Environmental distribution of Echinococcus- and Taenia spp.-contaminated dog feces in Kyrgyzstan.

Recently, there have been epidemics of human cystic echinococcosis (CE) and alveolar echinococcosis (AE) in Kyrgyzstan. This study investigated 2 districts for the presence of Echinococcus granulosus s.l. and Echinococcus multilocularis eggs; species identity was confirmed by polymerase chain reaction in dog feces and the level of environmental contamination with parasite eggs in 2017­2018 was also investigated. In the Alay district 5 villages with a high reported annual incidence of AE of 162 cases per 100 000 and 5 villages in the Kochkor district which had a much lower incidence of 21 cases per 100 000 were investigated. However, the proportion of dog feces containing E. granulosus s.l. eggs was ~4.2 and ~3.5% in Alay and Kochkor respectively. For E. multilocularis, the corresponding proportions were 2.8 and 3.2%. Environmental contamination of Echinococcus spp. eggs was estimated using the McMaster technique for fecal egg counts, weight and density of canine feces. The level of environmental contamination with E. multilocularis eggs was similar at 4.4 and 5.0 eggs per m2 in Alay and Kochkor respectively. The corresponding values for E. granulosus s.l. were 8.3 and 7.5 eggs per m2. There was no association between village or district level incidence of human AE or CE and the proportion of dog feces containing eggs of Echinococcus spp. or the level of environmental contamination. Increased contamination of taeniid eggs occured in the autumn, after the return of farmers with dogs from summer mountain pastures.

First isolation of Echinococcus granulosus sensu lato genotype 7 in the archipelago of Cape Verde.

There are no scientific data available on the occurrence of the Echinococcus granulosus sensu lato (s.l.) cluster in definitive hosts (domestic dogs), intermediate hosts (domestic livestock) nor humans in Cape Verde. In this pilot study, environmental dog fecal samples (n = 369) were collected around food markets, official slaughterhouses, as well as home and small business slaughter spots in 8 of the 9 inhabited islands from the Cape Verde archipelago, between June 2021 and March 2022. Additionally, during the same period, 40 cysts and tissue lesions were opportunistically collected from 5 islands, from locally slaughtered cattle (n = 7), goats (n = 2), sheep (n = 1) and pigs (n = 26). Genetic characterization by a multiplex polymerase chain reaction assay targeting the 12S rRNA gene confirmed the presence of E. granulosus s.l. in fecal and tissue material. In total, 17 cyst samples from Santiago (n = 9), Sal (n = 7) and São Vicente (n = 1) and 8 G6/G7-positive dog fecal samples from Santiago (n = 4) and Sal (n = 4) were identified as E. granulosus s.l. G7 by sequence analysis (nad2, nad5 and nad1 genes). This study discloses the transmission of E. granulosus s.l. G7, in pig, cattle and dog in Cape Verde.

Differences in clinical aspects of human cystic echinococcosis caused by Echinococcus granulosus sensu stricto and the G6 genotype in Neuquén, Argentina.

Most human cystic echinococcosis (CE) cases worldwide are attributed to Echinococcus granulosus sensu stricto (s.s), followed by the G6 and G7 genotypes. While E. granulosus s.s. has a cosmopolitan distribution, the G6 genotype is restricted to areas where camels and goats are present. Goats are the primary livestock in the Neuquén province in Argentina where the G6 genotype has been reported to be responsible for a significant percentage of CE human cysts genotyped. In the present study, we genotyped 124 Echinococcus cysts infecting 90 CE-confirmed patients. Echinococcus granulosus s.s. was identified in 51 patients (56.7%) with 81 cysts and the G6 genotype in 39 patients (43.3%) harbouring 43 cysts. Most CE cases ≤18 years were male suggesting pastoral work could be a risk factor for the infection. Echinococcus granulosus s.s. was significantly found more frequently in the liver (32/51 patients) and the G6 genotype in the lungs and extrahepatic localizations (27/39). The patients infected with E. granulosus s.s., presented up to 6 cysts while patients infected with G6 presented a maximum of 2. The diameter of lung cysts attributed to E. granulosus s.s. was significantly larger compared to lung cysts from G6. Following the WHO ultrasound classification of liver cysts, we observed inactive cysts in 55.6% of G6 cysts and only 15.3% of E. granulosus s.s cysts. In conclusion, we provide evidence of differences in clinical aspects of CE caused by E. granulosus s.s. and the G6 genotype of E. granulosus s.l. complex infecting humans.

Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species.

Alveolar (AE) and cystic echinococcosis (CE) are severe parasitic zoonoses caused by the larval stages of Echinococcus multilocularis and E. granulosus sensu lato, respectively. A panel of 7 monoclonal antibodies (mAbs) was selected against major diagnostic epitopes of both species. The binding capacity of the mAbs to Echinococcus spp. excretory/secretory products (ESP) was analyzed by sandwich-ELISA, where mAb Em2G11 and mAb EmG3 detected in vitro extravesicular ESP of both E. multilocularis and E. granulosus s.s. These findings were subsequently confirmed by the detection of circulating ESP in a subset of serum samples from infected hosts including humans. Extracellular vesicles (EVs) were purified, and the binding to mAbs was analyzed by sandwich-ELISA. Transmission electron microscopy (TEM) was used to confirm the binding of mAb EmG3 to EVs from intravesicular fluid of Echinococcus spp. vesicles. The specificity of the mAbs in ELISA corresponded to the immunohistochemical staining (IHC-S) patterns performed on human AE and CE liver sections. Antigenic small particles designated as ''spems'' for E. multilocularis and ''spegs'' for E. granulosus s.l. were stained by the mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2, while mAb Em2G11 reacted with spems and mAb Eg2 with spegs only. The laminated layer (LL) of both species was strongly visualized by using mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2. The LL was specifically stained by mAb Em2G11 in E. multilocularis and by mAb Eg2 in E. granulosus s.l. In the germinal layer (GL), including the protoscoleces, a wide staining pattern with all structures of both species was observed with mAb EmG3IgG1, mAb EmG3IgM, mAb AgB, mAb 2B2, and mAb Em18. In the GL and protoscoleces, the mAb Eg2 displayed a strong E. granulosus s.l. specific binding, while mAb Em2G11 exhibited a weak granular E. multilocularis specific reaction. The most notable staining pattern in IHC-S was found with mAb Em18, which solely bound to the GL and protoscoleces of Echinococcus species and potentially to primary cells. To conclude, mAbs represent valuable tools for the visualization of major antigens in the most important Echinococcus species, as well as providing insights into parasite-host interactions and pathogenesis.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Molecular characterization of Echinococcus granulosus sensu lato genotypes in dromedary camels from extreme Sahara of Algeria based on analysis of nad2 and nad5 genetic markers.

Cystic echinococcosis is parasitic disease caused by the metacestodes belonging to the Echinococcus granulosus sensu lato (s.l.) species complex. Cystic echinococcosis is of considerable economic and public health importance. It is endemic in both livestock and humans in North African countries, including Algeria. The present study aimed to characterize E. granulosus s.l. genotypes in dromedary camels (Camelus dromedarius) from the extreme Sahara of Algeria, using recently developed mitochondrial genetic markers (NADH dehydrogenase subunit 2 and NADH dehydrogenase subunit 5) for reliable identification of different genotypes. A total of 75 Echinococcus cysts were collected from 49 dromedary camels, including 65 and 10 cysts from 45 and four camels originating from two slaughterhouses of Tindouf and Illizi provinces, respectively. E. granulosus sensu stricto (s.s.) G1 and G3 were identified in camels from both areas based on nad5 (649 bp) gene sequences, whereas E. granulosus s.l. G6 was identified in camels from Tindouf region based on concatenated nad5 and nad2 gene sequences (total 1336 bp). Identified samples clustered into 11 different haplotypes (ALG1-ALG11), including four haplotypes (ALG8-ALG11) for E. granulosus s.s. G1, one haplotype (ALG7) for E. granulosus s.s. G3, and six haplotypes (ALG1-ALG6) for E. granulosus s.l. G6. The present study provides valuable molecular data, including genotyping and haplotypic variability, on E. granulosus s.l. in dromedary camels from two regions in the extreme Sahara of Algeria. Future characterization of the G1, G3, and G6 samples based on sequencing of complete mitochondrial genomes would be of considerable significance for a more comprehensive understanding of molecular epidemiology of CE in Algeria.

Serological Assays for Alveolar and Cystic Echinococcosis-A Comparative Multi-Test Study in Switzerland and Kyrgyzstan.

