Neospora caninum antibodies in bulk tank milk from dairy cattle herds in Italy in relation to reproductive and productive parameters and spatial analysis.

Among the available diagnostic techniques, antibody detection in bulk tank milk (BTM) represents a useful tool to estimate and monitor Neospora caninum herd prevalence. To evaluate the prevalence of N. caninum and the effect of parasite infection on herd performances, BTM samples collected from 586 dairy herds located in one of the largest dairy production areas in Italy (Lombardy) were analyzed by an indirect ELISA to detect anti-N. caninum specific antibodies. Generalized linear models (GLMs) were developed. A purely spatial analysis scanning for clusters with high or low rates for N. caninum using the Bernoulli model was performed. A maximum entropy approach was used to estimate the probability of distribution of the parasite based on occurrence records together with environmental variables. Overall, 180 herds resulted positive for N. caninum antibodies on bulk tank milk (P = 30.7 %). A higher risk of seropositivity was evidenced in the provinces of Milano, Cremona, Brescia, and Bergamo (P = 32-40 %); a lower risk was evidenced in Lodi, Pavia, and Mantova (P = 13-24 %). A higher risk of seropositivity was revealed for small-medium farms (101-300 animals) (O.R.=2.8) and for older animals with more than 4 years (O.R.=4.4). Regarding the effect of N. caninum infection on herd performances, the number of inseminations for conception was higher (> 3 inseminations), and the period from calving to conception was longer (> 150 days) for positive farms (O.R.=2.0 and O.R.=2.3, respectively); besides, lower head daily milk production (<20 kg and 21-25 kg) and mature equivalent milk yield (<11,000), and somatic cell counts higher than 300,000 cells/ml were observed for N. caninum positive herds (O.R.=0.4, O.R.=0.4 and O.R.=1.9 respectively). The geographical distribution of N. caninum positive farms with the highest level of probability covers the central sector of the Po Plain where a significant cluster for high risk of parasite infection was shown by spatial scan statistic and Maximum entropy ecological niche modelling. A further significant cluster of low risk occurred in the southern. The climatic and environmental variables with the highest training gain when used in isolation resulted altitude, land use/land cover, and other variables related to temperature and precipitation. Neosporosis is widely distributed in Italian dairy herds and an impact of the parasite on herd performances could be hypothesized. Even if the role of N. caninum in alterations of reproductive and productive parameters should be further explored, veterinarians and farmers should be aware of neosporosis, and control plans should be adopted.

Training in Honey Bee Veterinary Medicine in Italy: An Observational Study and Practical Proposals to Face Professional Challenges.

Honey bees, like other livestock, may be affected by infectious, parasitic, and abiotic diseases that need proper sanitary monitoring and control. Currently, there are limited opportunities for undergraduate students to receive education in Honey Bee Veterinary Medicine (HBVM) as part of their regular degree program, despite the professional requirements for veterinarians to carry out the increasing tasks related to honey bee health and production. Additionally, postgraduate training and specialization in HBVM is also underdeveloped. This study was an observational survey that evaluated the educational opportunities available in HBVM for current and future veterinarians in Italy. The survey analyzed both undergraduate and postgraduate programs, including Undergraduate Degree Programs in Veterinary Medicine (UDPVM), "Scuole di Specializzazione", Masters, and other postgraduate courses. The results indicate that the current training available for veterinarians in the field of apiculture, both before and after graduation, is also insufficient in Italy, as already reported in other EU- and extra-EU countries. Finally, a roadmap for veterinary training in HBVM is developed here describing objectives and teachings aimed at fulfilling the needs of the profession in the field of beekeeping, considering the existing rules and regulations governing public health and possible evolution of this legal framework in the future.

Monitoring Volatile Organic Compounds and Aroma Profile of Robinia pseudoacacia L. Honey at Different Storage Temperatures during Shelf Life.

