Outbreak of aflatoxicosis in a dairy herd induced depletion in milk yield and high abortion rate in Pakistan.

This case report investigated the outbreak of aflatoxicosis in a dairy herd in Pakistan, which resulted in 30 abortions of 40 confirmed (75%) pregnant cows in a period of 35 days and in 18.8% depression of farm average milk production for the entire herd. The analysis of the concentrate feed of the total mixed ration (TMR), using enzyme-linked immunosorbent assay (ELISA) procedures from two different local laboratories, indicated concentrations of 60 µg/kg dry matter (DM) of aflatoxin B1 (AFB1) and 100 µg/kg DM of total aflatoxins (AFs: sum of B1, B2, G1 and G2). Subsequently, a confirmatory analysis with a more sensitive and validated multi-metabolite liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was performed. This analysis detected a concentration of total AFs in the TMR of 166 µg/kg DM ± 3.5 (AFB1:134, AFB2:17.4 and AFM1:14.9 µg/kg DM). The concentrate feed (55% of the TMR DM) was confirmed as a source of contamination, presenting a concentration >29 times higher than the EU-maximum limit value (5.68 µg/kg DM). Additionally, the multi-mycotoxin analysis evidenced the co-occurrence of 81 other toxic and potentially toxic fungal metabolites in the fed TMR. After replacing the contaminated concentrate feed with feedstuffs of the same formulation but from a new charge of ingredients, the abortion episodes ceased, and milk production increased significantly. In conclusion, the data of this case report suggest that AFs may be associated with pregnancy losses in dairy cattle and milk production depression. From the public health perspective, the data also indicate the need for a more careful examination of dairy animal feed in Pakistan. Since the high concentration of AFB1 detected in feed and considering the literature-reported transfer rates (1-6%) of this toxin to AFM1 (carcinogen for humans) in milk, the milk produced during the outbreak period is expected to be contaminated with AFM1, which raises public health concerns.

Duration of Zearalenone Exposure Has Implications on Health Parameters of Lactating Cows.

There is a limited research focus on evaluating the detrimental effects of prolonged zearalenone (ZEN) intake on dairy cows' health under controlled conditions. This experiment was conducted to evaluate whether the length of exposure to a ZEN-contaminated total mixed ration (TMR) at a level of 9.45 mg per day can negatively influence animal health parameters, such as milk composition, rumen and fecal fermentation, and the chewing activity of lactating dairy cows. For this experiment, we used 18 lactating Simmental cows that were fed a diet of 60% forage and 40% concentrate (on dry matter basis) for 26 consecutive days. The first 4 days were for adaptation prior to the first sampling day (day 0). The sampling events took place on day 0 (baseline) without ZEN, followed by day 1, day 7, day 14, and day 21 (with toxin). Dry matter intake (DMI) and ruminating chews per minute increased on the third week of ZEN inclusion; meanwhile, ruminating, eating, and drinking times were not affected. Most milk composition variables were also unaffected. Rumen fluid osmolality increased on day 21 and total short-chain fatty acids (SCFA) of ruminal fluid decreased on day 7. Fecal SCFA increased on day 21 and the acetate-to-propionate ratio increased from day 1 onwards, showing the influence of toxin intake. Animal health parameters, like heart rate, respiratory rate, and body temperature, were negatively influenced by ZEN intake, all increasing consistently on days 4 and 6, 9 and 12, and 16 and 18, respectively. The liver enzyme glutamate dehydrogenase decreased in response to ZEN intake on day 7. A total daily ZEN intake at the level of 9.45 mg did not show detrimental effects on DMI. Nevertheless, certain health parameters were negatively affected, including body temperature, respiratory rate, and heart rate, starting from the 7th day of ZEN intake, with additional signs of possible loss of water balance on the last sampling day.

Correction: Penagos-Tabares et al. Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico. Toxins 2023, 15, 153.

In the original publication [...].

Changes in the nutrient profile and the load of mycotoxins, phytoestrogens, and pesticides in horse pastures during spring and summer in Austria.

