Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Identification of hard ticks (Acari: Ixodidae) and seroprevalence to Theileria parva in cattle raised in North Kivu Province, Democratic Republic of Congo.

This study aimed to identify tick species and to determine their relationship with the Theileria parva seroprevalence in cattle raised under an extensive farming system in North Kivu Province, Democratic Republic of Congo in two agro-ecological zones namely medium (1,000-1,850 m) and high (>1,850 m) altitude. Among the 3,215 ticks collected on 482 animals, from February to April 2009, Rhipicephalus appendiculatus (64.26 %), the main vector of T. parva, was the most abundant species followed by Rhipicephalus decoloratus (35.49 %) and Amblyomma variegatum (0.25 %). The mean burden of R. appendiculatus tick per infested animal appeared significantly higher at medium (6.5 ± 0.22 ticks) than at high (0.07 ± 0.3 ticks) altitude (P < 0.05). However, an indirect fluorescent antibody test carried out on 450 blood samples revealed a global T. parva seroprevalence of 43 % (95 % CI: 38-47) which was not significantly (P > 0.05) different between medium (48.4 %; 95 % CI: 38-49) and high (41.9 %; 95 % CI: 35-49) altitude. These relatively low seroprevalences suggest that there is a state of endemicity to T. parva infection in the study area. The presence of the tick vector on animals was associated with an increased risk of being seropositive to T. parva infection (odds ratio = 2.04; 95 % CI: 1.8-2.3; P < 0.001). The results suggest the need for a longitudinal study to investigate the seasonal dynamics of tick species and T. parva infection. The rate of tick infection should also be evaluated in order to determine the intensity of T. parva transmission to cattle.

Morphological and molecular characterization of lymnaeid snails and their potential role in transmission of Fasciola spp. in Vietnam.

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.

Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium.

Anaplasmosis and babesiosis are major tick-borne diseases with a high economic impact but are also a public health concern. Blood samples collected in the spring, summer, and autumn of 2010 from 65 cows in seven different farms in Belgium were monitored with an indirect immunofluorescence antibody test to assess seroprevalence against these pathogens. Seroprevalences to Babesia spp. were measured as 10.7%, 20%, and 12.3% in spring, summer, and autumn, respectively, whereas seroprevalences to Anaplasma phagocytophilum were 30.8%, 77%, and 56.9%, respectively. A total of 805 Ixodes ricinus ticks were collected at the same time from both cattle (feeding ticks) and grazed pastures (questing ticks). The infection level of ticks, assessed by PCR assay, for Babesia spp. DNA was 14.6% and 7.9% in feeding and questing ticks, respectively, whereas 21.7% and 3% of feeding and questing ticks were found be positive for A. phagocytophilum cDNA. Fifty-five PCR-positive samples were identified by sequencing as Babesia sp. EU1, of which five from feeding ticks were positive for both A. phagocytophilum and Babesia sp. EU1. The high density of wild cervids in the study area could explain these observations, as deer are considered to be the main hosts for adults of I. ricinus. However, the absence of Babesia divergens both in feeding and questing ticks is surprising, as the study area is known to be endemic for cattle babesiosis. Increasing cervid populations and comorbidity could play an import role in the epidemiology of these tick-borne diseases.

Human trichinellosis in Southeast Asia, 2001-2021.

To present the situation of human trichinellosis in Southeast Asia in the last 20th years we analyzed outbreak data and seroprevalence studies from 2001 to 2021 for this region. We queried PubMed (https://pubmed.ncbi.nlm.nih.gov) using keywords "Trichinella", "human" and "Southeast Asia". In addition, we described Trichinella species circulating in this region. In Southeast Asia, in communities eating pork, several cultural factors play important roles in the transmission of Trichinella to humans. The seroprevalences of Trichinella infection in humans are known for Laos and Vietnam to be 0-10.5% in some villages. Also, in Cambodia, Laos, Malaysia, Thailand and Vietnam relatively few human outbreaks (13) and cases (1604) have been recorded during the last 21st years. Their associated mortality rates were low (0.75%). Trichinella spiralis and T. papuae were transmitted after consumption of raw or undercooked pork from domesticated and wild pigs. T. papuae transmission was related to consumption of wild boar. In this region, trichinellosis was frequently subclinical and clinical or severe cases were sporadic and occurred more in male patients. Nevertheless, it is likely that trichinellosis is widely under-diagnosed and is an endemic disease.

Population genetics, invasion pathways and public health risks of the raccoon and its roundworm Baylisascaris procyonis in northwestern Europe.