Both alveolar (AE) and cystic echinococcosis (CE) are lacking pathognomonic clinical signs; consequently imaging technologies and serology remain the main pillars for diagnosis. The present study included 100 confirmed treatment-naïve AE and 64 CE patients that were diagnosed in Switzerland or Kyrgyzstan. Overall, 10 native Echinococcus spp. antigens, 3 recombinant antigens, and 4 commercial assays were comparatively evaluated. All native E. multilocularis antigens were produced in duplicates with a European and a Kyrgyz isolate and showed identical test values for the diagnosis of AE and CE. Native antigens and three commercial tests showed high diagnostic sensitivities (Se: 86-96%) and specificities (Sp: 96-99%) for the diagnosis of AE and CE in Swiss patients. In Kyrgyz patients, values of sensitivities and specificities were 10-20% lower as compared to the Swiss patients' findings. For the sero-diagnosis of AE in Kyrgyzstan, a test-combination of an E. multilocularis protoscolex antigen and the recombinant antigen Em95 appears to be the most suitable test strategy (Se: 98%, Sp: 87%). For the diagnosis of CE in both countries, test performances were hampered by major cross-reactions with AE patients and other parasitic diseases as well as by limited diagnostic sensitivities (93% in Switzerland and 76% in Kyrgyzstan, respectively).

Cystic echinococcosis of ruminant livestock in Namibia.

Cystic echinococcosis (CE) is widespread and locally frequent in southern Africa where it affects humans, livestock, and wild mammals. However, most data from the region are old and do not provide information on the causative Echinococcus species. For Namibian livestock only anecdotal records were available prior to this preliminary survey. Our retrospective analysis of slaughterhouse records of CE in cattle from the commercial farming area in central and southern Namibia resulted in 1.65% CE prevalence among 35,143 slaughtered cattle in the period 2015-2016. For comparison, carcasses of ruminant livestock were prospectively examined in the communal farming areas of northern Namibia, resulting in three CE cases among only 12 cattle, and no cases among nine goats. To determine the Echinococcus species affecting Namibian livestock, a total of 53 cysts were collected from all parts of the country and analysed for species and genotype by amplification and sequencing of the nad1 gene. All 50 cattle cysts (isolated from 40 cattle), both from the commercial and communal farming areas, were Echinococcus ortleppi (all fertile, and 42/50 from the lungs), while three opportunistically collected cysts from three sheep in southern Namibia were E. canadensis G7. Our data suggest that E. ortleppi is the only CE agent that is relevant for cattle infection in Namibia, and that low prevalence in the commercial farming areas contrasts with high CE burden in the northern traditional husbandry systems. The present data provide baseline information to stimulate epidemiological studies on the transmission pathways of various CE agents in livestock, wildlife, and humans in Namibia and neighbouring countries.

Evaluation of a structured treatment discontinuation in patients with inoperable alveolar echinococcosis on long-term benzimidazole therapy: A retrospective cohort study.

OBJECTIVES: Alveolar echinococcosis (AE) is an orphan zoonosis of increasing concern in endemic areas, including Europe. It frequently presents in an advanced, inoperable stage, that requires life-long parasitostatic benzimidazole therapy. In some patients, long-term therapy leads to negative anti-Em18 antibody ELISA and PET. It is disputed, whether these patients are truly cured and treatment can be safely discontinued. Our aim was to retrospectively assess long-term outcome of 34 patients with inoperable AE who participated in a previous study to determine feasibility of benzimidazole treatment cessation. METHODS: Retrospective analysis of medical charts was undertaken in all 34 AE patients who participated in our previous study. Of particular interest were AE recurrence or other reasons for re-treatment in patients who stopped benzimidazole therapy and whether baseline clinical and laboratory parameters help identify of patients that might qualifiy for treatment cessation. Additionally, volumetric measurement of AE lesions on contrast-enhanced cross-sectional imaging was performed at baseline and last follow-up in order to quantify treatment response. RESULTS: 12 of 34 patients stopped benzimidazole therapy for a median of 131 months. 11 of these patients showed stable or regressive AE lesions as determined by volumetric measurement. One patient developed progressive lesions with persistently negative anti-Em18 antibody ELISA but slight FDG-uptake in repeated PET imaging. At baseline, patients who met criteria for treatment cessation demonstrated higher lymphocyte count and lower total IgE. CONCLUSION: Treatment cessation is feasible in inoperable AE patients, who demonstrate negative anti-Em18 antibody ELISA and PET on follow-up. Close monitoring including sectional imaging is strongly advised.