Pastures are used for grazing and the production of conserved roughage in horses. Yet, the nutritional profile of the forage varies from spring to late summer, affecting equine nutrient supply and health. In addition, environmental factors may also favor plant contaminants such as mycotoxins. This study aimed to determine the nutritional profile and contaminant load of selected horse pastures from early spring till late summer. The nutrient composition (main macronutrients, macro elements and trace elements), as well as mycotoxins, metabolites, pesticides, and plant-derived compounds of seven horse pastures were analyzed. Each pasture was sampled three times and the samples were categorized according to the status of the pasture plants: ear emergence, early- till full bloom, and drought-damaged vegetation. Drought-damaged pastures demonstrated a rise in the acid to neutral detergent fiber ratio, calcium, iron, and magnesium but lower potassium contents. Mycotoxins and other contaminants were found in the pastures including 64 fungal compounds (ergot alkaloids (13) and metabolites from Fusarium (21), Aspergillus (2), Penicillium (8), Alternaria (8) and other fungal species (12), one bacterial metabolite (cereulide), twelve plant metabolites (including eight phytoestrogens and three cyanogenic glycosides (linamarin, lotaustralin and prunasin)), 11 nonspecific metabolites and six pesticides. Fusarium metabolites showed the highest concentrations among the fungal metabolites and drought-induced stress increased the contamination levels (range: 123-3873 µg/kg DM). In conclusion, there was a dominant effect of the developmental stages of the plants, botanical composition of the pastures and weather conditions on the nutritional composition and presence of contaminants on pastures.

Co-occurrence of mycotoxins and other fungal metabolites in total mixed rations of cows from dairy farms in Punjab, Pakistan.

After India and the USA, Pakistan is the third country leading in global dairy production, a sector of very high socioeconomic relevance in Asia. Mycotoxins can affect animal health, reproduction and productivity. This study analysed a broad range of co-occurring mycotoxins and fungal secondary metabolites derived from Alternaria, Aspergillus, Fusarium, Penicillium and other fungal species. To complete this, a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was employed, detecting 96 of > 500 tested secondary fungal metabolites. This first preliminary study demonstrated that total mixed rations (TMRs) (n = 30) from big commercial dairy cattle farms (> 200 lactating cows) in Punjab, Pakistan, presented ubiquitous contamination with mixtures of mycotoxins. The mean of mycotoxins per sample was 14, ranging from 11 to 20 mycotoxins among all TMR samples. Metabolites derived from other fungi and Fusarium spp. showed the highest levels, frequency and diversity among the detected fungal compounds. Among the most prevalent mycotoxins were Fusarium toxins like fumonisins B1 (FB1) (93%), B2 (FB2) (100%) and B3 (FB3) (77%) and others. Aflatoxin B1 (AFB1) was evidenced in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 µg/kg at 88% dry matter). No other mycotoxin exceeds the EU guidance values (GVs). Additionally, we found that dietary ingredients like corn grain, soybean meal and canola meal were related to increased contamination of some mycotoxins (like FB1, FB2 and FB3) in TMR from the province of Punjab, Pakistan. Among typical forage sources, the content of maize silage was ubiquitous. Individually, the detected mycotoxins represented relatively low levels. However, under a realistic scenario, long-term exposure to multiple mycotoxins and other fungal secondary metabolites can exert unpredictable effects on animal health, reproduction and productivity. Except for ergot alkaloids (73%), all the groups of metabolites (i.e. derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp. and other fungi) occurred in 100% of the TMR samples. At individual levels, no other mycotoxins than AFB1 represented a considerable risk; however, the high levels of co-occurrence with several mycotoxins/metabolites suggest that long-term exposure should be considered because of their potential toxicological interactions (additive or synergistic effects).

Evaluation of milk yield and composition, feed intake, chewing activities, and clinical variables in dairy cows under hot-humid climate of tropical zone.