The geographic range of the zoonotic raccoon roundworm (Baylisascaris procyonis) is expanding together with the range of its host, the raccoon (Procyon lotor). This creates a new public health risk in parts of Europe where this parasite was previously absent. In the Netherlands, a raccoon population is becoming established and incidental findings of B. procyonis have been reported. To assess the risk to public health, the prevalence of B. procyonis was determined in the province of Limburg, where currently the largest Dutch raccoon population is present, as well as in the adjoining region of southern Belgium. Furthermore, genetic methods were employed to assess invasion pathways of both the raccoon and B. procyonis to aid in the development of control measures. Macroscopic analysis of intestinal content and testing of faecal samples were performed to detect B. procyonis adults and eggs. The population genetics of both B. procyonis and its raccoon host were analysed using samples from central and northwestern Europe. B. procyonis was found in 14/23 (61%, 95% CI: 41%-78%) raccoons from Limburg, but was not detected in 50 Belgian raccoons. Genetic analyses showed that the majority of the Dutch raccoons and their roundworms were introduced through ex-captive individuals. As long as free-living raccoon populations originate from captivity, population control methods may be pursued. However, natural dispersal from the border regions will complicate prolonged population control. To reduce the public health risk posed by B. procyonis, public education to increase awareness and adapt behaviour towards raccoons is key.

Stunting, Beyond Acute Diarrhoea: Giardia Duodenalis, in Cambodia.

BACKGROUND: The adverse outcomes of malnutrition on the development of a child are well acknowledged as are the broad variety of contextual factors that may impact child nutritional status. Adequate nutrient intake and the adoption of appropriate water, sanitation and hygiene measures are largely documented for their positive influence on health. Improved sanitation and protection from human feces can significantly lower the incidence of diarrhea and environmental enteropathy. However, the impact of excessive exposure to animal feces on child health is less well documented. OBJECTIVES: This study tests the hypothesis that there is a positive association between exposure to animal feces, morbidity and anthropometric outcomes in children under 5 years of age, in Cambodia. It aims to improve insights that can contribute to discerning high-impact policies that promote children can develop to their full potential. METHODS: Data for this study was drawn from the third follow-up round of the MyHealth project cohort study that is conducted in six districts of three Cambodian provinces (Phnom Penh, Kratie and Ratanak Kiri). The analysis included a sample of 639 children under 5 years of age. RESULTS: The presence of livestock and more particularly, pigs near the main household dwelling was found a risk factor associated with Giardia duodenalis infection (23%). Giardia duodenalis infection was found to be a protective factor for acute diarrhea, yet, associated with stunting in the univariate model. CONCLUSIONS: Preventive measures that protect from extensive exposure to animal feces may be most effective to prevent infection with Giardia duodenalis and consequent stunting, thereby improving the potential for a healthy development in young Cambodian children. The results support the need for cross-sector policy measures that reinforce comprehensive early childhood interventions towards improving nutritional status as part of a wider set of child welfare and development measures.

Beta-lactam resistance among Enterobacteriaceae in Cambodia: The four-year itch.

Although antibiotics are too often used inappropriately in Cambodia, published data on antimicrobial resistance in this country are scarce. Epidemic dissemination and the transfer of resistance genes to other bacterial species put the population at risk. The aim of this study was to evaluate the frequency and characteristics of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) isolated in consecutive samples tested at Institut Pasteur du Cambodge over a 4-year period (2012-2015). Antimicrobial susceptibility testing was performed by disk diffusion on agar technique and the results were read automatically using an OSIRIS system. The Etest was used to determine minimum inhibitory concentrations (MIC) for some resistance phenotypes. The strain most commonly identified was Escherichia coli (63.9%). The proportion of ESBL-E increased gradually over the study period, from 23.8% to 38.4%. ESBL was detected in 42.7% of the E. coli strains and 33.7% of all Klebsiella pneumoniae isolated. The proportion of ESBL-producing E. coli increased significantly from 28.9% in 2012 to 48.2% in 2015, while the increase for K. pneumoniae remained non-significant. Multidrug resistance was high in this Cambodian series, with some strains displaying resistance to all antibiotics available in the country. There is currently no established system for the surveillance of antimicrobial resistance in Cambodia. Collecting samples from clinical settings throughout the country is critical to assess the impact of antimicrobial drug use in patients in Cambodia and in the Mekong Region.

First evidence of Besnoitia bennetti infection (Protozoa: Apicomplexa) in donkeys (Equus asinus) in Belgium.

