Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach.

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78-78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli-Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission.

Evidence of Dirofilaria immitis in Felids in North-Eastern Italy.

Dirofilaria immitis is a mosquito-borne nematode, causing heartworm (HW) disease in wild and domestic canids. HW can also affect felids with different clinical patterns from asymptomatic pictures to sudden death, making the monitoring and diagnosis complicated. Canine HW is endemic in North-eastern Italy; however, very little information has been recorded for felids. This study aims to provide new information on HW in felids in North-eastern Italy. Two hundred and six domestic cats from Veneto, Friuli-Venezia Giulia, Trentino Alto-Adige regions (North-eastern Italy), nine captive felids from zoological parks from Veneto, and nineteen European wildcats from Friuli Venezia Giulia were recruited. Sera/plasma was analysed for the detection of anti-HW antibodies (Ab) and HW antigens (Ag); positive blood samples were molecularly analysed, targeting the HW DNA (5S-rRNA gene). Twelve out of two hundred and six (5.8%) cats presented with Ab, and three out of two hundred and six (1.5%) presented with Ag, mainly those from the Veneto region, already known as a canine HW-endemic area. Among Ab-positive cats, two were from Belluno, a mountain province previously considered free, suggesting the expansion of HW into the northern areas. No cats were positive for both Ab and Ag. Three out of nineteen (15.8%) wildcats were Ag-positive, constituting the first HW report in Italy. No captive felids were positive. Dirofilaria immitis DNA was not amplified in positive samples, suggesting the low sensitivity of PCR on blood. This study provides new data on the occurrence of HW in domestic cats and wildcats in North-eastern Italy.

News Insights into the Host-Parasite Interactions of Amyloodiniosis in European Sea Bass: A Multi-Modal Approach.

Amyloodiniosis is a disease resulting from infestation by the ectoparasitic dinoflagellate Amyloodinium ocellatum (AO) and is a threat for fish species such as European sea bass (ESB, Dicentrarchus labrax), which are farmed in lagoon and land-based rearing sites. During the summer, when temperatures are highest, mortality rates can reach 100%, with serious impacts for the aquaculture industry. As no effective licensed therapies currently exist, this study was undertaken to improve knowledge of the biology of AO and of the host-parasite relationship between the protozoan and ESB, in order to formulate better prophylactic/therapeutic treatments targeting AO. To achieve this, a multi-modal study was performed involving a broad range of analytical modalities, including conventional histology (HIS), immunohistochemistry (IHC) and confocal laser scanning microscopy (CLSM). Gills and the oro-pharyngeal cavity were the primary sites of amyloodiniosis, with hyperplasia and cell degeneration more evident in severe infestations (HIS). Plasmacells and macrophages were localised by IHC and correlated with the parasite burden in a time-course experimental challenge. CLSM allowed reconstruction of the 3D morphology of infecting trophonts and suggested a protein composition for its anchoring and feeding structures. These findings provide a potential starting point for the development of new prophylactic/therapeutic controls.

Environmental Exposure of Wild Carnivores to Zoonotic Pathogens: Leptospira Infection in the First Free Living Wolf (Canis lupus Linnaeus, 1758) Found Dead in the Friuli Venezia Giulia Region.

Leptospirosis is a worldwide-spread zoonosis causing disease and death in dogs and in humans. A Leptospiral infection has been recorded in several wild carnivore species in Europe, but tissue pathological changes were not commonly described. The Grey wolf (Canis lupus) has been expanding its distribution range in north-eastern Italy during the last decade. A young wolf, representing the first individual handled in the region, was found road-killed and then submitted to necropsy. Pathological changes included erosive lesions of gingival mucosa, mild liver enlargement, and multifocal degenerative-necrotic areas along with hyperemic reactive lesions; multifocal interstitial nephritis and multifocal lung hemorrhages were observed. A Polymerase Chain Reaction (PCR) able to detect pathogenic species of Leptospira performed on a kidney sample was positive. Serological reactions for serogroup Gryppotyphosa (1:6400), Pomona (1:800), and Icterohaemorrhagiae (1:200) were evidenced by MAT. Genotyping by Multilocus Sequence Typing (MLST) performed on detected Leptospira characterized it as belonging to Sequence Type (ST) 117, which refers to L. kirschneri, serogroup Pomona, serovar Mozdok. Regardless of the role of Leptospira infection as an eventual predisposing factor to the road killing of this wolf, to the best of the authors' knowledge, this is the first report of Leptospira-induced pathology in a wolf in Europe. Surveys on Leptospira infection in free-ranging wildlife species should be pursued in order to achieve further epidemiological knowledge on the circulation of the Leptospira strain.

