Mentha villosa Hubs., M. x piperita and their bioactives against gastrointestinal nematodes of ruminants and the potential as drug enhancers.

Parasitic diseases caused by gastrointestinal nematodes (GIN) are responsible for a major impact on ruminant welfare. Although the available anthelmintics have a safe margin of toxicity to the animals, their indiscriminate use has increased the selection of resistant parasite populations. In this scenario, essential oils (EO) stand out as a promising ecofriendly therapeutic alternative against GIN. The objective of this work was to determine the effect of the EO of Mentha villosa Hubs (MVEO) collected in 2017 and 2018, M. x piperita (MPEO) and their main components, carvone and limonene, against the third stage larvae (L3) of Haemonchus spp. and Trichostrongylus spp. The solutions, including in nanoemulsion preparations, were tested in a range of concentrations using the larval migration inhibition test (LMIT). The EO and carvone were also tested in combination with nitroxynil (NTX) to determine their effect as drug enhancers (additive or synergy). MVEO/2017, MVEO/2018, MPEO and carvone showed 70.6 (73.4 mg/mL), 86.3 (74.9 mL/mL), 95.5 (143.6 mg/mL), and 88.2 % (38.3 mg/mL) efficacy against L3, respectively. Carvone alone had approximately a 3-fold higher efficacy when compared to its concentration in each EO: 68.8 % in MVEO/2017 and 83.9 % in MVEO/2018. Limonene did not show any significant effect on inhibiting L3 migration. The combination of MPEO and NTX, and carvone and NTX showed a statistically significantly (P <  0.05) synergic and additive effect, respectively, when compared to the isolated treatment. The nanoemulsion of MVEO/2017 at 0.367 mg/mL, inhibited L3 migration by 83.1 %, demonstrating to be highly effective (concentration ratio of 1:0.004), when compared to the MVEO/2017 (70.6 % at 73.4 mg/mL) extraction. The in vitro data from the combination of MPEO or carvone plus NTX suggest that these products can be considered for in vivo experiments against the most important GIN of ruminants as drug enhancers, possibly reducing the final concentration of NTX`. The efficacy of carvone was higher (EC50 = 1.96 mg/mL) than its expected efficacy, based on its concentrations on both EO. Therefore, this component does not need the entire EO composition to exert its L3 motility action. The remarkable efficacy demonstrated by the MVEO/2017/nanoemulsion (EC50 = 0.10 mg/mL), supports its potential to be a candidate to the next-generation therapy to alleviate clinical parasite infections and combat GIN resistant populations.

Regulation of the Fasciola hepatica newly excysted juvenile cathepsin L3 (FhCL3) by its propeptide: a proposed 'clamp-like' mechanism of binding and inhibition.

BACKGROUND: The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues. RESULTS: Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site. CONCLUSIONS: The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a "clamp-like" mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

Fascioliasis in buffaloes: A 5-year forecast analysis of the disease based on a 15-year survey in Brazil.

In South America, fascioliasis caused by the trematode Fasciola hepatica is an anthropozoonosis disease associated with significant economic losses and poor animal welfare. The objective of this study was to determine the prevalence of F. hepatica in the liver of buffaloes slaughtered from 2003 to 2017 in Brazil, and to perform a forecast analysis of the disease for the next five years using the Autoregressive Integrated Moving Average (ARIMA) model. Data analysis revealed an incidence of 7,187 cases out of 226,561 individuals. The disease presented a considerable interannual variation (p<0.005). Fasciola hepatica was more prevalent in the southern states of Brazil; Paraná, Rio Grande do Sul, and Santa Catarina, presenting 11.9, 7.7, and 3.2% of infected livers, respectively. The high frequency of liver condemnation in Paraná was influenced by weather conditions. The ARIMA models calculated a constant trend of the disease, depicting an average of its future prevalence. The models also described a worse-case and a positive-case scenario, calculating the effects of intervention measurements. In reality, there is an urgent need for regular diagnostic in the animals (fecal and immune diagnose) and in the environment (intermediate host), in order to avoid the high rates of infection.

First reported case of clinical fascioliasis in Santa Catarina, Brazil.

Fascioliasis is a food-borne anthropozoonotic disease caused by Fasciola hepatica that affects multiple hosts, including humans. We herein report the first case of human fascioliasis in the state of Santa Catarina, Brazil. A 57-year-old female patient complaining of abdominal pain was admitted to the hospital for a clinical investigation. The diagnosis of F. hepatica was confirmed by ultrasound and indirect enzyme-linked immunosorbent assay. Authorities of the Northern coast of Santa Catarina were notified to investigate other cases and risk factors for contamination. The disease is also prevalent in cattle, which could pose as a potential route for infection.