BACKGROUND: Besnoitiosis is caused by different species of intracellular protozoan parasites belonging to the family Sarcocystidae and affecting multiple host species worldwide. Including B. besnoiti, ten species are described infecting animals. Among ungulates, Besnoitia bennetti infects horses, donkeys and zebras and was described in Africa and in the USA where donkey besnoitiosis is considered as an emerging disease. CASE PRESENTATION: A two-year-old male donkey was purchased in May 2016 in poor body condition (cachexia, alopetic areas and pruritus mainly on neck and head) by the present owner in Le Roeulx (Belgium) from a milk producing donkey farm in Frasnes-lez-Buissenal (Belgium). Shortly after its purchase and shearing, the donkey presented with crusts, hyperkeratosis (both flanks and neck) anorexia and cachexia. A treatment with phoxim was given with no improvement. A cutaneous biopsy of hyperkeratotic skin was performed in July. It showed a perivascular eosinophilic infiltrate with a large thick walled cyst located in the dermis containing numerous bradyzoites. This was highly suggestive of besnoitiosis. Several skin biopsy samples were obtained for qPCR analysis and confirmed the presence of Besnoitia spp. DNA. Further laboratory diagnosis tests were performed (western blot and rDNA sequencing) confirming Besnoitia bennetti aetiology for the male. For the female, the punch-biopsy, haematology and qPCR were negatives but the western blot showed the presence of antibodies directed to Besnoitia spp. Further clinical examination performed in August highlighted scleral pinhead sized cysts (pearl) in the right eye and between nares. Another ten-year-old female donkey purchased in France and sharing the same accommodation showed a good clinical condition, but a thorough clinical examination showed the presence of numerous cysts on the inner face of upper labial mucosa. A daily treatment based on sulfamethaxzole and trimethoprim (Emdotrim 60% Mix®, 30 mg/kg) was given orally and some improvement was noticed. CONCLUSION: This is the first evidence of Besnoitia bennetti infection (Protozoa: Apicomplexa) in donkeys (Equus asinus) in Belgium.

Swimmer's Itch in Belgium: First Recorded Outbreaks, Molecular Identification of the Parasite Species and Intermediate Hosts.

BACKGROUND: Cercarial dermatitis or swimmer's itch is a skin condition in humans due to the larval forms of bird schistosomes of some species of the genus Trichobilharzia. The life cycle of these schistosomes requires freshwater snails (intermediate host) and waterfowl (definitive host). Repeated exposures to cercariae can lead to skin sensitization with the induction of pruritic skin lesions. METHODS: We describe, in this study, two outbreaks of human cercarial dermatitis at the Eau d'Heure Lakes, Belgium. In July and August 2012, a total of, respectively, 78 and 10 people reported a sudden skin rash accompanied by pruritus following recreational activities in the Plate Taille Lake. However, no ocellate furcocercariae were detected following light exposure of the snails collected between September 2012 and September 2013 (n = 402). No outbreaks were recorded in 2013 and 2014. In August 2015, about 30 new cases were recorded. Snails were collected (n = 270) in different locations around the lake. PCR was used to identify accurately the intermediate hosts and the parasite species involved. RESULTS: After light exposure, seven Radix spp. (2.6%) shed ocellate furcocercariae. Molecular identification based on the rDNA internal transcribed spacer 2 sequence ascribed the infected snails to R. balthica ( = R. peregra = R. ovata) (6/7) and R. auricularia (1/7). Based on the amplification of the D2 domain of the 28S rDNA, the cercariae were shown to belong to two different haplotypes of Trichobilharzia franki. CONCLUSIONS: This is the first record in Belgium of T. franki and associated skin condition.

Is Galba schirazensis (Mollusca, Gastropoda) an intermediate host of Fasciola hepatica (Trematoda, Digenea) in Ecuador?

