Host drivers of canine dirofilariosis in an arid environment of western Argentina.

Canine dirofilariosis is a mosquito-borne zoonotic disease largely caused by Dirofilaria immitis. Mainly present in temperate, semitropical, and tropical areas, its worldwide emergence and spread are causing concern. In Argentina, most cases have been reported in humid regions of the center and northeast of the country. The occurrence of canine dirofilariosis in an arid environment of western Argentina was investigated by blood smears, microhematocrit tube test, and Knott's technique. Association and odds ratio were determined in relation to dog characteristics. Thirty-three dogs (51.6%) had microfilariae, morphologically identified as D. immitis. Knott's test was more sensitive in detecting the positive animals. The odds of harboring microfilariae were 12, 29, and 66 if the dog was male, adult (three to 6 years old), or older (> 7 years old), respectively. The prevalence of canine dirofilariosis herein reported outstands among the highest in Argentina and extends to the west its geographic distribution in the country. Far from being an exception, this epidemiological situation might reflect similar circumstances in several arid locations in the west and center of Argentina. Being this a disease of recent appearance in the region, both veterinarians and physicians should be aware of its potential to cause disease in animals and humans.

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs.

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

Blood parameters and parasite burden in cattle with chronic fascioliasis.

Fascioliasis is a trematodiasis that affects domestic and wild animals as well as humans worldwide. It is a well-recognized disease in livestock, were it produces serious economic losses. Yet in cattle, there is limited information about the burden of liver flukes and its relation to the eggs per gram shed to the environment. There is also lack of knowledge on the effect of parasite load in blood parameters of infected animals, which is important to evaluate the severity and progression of the disease. The objective of this work was to gain insight in these aspects. Cattle from Mendoza province, Argentina, were inspected at a farm and at the abattoir determining the presence or absence of Fasciola hepatica. Each animal was sampled for blood and feces and in the slaughterhouse the livers were inspected. Hematology and blood chemistry parameters were determined, feces were examined for F. hepatica eggs by a quantitative sedimentation technique and livers were thoroughly inspected to determine the number of flukes. Infected cattle presented a mild burden of liver flukes per animal, strongly correlated (r = 0.72) to the number of eggs per gram of feces. The total number of eggs (X̄=35,100) shed per animal to the environment and the type of livestock management techniques in the region exacerbate the role of cattle as efficient reservoirs of this disease. Statistically significant lower red blood cell, lymphocyte and neutrophil counts were observed in infected compared to uninfected animals. All hepatic parameters tested showed highly statistically significant differences (p < 0.001) as well as proteins by cause of rise of globulins in infected cattle. The correlation between the amount of flukes in the liver and the number of eggs per gram of faces indicates coprology as a reliable and cost-effective method to infer parasite burden. The impact of fascioliasis on blood parameters can be of aid for the veterinary practitioner on the assessment of this disease on cattle.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda).

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

A Single Nucleotide Polymorphism Translates into a Radical Amino Acid Substitution at the Ligand-Binding Site in Fasciola hepatica Carboxylesterase B.

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants.

Natural Fasciola hepatica infection in nutria (Myocastor coypus) in Uruguay.

Fascioliasis, the zoonotic disease caused by the trematode Fasciola hepatica, is expanding worldwide, with a 17 million people at risk. Rodents, often recognized as a major source of zoonotic diseases, are affected by F. hepatica, with some species playing important roles in the disease epidemiology. The case reported here in a nutria or kiyá (Myocastor coypus) is the first documented case of F. hepatica in this species in Uruguay. Parasitic burden and total egg production detected are markedly higher than reported previously for this species, confirming its potential role as an effective reservoir and disseminator of liver flukes. Although further research is needed, nutria should be considered when designing effective control programs for fascioliasis.

Hematology and serum chemistry values in captive and wild pichis, Zaedyus pichiy (Mammalia, Dasypodidae).

