A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus.

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Increased prevalence of canine echinococcosis a decade after the discontinuation of a governmental deworming program in Tierra del Fuego, Southern Chile.

Hydatid disease is a neglected zoonotic parasitic disease caused by cysts of the tapeworm Echinococcus granulosus. Canids, especially domestic dogs, are definitive hosts of the parasite and are the most pragmatic targets for control programs. A governmental dog deworming campaign was established in 1979 to control hydatidosis in southern Chile, which succeeded in reducing the prevalence of canine echinococcosis in Tierra del Fuego province from 68.4% (in 1978) to 1.2% (in 2002). In 2004, however, the program was dismantled to reduce costs, and since then, no follow-up echinococcosis monitoring has been conducted. We surveyed 356 domestic dogs and interviewed owners or workers at 45 ranches in Chilean Tierra del Fuego during the summer of 2015-2016. Faecal flotation was employed to detect Taeniidae eggs, and PCR was used to test faecal samples for Echinococcus granulosus. Taeniidae eggs and Echinococcus sp. DNA were detected in the faeces of 45.4% (147/324) and 6.9% (23/331) of dogs, respectively. Infrequent dog deworming and the presence of culpeo foxes (Lycalopex culpaeus) were significant predictors of the prevalence of Echinococcus sp. DNA and Taeniidae eggs. Furthermore, the presence of introduced chilla foxes (Lycalopex griseus), the municipality, and several operational characteristics of ranches (number of sheep, frequency of sheep slaughter, number of dogs, frequency of removal of dog faeces, feeding of dogs with sheep viscera) were also predictive of the prevalence of Taeniidae eggs. Our findings reveal an ongoing risk of echinococcosis with pathogen maintenance in ranch dogs in Chilean Tierra del Fuego, and in the absence of adequate control programmes, there is a tangible risk of re-emergence of hydatid disease as a public health concern.

Lead exposure in dogs fed game meat and offal from culled invasive species in El Palmar National Park, Argentina.

This is the first field study to evaluate lead exposure in dogs fed game meat and offal and, to our knowledge, the first study exploring associations between game consumption and dog health status. We quantified lead concentrations in blood and hair and haematological parameters of 31 dogs fed game meat and offal from wild boar (Sus scrofa) and axis deer (Axis axis) culled with lead ammunition in El Palmar National Park, Argentina. Despite variable weekly frequency in game consumption, dogs had detectable blood and hair lead levels, demonstrating recent and chronic exposure. Lead geometric mean and SD were 18.91 ± 346.85 µg/dL w.w. in blood (range = 0.53-216.58), and 0.721 ± 6.444 µg/gr d.w. in hair (range = 0.007-34.800). Hair lead levels were relatively low in most samples, except for the oldest dog which had an atypically high value concurrent with anaemia, a common outcome of chronic lead exposure. Dog's owner was significantly predictive of both blood and hair lead levels, which reflects the same feeding patterns for all dogs owned by the same person. Body condition was associated with hair lead, with dogs in good condition presenting higher lead levels. This could be related to greater game consumption by those dogs, resulting in higher lead ingestion. Dogs fed game meat and offal at very low or low frequency (≤4 times per week) showed higher blood lead levels, suggesting there might not be a risk-free frequency for game provision to dogs. Considering the risks of dietary lead exposure, avoiding feeding dogs lead-killed game and replacing lead ammunition with non-toxic alternatives are recommended. This would allow using hunted game as a valuable food resource without unnecessary risk for the health of consumers and the environment.

Lead exposure in consumers of culled invasive alien mammals in El Palmar National Park, Argentina.

Consumption of meat from animals hunted with Pb ammunition can cause toxic accumulation with consequent health risks, even if relatively small amounts are consumed in each exposure. In El Palmar National Park, Argentina, invasive alien mammals, wild boar (Sus scrofa) and axis deer (Axis axis), are culled with Pb ammunition and their meat is consumed. In this study, we evaluated blood Pb concentrations in 58 consumers of culled game and examined Pb exposure risk according to their demographics, duty, and consumption habits. Likewise, the likelihood of exposure was evaluated by quantifying Pb concentrations in meat samples of seven culled axis deer. Twenty-seven participants (46%) had detectable blood Pb levels (limit of detection = 3.3 µg/dL), with an average 4.75 ± 1.35 µg/dL (geometric mean ± geometric S.D.); the average for all participants was 3.25 ± 1.51 µg/dL. Blood Pb concentrations were significantly higher in hunters, in participants who reported consuming game meat more than 5 times per week, and in participants who reported frequently consuming cured game meat (compared to cooked or pickled). Pb concentration varied significantly along the trajectory of the bullet in deer muscle, being highest at mid-point but with detectable Pb levels even in distant tissue samples (control), suggesting potential for dietary intake by consumers. These findings provide evidence of Pb exposure risk in consumers and emphasize the relevance of replacing Pb ammunition with non-toxic alternatives. This change would reduce dietary exposure in frequent consumers and allow the use of game meat as safe food for people whilst eliminating collateral risks to wild animals and the environment.