Fasciolosis is a widely distributed disease in livestock in South America but knowledge about the epidemiology and the intermediate hosts is relatively scarce in Ecuador. For three months, lymnaeid snails were sampled (n = 1482) in Pichincha Province at two sites located in a highly endemic area. Snails were identified (based on morphology and ITS-2 sequences) and the infection status was established through microscopic dissection and a multiplex polymerase chain reaction (PCR)-based technique. Techniques based on morphology were not useful to accurately name the collected snail species. Comparison with available DNA sequences showed that a single snail species was collected, Galba schirazensis. Live rediae were observed in 1.75% (26/1482) and Fasciola sp. DNA was detected in 6% (89/1482) of collected snails. The COX-1 region permitted identification of the parasite as Fasciola hepatica. The relative sensitivity and specificity of the microscope study, compared to PCR results, were 25.84% and 99.78%, respectively. The mean size of the snails recorded positive for F. hepatica through crushing and microscopy was significantly higher than the mean size of negative snails, but there was no such difference in PCR-positive snails. The role of G. schirazensis as an intermediate host of F. hepatica in Ecuador is discussed and the hypothesis of an adaptation of the parasite to this invasive snail is proposed. For the first time, an epidemiological survey based on molecular biology-based techniques assessed the possible role of lymnaeid snails in the epidemiology of fasciolosis in Ecuador.

First report of a fatal autochthonous canine Angiostrongylus vasorum infection in Belgium.

Canine angiostrongylosis is considered as an emergent disease in Europe and Canada. A fatal case of Angiostrongylus vasorum infection is described in a four and a half month old puppy born in Belgium. The dog was presented with marked neurological disorders, body weight loss, a profound weakness and mild respiratory signs. The dog was given antibiotics and mucolytic compounds with very little improvement and consequently was referred to a specialist for additional examinations. As the general condition of the dog was rapidly declining, the animal was euthanized shortly after on its owners' request and a necropsy was carried out. Extensive gross pulmonary lesions were observed and histopathological examination revealed the presence of numerous larvae with morphology compatible with A. vasorum. Larvae were also found in the product of a bronchoalveolar lavage but fecal material was not examined. The presence of A. vasorum circulating serum antigen was demonstrated through ELISA; additionally an A. vasorum specific PCR was performed on brain material and yielded a positive result. This case confirms that the clinical diagnosis of canine angiostrongylosis can be very challenging especially when respiratory signs are absent or very mild such in the present case. This is the first reported case of canine angiostrongylosis in Belgium.

New insight in lymnaeid snails (Mollusca, Gastropoda) as intermediate hosts of Fasciola hepatica (Trematoda, Digenea) in Belgium and Luxembourg.

BACKGROUND: The present study aims to assess the epidemiological role of different lymnaeid snails as intermediate hosts of the liver fluke Fasciola hepatica in Belgium and Luxembourg. METHODS: During summer 2008, 7103 lymnaeid snails were collected from 125 ponds distributed in 5 clusters each including 25 ponds. Each cluster was located in a different biogeographic area of Belgium and Luxembourg. In addition, snails were also collected in sixteen other biotopes considered as temporary wet areas. These snails were identified as Galba truncatula (n = 2474) (the main intermediate host of F. hepatica in Europe) and Radix sp. (n = 4629). Moreover, several biological and non-biological variables were also recorded from the different biotopes. DNA was extracted from each snail collected using Chelex® technique. DNA samples were screened through a multiplex PCR that amplifies lymnaeid internal transcribed spacer 2 gene sequences (500-600 bp) (acting as an internal control) and a 124 bp fragment of repetitive DNA from Fasciola sp. RESULTS: Lymnaeid snails were found in 75 biotopes (53.2%). Thirty individuals of G. truncatula (1.31%) and 7 of Radix sp. (0.16%) were found to be positive for Fasciola sp. The seven positive Radix sp. snails all belonged to the species R. balthica (Linnaeus, 1758). Classification and regression tree analysis were performed in order to better understand links and relative importance of the different recorded factors. One of the best explanatory variables for the presence/absence of the different snail species seems to be the geographic location, whereas for the infection status of the snails no obvious relationship was linked to the presence of cattle. CONCLUSIONS: Epidemiological implications of these findings and particularly the role of R. balthica as an alternative intermediate host in Belgium and Luxembourg were discussed.

Wild cervids are host for tick vectors of babesia species with zoonotic capability in Belgium.

