RFLP-PCR of the nad1 gene and sequencing. 38 of these samples contained eggs of Echinococcus spp., which were identified as either Echinococcus felidis (n=27) or Echinococcus granulosus sensu stricto (n=12); one sample contained eggs from both taxa. E. felidis was found in faeces from lions (n=20) and hyenas (n=5) while E. granulosus in faeces from lions (n=8), leopards (n=1) and hyenas (n=3). The host species for two samples containing E. felidis could not be identified with certainty. As the majority of isolated eggs could not be analysed with the methods used (no amplification), we do not attempt to give estimates of faecal prevalences. Both taxa of Echinococcus were found in all conservation areas except Meru (only E. felidis) and Tsavo West (only E. granulosus). Host species identification for environmental faecal samples, based on field signs, was found to be unreliable. All samples with taeniid eggs were subjected to a confirmatory host species RLFP-PCR of the cytochrome B gene. 60% had been correctly identified in the field. Frequently, hyena faeces were mistaken for lion and vice versa, and none of the samples from jackals and wild dogs could be confirmed in the tested sub-sample. This is the first molecular study on the distribution of Echinococcus spp. in Kenyan wildlife. The presence of E. felidis is confirmed for lions and newly reported for spotted hyenas. Lions and hyenas are newly recognized hosts for E. granulosus s.s., while the role of leopards remains uncertain. These data provide the basis for further studies on the lifecycles and the possible link between wild and domestic cycles of cystic echinococcosis in eastern Africa.

Salmonella Enteritidis phage type 21 outbreak in Austria, 2005.

We report an outbreak of gastroenteritis due to Salmonella Enteritidis PT 21 associated with attending an annual traditional fair in a small Austrian village on 4 May 2005. The outbreak lasted from 4 to 8 May. Descriptive and analytical epidemiological investigations were conducted in order to determine the extent of the outbreak and to identify outbreak risk factors. Of the 115 persons who visited the fair, 85 persons fulfilled the criteria of an outbreak case (attack rate = 73.9%). Stool specimens from 52 patients, including two kitchen staff, were tested for salmonella, and 20 specimens were positive for Salmonella Enteritidis PT 21. The cohort study revealed mixed salad (which included potatoes) as the likely cause of the outbreak (RR: 10.4, 95%CI 2.8 - 39.1; P = < 0.001). The causative agent of the outbreak was cultured from the stock of eggs used at the fair and from all three drag swabs and one barn dust sample collected from the responsible egg laying flock. Molecular subtyping by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion showed that isolates from eggs, from the flock and from humans were indistinguishable. We hypothesise that cross contamination from eggs to boiled potatoes occurred in the kitchen area, where raw eggs were handled by village residents preparing a traditional Viennese egg dressing. Unrefrigerated storage of peeled potatoes may have favoured bacterial growth. Eggs from small rural flocks of laying hens kept in a traditional 'natural' way should not be assumed to be salmonella-free.

Salmonella Enteritidis phage type 21 outbreak in Austria, 2005.

We report an outbreak of gastroenteritis due to Salmonella Enteritidis PT 21 associated with attending an annual traditional fair in a small Austrian village on 4 May 2005. The outbreak lasted from 4 to 8 May. Descriptive and analytical epidemiological investigations were conducted in order to determine the extent of the outbreak and to identify outbreak risk factors. Of the 115 persons who visited the fair, 85 persons fulfilled the criteria of an outbreak case (attack rate=73.9%). Stool specimens from 52 patients, including two kitchen staff, were tested for salmonella, and 20 specimens were positive for Salmonella Enteritidis PT 21. The cohort study revealed mixed salad (which included potatoes) as the likely cause of the outbreak (RR: 10.4, 95%CI 2.8 - 39.1; P=<0.001). The causative agent of the outbreak was cultured from the stock of eggs used at the fair and from all three drag swabs and one barn dust sample collected from the responsible egg laying flock. Molecular subtyping by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion showed that isolates from eggs, from the flock and from humans were indistinguishable. We hypothesise that cross contamination from eggs to boiled potatoes occurred in the kitchen area, where raw eggs were handled by village residents preparing a traditional Viennese egg dressing. Unrefrigerated storage of peeled potatoes may have favoured bacterial growth. Eggs from small rural flocks of laying hens kept in a traditional 'natural' way should not be assumed to be salmonella-free.