Dairy cows increase heat loads when the temperature-humidity index (THI) value is elevated in the ambient environments. This condition often occurs in the tropical areas due to a higher THI rate throughout seasons. The major objective of the study was to investigate the different responses in milk yield and composition, chewing activities, and health parameters in dairy cows under the dry and wet seasons of tropical climate zone in Indonesia. Twenty mid-lactating Indonesian Holstein-Friesian cows (139.3 ± 24.63 DIM; 10 primiparous and 10 multiparous; 441 ± 21.5 kg BW) were randomly subjected to 2 groups, dairy cows under dry (n = 10) and wet season (n = 10). Both groups received the same diets throughout the experiment. To determine the heat stress condition, the THI values were recorded daily. Overall, a higher number of THI was more pronounced in wet season. A lower dry matter intake (DMI) and milk yield were observed in wet season group. A tendency towards higher milk protein contents was found in dairy cows under dry season compared to cows under wet season. The other milk compositions such as fat, lactose, and SNF remained unchanged in both dry and wet season groups. The comparison between both groups at several time points of eating and ruminating time revealed significantly higher in cows under dry season. Overall, a higher chewing per bolus was observed in cows under dry season than their counterparts. Furthermore, a tendential greater extent rectal temperature pointed in the wet season group compared to the dry season group relatively. Data suggest that a stronger heat stress condition in wet season was more pronounced compared to dry season, with adversely affecting stronger declined DMI, milk yield, and chewing activities of dairy cows.

Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico.

Mycotoxins and endocrine disruptors such as phytoestrogens can affect cattle health, reproduction, and productivity. Most studies of mycotoxins in dairy feeds in Mexico and worldwide have been focused on a few (regulated) mycotoxins. In contrast, less known fungal toxins, phytoestrogens, and other metabolites have been neglected and underestimated. This study analyzed a broad spectrum (>800) of mycotoxins, phytoestrogens, and fungal, plant, and unspecific secondary metabolites in whole-plant corn silages (WPCSs) and total mixed rations (TMRs) collected from 19 Mexican dairy farms. A validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was used. Our results revealed 125 of >800 tested (potentially toxic) secondary metabolites. WPCSs/TMRs in Mexico presented ubiquitous contamination with mycotoxins, phytoestrogens, and other metabolites. The average number of mycotoxins per TMR was 24, ranging from 9 to 31. Fusarium-derived secondary metabolites showed the highest frequencies, concentrations, and diversity among the detected fungal compounds. The most frequently detected mycotoxins in TMRs were zearalenone (ZEN) (100%), fumonisin B1 (FB1) (84%), and deoxynivalenol (84%). Aflatoxin B1 (AFB1) and ochratoxin A (OTA), previously reported in Mexico, were not detected. All TMR samples tested positive for phytoestrogens. Among the investigated dietary ingredients, corn stover, sorghum silage, and concentrate proportions were the most correlated with levels of total mycotoxins, fumonisins (Fs), and ergot alkaloids, respectively.

Residues of pesticides and veterinary drugs in diets of dairy cattle from conventional and organic farms in Austria.

Modern agriculture depends highly on pesticides and pharmaceutical preparations, so controlling exposure to these substances in the feed and food chain is essential. This article presents the first study on residues of a broad spectrum of pesticides and veterinary drugs in the diets of dairy cattle. One hundred and two representative samples of the complete diets, including basal feed rations and additional fed concentrate, were collected in three Austrian provinces (Styria, Lower and Upper Austria) in 2019 and 2020. The samples were tested for >700 pesticides, veterinary drugs and related metabolites using a validated method based on liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). In total, 16 residues (13 pesticides and three veterinary drug residues) were detected. > 90% of the diets contained pesticide residues and <10% veterinary drug residues, whereas banned pesticides were not found. The most frequent pesticide residues were fluopyram (62%), piperonyl butoxide (39%) and diethyltoluamide (35%). The following pesticides exceed the default EU maximum residue level (MRL) (10 µg kg-1) for products exclusively used for animal feed production: Benzovindiflupyr (proportion of samples > MRLs: 1%), bixafen (2%), fluopyram (6%), ipconazole (1%) and tebuconazole (3%). Three residues (dinitrocarbanilide, monensin and nicarbazin) of veterinary drugs were identified, all below the MRLs. Over 60% of the evaluated samples contained mixtures of two to six residues/sample. Only one pesticide (diethyltoluamide) presented a significant difference among regions, with higher concentrations in Upper Austria. Brewery's spent grains were the dietary ingredient that showed the strongest correlation to pesticide residues. These findings evidence the realistic scenario of highly occurrent low doses of pesticides cocktails in the feed/food chain, which may affect the animal, human and environmental health. Since the risk assessments are based on single pesticides, the potential synergistic effect of co-occurring chemicals ("cocktail effect") requires further investigations.