Innate immune-gene expression during experimental amyloodiniosis in European seabass (Dicentrarchus labrax).

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 106/tank; 70 dinospores/mL) under controlled conditions (26-28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.

Red mark syndrome (RMS) in farmed rainbow trout: First report of outbreaks in Bosnia and Herzegovina.

Red mark syndrome (RMS) is a non-lethal inflammatory skin disorder spreading in farmed adult rainbow trout (Oncorhynchus mykiss) and reported worldwide. The aetiology is still uncertain, but positive correlation was found between Midichloria-like organism and RMS-affected fish. Here, we describe the first cases of RMS in Bosnia and Herzegovina. The outbreaks under study occurred in two intensive farms during the late winter and spring of 2020. Affected fish showed signs of disease ascribable to RMS, confirmed by pathological and molecular examination.

Histological and immunohistochemical characterization of 17 gonadal tumours in koi carp (Cyprinus carpio koi).

Reports on abdominal tumours in koi carp are scarce and most are from the gonads. Their histological diagnosis is challenging due to the occurrence of mixed populations of neoplastic cells and the few availability of cross-reactive antibodies in fish tissues. The present study aims to provide a histopathological characterization of seventeen gonadal tumours, enriched by a wide antibody panel (vimentin, CD117, placental alkaline phosphatase-PLAP, AE1/AE3 cytokeratin, E-cadherin, proliferating cell nuclear antigen-PCNA, müllerian-inhibiting substance-MIS, GATA4 and Inhibin-α) applied on whole and tissue microarray (TMA) sections. Abdominal enlargement was associated with tumours filling 30%-80% of the abdominal cavity; frequently, the gonads had been completely replaced by neoplastic tissue. Twelve cases were characterized as sex cord-stromal tumours (SCSTs), three as germ cell tumours (GCTs), one as mixed germ cell sex cord-stromal tumour (MGCSCST) and one as carcinoma. By immunohistochemistry, PLAP enabled confirmation of GCTs, ovarian carcinoma and the objective identification of a further cell component in 8 out of the 12 SCSTs that were reclassified as mixed tumours. The use of an immunohistochemical panel can help in refining the histological diagnosis, but the morphological diagnosis still represents the main tool for the characterization of these tumours in koi carp.

Transcriptome Analysis of Amyloodinium ocellatum Tomonts Revealed Basic Information on the Major Potential Virulence Factors.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll® density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq® Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines.

Host-Parasite Interaction between Parasitic Cymothoid Ceratothoa oestroides and Its Host, Farmed European Sea Bass (Dicentrarchus labrax).

Parasitic isopod Ceratothoa oestroides (Cymothoidea, Isopoda) is a common and generalist buccal cavity-dweller in marine fish, recognised for its detrimental effect in fingerling and juvenile farmed European sea bass (Dicentrarchus labrax). Although distributed throughout the Mediterranean, the isopod provokes acute outbreaks mainly limited to particular endemic areas in Croatia (Adriatic Sea) and Greece (Aegean Sea). While numerous studies have previously evidenced its gross effect on farmed fish (i.e. decreased condition index, slower growth rate, lethargy and mortality), details on the host-parasite interaction are still lacking. Therefore, using a multimethodological approach, we closely examined the structure and appearance of isopod body parts acting in the attachment and feeding (stereomicroscopy, scanning and transmission electron microscopy), and the extent of host tissues damage (histology, immunohistochemistry, micro-computational tomography) induced by parasitation. Interestingly, while hematophagous nature of the parasite has been previously postulated we found no unambiguous data to support this; we observed host tissues fragmentation and extensive hyperplasia at the parasitation site, and no structures indicative of heme detoxifying mechanisms in the parasite gut, or other traces of a blood meal. The bacterial biofilm covering C. oestroides mouthparts and pereopods suggests that the isopod may play a role in conveying secondary pathogens to the infected host, or alternatively, it serves the parasite in normal interaction with its environment.