Bee honey has different volatile organic compound profiles that depend on the botanical origin and the state of conservation and which are mainly responsible for its specific aroma. During honey storage, the profile of these molecules and other indicators, such as 5-hydroxymethylfurfural and the diastatic index, can change depending on temperature and time. This study analyzed the variations that these parameters in acacia honey stored at three different temperatures for a total period of 550 days, using gas chromatography coupled with mass spectrometry and an electronic nose equipped with 10 different sensors. The results confirm that the composition of acacia honey varies over time due to both the reduction in the concentration of volatile molecules (e.g., formic acid, a natural acaricide) and the increase in compounds resulting from heat-dependent degradations (e.g., 5-hydroxymethylfurfural). This study supports the usefulness of the electronic nose for the early detection of aromatic alterations in honey subjected to high-temperature storage.

Molecular Prevalence of Selected Tick-Borne Pathogens in Dermacentor reticulatus Collected in a Natural Park in Italy.

Dermacentor reticulatus is one of the most important vectors of tick-borne pathogens (TBPs) in Europe causing diseases in animals and humans. A longitudinal study was planned, aimed to detect the molecular prevalence of tick-borne pathogens, i.e., Babesia spp. and the spotted fever group Rickettsiae, and its seasonal variation in D. reticulatus questing ticks to define the temporal infection risk. Ticks were collected monthly over a period of 15 months in a peri-urban park in Lombardy, Italy. DNA extraction and molecular analyses were performed. Statistical analysis was carried out. Out of 488, 53 (P = 10.9%) adult questing ticks were positive for Babesia DNA. A higher prevalence was revealed in male (32/241, P = 13.3%) than in female (21/247, P = 8.5%) ticks. Positive ticks were mostly collected in winter months (P = 13.3%) compared to early (P = 7.9) and late (P = 12.8) spring months. A similar percentage of positive ticks was evidenced in transects 1 and 3 (5.8% and 6.5%, respectively); instead, a significant higher prevalence was recorded in transect 2 (P = 16.0%). Obtained sequences confirmed a homology of 100% with B. canis sequences deposited in GenBank. No ticks tested positive for Rickettsia spp. DNA (0/488, P = 0%). The conspicuous circulation of B. canis infection in D. reticulatus adult questing ticks confirms their role in the epidemiology of canine babesiosis and requires preventive measures for dogs in this recreational area. Even if no tick was positive for the spotted fever group Rickettsia, its capacity as a vector of zoonotic pathogens should not be neglected.

The Prophylactic Effect of Ivermectin Treatments on Nematode Infections of Mammals in a Faunistic Park (Northern Italy).

Nematode infections of mammals can spread in zoos and faunistic parks and lead to disease in humans and animals. Group treatment strategies with anthelminthic drugs are common. Still, their effectiveness should be verified by sensitive and specific copromicroscopic analyses. This study assessed longitudinal parasitological monitoring, by FLOTAC® dual technique, in mammals housed in an Italian faunistic park, in order to verify the effectiveness of the two adopted ivermectin prophylactic treatments. Twenty-one species of herbivorous mammals from ten families were treated twice per year with ivermectin in an in-feed formulation (medicated feed containing 1.7 g/ton ivermectin daily, for 30 days in March and November), while 13 species of carnivores and primates from five families were treated once a month with oral or subcutaneous administrations of ivermectin (200 µg/kg body weight (b.w.), from March to November). Fecal samples were collected in June-July and October 2019 (late spring-early summer and autumn sampling groups, respectively). All nematode infections, sustained by Nematodirus spp., Capillaria spp., Trichuris spp., Parascaris spp. and Strongylida, were detected in samples collected from herbivores, presenting prevalence rates of infection of 17.3% (9/52), 15.4% (8/52), 15.4% (8/52), 5.8% (3/52), and 3.8% (2/52), respectively. All carnivores and primates tested negative. The general linear mixed model showed that nematode eggs' excretion in herbivores were influenced by sampling and sampling-host family interaction. Results showed that frequency and dose of prophylactic treatments in herbivores should be improved according to host and parasite taxonomic groups. The treatment adopted in carnivores and primates, together with hygienic management, was effective in nematode control.

