Lisa Guardone

Serological detection of circulating Angiostrongylus vasorum antigen and specific antibodies in dogs from central and northern Italy.

The most frequently employed method for the diagnosis of Angiostrongylus vasorum in dogs is the detection of first stage larvae (L1) in faeces. The sensitivity of coproscopy, however, is limited in case of low parasite load, intermittent larval excretion, and during pre-patency. An epidemiological survey on dogs was conducted applying serological methods in two Italian regions where angiostrongylosis is endemic in foxes. 265 dog serum samples from Tuscany (central Italy - site A) and 447 from Liguria (north-western Italy - site B) were tested with a sandwich-ELISA for detection of circulating antigen, and with an ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies for specific antibody detection. During previous examinations dogs naturally infected with Leishmania infantum (n=149), Dirofilaria immitis (n=40), Dirofilaria repens (n=30), Acanthocheilonema reconditum (n=27), Crenosoma vulpis (n=1), A. vasorum (n=2), Capillaria aerophila (n=35), Capillaria boehmi (n=3), Toxocara canis (n=68), Toxascaris leonina (n=5), hookworms (n=37) and Trichuris vulpis (n=39) were detected. Sera of these dogs were used to evaluate cross reactions. In site A, 2 dogs (0.8%) were seropositive for antibody and antigen detection and 4 (1.5%) for antibody detection only. From site B, 4 dogs (0.9%) were seropositive for both tests, while other 4 dogs (0.9%) for antigen detection only and 9 dogs (2%) for antibody detection only. Considering a subgroup of 347 dogs from site B which had also been tested with the Baermann technique, 2 (0.6%) were positive for both tests, 4 (1.2%) for antigen detection only and 9 (2.6%) for antibody detection only. The two dogs which were positive for both serological tests were also positive for A. vasorum L1 in the faeces. No significant difference in seropositivities was observed in the group of dogs with other proven parasitic infections. A. vasorum serology presents significant advantages (diagnosis before patency, single serum sample instead of repeated faecal samples, rapidity and affordability particularly in case of large number of samples) and it can be considered a valid alternative for diagnosis in individuals and in epidemiological studies.

Extraintestinal nematodes of the red fox Vulpes vulpes in north-west Italy.

Extraintestinal nematodes of the red fox (Vulpes vulpes) are a wide group of parasites that infect wild and domestic carnivores and occasionally humans. Nematodes in the cardiopulmonary system, stomach, urinary apparatus and muscle tissue of 165 red foxes (Vulpes vulpes) from north-west Italy (Liguria and Piedmont) were investigated between 2009 and 2012. Of the cardiopulmonary nematodes, a high prevalence of Angiostrongylus vasorum and Eucoleus aerophilus (syn. Capillaria aerophila) was found, 78.2% and 41.8% respectively; Crenosoma vulpis (15.8%) and Filaroides spp. (4.8%) were also found. Spirocerca lupi (23.5%), Aonchotheca putorii (syn. Capillaria putorii) (8.6%) and Physaloptera spp. (2.5%) were detected in the stomach and Pearsonema plica (syn. Capillaria plica) (56.8%) in the bladder. Eucoleus boehmi (syn. Capillaria boehmi) was also detected in the nasal cavities of one of the two foxes examined. A coprological examination revealed eggs of E. aerophilus, A. putorii, S. lupi, Physaloptera spp. and eggs of intestinal parasites. Filarial worms were absent in all the 165 animals examined, nor was there evidence of Trichinella spp. in any of the foxes. The foxes were found to host a high prevalence of many species of extraintestinal nematodes. The prevalence of A. vasorum in foxes found in the present study is among the highest in Europe. In addition, to the best of our knowledge, E. boehmi and Filaroides spp. have never been reported before in this host in Italy.

First report of Eucoleus boehmi (syn. Capillaria boehmi) in dogs in north-western Italy, with scanning electron microscopy of the eggs.

Dogs can be infected by several nematodes of the Trichuridae family. Trichuridae eggs are all similar, barrel shaped with polar plugs, and misdiagnosis among different species can occur. The most common species is Trichuris vulpis, while the respiratory parasites Eucoleus boehmi (syn. Capillaria boehmi) and Eucoleus aerophilus (syn. Capillaria aerophila) are rarely observed in pets. E. boehmi is reported for the first time in this study in north-western Italy with other Trichuridae. Dog faecal samples (270) were examined by flotation. E. boehmi (2.2%), E. aerophilus (4.4%) and T. vulpis (12.2%) were found; identification was done with measurements and through observation of morphological characters already known. The specific identification of E. boehmi was confirmed using scanning electron microscopy: its egg shell shows a dense network with a fine mesh, surrounding small pits, on the contrary E. aerophilus eggs present a thick mesh with wide depressions, while T. vulpis eggs surface is smooth.

Canine filarial infections in Tuscany, central Italy.

This study was conducted in order to investigate the epidemiology of filarial species in a region of central Italy (Tuscany) in dogs that did not undergo prophylaxis for filariasis. From 2007 to 2009, 630 blood samples were collected from 40 kennels throughout the regional territory. Samples were analysed with Knotts modified test and with an enzyme-linked immunosorbent assay (ELISA) for the detection of Dirofilaria immitis antigens, those positive for microfilariae were also subjected to histochemical staining for acid phosphatase activity to validate the identification of the species. An overall elevated prevalence of filariasis (23.2%) was found. Dirofilaria immitis and D. repens were found to be the dominant species, with similar prevalences (12.5% and 12.1% respectively), while Acanthocheilonema reconditum was less common (1.9%). Results of this study indicate that monitoring for filariasis should be kept at a high level. A constant monitoring of the canine sanitary status should also be carried out for the protection of human health, considering the zoonotic potential of filarial worms.