Fascioliasis in buffaloes: A 5-year forecast analysis of the disease based on a 15-year survey in Brazil / Fasciolose em búfalos: uma análise de previsão de 5 anos da doença com base em uma pesquisa de 15 anos no Brasil

Abstract In South America, fascioliasis caused by the trematode Fasciola hepatica is an anthropozoonosis disease associated with significant economic losses and poor animal welfare. The objective of this study was to determine the prevalence of F. hepatica in the liver of buffaloes slaughtered from 2003 to 2017 in Brazil, and to perform a forecast analysis of the disease for the next five years using the Autoregressive Integrated Moving Average (ARIMA) model. Data analysis revealed an incidence of 7,187 cases out of 226,561 individuals. The disease presented a considerable interannual variation (p<0.005). Fasciola hepatica was more prevalent in the southern states of Brazil; Paraná, Rio Grande do Sul, and Santa Catarina, presenting 11.9, 7.7, and 3.2% of infected livers, respectively. The high frequency of liver condemnation in Paraná was influenced by weather conditions. The ARIMA models calculated a constant trend of the disease, depicting an average of its future prevalence. The models also described a worse-case and a positive-case scenario, calculating the effects of intervention measurements. In reality, there is an urgent need for regular diagnostic in the animals (fecal and immune diagnose) and in the environment (intermediate host), in order to avoid the high rates of infection.

Resumo Na América do Sul, a fasciolose causada pelo Trematoda Fasciola hepatica é uma antropozoonose associada a perdas econômicas significativas e baixo grau de bem-estar animal. O objetivo deste estudo foi determinar a prevalência de F. hepatica no fígado de búfalos abatidos entre 2003 a 2017 e realizar uma análise de previsão da doença para os próximos cinco anos, utilizando o modelo Auto-Regressivo Integrado de Médias Móveis (ARIMA). A análise dos dados revelou uma incidência total de 7.187 casos em 226.561 indivíduos. Houve um acentuado grau de variação interanual nas taxas de prevalência (p<0,005). Fasciola hepatica foi mais prevalente nos estados do sul do Brasil; Paraná, Rio Grande do Sul e Santa Catarina, com 11,9; 7,7; e 3,2% de fígados condenados, respectivamente. A alta incidência de condenação de fígado no Paraná foi influenciada pelo fator climático. Os modelos ARIMA indicaram uma tendência constante na ocorrência da doença, destacando um padrão futuro da doença. Os modelos também descreveram cenários de piora e de possível melhoria, calculando os efeitos de medidas de intervenção. Assim, existe a urgência de realizar diagnóstico constante nos animais (coprológico e diagnóstico imunológico) e no ambiente, para que se evite os altos índices de infecção.

The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola.

Fasciolosis caused by trematode parasites of the genus Fasciola is a global disease of livestock, particularly cattle, sheep, water buffalo and goats. It is also a major human zoonosis with reports suggesting that 2.4-17 million people are infected worldwide, and 91.1 million people currently living at risk of infection. A unique feature of these worms is their reliance on a family of developmentally-regulated papain-like cysteine peptidases, termed cathepsins. These proteolytic enzymes play central roles in virulence, infection, tissue migration and modulation of host innate and adaptive immune responses. The availability of a Fasciola hepatica genome, and the exploitation of transcriptomic and proteomic technologies to probe parasite growth and development, has enlightened our understanding of the cathepsin-like cysteine peptidases. Here, we clarify the structure of the cathepsin-like cysteine peptidase families and, in this context, review the phylogenetics, structure, biochemistry and function of these enzymes in the host-parasite relationship.

First reported case of clinical fascioliasis in Santa Catarina, Brazil

Abstract Fascioliasis is a food-borne anthropozoonotic disease caused by Fasciola hepatica that affects multiple hosts, including humans. We herein report the first case of human fascioliasis in the state of Santa Catarina, Brazil. A 57-year-old female patient complaining of abdominal pain was admitted to the hospital for a clinical investigation. The diagnosis of F. hepatica was confirmed by ultrasound and indirect enzyme-linked immunosorbent assay. Authorities of the Northern coast of Santa Catarina were notified to investigate other cases and risk factors for contamination. The disease is also prevalent in cattle, which could pose as a potential route for infection.

Chemical Characterization of Opuntia ficus-indica (L.) Mill. Hydroalcoholic Extract and Its Efficiency against Gastrointestinal Nematodes of Sheep.