Advances in the treatment, diagnosis, control and scientific understanding of taeniid cestode parasite infections over the past 50 years.

In the past 50 years, enormous progress has been made in the diagnosis, treatment and control of taeniid cestode infections/diseases and in the scientific understanding thereof. Most interest in this group of parasites stems from the serious diseases that they cause in humans. It is through this lens that we summarize here the most important breakthroughs that have made a difference to the treatment of human diseases caused by these parasites, reduction in transmission of the taeniid species associated with human disease, or understanding of the parasites' biology likely to impact diagnosis or treatment in the foreseeable future. Key topics discussed are the introduction of anti-cestode drugs, including benzimidazoles and praziquantel, and the development of new imaging modalities that have transformed the diagnosis and post-treatment monitoring of human echinococcoses and neurocysticercosis. The availability of new anti-cestode drugs for use in dogs and a detailed understanding of the transmission dynamics of Echinococcus granulosus sensu lato have underpinned successful programs that have eliminated cystic echinococcosis in some areas of the world and greatly reduced the incidence of infection in others. Despite these successes, cystic and alveolar echinococcosis and neurocysticercosis continue to be prevalent in many parts of the world, requiring new or renewed efforts to prevent the associated taeniid infections. Major advances made in the development of practical vaccines against E. granulosus and Taenia solium will hopefully assist in this endeavour, as might the understanding of the parasites' biology that have come from an elucidation of the nuclear genomes of each of the most important taeniid species causing human diseases.

Alveolar echinococcosis: what triggers emergence in North America, Central Europe and Asia?

PURPOSE OF REVIEW: Infection with the larval (metacestode) stage of Echinococcus multilocularis causes alveolar echinococcosis (AE), a serious hepatic disorder. The parasite has increased its infection extensity in wildlife and domestic dogs, mainly due to urbanization and spatial extension of wildlife hosts in Europe, Asia as well as North America, resulting in emerging infection risk for humans. RECENT FINDINGS: In hyperendemic areas such as Kyrgyzstan and China, ecological and socioeconomic changes have been associated with the unpredictable increase of AE cases. In North America, the appearance of the European-like genotype is of concern. In Europe, the annual increase of human case numbers reached a plateau even in hyperendemic situations. Therefore, we conclude that most of the exposed individuals are resistant to parasite invasion and/or to disease development. Thus, AE develops in a few healthy individuals, but preferentially in immunosuppressed patients. SUMMARY: In the future, improved diagnostic strategies will allow more precise estimations of transmission routes including the role of food, water and direct dog contact, which should yield improved public health recommendations. Finally, understanding protective innate and acquired immune mechanisms as well as parasite-driven immune-evasion processes will be essential to develop curative therapies in nonoperable patients and, futuristically, appropriate vaccines.

Past and present of cystic echinococcosis in Bolivia.

Viable eggs of the canine intestinal tapeworm Echinococcus granulosus sensu lato (s.l.) infect various intermediate hosts causing cystic echinococcosis (CE). Furthermore, CE represents a serious zoonosis causing a significant global burden of disease. CE is highly endemic in South America, including Argentina, Brazil, Chile, Uruguay, and Peru. For Bolivia, no official data concerning the incidence in humans or the number of livestock and dogs infected are available. However, it is well known that CE occurs in Bolivia. We aim here to fill the gap in the current knowledge of the epidemiological situation of CE in Bolivia, providing a historical overview of documents published within the country, which have never been comprehensively reviewed. The very first documentation of E. granulosus infection in animals dates in 1910, while the first human case was reported in 1913. In total, 876 human CE cases have been reported in the scientific literature, with an apparent increase since the 1970s. In the absence of other epidemiological studies, the highest prevalence in human comes from Tupiza, Potosí Department, where 4.1% (51/1,268) of the population showed signs of CE at mass ultrasound screening in 2011. In the same report, 24% of dog faecal samples were positive for coproantigens of E. granulosus s.l. in ELISA. The highest prevalence in intermediate hosts reported at abattoir reached 37.5% in cattle from Potosí, followed by 26.9% in llamas from Oruro, 2.4% in pigs and 1.4% in sheep from La Paz. Finally, Echinococcus granulosus sensu stricto (s.s.), Echinococcus ortleppi (G5), and Echinococcus intermedius (G7) have been identified in Bolivia. Data reviewed here confirm that E. granulosus s.l. is circulating in Bolivia and that a proper prospective nationwide epidemiological study of CE is urgently needed to define transmission patterns as a basis for the planning and implementation of future control measurements.