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae).

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2',4'-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2',4'-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2',4'-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2',4'-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

Occurrence of canine and feline extra-intestinal nematodes in key endemic regions of Italy.

Extra-intestinal nematodes of companion animals are of growing concern in veterinary medicine for their pathogenic potential and the current expansion throughout Europe. The present study has evaluated the occurrence of major canine and feline extra-intestinal nematodes in regions of Italy having epidemiological relevance. Associations of various recorded parameters related to the examined animals have been statistically evaluated, along with a comparative analysis with the most recent epidemiological data. Overall, 1055 dogs and 1000 cats were tested. Among extra-intestinal nematodes Angiostrongylus vasorum was the most common in dogs followed by Capillaria aerophila and Dirofilaria spp.; Aelurostrongylus abstrusus was the most recorded parasite in cats, followed by C. aerophila and Troglostrongylus brevior. The statistical analysis revealed that outdoor access is associated with A. vasorum, A. abstrusus and T. brevior infections, that were also more prevalent in animals with cardio-respiratory signs. Moreover, cats aged less than 12 months had more chances to be infected by lungworms. The data herein presented confirm the occurrence and the possible risk of expansion of different extra-intestinal parasitoses of dogs and cats in Italy, underlining the importance of a constant epidemiologic vigilance and of appropriate control methods.

Expression of infection-related immune response in European sea bass (Dicentrarchus labrax) during a natural outbreak from a unique dinoflagellate Amyloodinium ocellatum.

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.

Shrub growth and plant diversity along an elevation gradient: Evidence of indirect effects of climate on alpine ecosystems.

Enhanced shrub growth and expansion are widespread responses to climate warming in many arctic and alpine ecosystems. Warmer temperatures and shrub expansion could cause major changes in plant community structure, affecting both species composition and diversity. To improve our understanding of the ongoing changes in plant communities in alpine tundra, we studied interrelations among climate, shrub growth, shrub cover and plant diversity, using an elevation gradient as a proxy for climate conditions. Specifically, we analyzed growth of bilberry (Vaccinium myrtillus L.) and its associated plant communities along an elevation gradient of ca. 600 vertical meters in the eastern European Alps. We assessed the ramet age, ring width and shoot length of V. myrtillus, and the shrub cover and plant diversity of the community. At higher elevation, ramets of V. myrtillus were younger, with shorter shoots and narrower growth rings. Shoot length was positively related to shrub cover, but shrub cover did not show a direct relationship with elevation. A greater shrub cover had a negative effect on species richness, also affecting species composition (beta-diversity), but these variables were not influenced by elevation. Our findings suggest that changes in plant diversity are driven directly by shrub cover and only indirectly by climate, here represented by changes in elevation.

Evaluation of the physicochemical properties of gluten-free pasta enriched with resistant starch.

BACKGROUND: The objective was to examine the potential use of resistant starch (RS) as a fibre-enriching ingredient in gluten-free pasta. Pasta was enriched with commercial RS type II (Hi-Maize™ 260) at 100-200 g kg-1 substitution of rice flour. The effects on the rheological properties of dough and pasta quality as a result of RS addition and the loss in RS due to the process were evaluated. RESULTS: Dough water absorption was not influenced by the addition of RS. The cooking loss (CL) of RS-enriched samples was 30% lower than reference without fibre. The addition of RS significantly increased firmness of cooked pasta, and above 100 g kg-1 RS level of substitution samples showed a lower stickiness value. Dynamic rheological tests on pasta dough showed a higher storage modulus for fibre samples, indicating a higher number of elastically physical interactions. Loss in RS in uncooked pasta was about 31% compared with the initial amount added to the product. CONCLUSIONS: The addition of RS improved the quality of gluten-free pasta owing to its ability to increase the firmness and decrease the CL and stickiness of cooked pasta. The product enriched with 200 g kg-1 RS can be considered a source of DF. © 2016 Society of Chemical Industry.