Prevalence of Neospora caninum antibodies in fattening pigs and sows from intensive farms in northern Italy.

Neospora caninum (Apicomplexa, Sarcocystidae) is a major cause of reproductive failure in cattle. In pigs, only a few studies investigated the effects of this parasite on reproductive efficiency. Considering the relevance of swine farms in northern Italian regions, an epidemiological survey was designed to investigate the spread of N. caninum infection. Three hundred seventy fattening pigs and sows from 23 intensive farms in Lombardy were sampled. Sera were analyzed by a commercial immunofluorescence antibody test. Statistical analysis through univariate and multivariate generalized linear models was conducted to detect farm management practices enhancing the risk of infection. At the farm level, 52.1% (12/23) of the selected farms, 72.7% housing sows and 40% fattening pigs, scored positive. At the individual level, 25 animals (25/370, P = 6.7%) were positive to N. caninum antibodies: one fattening pig and two sows showed an antibody titer of 1:100, and in two sows, an antibody titer of 1:400 and 1:6400 was evidenced. A higher seroprevalence was detected in sows (17/151, P = 11.2%) if compared to fattening pigs (8/219, P = 3.6%) (OR = 1.19, P value = 0.000 in sows). Moreover, a higher seroprevalence was recorded in farms with low and moderate sanitary score (P = 100% and P = 64.2%, respectively) if compared to farms with high sanitary score (P = 22.2%) (OR = 1.24, P value = 0.007 in score = 1 and OR = 1.10, P value = 0.050 in score = 2). This study provides the first data on the circulation of N. caninum in intensive swine farms in Italy, demonstrating the spread of the parasite in fattening pigs and sows in Lombardy region.

Spatial distance between sites of sampling associated with genetic variation among Neospora caninum in aborted bovine foetuses from northern Italy.

BACKGROUND: Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. Available N. caninum strains show considerable variation in vitro and in vivo, including different virulence in cattle. To which extent sexual recombination, which is possible in the intestines of domestic dogs and closely related carnivores as definitive hosts, contributes to this variation is not clear yet. METHODS: Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum samples were subjected to multilocus-microsatellite genotyping using ten previously established microsatellite markers. In addition to our own data, those from a recent study providing data on five markers from other northern Italian regions were included and analysed. RESULTS: Of the 55 samples finally subjected to genotyping, 35 were typed at all or 9 out of 10 loci and their individual multilocus-microsatellite genotype (MLMG) determined. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P < 0.001). Including data from this and a previous North Italian study into eBURST analysis revealed that several of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. Variations observed between MLMGs were not equally distributed over all loci, but predominantly observed in MS7, MS6A, or MS10. CONCLUSIONS: Our findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by microsatellite typing. Results suggest that multi-parental recombination in N. caninum is a rare event, but does not exclude uniparental mating. More comprehensive studies on microsatellites in N. caninum and related species like Toxoplasma gondii should be undertaken, not only to improve genotyping capabilities, but also to understand possible functions of these regions in the genomes of these parasites.

Detecting antibodies to Leishmania infantum in horses from areas with different epizooticity levels of canine leishmaniosis and a retrospective revision of Italian data.