Babesiosis is a tick-borne disease caused by different species of intraerythrocytic protozoan parasites within the genus Babesia. Different species of Babesia are described as potentially zoonotic and cause a malaria-like disease mainly in immunocompromised humans. Interest in the zoonotic potential of Babesia is growing and babesiosis has been described by some authors as an emergent zoonotic disease. The role of cervids to maintain tick populations and act as a reservoir host for some Babesia spp. with zoonotic capability is suspected. To investigate the range and infection rate of Babesia species, ticks were collected from wild cervids in southern Belgium during 2008. DNA extraction was performed for individual ticks, and each sample was evaluated for the absence of PCR inhibition using a PCR test. A Babesia spp. genus-specific PCR based on the 18S rRNA gene was applied to validated tick DNA extracts. A total of 1044 Ixodes ricinus ticks were collected and 1023 validated samples were subsequently screened for the presence of Babesia spp. DNA. Twenty-eight tick samples were found to be positive and identified after sequencing as containing DNA representing: Babesia divergens (3), B. divergens-like (5), Babesia sp. EU1 (11), Babesia sp. EU1-like (3), B. capreoli (2), or unknown Babesia sp. (4). This study confirms the presence of potentially zoonotic species and Babesia capreoli in Belgium, with a tick infection rate of 2.7% (95% CI 1.8,3.9%). Knowledge of the most common reservoir source for transmission of zoonotic Babesia spp. will be useful for models assessing the risk potential of this infection to humans.

First molecular evidence of potentially zoonotic Babesia microti and Babesia sp. EU1 in Ixodes ricinus ticks in Belgium.

We report the first molecular evidence of the presence of Babesia sp. EU1 and Babesia microti in Ixodes ricinus ticks in Belgium. A 1-year national survey collected 1005 ticks from cats and dogs. A polymerase chain reaction technique amplifying a part of the 18S rRNA gene detected Babesia spp. in 11 out of 841 selected and validated tick extracts. Subsequent sequencing identified Ba. microti (n=3) and Babesia sp. EU1 (n=6). This study has demonstrated a low infection rate (1.31% with 95% CI: 0.65-2.33) of Babesia spp. carriage in I. ricinus ticks in Belgium but, for the first time, reports two potentially zoonotic species belonging to this genus. Coinfection with Ba. microti and Borrelia burgdorferi sensu stricto also was demonstrated. In addition, this study clearly demonstrates that inhibitors of polymerase chain reaction amplification are present in engorged ticks.

Towards assessing fine-scale indicators for the spatial transmission risk of Fasciola hepatica in cattle.

In order to improve the spatial resolution of current risk maps for fasciolosis in cattle, more knowledge is needed with respect to farm-level factors that determine infection risk. In this study, we visited 39 dairy farms within a predefined low- and high-risk area for fasciolosis in Belgium and assessed their infection status by an indirect bulk tank milk enzyme-linked immunosorbent assay (ELISA). Management factors were collected and all pastured lands of the farms were visited to identify and georeference potential snail habitats. The habitats were visually characterised, investigated for the presence of the intermediate host snails of Fasciola hepatica (i.e. Galba truncatula and Radix spp) and used in a geographical information system (GIS) to construct overlays including information on soil and hydrology. A linear regression model was used to evaluate associations between bulk tank milk ELISA results and farm level management and habitat factors. A logistic, mixed model was used to identify possible risk factors for the presence of intermediate host snails on a potential habitat. Potential snail habitats were found in 35 out of 39 farms. A total of 87 potential habitats were identified and on 29% of these, intermediate host snails were found. The number of potential habitats, the presence of snails, drainage of pastures, month of turnout of the cows, stocking rate, type of watering place and risk area were significantly associated with the bulk tank milk ELISA result and explained 85% of the observed variation. Intermediate host snails were more likely to be present with increasing surface of the potential habitat and on loamy soils. This study confirms the importance of farm management factors in the infection risk for F. hepatica in cattle and highlights that the combination of management factors with characterization of snail habitats is a powerful means to predict the infection risk with F. hepatica at the individual farm level. Further research is needed to investigate how this knowledge can be incorporated in nation-wide spatial distribution models of the parasite.

The detection and quantification of a digenean infection in the snail host with special emphasis on Fasciola sp.

In this review, ten methods used to study digenean infections in their intermediate hosts were compared to determine which one should be used either in the field or in the lab to establish the prevalence and intensity of infections in snails. Snail crushing and snail dissection allow quick establishing of prevalence in natural or experimental infections, whereas histology is considered as the most accurate approach to assess the intensity of infection. The follow-up of cercarial shedding only gave an idea on cercarial production. Among recently developed techniques, polymerase chain reaction (PCR) brings the most accurate information and shows high sensitivity and specificity levels when compared to blotting techniques. The easiness and relatively low cost of the basic PCR protocol make it interesting to investigate the epidemiology of the liver fluke in a lab with limited financial resources. Nevertheless, if this technique allows a relatively good estimation of the prevalence, information concerning the intensity of infection is best obtained through real time PCR. However, at the time being this technique is too expensive to be used routinely in the field. The choice between classical or new techniques is usually based on a compromise, as each technique has its advantages and drawbacks.