Mixtures of mycotoxins, phytoestrogens and pesticides co-occurring in wet spent brewery grains (BSG) intended for dairy cattle feeding in Austria.

Spent brewery grains (BSG) are the main by-product of beer production and are incorporated in rations of food-delivering animals, mainly dairy cows. Like other agricultural commodities, BSG can be contaminated by a broad spectrum of natural and synthetic undesirable substances, which can be hazardous to animal and human health as well as to the environment. The co-occurrence of mycotoxins, phytoestrogens, other fungal and plant secondary metabolites, along with pesticides, was investigated in 21 BSG samples collected in dairy farms in Austria. For this purpose, a validated multi-metabolite liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) was employed. Metabolites derived from Fusarium, Aspergillus, Alternaria and pesticide residues, were ubiquitous in the samples. Zearalenone (ZEN), T-2 and HT-2 toxins were the only regulated mycotoxin detected, albeit at concentrations below the European guidance values for animal feeds. Ergot alkaloids, Penicillium-derived metabolites, and phytoestrogens had occurrence rates of 90, 48 and 29%, respectively. Penicillium metabolites presented the highest levels among the fungal compounds, indicating contamination during storage. Aflatoxins (AFs), ochratoxins and deoxynivalenol (DON) were not detected. Out of the 16 detected pesticides, two fungicides, ametoctradin (9.5%) and mandipropamid (14.3%) revealed concentrations exceeding their respective maximum residue level (MRL) (0.01 mg kg-1) for barley in two samples. Although based on European guidance and MRL values the levels of the detected compounds probably do not pose acute risks for cattle, the impact of the long-time exposure to such mixtures of natural and synthetic toxicants on animal health and food safety are unknown and must be elucidated.

Cocktails of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Diets of Dairy Cows in Austria: Inferences from Diet Composition and Geo-Climatic Factors.

Dairy production is a pivotal economic sector of Austrian and European agriculture. Dietary toxins and endocrine disruptors of natural origin such as mycotoxins and phytoestrogens can affect animal health, reproduction, and productivity. This study characterized the profile of a wide spectrum of fungal, plant, and unspecific secondary metabolites, including regulated, emerging, and modified mycotoxins, phytoestrogens, and cyanogenic glucosides, in complete diets of lactating cows from 100 Austrian dairy farms. To achieve this, a validated multi-metabolite liquid chromatography/electrospray ionization−tandem mass spectrometric (LC/ESI−MS/MS) method was employed, detecting 155 of >800 tested metabolites. Additionally, the most influential dietary and geo-climatic factors related to the dietary mycotoxin contamination of Austrian dairy cattle were recognized. We evidenced that the diets of Austrian dairy cows presented ubiquitous contamination with mixtures of mycotoxins and phytoestrogens. Metabolites derived from Fusarium spp. presented the highest concentrations, were the most recurrent, and had the highest diversity among the detected fungal compounds. Zearalenone, deoxynivalenol, and fumonisin B1 were the most frequently occurring mycotoxins considered in the EU legislation, with detection frequencies >70%. Among the investigated dietary factors, inclusion of maize silage (MS) and straw in the diets was the most influential factor in contamination with Fusarium-derived and other fungal toxins and metabolites, and temperature was the most influential among the geo-climatic factors.