BACKGROUND: Leishmania infantum is a vector-borne pathogen endemic in countries in the Mediterranean basin, including Italy. Dogs act as the primary reservoir for this parasite, but other animal species may also be infected. Low-to-moderate seroprevalence levels of infection have been reported in apparent healthy equine populations in southern Europe, reinforcing the importance of exploring those species, including horses, that act as a food source for vectors and may thus participate in the epizoological scenario of canine leishmaniosis (CanL) and zoonotic visceral leishmaniosis (ZVL). Since little is known regarding the exposure to L. infantum in horses in Italy, we assessed the seroprevalence in healthy equine populations from different CanL endemic areas. METHODS: The survey was conducted on 660 apparently healthy horses distributed throughout central and northern regions of Italy between 2016 and 2019. Blood samples were collected and the presence of anti-Leishmania antibodies (IgG) was investigated by the immunofluorescence antibody test. Information on the location and altitude of the stables, along with the horses' breed, age, sex, and reproductive status was obtained by filling in a questionnaire. This was then used for statistical analysis by generalized linear models to explore risk factors associated with seroreactivity to L. infantum. RESULTS: An average seroprevalence of 13.9% was detected for L. infantum in the equine populations investigated, with statistically significant associations between seroprevalence, geographical variables (northern vs central Italy, origin and altitude) and individual factors (i.e. age and breed morphotype). CONCLUSIONS: Our results highlight that horses are frequently exposed to L. infantum. Further prevalence surveys in horses, also using direct methods (e.g. PCR), are warranted to clarify the role of these hosts in the epidemiology of Leishmania in Italy.

Raw milk and fecal microbiota of commercial Alpine dairy cows varies with herd, fat content and diet.

The factors that influence the diversity and composition of raw milk and fecal microbiota in healthy commercial dairy herds are not fully understood, partially because the majority of metataxonomic studies involve experimental farms and/or single factors. We analyzed the raw milk and fecal microbiota of 100 healthy cows from 10 commercial alpine farms from the Province of Trento, Italy, using metataxonomics and applied statistical modelling to investigate which extrinsic and intrinsic parameters (e.g. herd, diet and milk characteristics) correlated with microbiota richness and composition in these relatively small traditional farms. We confirmed that Firmicutes, Ruminococcaceae and Lachnospiraceae families dominated the fecal and milk samples of these dairy cows, but in addition, we found an association between the number of observed OTUs and Shannon entropy on each farm that indicates higher microbiota richness is associated with increased microbiota stability. Modelling showed that herd was the most significant factor affecting the variation in both milk and fecal microbiota composition. Furthermore, the most important predictors explaining the variation of microbiota richness were milk characteristics (i.e. percentage fat) and diet for milk and fecal samples, respectively. We discuss how high intra-herd variation could affect the development of treatments based on microbiota manipulation.

Honeybee pupal length assessed by CT-scan technique: effects of Varroa infestation, developmental stage and spatial position within the brood comb.

Honeybee pupae morphology can be affected by a number of stressor, but in vivo investigation is difficult. A computed tomography (CT) technique was applied to visualize a comb's inner structure without damaging the brood. The CT scan was performed on a brood comb containing pupae developed from eggs laid by the queen during a time window of 48 hours. From the CT images, the position of each pupa was determined by recording coordinates to a common reference point. Afterwards, every brood cell was inspected in order to assess the developmental stage of the pupa, the presence of Varroa destructor, the number and progeny of foundress mites. Using data on 651 pupae, the relationships between varroa infestation status, developmental stage and spatial position of the pupa within the brood comb, and its length were investigated. Pupae at 8 post-capping days were shorter than pupae at 7 post-capping days. Pupae in infected cells were significantly shorter than those in varroa-free cells and this effect was linked both to mite number and stage and to the position in the comb. Overall, the results suggest that the CT-scan may represent a suitable non-invasive tool to investigate the morphology and developing status of honeybee brood.

Risk prioritization as a tool to Guide Veterinary Public Health activities at the regional level in Italy.