First detection of Cytauxzoon spp. infection in European wildcats (Felis silvestris silvestris) of Italy.

Cytauxzoonosis is an emerging, tick-transmitted, protozoan disease affecting domestic and wild felids and caused by Cytauxzoon felis, Cytauxzoon manul and Cytauxzoon spp. This study aimed to determine the presence of infection with Cytauxzoon spp. in Felis silvestris silvestris in Italy, in order to enhance the comprehension of its pattern distribution among domestic cat populations. In addition, wildcats were tested for other endemic vector-borne pathogens in Italy. The carcasses of 21 F. s. silvestris were collected from central and northern regions of Italy. All the animals were submitted to necropsy and samples of the spleens were collected. Cytauxzoon infection was surveyed by a conventional PCR amplifying a portion of the SSU-rDNA of species of Piroplasmida. The samples were also screened for Anaplasma spp., Ehrlichia spp., Rickettsia spp., Babesia spp., Theileria spp., and Leishmania spp. using SYBR Green Real-Time PCR (rPCR) assays. Four animals (19%) were positive for Piroplasmida-PCR assay and three sequenced amplicons were obtained (14.3%), clustering with the Italian, Spanish, French and Romanian Cytauxzoon spp. isolates and with C. manul found in Mongolia. The samples were negative for the other pathogens screened. The present results showed that Cytauxzoon spp. may infect both F. s. silvestris and F. s. catus.

Respiratory nematodes in cat populations of Italy.

The occurrence of common respiratory parasites of domestic cats (the metastrongyloid "cat lungworm" Aelurostrongylus abstrusus and the trichuroid Capillaria aerophila) and of neglected respiratory nematodes of felids (Troglostrongylus brevior, Angiostrongylus chabaudi and Oslerus rostratus) was here evaluated in two and three geographical sites of Northern and Central Italy, respectively. In 2014-2015, individual fecal samples of 868 domestic cats were examined microscopically and genetically, and epidemiological data related to parasitic infections were evaluated as possible risk factors by binary logistic regression models. The most common parasite was A. abstrusus in both mono- and poli-specific infections, followed by T. brevior and C. aerophila, while cats scored negative for other parasites. Cats positive for A. abstrusus (1.9-17 % infection rate) and C. aerophila (0.9-4.8 % infection rate) were found in all examined sites, while cats scored positive for T. brevior (1-14.3 % infection rate) in four sites. Also, T. brevior was here found for the first time in a domestic cat from a mountainous area of Northern Italy. The occurrence of lungworms was statistically related to the presence of respiratory signs and more significant in cats with mixed infection by other lungworms and/or intestinal parasites. Cats living in site C of Central Italy resulted statistically more at risk of infection for lungworms than cats living in the other study sites, while animals ageing less than 1 year were at more risk for troglostrongylosis. Finally, the presence of lungworms was more significant in cats with mixed infection by other lungworms and/or intestinal parasites. These results are discussed under epidemiological and clinical points of views.

Copromicroscopic and molecular investigations on intestinal parasites in kenneled dogs.