Fungal species and mycotoxins in mouldy spots of grass and maize silages in Austria.

Fungi and mycotoxins in silage can have detrimental consequences for both cattle and human health. This pilot study identified, via the routinary direct plating method, the dominant cultivable fungi in mouldy grass silages (GS) (n = 19) and maize silages (MS) (n = 28) from Austria. The profiles of regulated, modified, and emerging mycotoxins together with other fungal metabolites were analysed via LC-(ESI)MS/MS. Penicillium roqueforti, Saccharomyces spp., Geotrichum candidum, Aspergillus fumigatus and Monascus ruber were the most frequent fungal organisms identified. Other species including Mucor circinelloides, Fusarium spp. and Paecilomyces niveus were detected at lower frequencies. The presence of complex mixtures of toxic and potentially toxic compounds was evidenced by high levels and occurrences (≥ 50%) of Penicillium-produced compounds such as mycophenolic acid (MPA), roquefortines (ROCs), andrastins (ANDs) and marcfortine A. Mouldy silages contained toxins commonly produced by genus Fusarium (e.g. zearalenone (ZEN) and trichothecenes), Alternaria (like tenuazonic acid (TeA) and alternariol (AHO)) and Aspergillus (such as sterigmatocystin (STC)). Compared to those in GS, mouldy spots in MS presented significantly higher fungal counts and more diverse toxin profiles, in addition to superior levels of Fusarium spp., Penicillium spp. and total fungal metabolites. Generally, no correlation between mould counts and corresponding metabolites was detected, except for the counts of P. roqueforti, which were positively correlated with Penicillium spp. metabolites in mouldy MS. This study represents a first assessment of the fungal diversity in mouldy silage in Austria and highlights its potential role as a substantial contributor to contamination with complex mycotoxin mixtures in cattle diets.

Mycotoxins, Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences, Contamination Levels and Implications of Geo-Climatic Factors.

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16-138 and 107-505 µg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.

Prevalence of Toxocara spp. in dogs and cats in South America (excluding Brazil).

Toxocariasis is a worldwide anthropozoonosis caused by Toxocara spp. nematodes. High prevalences of the disease has been found in developing countries, particularly in regions with poor sanitary conditions. The definitive hosts of the nematodes are dogs and cats, which play a vital role in the transmission of this parasite as humans are considered a paratenic host. The epidemiology of the disease in South America is not clear as it is usually not diagnosed and is not a notifiable disease. This review summarizes information regarding prevalence reports of Toxocara spp. in dog and cats in South America (excluding Brazil). Additionally, and in accordance with the one health approach, reports of contaminated soil in public zones and parks as well as infection/prevalence reports in wildlife species by geographical regions are also included. The findings show the importance of awareness among veterinarians and public health authorities about Toxocara spp. as neglected disease.

Occurrence of canine and feline lungworms in Arion vulgaris in a park of Vienna: First report of autochthonous Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior in Austria.

So far, neither the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior nor the canine lungworm Angiostrongylus vasorum was reported in wildlife or intermediate hosts from Austria. The slug Arion vulgaris represents an invasive species in Europe and serves as intermediate host for several lungworm species. This study aimed to analyse the occurrence of metastrongyloid lungworm larvae in slugs in Vienna, Austria. Therefore, 193 A. vulgaris were collected in the central Prater park in summer 2016. Specimens were artificially digested, analysed microscopically for lungworm larvae, and species were confirmed via PCR and sequencing. Out of 193, five slugs were positive to lungworms (2.6%), one for A. vasorum, two for A. abstrusus (genotypes A and B) and one for T. brevior, and one slug had a mixed infection of A. abstrusus and T. brevior larvae. The current study is the first evidence on the endemicity of these metastrongyloid lungworm species in Austria.