In this study we developed a model for risk prioritisation and characterisation focused on zoonoses and food safety for diseases of interest in veterinary public health at a regional level in Italy. A previous model (Discontools) based on scorecards was used as a basis to develop the new model. A Formalised Consensus Process approach involving academics and veterinary officers was used to develop scorecards and relative form and guidelines. Scorecards include several areas of interest, with different categories and coefficient of importance. The following areas were identified: relevance of the disease, socio-economic impact, impact on public health, impact on trade, impact on animal welfare, control tools. A guide and a form were finalised in order to fill scorecards. Scorecards were filled by consulting available data, literature, and expert opinions. Among bovine diseases, mastitis (Salmonella aureus) showed the highest score; Q fever was the highest among small ruminants; among swine diseases the highest was salmonellosis; while among other animal diseases, toxoplasmosis had the highest score. The approach described in this study is designed to aid professionals in risk prioritisation, decision-making, and to improve disease control systems at a regional level in Italy. It also facilitates risk characterisation in different backgrounds and the identification of data holes in specific areas of interest for the diseases considered. This approach is conceived to aid professionals in risk prioritization, decision-making and to improve disease control systems at a regional level. It also allows to perform risk characterization in different backgrounds and to identify lacks of data in specific areas of interest for the diseases considered.

Evaluation of virulence factors profiles and antimicrobials resistance of Escherichia coli isolated from bulk tank milk and raw milk filters.

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern. One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates. None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes. Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance.

Nuclear competence and genetic expression of growth differentiation factor-9 (GDF-9) of canine oocytes in 3D culture.

To evaluate the ability of a 3D culture system in improving the nuclear and molecular competence of canine oocytes, barium alginate microcapsules were used for in vitro maturation (IVM) and the expression profile of one selected oocyte-secreted factor, the growth differentiation factor-9 (GDF-9) was analysed. In Experiment I, canine grade I cumulus-oocyte complexes (COCs) were in vitro matured in 3D microcapsules in a controlled atmosphere for 72 hr, and meiosis resumption rates were compared to those of oocytes cultured in traditional 2D microdrops of medium. In Experiment II, a primer pair specific for canine GDF-9 was designed, and preliminary tested in conventional PCR on genomic DNA. Total RNA content was isolated from oocytes at different time intervals (T0-T24-T48-T72) during in vitro 3D culture, and a reverse transcription to cDNA was performed. The expression of target gene was assessed by quantitative Reverse Transcription Real-Time PCR (qRT-PCR), and the obtained amplicons were sequenced to check the specificity of the analysis. Canine COCs resumed meiosis at higher rates in 3D microcapsules than in 2D microdrops (p < 0.05), even though no significant differences in the proportions of oocytes achieving full maturational stages were obtained. A significant dynamic decrease in GDF-9 expression was recorded during culture: after 72 hr of IVM, the GDF-9 transcription significantly dropped (p = 0.018) compared to 24 and 48 hr. In conclusion, in vitro 3D culture represents an efficient system for IVM of canine oocytes, and the expression profile of GDF-9 well reflects temporal dynamics for the acquisition of developmental competence in this species.

Effects of herd and physiological status on variation of 16 immunological and inflammatory parameters in dairy cows during drying off and the transition period.

During drying off and transition period, cows are subject to changes in endocrine status, metabolic stressors and altered immune functions, which could lead to an increased risk of disease. To expand our knowledge on the immune/inflammatory status and to identify markers to define cow status during this interval, the pattern of 9 different cellular parameters, 5 cytokines, 2 enzymes and 3 cellular ratios in blood samples were assessed in 15 primiparous cows belonging to three different dairy herds in Lombardy. Our data showed that the variation of almost all parameters was influenced by the physiological period in which the samples were collected, except for apoptosis, IL-1ß, IL-6, lysozyme and granulocyte/monocyte ratio. Several markers were directly correlated either to the herd alone (IL-1ß, IL-6, lysozyme, granulocyte/lymphocyte ratio and granulocyte/monocyte ratio) or in association with the sampling time (white blood cell count, necrosis, lymphocytes count, CD4+ lymphocytes proportion). Hierarchical cluster analysis identified three herd-associated sample clusters showing different frequency along the follow-up period. The results of this field study highlight the importance of the herd factor in the immune/inflammatory response. Furthermore, these results suggest that cellular parameters are probably the most suitable markers to define cow status during drying-off and the peripartum period.

A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status.