Construccion de una genoteca de cDNA de Plasmodium falciparum y busqueda posterior del gen de calmodulina / Contruction of cDNA library of plasmodium falciparum and search of hibrization of the calmodium gene

Se construyeron dos genotecas de cDNA usando el método de Gubler y Hoffman, la primera partiendo del mRNA del parásito Pfalciparum, la segunda a partir del RNA total, ambas en estadio de trofozoítos maduros y esquizontes. Se enfatizó en la búsqueda por hibridización de clonos que tuvieran el gen de la calmodulina, proteína mediadora que sirve como receptor intracelular de calcio, regula una variedad de enzimas y posiblemente está relacionada con los procesos de invasión del eritrocito. Se buscaron otros genes de interés de las proteínas miosina y Amasa, también involucradas en el proceso de invasión del eritrocito por el parásito. J,os títulos de las genotecas de cDNA fueron 1,7 x lo4 ufp/pg de DNA, a partir de mRNA y 3,4 x lo4 ufp/pg de DNA a partir de RNA total. Los rendimientos de la síntesis de primera cadena de cDNA fueron entre 15 y 20 por ciento mayores a partir de RNA total que a partir de RNA mensajero (mRNA). Se obtuvieron trece clonos que hibridizaron con una sonda de calmodulina, dos de la genoteca de cDNA a partir del mRNA y once a partir del RNA total del parásito. También ocho clonos que hibridizaron con una sonda de miosina, uno de la genoteca de cDNA a partir del mRNA y siete a partir del RNA total del parásito; y cinco clonos que hibridizaron con una sonda de ATPasa, una a partir de la genoteca de cDNA a partir del mRNA y cuatro a partir del RNA total. Como alternativa para la construcción de genotecas de cDNA, se plantea la utilización de RNA total como material de partida. Sin embargo, es necesario validar el método en éste y otros organismos

Serum levels of polychlorinated biphenyls and some organochlorine insecticides in women with recent and former missed abortions.

Polychlorinated biphenyls and some organochlorine insecticides (DDT isomers and their metabolites, lindane (gamma-BHC), dieldrin, heptachlor epoxide) were assessed in the serum of 17 women with recent missed abortions, 7 women who experienced one or several missed abortions in their past, and 7 women with normal, second trimester pregnancy. Fifty-three percent of the cases of recent missed abortion had PCB serum levels higher than the mean plus two standard deviations of the control group. The mean PCB serum level in women with recent missed abortion and relatively high PCB serum levels was significantly different from that of the control group (103.04 versus 20.69 ppb, P less than 0.001). The mean PCB serum level of the former missed abortions group was also significantly different from that of the control group (82.00 versus 20.69 ppb, P less than 0.001). The quantity of the higher chlorinated biphenyl homologues (penta- and hexachlorobiphenyls) was increased in the high PCB level, missed abortion group and in the former missed abortion group, while the lower chlorinated biphenyl homologues were decreased in these groups in comparison with the control group. The percentage of total o,p'-DDT serum level, as related to total DDT, showed an increase in the high PCB level, recent missed abortion and in the former missed abortion groups, in comparison with the control group (36.18 and 48.98 versus 25.57, respectively). The fact that the former missed abortion group showed increased PCB serum levels similar to those found in the high PCB level, recent missed abortion group confirms the existence of an association between relatively high PCB serum levels and the occurrence of missed abortion. The possible role of PCBs in the occurrence of missed abortion is discussed.

Mesothelioma in children.

A literature review of mesothelioma in children is presented. The role of nonoccupational exposure to asbestos fibres in the etiology of mesothelioma, and the relatively short latent period of the disease in children are discussed. The transplacental transfer of asbestos fibres and the physiology of the immunological system during intrauterine life and infancy may explain some of the differences between mesotheliomas in children and those in adults.

World PCBs map: storage and effects in man and his biologic environment in the 1970s.

PCB residues are found in biota all over the world. A biologic magnification of PCBs has been demonstrated in the food chain: plankton-fish-fish eating birds. A world map of the PCBs residues in biota and some of their biologic effects are given in this study. The biologic effects of PCBs are varied and may generally be explained by the induction or inhibition of the activity of a large number of enzymes, which upsets quantitatively, normal biochemical processes. Harm to reproduction, growth, development, defense systems, tissues, and organs appeared in susceptible organisms as a result of such changes or as a chain reaction to heterostases. The adverse health effects, observed in persons occupationally exposed and in those accidentally poisoned, point to the risk for the general population of an ever-increasing environmental pollution by PCBs. There is need for an integrated approach, consisting of epidemiologic studies on high risk groups in the general population and in persons occupationally exposed, as well as periodic assessment of PCB residues in man, his food, and feed supplies.