Association between environmental and climatic risk factors and the spatial distribution of cystic and alveolar echinococcosis in Kyrgyzstan.

BACKGROUND: Cystic and alveolar echinococcosis (CE and AE) are neglected tropical diseases caused by Echinococcus granulosus sensu lato and E. multilocularis, and are emerging zoonoses in Kyrgyzstan. In this country, the spatial distribution of CE and AE surgical incidence in 2014-2016 showed marked heterogeneity across communities, suggesting the presence of ecological determinants underlying CE and AE distributions. METHODOLOGY/PRINCIPAL FINDINGS: For this reason, in this study we assessed potential associations between community-level confirmed primary CE (no.=2359) or AE (no.=546) cases in 2014-2016 in Kyrgyzstan and environmental and climatic variables derived from satellite-remote sensing datasets using conditional autoregressive models. We also mapped CE and AE relative risk. The number of AE cases was negatively associated with 10-year lag mean annual temperature. Although this time lag should not be considered as an exact measurement but with associated uncertainty, it is consistent with the estimated 10-15-year latency following AE infection. No associations were detected for CE. We also identified several communities at risk for CE or AE where no disease cases were reported in the study period. CONCLUSIONS/SIGNIFICANCE: Our findings support the hypothesis that CE is linked to an anthropogenic cycle and is less affected by environmental risk factors compared to AE, which is believed to result from spillover from a wild life cycle. As CE was not affected by factors we investigated, hence control should not have a geographical focus. In contrast, AE risk areas identified in this study without reported AE cases should be targeted for active disease surveillance in humans. This active surveillance would confirm or exclude AE transmission which might not be reported with the present passive surveillance system. These areas should also be targeted for ecological investigations in the animal hosts.

Occurrence of Echinococcusgranulosussensulato and Other Taeniids in Bhutan.

The present research shows the results of a national study documenting the occurrence and genetic diversity of Echinococcus and Taenia species across Bhutan. Environmental dog faecal samples (n = 953) were collected from 2016 to 2018 in all 20 Bhutanese districts, mainly in urbanised areas. Cystic echinococcosis cysts were isolated from 13 humans and one mithun (Bos frontalis). Isolation of taeniid eggs from faeces was performed by sieving/flotation technique, followed by DNA isolation, PCR and sequence analyses for species identification (gene target: small subunit of ribosomal RNA). Genetic diversity of E. granulosuss.s. was based on the sequence (1609 bp) of the cox1 gene. A total of 67 out of 953 (7%) dog faecal samples were positive for at least one taeniid species. From the 670 free-roaming dog faecal samples, 40 (5.9%) were positive for taeniid DNA, 22 (3.2%) of them were identified as E. granulosuss.s. and four (0.5%) as E. ortleppi (G5). From the 283 faecal samples originating from yak-grazing areas, 27 (9.5%) were taeniid positive, including eight (2.8%) infected with E. granulosuss.s. and four (1.4%) with E. ortleppi. E. granulosuss.s. was identified in all isolates from human and the cyst from mithun. A haplotype network (cox1 gene) from E. granulosuss.s, including isolates from 12 dogs, two human and one mithun, revealed eight different haplotypes. The most common cox1 haplotype was the globally distributed Eg01, followed by Eg40 and Eg37 (previously described in China). Five new cox1 haplotypes (EgBhu1-5) originated from human, dogs, and a mithun were identified. The study indicated the contamination of urban areas and pastures with Echinococcus eggs in seven districts in Bhutan. The molecular characterisation of E. granulosuss.l. revealed different E. granulosuss.s. haplotypes as well as E. ortleppi. The transmission of T. multiceps was documented only in the western part of the country. Considering the zoonotic feature of E. granulosus s.s. and E. ortleppi and the economic impact of coenurosis caused by T. multiceps (also known as gid) in Bhutan, the findings of this study represent a significant contribution towards an epidemiological baseline for the establishment of a national control programme.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Agranulocytosis leads to intestinal Echinococcus multilocularis oncosphere invasion and hepatic metacestode development in naturally resistant Wistar rats.