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceability.

Canine filarial infections in Liguria, north-west Italy

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knotts test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knotts test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

Eucoleus aerophilus (syn. Capillaria aerophila) and Other Trichinelloid Nematodes in Dogs from Liguria (Northwest Italy)

As a part of a wider study that examined endoparasites of carnivores in northwest Italy, 270 canine fecal samples were analyzed with flotation techniques. Overall prevalence was 7.4 % for Capillaria spp. and 12.2 % for Trichuris vulpis. This article stresses the importance of an accurate morphological and morphometrical analysis to distinguish eggs of T. vulpis from eggs of the genus Capillaria. We also suggest that different Capillaria spp. are present in the Italian dog population.

Cardiorespiratory and gastrointestinal parasites of dogs in north-west Italy

Summary The present study was conducted to evaluate the prevalence of gastrointestinal and cardio-respiratory parasites in dogs in Liguria (northwest Italy) which is a region that has scarcely been studied. A total of 450 dogs (260 males and 190 females) were enrolled in the study from 2009 and 2013. All dogs lived in a rural or semi-rural environment in the provinces of Imperia (n = 352) or Savona (n = 98), Liguria. Coprological examinations showed that 197 dogs (43.8 %, CI 38.7-48.9 %) were infected by parasites. Specifically, 3.3 % of the dogs presented only cardio-respiratory species, 32.4 % only intestinal species, and 8.0 % presented a mixed infection. The most frequent intestinal parasites were Toxocara canis (20 %), Trichuris vulpis (17.8 %), Ancylostomatidae (12 %), Coccidia (2.7 %), Aonchotheca putorii (1.8 %) and Toxascaris leonina (1.8 %). The cardio-respiratory species found were Eucoleus aerophilus (9.6 %), Eucoleus boehmi (1.6 %), Angiostrongylus vasorum (0.7 %), and Crenosoma vulpis (0.2 %). A total of 116 dogs (25.8 %) were parasitized by a single species, multiple infections were observed in 81 dogs (18.0 %) up to a six-order infection. This preliminary study highlighted that the prevalence of intestinal parasites in investigated area is high. Cardio respiratory parasites were detected in an area that has not been investigated before. The creation of a more extensive sampling programme of the area, on a provincial basis in order to build a more detailed map of prevalences for different species of dog parasites throughout Liguria, a more extensive sampling programme of the area needs to be created - ideally for each province.

Intestinal helminths of red foxes (Vulpes vulpes) in north-west Italy

Summary A total of 180 foxes (Vulpes vulpes) from an area scarcely investigated of north-west Italy, were examined for intestinal helminths using sedimentation and counting technique (SCT). Faecal samples were submitted to centrifugation with 50 % zinc sulphate used as flotation solution. No fox was found completely negative for intestinal helminths. The most frequently identified nematodes were Uncinaria stenocephala (70.0 %), Molineus legerae (27.2 %), Toxocara canis (26.7 %), Toxascaris leonina (25.6 %), Trichuris vulpis (21.1 %), Aonchotheca putorii (8.9 %), Pterygodermatites affinis (5.6 %). Genus Mesocestoides (81.7 %), family Dilepididae (29.4 %) and Taenia spp. (8.3 %) were the most prevalent cestodes. All foxes were negative for E. multilocularis and E. granulosus. In two foxes trematodes belonging to the family Plagiorchidae were found. The study highlighted that foxes are hosts of intestinal helminths of veterinary and medical importance which may be transmitted to dogs and humans.

DNA barcoding for the verification of supplier’s compliance in the seafood chain: How the lab can support companies in ensuring traceability

Food Business Operators (FBOs) rely on laboratory analysis to ensure seafood traceability. DNA barcoding and Forensically Informative Nucleotide Sequencing may represent a support within self-checking programs finalized to suppliers’ qualification and products identity certification. The present study aimed at verifying the usefulness of a decisional procedure (decision tree) set up at the FishLab (Department of Veterinary Sciences, University of Pisa, Italy) for seafood species identification by DNA analysis, to cope with FBOs’ needs. The decision tree was applied to the analysis of 182 seafood (fish and molluscs) products, conferred to the FishLab by different FBOs between 2014 and 2015 as result of their self-checking activities. The analysis relied on a standard COI gene fragment eventually integrated by the analysis of alternative or supportive molecular targets (cytb and 16S rRNA). It also included a mini-DNA barcoding approach for processed products. Overall, 96.2% of the samples were unambiguously identified at species level using the elective target alone (92.4%) or a multi-target approach (3.8%). The lack of species identification (3.8%) was attributable to the absence of reference sequences or to the low resolution of the molecular targets. Nonetheless, all the molecular results were deemed adequate to evaluate the sample’s compliance to the label information. Non-compliances were highlighted in 18.1% of the products. The protocol was proven as an effective supportive tool for the seafood identity verification within the supply chain self-checking activities. In addition, a considerable fraud rate was confirmed and the species most frequently involved in substitution were pointed out.