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

Identification of Ixodes ricinus blood meals using an automated protocol with high resolution melting analysis (HRMA) reveals the importance of domestic dogs as larval tick hosts in Italian alpine forests.

BACKGROUND: In Europe, Ixodes ricinus L. is the main vector of a variety of zoonotic pathogens, acquired through blood meals taken once per stage from a vertebrate host. Defining the main tick hosts in a given area is important for planning public health interventions; however, until recently, no robust molecular methods existed for blood meal identification from questing ticks. Here we improved the time- and cost-effectiveness of an HRMA protocol for blood meal analysis and used it to identify blood meal sources of sheep tick larvae from Italian alpine forests. METHODS: Nine hundred questing nymphs were collected using blanket-dragging in 18 extensive forests and 12 forest patches close to rural villages in the Province of Trento. Total DNA was either extracted manually, with the QIAamp DNA Investigator kit, or automatically using the KingFisher™ Flex Magnetic Particle Processors (KingFisher Cell and Tissue DNA Kit). Host DNA was amplified with six independent host group real-time PCR reactions and identified by means of HRMA. Statistical analyses were performed in R to assess the variables important for achieving successful identification and to compare host use in the two types of forest. RESULTS: Automating DNA extraction improved time- and cost-effectiveness of the HRMA protocol, but identification success fell to 22.4% (KingFisher™) from 55.1% (QIAamp), with larval hosts identified in 215 of 848 questing nymphs; 23 mixed blood meals were noted. However, the list of hosts targeted by our primer sets was extended, improving the potential of the method. Host identification to species or genus level was possible for 137 and 102 blood meals, respectively. The most common hosts were Rodentia (28.9%) and, unexpectedly, Carnivora (28.4%), with domestic dogs accounting for 21.3% of all larval blood meals. Overall, Cetartiodactyla species fed 17.2% of larvae. Passeriformes (14.6%) fed a significantly higher proportion of larvae in forest patches (22.3%) than in extensive forest (9.6%), while Soricomorpha (10.9%) were more important hosts in extensive forest (15.2%) than in forest patches (4.3%). CONCLUSIONS: The HRMA protocol for blood meal analysis is a valuable tool in the study of feeding ecology of sheep ticks, especially with the cost- and time- reductions introduced here. To our knowledge, we show for the first time that domestic dogs are important larval hosts in the Alps, which may have possible implications for tick-borne disease cycles in urbanized areas.

Identifying the last bloodmeal of questing sheep tick nymphs (Ixodes ricinus L.) using high resolution melting analysis.

The sheep tick, Ixodes ricinus L., is an important hematophagous vector of zoonotic disease of both veterinary and public health importance in Europe. Risk models for tick-borne diseases can be improved by identifying the main hosts of this species in any given area. However, this generalist tick stays on a host for only a few days a year over its life cycle, making the study of its feeding ecology difficult. In contrast, ticks can easily be collected from vegetation when they are questing. Molecular methods have proved to be a reliable alternative to field observation, but most current methods have low sensitivity and/or low identification success (i.e. hosts are only identified to taxonomic levels higher than species). In this study we use Real-time PCR coupled with High Resolution Melting Analysis (HRMA) to identify the source of the last bloodmeal in questing tick nymphs. Twenty of the most important tick hosts were grouped taxonomically and six group-specific primer sets, targeting short mitochondrial DNA regions, were designed de novo. Firstly, we show that these primers successfully amplify target host DNA (from host tissue or engorged ticks), and that HRMA can be used to reliably identify hosts to species (or genera in the case of Sorex and Apodemus). Secondly, the new protocol was tested on field-collected questing nymphs. Bloodmeal source was identified in 65.4% of 52 individuals. In 83.3% of these, the host was identified to species or genera using HRMA alone. Moreover, the primer sets designed here can unequivocally identify mixed bloodmeals. The combination of sensitivity and identification success together with the closed-tube and single step approach that minimizes contamination, make Real-time HRMA a good alternative to current methods for bloodmeal identification.

Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.

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.