As part of an ongoing study on the health status of pichis, Zaedyus pichiy (Mammalia, Dasypodidae), blood was collected under manual restraint from 72 free-ranging pichis captured in Mendoza Province, Argentina, between November 2001 and December 2006, and from 22 captive-kept pichis in January 2007. Reference values were established for hematology and serum chemistry. Pichis had lower leukocyte counts and higher mean corpuscular volumes than most other mammals. Blood values were similar for captive and wild pichis, and only a few significant differences were found among genders or age classes.

Report of Amblyomma parvitarsum infesting goats in Midwestern Argentina.

Adult ticks were collected from goats on September 2012 in the locality of Trintrica (35° 17' 19″ S - 68° 44' 6″ W, 1430 m.a.s.l.), Mendoza, Argentina. The specimens were identified as seven females and three males of Amblyomma parvitarsum Neumann, 1901. This record represents the new occurrence of A. parvitarsum in the province of Mendoza, as well as the first report of the parasitism in goats along the geographical range of A. parvitarsum.

Fasciola hepatica infection and association with gastrointestinal parasites in Creole goats from western Argentina.

Goats, called "the cow of the poor", are the livestock species with the most significant population growth worldwide in recent years. Gastrointestinal parasitism constitutes one of the main constraints to its outdoor and extensive breeding in temperate and tropical countries. Despite a Creole goat population of nearly 4 million heads, local reports on parasitological prevalence are scarce, and while Fasciola hepatica infection is spread all over Argentina, the goat is usually neglected as a reservoir and economic losses are not considered. To evaluate gastrointestinal parasitism prevalence and associations between parasite genera and species, with emphasis on fascioliasis, Creole goats from the plateau and Andean regions from western Argentina were investigated by coprological techniques, and associations were statistically assessed. Eighty-five percent (85%) of the animals harbored one or more parasite types, while 46% showed mixed infections. Significant positive associations between F. hepatica + Strongyle eggs, Eimeria sp. + Nematodirus sp. and Nematodirus sp. + Trichuris ovis were detected. Further studies are required to define the causality of these associations and their relevance in epidemiology. F. hepatica is rarely considered as goat parasite in the country, but a 33% prevalence poses an interrogation on the role goats play on the transmission and dissemination of this zoonotic trematode.

Analysis of climate and extrinsic incubation of Dirofilaria immitis in southern South America.

Dirofilariosis, caused by Dirofilaria immitis and D. repens, is spreading in several geographic regions. The development of infective larvae in the mosquito vector (extrinsic incubation) needs an accumulated total of 130 degree-days above the 14 °C threshold, normally expressed as heartworm development units (HDUs). Based on this information, temperature- based models have been developed and applied to evaluate the distribution and spread of Dirofilaria infections in various countries and continents. Despite the confirmed presence of D. immitis in most South American countries, the available information about its epidemiology remains scarce. We analysed the temporal and spatial extrinsic incubation of this parasite in Argentina, Chile and Uruguay, taking into account daily temperatures from 49 meteorological stations during a 30-year period (1982-2012). The theoretically possible number of D. immitis generations was calculated based on the number of meteorological stations that reached the 130-HDUs threshold. The resulting information was spatially interpolated using the inverse weighted distance (IWD) model to produce thematic maps. The model shows that 41 of the meteorological stations reach the threshold needed and that D. immitis transmission is markedly seasonal with a peak in late spring (December), stable during summer (January to March) and declining in the autumn (April and May). Suitable temperatures exist in Uruguay and most of Argentina, whereas D. immitis transmission in Chile is only possible in the north and in the central inlands. The results suggest that the climatic impact on D. immitis transmission must have been minimal in the countries investigated since the annual meteorological records did not change much during the 30-year period analysed.

Detection of Dirofilaria immitis in mid-western arid Argentina.

Dirofilariosis, caused by Dirofilaria immitis and D. repens, is (re-) emerging worldwide. Dogs are the main reservoirs, while human infection has recently become an important focus of interest and attention. In Argentina, canine D. immitis infection has been described in eastern and northern subtropical and temperate humid regions, but never reported in mid-western arid regions so far. In this research note we report for the first time the occurrence of autochthonous human and canine D. immitis infection in the region.

Human fascioliasis in Argentina: retrospective overview, critical analysis and baseline for future research.