Intestinal parasites are common in dogs worldwide, and their importance has recently increased for a renewed awareness on the public health relevance that some of them have. In this study, the prevalence of helminths and protozoa was evaluated by microscopy in 318 canine faecal samples collected from eight rescue shelters in the North-eastern Italy; 285 of them were also submitted to the molecular characterization of Giardia duodenalis and Cryptosporidium spp. isolates. An analysis was performed to evaluate the prevalence rates in relation to canine individual data, shelter provenance and anthelmintic treatments. Overall, 52.5% (167/318) of faecal samples were positive for at least one parasite. Trichuris vulpis showed the highest overall prevalence rate (29.2%), followed by G. duodenalis (15.1%), Toxocara canis (9.7%), ancylostomatids (8.2%) and Cystoisospora (5.7%). The prevalence of G. duodenalis, evaluated by real-time PCR, was 57.9% (165/285), and 79 isolates were characterized by nested PCR on the ß-giardin gene. The assemblages found were mainly the host-specific genotypes C and D, while only one assemblage was identified as the human-specific genotype B1. Isolates of Cryptosporidium spp., recorded in 3/285 (1.1%) stool samples, were Cryptosporidium parvum based on the characterization of the Cryptosporidium oocyst wall protein (COWP) gene. Although the results describe a relatively limited risk of dog-originating zoonoses, there is the need to improve the quality of shelter practices towards better health managements for safe pet-adoption campaigns and a minimization of the environmental faecal pollution with canine intestinal parasites.

Decreased strongyle egg re-appearance period after treatment with ivermectin and moxidectin in horses in Belgium, Italy and The Netherlands.

The objective of the present study was to evaluate the efficacy of an oral treatment with ivermectin (IVM) or moxidectin (MOX) against gastro-intestinal strongyles in naturally infected horses by performing a faecal egg count reduction test (FECRT) and by monitoring the egg reappearance period (ERP) after treatment. Therefore, a field efficacy study with a randomised complete block design for each study site was conducted, with the individual animal as the experimental unit. At least 10 study sites in Italy, Belgium and The Netherlands were selected and animals were allocated to one of the two treatment groups based on the pre-treatment faecal egg counts (FEC). Animals were treated on Day 0 with an oral paste containing either IVM (at 0.2mg/kg bodyweight) or MOX (at 0.4 mg/kg bodyweight). After treatment, faecal samples were collected at least every fortnight during 56 days after treatment with IVM and during 84 days after MOX treatment. In total, 320 horses on 32 farms were examined. The FECRT on Day 14 indicated a 100% efficacy in 59 of the 64 treatment groups and >92% efficacy in the remaining 5 groups. The ERP was decreased for at least one of the anthelmintics on 17 out of 32 study sites (15 sites or 47% for MOX and 17 sites or 53% for IVM) and on 9 sites (28%) the ERP was decreased for both anthelmintics. On some of these study sites the efficacy declined at the end of the expected ERP, often with good efficacy 2 weeks earlier. Nevertheless, on 1, 3 and 5 study sites in Italy, Belgium and The Netherlands respectively, an efficacy below 90% for IVM and MOX was identified as soon as Day 42 or Day 56. In The Netherlands, the efficacy of IVM was below 90% from Day 28 or Day 35 after treatment on 1 site each. The present study reports a high efficacy of MOX and IVM in a FECRT 14 days after treatment, yet does indicate a shortened ERP for these treatments in more than half of the selected study sites.

Cryopreservation of roe deer abomasal nematodes for morphological identification.

Conventional methods to preserve adult nematodes for taxonomic purposes involve the use of fixative or clearing solutions (alcohol, formaldehyde, AFA and lactophenol), which cause morphological alterations and are toxic. The aim of this study is to propose an alternative method based on glycerol-cryopreservation of nematodes for their subsequent identification. Adults of trichostrongylid nematodes from the abomasum of roe deer (Capreolus capreolus Linnaeus) were glycerol-cryopreserved and compared with those fixed in formaldehyde, fresh and frozen without cryoprotectans. Morphology, transparency and elasticity of the anterior and posterior portion of male nematodes were compared, especially the caudal cuticular bursa and genital accessories. The method presented is quick and easy to use, and the quality of nematode specimens is better than that of nematodes fixed by previously used fixatives. Moreover, glycerol cryopreserved nematodes can be stored for a long time at -20 degrees C in perfect condition and they could be suitable for further analyses, such as histological or ultrastructural examinations.