Susceptibility to Echinococcus multilocularis infection considerably varies among intermediate (mostly rodents) and dead-end host species (e.g. humans and pig), in particular regarding intestinal oncosphere invasion and subsequent hepatic metacestode development. Wistar rats are highly resistant to infection and subsequent diseases upon oral inoculation with E. multilocularis eggs, however, after immunosuppressive treatment with dexamethasone, rats become susceptible. To address the role of the cellular innate immunity, Wistar rats were individually or combined depleted of natural killer (NK) cells, macrophages (MΦ) and granulocytes (polymorphonuclear cells, PMN) prior to E. multilocularis egg inoculation. Although NK cell and MΦ depletion did not alter the resistance status of rats, the majority of PMN-depleted animals developed liver metacestodes within 10 weeks, indicating that PMN are key players in preventing oncosphere migration and/or development in Wistar rats. In vitro studies indicated that resistance is not caused by neutrophil reactive oxygen species or NETosis. Also, light microscopical examinations of the small intestine showed that oral inoculation of E. multilocularis eggs does not elicit a mucosal neutrophil response, suggesting that the interaction of oncospheres and neutrophils may occur after the former have entered the peripheral blood. We suggest to consider granulocytes as mediators of resistance in more resistant species, such as humans.

Antibody kinetics and exposure to Toxoplasma gondii in cats: a seroepidemiological study.

Domestic cats are the most important definitive hosts for Toxoplasma gondii, the agent of an important global zoonosis. Serial sera from cats orally inoculated either withT. gondii tissue cysts (n = 3) or sporulated oocysts (n = 3) and from 65 client-owned cats, plus sera from 1,757 client-owned cats presented to veterinarians in Switzerland were analysed for an antibody response to T. gondii by ELISA. Risk factors for seropositivity and prevalence were estimated with a generalised linear and beta regression model. The first model examined the association of an OD405 value as the dependent variable, with gender, age, and outside access as possible independent variables. In the second model, we first analysed the data assuming a bimodal distribution representing two overlapping distributions of OD405 values from positive and negative cats, enabling the assignment of a probability of true infection status to each cat. Mean probabilities of true infection status across groups represent an estimate of true prevalence. These probabilities were then regressed against age, gender and outside access. Antibody kinetics in cats orally inoculated with tissue cysts, shedding oocysts, did not differ significantly from those of cats inoculated with sporulated oocysts without detectable oocyst excretion, suggesting extraintestinal parasite invasion and exposure to tachyzoites in both situations at an early stage of infection. Analysis of serial serum samples suggested a persisting long-term humoral immune response. Of the client-owned cats, 42.4% (95% confidence interval (CI): 40.1-44.6) had a positive true infection status. This was higher (56.3% (95% CI: 53.2-59.6)) in cats with outside access than in those without (22.1% (95% CI: 18.9-25.4)). In the first model, the factors age (P < 0.0001), gender (male: P = 0.046), and outside access (P < 0.0001) were independently associated with significantly higher OD405 values. In the second model, the probability of having a positive true infection status increased with age (P < 0.0001), was higher with outside access (P < 0.0001) and in outdoor male cats (P = 0.0006).

Immunohistological detection of small particles of Echinococcus multilocularis and Echinococcus granulosus in lymph nodes is associated with enlarged lymph nodes in alveolar and cystic echinococcosis.

BACKGROUND: Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. CONCLUSIONS/SIGNIFICANCE: mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp.

New Insights Into the Peculiar World of the Shepherd-Dog Parasites: An Overview From Maremma (Tuscany, Italy).