In Argentina, human fascioliasis has never been adequately analysed, although having a physiography, climate, animal prevalences and lymnaeids similar to those of countries where the disease is endemic such as Bolivia, Peru and Chile. We performed a literature search identifying 58 reports accounting for 619 cases, involving 13 provinces, their majority (97.7%) from high altitudes, in central mountainous areas and Andean valleys, concentrated in Cordoba (430 cases), Catamarca (73), San Luis (29) and Mendoza (28), the remaining provinces being rarely affected. This distribution does not fit that of animal fascioliasis. Certain aspects (higher prevalence in females in a local survey, although a trend non-significant throughout Argentina) but not others (patient's age 3-95 years, mean 37.1 years) resemble human endemics in Andean countries, although the lack of intensity studies and surveys in rural areas does not allow for an adequate evaluation. Human infection occurs mainly in January-April, when higher precipitation and temperatures interact with field activities during summer holidays. A second June peak may be related to Easter holidays. The main risk factor appears to be wild watercress ingestion (214) during recreational, weekend outings or holiday activities, explaining numerous family outbreaks involving 63 people and infection far away from their homes. Diagnosis mainly relied on egg finding (288), followed by serology (82), intradermal reaction (63), surgery (43), and erratic fluke observation (6). The number of fascioliasis-hydatidosis co-infected patients (14) is outstanding. Emetine appears as the drug most used (186), replaced by triclabendazole in recent years (21). Surgery reports are numerous (27.0%). A long delay in diagnosis (average almost 3.5 years) and high lithiasis proportion suggest that many patients are frequently overlooked and pose a question mark about fascioliasis detection in the country. High seroprevalences found in recent random surveys suggest human endemic situations. This analysis highlights that human fascioliasis may have been overlooked in the past and its real epidemiological situation in high risk rural, mainly altitudinal areas, may currently be underestimated. Results provide a valuable baseline on which to design appropriate multidisciplinary studies on humans, animals and lymnaeids to assess up to which level and in which areas, human fascioliasis may represent a health problem in Argentina.

Characterisation of fascioliasis lymnaeid intermediate hosts from Chile by DNA sequencing, with emphasis on Lymnaea viator and Galba truncatula.

In South America, Fasciola hepatica infection poses serious health problems in both humans and livestock. In Chile, the medical impact appears yearly stable and mainly concentrated in central regions, where the veterinary problem is highlighted by higher animal prevalences. Studies were undertaken by rDNA ITS-2 and ITS-1 and mtDNA cox1 sequencing to clarify the specific status of the lymnaeids, their geographical distribution and fascioliasis transmission capacity in Chile, by comparison with other American countries and continents. Results change the lymnaeid scenario known so far. The lymnaeid fauna of mainland Chile shows to be poor, including only two authochthonous species, Lymnaea viator and Pectinidens diaphana, and a third introduced species of Palaearctic origin Galba truncatula. Both Lymnaea lebruni and Lymnaea patagonica proved to be synonyms of P. diaphana. G. truncatula appears to have always been confused with L. viator and seems distributed from Región VI to Región IX, overlapping with human endemic areas. DNA sequencing results suggest that the absence of correlation between remote sensing data and disease prevalences could be due to transmission capacity differences between L. viator and G. truncatula. Results furnish a new baseline on which to undertake future appropriate studies on transmission, epidemiology and control.

Fascioliasis transmission by Lymnaea neotropica confirmed by nuclear rDNA and mtDNA sequencing in Argentina.

