Figure 1: The Geographical Location of the Futaleufu Department inside the Chubut Province, Southern Argentina, Where the study has been conducted. Red circles indicate the sampling locations (Image from Google Earth).
Images used are in the public domain and have been downloaded from: National Geographic Information Center, Madrid, Spain.
Figure 2: Shell of Chilina spp (up), Physa spp (middle) and Lymnaea spp (below) from Futaleufu Department, Southern Argentina. In each case: a) Ventral views, b) Dorsal views. Photographs credits: Débora Saunders.
Figure 3: Stages of larval trematode founded in snails from Futaleufu Department, Southern Argentina.
a). Redia with immature cercariae of Notocotylidae family in Physa spp.,
b). Monostome type cercaria of Notocotylidae family in Chilina spp.,
c). Monostome type cercaria Notocotylidae family in Lymnaea spp.,
d). Fasciolidae type cercaria in Physa spp (40x Magnification). Photographs credits: Debora Saunders,
e). Encysted metacercarial stages of Notocotylidae founded in watercress (10x Magnification). Photographs credits: Viviana Hechem,
f). Adult Fasciola hepatica recovered from liver sheep (40x Magnification). Photographs credits: Elena Sanero,
g). F. hepatica egg recovery from faecal sample of hares,
h). F. hepatica egg recovery from faecal sample of cattle (40x Magnification). Photographs credits: Elena Sanero.
The aquatic and semiaquatic plants studied were identified as Veronica anagallis-aquatica (locally known as berro, watercress), Mimulus glabratus (locally known as berro amarillo, yellow watercress) Myriophyllum quitense (locally known as hinojo, fennel), and Dysphamia ambrosioides (locally known as paico). Only V. anagallis-aquatica was found positive for the presence of the encysted metacercarial stage of Notocotylidae-like trematode family (n = 3) and F. hepatica (n = 1) (Table 1, Figure 3).
F. hepatica eggs were detected in 14/60 (23.3%) samples of faecal material from different domestic and wild animal species (Table 2, Figure 3).
As seen in Table 3, cattle were the type of animal most affected by fascioliasis over the 2009-2012 periods with a relative frequency of liver condemnation due to fascioliasis of 31.4% in cattle and 6.1% in sheep. Further to that, adult stages of the parasite were detected in condemned liver from both animal species (Figure 3). The direct costs derived from liver condemnation due to fascioliasis were estimated to be about USD 190,887.5 USD and USD 19,920 in bovine and ovine respectively.
This study provides the first extensive description of the diversity and frequency of trematodes from Futaleufu Department, including Los Alerces National Park, a region of Argentina with ecological importance due its value as natural fresh water resource and flora and fauna sanctuary. Ours results confirm the presence of F. hepatica and report the finding of Notocotylidae-like family in different natural environments from Patagonian. In consequence, this investigation complements and expands the limited data on the epidemiology of trematodes in Southern Andean Argentinian region. It’s important pointing out that F. hepatica, as causal agent of water related diseases, inflict considerable morbidity on healthy immunocompetent people, and may cause life-threatening diseases among immunocompromised and immunosuppressed populations . On the other hand, it is noteworthy that the Notocotylidae is an intestinal parasite family of birds and mammals.
In terms of vectors, at least three genera of the aquatic pulmonate gastropod mollusks were identified and we documented the presence of naturally infected snails of Chilina spp., Physa spp. and Lymnaea spp. with Notocotylidae-like and, Lymnaea spp. as well as Physa spp. with F. hepatica. Chilina spp. has been the most ubiquitous genus. It has been established that fasciolid life cycle and lymnaeid population dynamics are markedly dependent on climate, mainly temperature and rainfall . Ours findings suggest that the climate conditions of the region appear to be appropriate for the fluke’s biological cycle development and for fascioliasis transmission, with maximum and minimum registers of 31.4 and 8ºC of temperature, of 92% and 22% of RH and of 182 mm and 52 mm of accumulated precipitations.
One of the most intriguing finding of this study was the detection of some sporocysts of F. hepatica in Physa spp. Arguing in favor of the occurrence of F. hepatica in this intermediate host it was reported that P. acuta could be an unusual snail species involved in the transmission of this trematode in watercress beds in central France, with a prevalence of the snail infection less than 1% . Even been Physa spp. a poor host for this fluke, it is interesting to carry out more exhaustive studies about its role in the transmission of this parasite under natural environmental Patagonian conditions. Nevertheless, this result should be confirmed in the future due to its rarity and the F. hepatica specify characteristics worldwide.
In Argentina, cattle and sheep are natural hosts for F. hepatica  and fascioliasis in wild animals such as Lagomorpha, Cervidae and Rheidae species has also been reported . Our study corroborates the presence of F. hepatica eggs in faecal material belonging to cows, sheep and hares collected in different biotopes of the studied region. The finding of F. hepatica eggs in feces from hares (Lepus europaeus) supports the hypothesis that the liver fluke naturally infects the European brown hare also in Southern Patagonia . Noteworthy, L. europaeus may play a significant role in the trematode transmission cycle given that it shares habitats with livestock and snails and its abundance is high.