Several developments have been recently achieved to understand pet-dog parasites and their relationship with hosts; however, parasites' presence and distribution in shepherd-dog have been mainly neglected; this knowledge gap is of critical sanitary importance, as shepherd-dogs could harbor zoonotic helminths including Echinococcus granulosus sensu lato. The related human disease, cystic echinococcosis, is a worldwide neglected disease, with high endemicity in the Mediterranean Basin. To evaluate the presence of E. granulosus and other parasites, a sheep-dog population from the province of Grosseto (Tuscany, Italy) has been investigated. Overall, 648 dog fecal samples obtained from 50 modern sheep farms, having a total of 216 dogs, were collected. Specimens were analyzed using a standardized centrifugal flotation method (specific gravity = 1.3). Taeniid eggs detected were further isolated using a sieving/flotation technique. DNA was isolated from eggs for PCR and sequence analyses for species identification (gene target: 12S rRNA and nad1). Thirty-nine (78%) farms tested positive for at least one parasite species or genus. The most represented intestinal helminths were Toxocara spp. in 64% of farms, followed by Ancylostomatidae (58%), Trichuris vulpis (50%), Capillaria spp. (34%), and taeniids (32%). Sequence analyses confirmed the presence of Taenia hydatigena in seven farms, Taenia (syn. Multiceps) multiceps in five farms, and T. pisiformis in one farm. No DNA was extracted from four previously taeniid egg-positive farms. No amplification of amplicon corresponding to E. granulosus was achieved in the investigated farms. Although not entirely expected, Spearman's test showed a positive correlation between flock size and the number of dogs per farm (ρ = 0.588, P < 0.001). The quantitative analysis reported that the home slaughter practice was affected neither by the flock size nor by the number of dogs per farm. The probability to diagnose farms positive for taeniids had been increased by about 35% for each dog unit increase [odds ratio (OR) = 1.35, P = 0.012]. In conclusion, the wide distribution of T. hydatigena and T. multiceps detected in the present study clearly reveals that dogs have still access to raw offal, a major risk for the transmission of E. granulosus. Home slaughtering is an unavoidable practice, and more efforts must be undertaken by the public health system to prevent and control potential zoonotic taeniids.

Genetic diversity of Echinococcus multilocularis and Echinococcus granulosus sensu lato in Kyrgyzstan: The A2 haplotype of E. multilocularis is the predominant variant infecting humans.

Alveolar and cystic echinococcosis (AE, CE) caused by E. multilocularis and E. granulosus s.l., respectively, are considered emerging zoonotic diseases in Kyrgyzstan with some of the world highest regional incidences. Little is known regarding the molecular variability of both species in Kyrgyzstan. In this study we provide molecular data from a total of 72 parasite isolates derived from humans (52 AE and 20 CE patients) and 43 samples from dogs (23 infected with E. multilocularis and 20 with E. granulosus s.l.).Genetic variability in E. multilocularis was studied using the concatenated complete sequences of the cob, nad2 and cox1 mitochondrial genes adding a total of 3,558bp per isolate. The cob/nad2/cox1 A2 haplotype was identified in 63.4% of the human and in 65.2% of the dog samples. This haplotype was originally described in samples from Kazakhstan and St. Lawrence Island (Alaska, USA). We also describe here 16 non-previously defined variants of E. multilocularis (called A11-A26). All haplotypes cluster together within the Asian group in the haplotype network. Based on Fst values, low level of genetic differentiation was found between the populations of E. multilocularis isolated from different regions within the country. However, high degree of differentiation was found when all the concatenated sequences from Kyrgyzstan are considered as a single population and compared with the population of the parasite from the neighbouring country China. In the case of E. granulosus s.l. the analysis was based in 1,609bp of the cox1 gene. One isolate from a dog was identified as E. equinus, while all the other sequences were identified belonging to E. granulosus s.s. In total, 24 cox1 haplotypes of E. granulosus s.s. were identified including the already described variants: Eg01 (in 6 samples), Eg33 (in 4 samples), EgCl04 (in 2 samples), Eg03 (in 1 sample) and Eg32 (in 1 sample). From the twenty-five other isolates of E. granulosus s.s. a total of 19 non-previously described cox1 haplotypes were identified and named as EgKyr1 to EgKyr19. The most common haplotype infecting human is the EgKyr1 which was found in 5 isolates.The cob/nad2/cox1 A2 haplotype of E. multilocularis is responsible for the majority of human infections in Kyrgyzstan and is also found in the majority of dogs included in this study. Further similar studies in different parts of Asia could elucidate if it is also the most common variant infecting humans in other countries. It remains unknown if this particular haplotype presents differences in virulence which could have contributed to the emergency of alveolar echinococcosis in Kyrgyzstan. In the case of E. granulosus s.s. it seems that there is no dominant haplotype infecting humans in Kyrgzstan. Further characterization of biological or antigenic features of dominant mitochondrial haplotypes could help to elucidate if they present differences which could be relevant in the diagnostic, pathogenicity or in the host/parasite interaction when infecting humans.