The invasive giant African snail Lissachatina fulica as natural intermediate host of Aelurostrongylus abstrusus, Angiostrongylus vasorum, Troglostrongylus brevior, and Crenosoma vulpis in Colombia.

BACKGROUND: Several metastrongyloid lungworms are unreported pathogens in Colombia. Angiostrongylus vasorum and Crenosoma vulpis target the cardiopulmonary system of domestic and wild canids. Aelurostrongylus abstrusus and Troglostrongylus brevior infect felids and considering that six wild felid species exist in Colombia, knowledge of feline lungworm infections is important for their conservation. The zoonotic metastrongyloids Angiostrongylus costaricensis and Angiostrongylus cantonensis can cause severe gastrointestinal and neurological diseases. Angiostrongylus costaricensis has been reported in Colombia, while Ang. cantonensis is present in neighbouring countries. Research on the epidemiology of metastrongyloids in Colombia and South America more broadly requires evaluating the role that gastropods play as intermediate hosts in their life cycles. This study assessed the prevalence of metastrongyloid larvae in populations of the invasive giant African snail, Lissachatina fulica, in Colombia. METHODOLOGY/PRINCIPAL FINDINGS: A total of 609 Lissachantina fulica were collected from 6 Colombian municipalities. The snails were then cryo-euthanized, artificially digested and the sediments examined microscopically for the presence of metastrongyloid larvae. Based on morphological characteristics 53.3% (56/107) of the snails from Puerto Leguízamo (Department of Putumayo) were infected with Ael. abstrusus larvae, 8.4% (9/107) with Ang. vasorum larvae, 6.5% (7/107) with T. brevior larvae and 5.6% (6/107) with C. vulpis larvae, being the region with highest prevalences of the four species. Snails from Andes (Department of Antioquia) and Tulúa (Department of Valle del Cauca) were positive for Ang. vasorum larvae with a prevalence of 4.6 (11/238) and 6.3% (4/64), respectively. Species identifications were confirmed by PCR and sequencing. CONCLUSIONS/SIGNIFICANCE: This epidemiological survey reports for first time the presence of Ael. abstrusus, T. brevior, C. vulpis and Ang. vasorum in L. fulica in a number of regions of Colombia.

Angiostrongylus vasorum and Aelurostrongylus abstrusus: Neglected and underestimated parasites in South America.

The gastropod-borne nematodes Angiostrongylus vasorum and Aelurostrongylus abstrusus are global causes of cardio/pulmonary diseases in dogs and cats. In the last decade, the number of reports on canine and feline lungworms has increased in several areas of Europe and North America. The unspecific clinical signs and prolonged course of these diseases often renders diagnosis challenging. Both infections are considered as emerging and underestimated causes of disease in domestic pets. In South America, little information is available on these diseases, apart from occasional reports proving the principle presence of A. vasorum and A. abstrusus. Thus, the purpose of this review is to summarize reports on infections in both domestic and wildlife animals in South America and to increase the awareness on gastropod-borne metastrongyloid parasites, which also include important zoonotic species, such as A. cantonensis and A. costaricensis. This review highlights the usefulness of diagnostic tools, such as the Baermann funnel technique, serology and PCR, and proposes to include these routinely on cases with clinical suspicion for lungworm infections. Future national epidemiological surveys are recommended to be conducted to gain a deeper insight into the actual epidemiological situation of gastropod-borne parasitoses in South America.

Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

BACKGROUND: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. RESULTS: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. CONCLUSIONS: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Pharmacological overview of galactogogues.

Galactogogues are substances used to induce, maintain, and increase milk production, both in human clinical conditions (like noninfectious agalactias and hypogalactias) and in massification of production in the animal dairy industry. This paper aims to report the state of the art on the possible mechanisms of action, effectiveness, and side effects of galactogogues, including potential uses in veterinary and human medicine. The knowledge gaps in veterinary clinical practice use of galactogogues, especially in the standardization of the lactogenic dose in some pure drugs and herbal preparations, are reviewed.