Transmission of trematodes is enhanced by cultural practices of eating raw or inadequately cooked food, socio-economic factors, and the availability of wide zoonotic and sylvatic reservoirs . The inhabitants of the area use all the plant species studied for human consumption. It is important to highlight the presence of the metacercariae stage of F. hepatica in specimens of V. anagallisaquatica growing naturally along the river and streambeds. Ingestion of watercress carrying this infective form of the parasite is known to constitute the main fascioliasis infection source in humans worldwide. In particular, in Argentina previous watercress ingestion has been described in 214 patients . To the best of our knowledge, Just one study included the assessment of plant borne contamination in Patagonian region , been the present the first and Southernmost report of watercress contamination with metacercarial stages of F. hepatica and of Notocotylidae.
To the Chubut province, it has been reported prevalence’s of 18.3% in bovines and 2.7% in ovines for the 2006-2009 period , using data from the Agriculture and Food Quality National Service. In our study the true prevalence of F. hepatica infection was estimated at 31.4% (CI95%: 31.1%, 31.8%) in bovines and 6.0% (CI95%: 5.9%, 6.2%). Moreover, Fasciola spp. is the most widespread liver fluke found in cow and sheep and is more dominant in cows than sheep. Our abattoir survey generally reflected the infection situation in the Futaleufu Department. These results indicate that the prevalence in bovine and ovine fascioliasis is higher than previously reported to the whole province. These findings could be attributed to at least two causes, a real increase of the frequency of appearance of the infection in livestock or/and a notification bias from slaughterhouse records to governmental control authorities.
Argentina has a large livestock industry in which F. hepatica causes serious economic impact due to reduction in milk and meat production, liver condemnation, reproductive failure and mortality. We estimated that for the 2009-2012 period direct economic loss due to fascioliasis bovine and ovine condemnation amounts 210,807.5 USD in losses. In order to mitigate the effect of fascioliasis on regional economics, the control of the trematodiasis mainly relies on timely treatment with triclabendazole of livestock and veterinary slaughterhouses inspection. However, this timely treatment is the quickest way to control morbidity associated with fascioliasis, it should be complemented by implementing measures that aim to reduce transmission rates. Nevertheless, there is no a special control program of fascioliasis conducted by local or regional sanitary authorities and/or planned information about the disease that is accessible to the community. In addition, triclabendazole resistance detected in Argentinian cattle in the Patagonian province of Neuquen  supposes a serious question mark on disease transmission control in Argentina. Although the types of control measures to be complemented depend on the setting it would be recommendable to include measures at three different fields . One would be education and communication promoting cultivation of vegetables in water free from faecal pollution as well as thorough cooking of vegetables before human consumption. Related to the veterinary field treating domestic animals and enforcing separation between husbandry and humans should be taken into account. Finally, environmental measures such as containment of the snail intermediate hosts and drainage of grazing land should be implemented. Our epidemiological survey is added to the evidence from other exhaustive studies that have been performed mainly in central and northern Patagonia [19,21-24]. This kind of findings regarding the ecology of definitive and intermediate hosts would not only help to understand the flow patterns of larval digeneans in Patagonian freshwater environments but also provide a valuable baseline on which to design adequate control strategies on fascioliasis in animal reservoirs and snails vectors in this very large and environmentally heterogeneous region of the country.
Our work has some limitations, we have no the possibility to perform experimental infections in laboratory animals to obtain the trematode adult stages and verify their systematic belonging and, molecular studies could not be carried out for specimen classification purposes in snails, with the exception of Physa spp. In this last case we expanded the preliminary finding further. Thus, the presence of Physa acuta was assessed by molecular methods. More information of some life cycle stages and on the ecology of the intermediate hosts is needed to clarify the taxonomic status of the Notocotylidae like parasites. It is surprising that Paramphistomids, that are usual parasites of ruminants everywhere in the world, were not founded in this study. Considering that Paramphistomid larval stages also present eye spots in their larval stages, we used characteristic as ventral sucker absent and the presence of many cystogenous glands in body to differentiate with Notocotylidae type cercaria. Also, the opperculate eggs of this specie of ruminants parasites could be confused with F. hepatica eggs. Again, the impossibility of carrying out molecular studies constitutes a limitation to our results. Therefore, they should be included in future research conducted in this region.
1 Located in Los Alerces National Park. m. a. s. l: meters above sea level, NR: not recorded
Table 1: Collection sites, location data, number of snails sampled and number of naturally infected specimens, presence of contaminated vegetables with metacercarial stages and presence of Fasciola hepatica eggs in animal feces in Patagonian freshwater biotopes of Futaleufu Department, Southern Argentina.
Table 2: Frequency per faecal pellet of positive samples of domestic and wild animals to Fasciola hepatica eggs. Futaleufu Department, Southern Argentina
1Total period prevalence 2009-2012.
Table 3: Annual records of livestock slaughtered in official slaughter houses during 2009-2012 period and related prevalence of fascioliasis. Futaleufu Department, Southern Argentina.
Water for life is the predominant natural resource, which is at the dangerous edge today and it is necessary to protect the resources for future generations. In this sense, we may conclude that this study is of sanitary importance as Futaleufu Department, a key freshwater resource at national level, may be regarded as an endemic region for animal fascioliasis. Ours results would be useful for the application of more effective control strategies against the mentioned water related trematodiasis in the studied region and, in consequence, decrease the economic losses due to condemnation of infected livers with F. hepatica. Likewise, it contributes to expanding the knowledge of the parasitic fauna in this important Southern Argentinean area.