Opuntia ficus-indica (L.) Mill. is a xerophylous plant that originated in tropical and subtropical America. This plant is popularly known in Brazil as "palma forrageira" (cactus pear) and plays a fundamental role in animal nutrition, mainly in the Northeastern semi-arid region of the country. The plant has several uses since it presents bioactive compounds that confer biological and pharmacological properties. In this context, the cactus pear can also be considered a potential product to combat parasite infections. The objective of this study was to chemically characterize the O. ficus-indica hydroalcoholic extract (OFIEOH) and to determine its efficacy against gastrointestinal parasites using in vitro tests. Initially, the hydroalcoholic extract from cladode peels of O. ficus-indica was produced by maceration for 21 days. For the chemical characterization, colorimetric dosages were performed for carbohydrates, proteins, phenols and condensed tannins. Liquid chromatography coupled to mass spectrometry/electron spray ionization (LC-MS/ESI) was used to characterize the polyphenolic profile of the OFIEOH extract. Fifteen compounds were identified in the OFIEOH extract, such as methyl, glycosylated and aglycone quercetin derivatives and aglycone and glycosylated kaempferol derivatives. Tri-glycosylated methyl quercetin derivatives were the main compounds identified. In vitro egg hatch (EHT) and larval migration tests (LMT) were used in a range of concentrations of OFIEOH from 12.5 to 100 mg/mL for EHT and 12.5 to 200 mg/mL for LMT. In addition, the LMT was used to test ivermectin (IVM) (from 11.4 to 57.1 µM), associated with the inhibitory concentration of 50% (IC50) for OFIEOH. The combination of OFIEOH (12.5 to 200 mg/mL) plus the IC50 of IVM was also tested. The efficacy of OFIEOH alone varied from 19.33 to 90.0% using the EHT. The LMT revealed an efficacy of 5.78 to 77.26% for the extract. Both tests showed a concentration-dependence inhibitory effect. We found a drug-extract antagonistic neutralizing effect when doses of IVM were added to OFIEOH (maximum efficacy of 73.78%), while a positive additive effect was observed when OFIEOH was added to the IC50 of IVM (IC50 of 82.79 for OFIEOH alone against an IC50 of 55.08 of OFIEOH + IVM). The data from this work indicate that OFIEOH alone may be considered as a suitable ecofriendly product to control gastrointestinal parasites of sheep, offering a more holistic approach to improve animal farming and welfare. The drug-extract interaction is also a promising therapeutic alternative, reducing the final dose to the host, with an optimum combination effect.

Recount of reported cases of human fascioliasis in Brazil over the last 60 years

Fascioliasis is an important anthropozoonotic disease caused by the ubiquitous trematode helminth, Fasciola spp. Here, as elsewhere, it is thought that the disease lacks proper reporting, and the available literature does not reflect unreported cases found in the Brazilian population, or new recently reported cases. The purpose of this work was to perform a recount of human fascioliasis (HF) cases in Brazil. For this, we considered all positive cases published in local and international official Journals, from 1950 to 2016. A theoretical-conceptual research method based on a systematic bibliographic review was applied to identify, select and index articles using the Endnote Basic Software. Here, only 48 cases of HF were found, of which 21 (43.7%) occurred in the South of the country. The small number of reported cases reflects the difficulty in diagnosing HF correctly (clinical and fecal tests). This work provides a real figure of HF reported cases in Brazil and has also corrected inaccurate information found in the literature by conducting a historical survey of the disease. Fasciola hepatica is highly endemic in ruminants and, hypothetically, the number of human cases should also be considerably higher than that reported in the literature. These findings call for more attention in regard to this neglected disease in Brazil.

Bovine fascioliasis in Brazil: Economic impact and forecasting.

Fasciola hepatica causes liver damage and poor growth in cattle and other animals, including humans. Although the disease occurs throughout the country, it is hyperendemic in cattle in the South of Brazil. This work aimed to determine the economical loss by carcass weight variance using data from all the states where F. hepatica is found, as well as to run a disease forecast analysis for the Rio Grande do Sul State. We found a direct loss of approximately US$ 210 million/year from infected cattle in Brazil and the ARIMA model analysis revealed that an increase of fascioliasis is most probable if no parasite control program is adopted.

Ocorrência de geohelmintos em areia de locais públicos municipais de Videira e Itá SC, Brasil / Occurrence of parasites in public places of sand municipal Videira and Itá, SC, Brazil

A areia das áreas de lazer presentes em praças públicas pode constituir uma via de transmissão de doenças, com potencial risco para as crianças. Um grande número de espécies que podem ser encontradas na areia são potentes agentes patogênicos. Por este motivo, foi expressa a preocupação de que estes locais possam atuar como reservatórios de doenças. Portanto, este estudo objetiva avaliar a qualidade da areia de praças nas cidades de Videira e Itá SC, sob o ponto de vista parasitológico, utilizando os métodos de Lutz e Rugai. No presente trabalho, através da análise microscópica do material coletado, foi possível observar que 40 % das praças analisadas apresentavam-se contaminadas. Foram encontrados ovos de Ascaris lumbricoides e larvas de ancilostomídeos, indicando que a população que frequenta estes locais apresenta risco de contrair estas parasitoses se não forem tomadas medidas de sanitização e prevenção.

The sand of recreational areas found in public squares can be a route of transmission of disease, with potential risk to children. A large number of species that can be found in the sand are potent pathogenic agents. For this reason, concern has been expressed that these places can act as reservoirs or disease vectors. Therefore, this study aims to evaluate the quality of the sand public squares in the cities of Videira and Ita SC, under the parasitological point of view, using Lutz and Rugai methods. In this study, through the microscopic analysis of the collected material, it was observed that 40% of the analyzed squares had become contaminated. Ascaris lumbricoides and hookworm larvae were found, indicating that the population that frequents these places is at risk of contracting these parasites if not taken measures of sanitation and prevention.