Fascioliasis is widespread in livestock in Argentina. Among activities included in a long-term initiative to ascertain which are the fascioliasis areas of most concern, studies were performed in a recreational farm, including liver fluke infection in different domestic animal species, classification of the lymnaeid vector and verification of natural transmission of fascioliasis by identification of the intramolluscan trematode larval stages found in naturally infected snails. The high prevalences in the domestic animals appeared related to only one lymnaeid species present. Lymnaeid and trematode classification was verified by means of nuclear ribosomal DNA and mitochondrial DNA marker sequencing. Complete sequences of 18S rRNA gene and rDNA ITS-2 and ITS-1, and a fragment of the mtDNA cox1 gene demonstrate that the Argentinian lymnaeid belongs to the species Lymnaea neotropica. Redial larval stages found in a L. neotropica specimen were ascribed to Fasciola hepatica after analysis of the complete ITS-1 sequence. The finding of L. neotropica is the first of this lymnaeid species not only in Argentina but also in Southern Cone countries. The total absence of nucleotide differences between the sequences of specimens from Argentina and the specimens from the Peruvian type locality at the levels of rDNA 18S, ITS-2 and ITS-1, and the only one mutation at the mtDNA cox1 gene suggest a very recent spread. The ecological characteristics of this lymnaeid, living in small, superficial water collections frequented by livestock, suggest that it may be carried from one place to another by remaining in dried mud stuck to the feet of transported animals. The presence of L. neotropica adds pronounced complexity to the transmission and epidemiology of fascioliasis in Argentina, due to the great difficulties in distinguishing, by traditional malacological methods, between the three similar lymnaeid species of the controversial Galba/Fossaria group present in this country: L. viatrix, Galba truncatula and L. neotropica. It also poses a problem with regard to the use, for lymnaeid vector species discrimination, of several molecular techniques which do not show sufficient accuracy, as those relying on the 18S rRNA gene or parts of it, because both L. neotropica and L. viatrix present identical 18S sequence.

Fasciola hepatica infection and association with gastrointestinal parasites in Creole goats from western Argentina / Fasciola hepatica infecção e associação com parasitas gastrintestinais em caprinos crioulos do oeste da Argentina

Goats, called "the cow of the poor", are the livestock species with the most significant population growth worldwide in recent years. Gastrointestinal parasitism constitutes one of the main constraints to its outdoor and extensive breeding in temperate and tropical countries. Despite a Creole goat population of nearly 4 million heads, local reports on parasitological prevalence are scarce, and while Fasciola hepatica infection is spread all over Argentina, the goat is usually neglected as a reservoir and economic losses are not considered. To evaluate gastrointestinal parasitism prevalence and associations between parasite genera and species, with emphasis on fascioliasis, Creole goats from the plateau and Andean regions from western Argentina were investigated by coprological techniques, and associations were statistically assessed. Eighty-five percent (85%) of the animals harbored one or more parasite types, while 46% showed mixed infections. Significant positive associations between F. hepatica + Strongyle eggs, Eimeria sp. + Nematodirus sp. andNematodirus sp. + Trichuris oviswere detected. Further studies are required to define the causality of these associations and their relevance in epidemiology. F. hepaticais rarely considered as goat parasite in the country, but a 33% prevalence poses an interrogation on the role goats play on the transmission and dissemination of this zoonotic trematode.

As cabras, nomeadas como "a vaca dos pobres", são as espécies de gado com o crescimento populacional mais significativo nos últimos anos em todo o mundo. O parasitismo gastrintestinal constitui uma das principais limitações à sua criação extensiva em clima temperado e tropical. Na Argentina, apesar de uma população de caprinos crioulos de cerca de quatro milhões de cabeças, são escassos os relatórios locais de prevalências parasitológicas. Embora a infecção por Fasciola hepatica esteja espalhada em todo o país, as cabras são geralmente negligenciadas como um reservatório, e as perdas econômicas não são consideradas. Para avaliar a prevalência do parasitismo gastrintestinal e associações entre os gêneros e espécies de parasitos, com ênfase na fasciolose, caprinos crioulos da região andina e do planalto do oeste de Argentina foram avaliados por meio de técnicas coprológicas. Oitenta e cinco por cento dos animais hospedaram um ou mais tipos de parasitos, enquanto 46% hospedaram infecções mistas. Foram encontradas associações significativas entre F. hepatica+ ovos de estrongilídeos, Eimeria sp. +Nematodirus sp. e Nematodirus sp. +Trichuris ovis. Mais estudos são necessários para definir a causalidade dessas associações e sua relevância na epidemiologia. Raramente F. hepatica é considerada como um parasito de cabra no país, mas uma prevalência de 33% suscita uma interrogação sobre o papel dos caprinos na transmissão e disseminação desse trematódeo zoonótico.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.