Roberta Galuppi

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.

Detection of Babesia EU1 in Ixodes ricinus ticks in northern Italy.

Babesia EU1, a potentially important emerging zoonotic pathogen, already detected in ticks and wild ruminants of different European Countries, was found in three pools of Ixodes ricinus nymphs in three different sites located in a single District of north-eastern Italy. Totally 356 ticks (60 pools) were collected from the environment during a surveillance activity in the year 2006. Babesia EU1 estimated individual tick prevalence in the area is 0.85%. The finding that also in northern Italy the tick population is carrying Babesia EU1 suggests a wide geographical spreading of this zoonotic pathogen in Europe.

Seroprevalence to chlamydiae in pigs in Italy

ACCORDING to the current taxonomy (Everett and others 1999), four chlamydial species, namely Chlamydia suis (formerly the porcine serovar of Chlamydia trachomatis), Chlamydophila psittaci (formerly avian serovars of Chlamydia psittaci), Chlamydophila abortus (formerly ovine serovars of C psittaci) and Chlamydophila pecorum (formerly Chlamydia pecorum) have been isolated from pigs. Chlamydiae in pigs can cause asymptomatic infections and have been associated with pneumonia, polyarthritis, pericarditis, conjunctivitis, enteritis, reproductive disorders and increased perinatal mortality (Martinov and others 1985, Woolen and others 1990, Rogers and others 1993, Andersen 1994, Zahn and others 1995, Thoma and others 1997, Guscetti and others 2000, Becker and others 2004, Sachse and others 2004). It is widely believed that chlamydiae may act together with other agents in multifactorial infectious diseases (Pospischil 2004). Chlamydial infections in pigs have been reported mainly in eastern European countries, Austria, Germany, Switzerland and Belgium. In Italy, chlamydiae were detected in fetal organs by direct immunofluorescence, and seropositivity was shown by the complement fixation test in 26 of 100 sows with reproductive problems (Sala 2003). This short communication describes a study to determine the seroprevalence of C suis, C pecorum, C abortus and C psittaci by the microimmunofluorescence test (MIF) in 337 pigs from 11 intensive farms located in northern Italy. Serum samples were collected randomly from finishing pigs at an abattoir in 2004. The MIF was performed using purified elementary bodies (EBs) of the four different species as antigens: the Italian C suis isolate MS04, obtained from a conjunctival swab from a pig; the Italian C pecorum isolate PV5268, obtained from a bovine cervical swab; the ovine reference strain S26/3 of C abortus; and the avian reference strain 6BC of C psittaci. MS04 and PV5268 had been characterised by molecular analysis. The EBs were purified by sucrose density-gradient ultracentrifugation by the method of Fukushi and Hirai (1988). The presence of chlamydial antibodies was assessed using fluorescein-conjugated goat anti-pig immuno globulin G serum (Euroclone). The sera were screened at 1:32 dilution in phosphate-buffered saline supplemented with 2 per cent fetal calf serum. The test was performed according to the method of Wang and Grayston (1986). Serial twofold dilutions of the sera that tested positive at 1:32 dilution were tested to determine the antibody titre. The reciprocal of the highest serum dilution showing an apple-green fluorescence of EBs was considered to be the antibody titre. Antibodies to C suis were detected in 214 of the 337 (63·5 per cent) samples tested, with antibody titres ranging from 32 to 512. Seropositivity to C suis was observed in pigs from all 11 farms investigated, ranging from 20 per cent to 100 per cent (mean 61 per cent). Only a few sera with high antibody titres to C suis reacted weakly at 1:32 dilution with the other chlamydial species. No antibody titres above 32 were detected in any sera to C pecorum, C abortus or C psittaci. The high chlamydial seroprevalence is consistent with the results of serological surveys performed in other countries. Wendt and others (1998) reported a chlamydial seropositivity ranging from 4 per cent to 72 per cent in breeding sows in Germany, in a study that used an ELISA for antibodies to the chlamydial-specific lipopolysaccharide (LPS) antigen; significantly higher percentages of seropositive sows were found in herds with reproductive disorders. Vanrompay and others (2004) reported seropositivity in 96·5 per cent of 258 closed pig breeding farms in Belgium, tested by an indirect ELISA using C psittaci recombinant major outer membrane protein as antigen. Camenisch and others (2004) reported that 61·7 per cent of 193 sera taken from Swiss herds of sows with or without reproductive problems were positive for antibodies to LPS of Chlamydiaceae, with no significant difference between the two groups of herds. In those studies, family-specific antibodies were detected. In the present study, the MIF performed with EBs of the different chlamydial species allowed the evaluation of species-specific seroreactivity and showed a high seroprevalence for C suis. Since antichlamydial vaccines are not administered in pig herds, this seropositivity suggests that the pigs had extensive contact with C suis. The association of C suis with the porcine intestinal tract (Schiller and others 1997a, Hoelzle and others 2000) and its shedding in faeces could increase its spread on farms. C suis is associated with conjunctivitis, enteritis and pneumonia (Rogers and Andersen 2000, Merialdi and others 2003, Sachse and others 2004); it has also been detected in fetal organs from porcine abortions together with C pecorum (Schiller and others 1997b), and in cervical swabs of sows with reproductive problems in association with C abortus (Hoelzle and others 2000). In the present study, the history of the herds described respiratory and reproductive problems on only two farms and on these farms seroprevalences of 75 per cent and 66 per cent against chlamydiae were detected. Since these farms did not test for antibodies to other infectious agents, the association between the C suis seropositivity and the clinical signs is not clear. The remaining nine farms, which showed seropositivity ranging from 20 per cent to 100 per cent (mean 59 per cent), did not report clinical signs suggestive of chlamydiosis. The seropositivity on these farms could be due to asymptomatic infections; for example, intestinal chlamydiosis seems to be a mainly subclinical condition (Nietfeld and others 1997). Further investigations are needed to assess the role of C suis as a bacterial pathogen in pigs.

Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

Abundance of questing ticks and molecular evidence for pathogens in ticks in three parks of Emilia-Romagna region of Northern Italy.

INTRODUCTION AND OBJECTIVE Infectious and parasitic diseases transmitted by ticks, such as Lyme diseases, granulocytic anaplasmosis and piroplasmosis, have been frequently reported in Europe, with increasing attention to them as an emerging zoonotic problem. The presented study was performed to assess the distribution and the density of questing ticks in three regional parks of Emilia-Romagna region of Northern Italy, and to seek molecular evidence of potential human pathogens in tick populations. MATERIALS AND METHODS In the period April-October 2010, 8,139 questing ticks were collected: 6,734 larvae, 1,344 nymphs and only a few adults - 28 females and 33 males. The abundance of Ixodes ricinus questing ticks was compared among different sampling sites and related to microclimate parameters. 1,544 out of 8,139 ticks were examined for the presence of pathogens: PCR was used to detect piroplasms DNA and Real time Taqman PCR for Anaplasma phagocytophilum and Borrelia burgdorferi s.l. RESULTS The predominant species was I. ricinus (overall abundance 1,075.9/100 m(2) ); more rarely, Dermacentor marginatus (n = 37 - 0.45%), Scaphixodes frontalis (n = 13 - 0.16%), Hyalomma spp. (n = 6 - 0.07%) and Ixodes acuminatus (n = 3 - 0.04%) were also found. 28 out of 324 (8.6%) samples of ticks were PCR-positive for piroplasm DNA. 11 amplicons of 18S rRNA gene were identical to each other and had 100% identity with Babesia EU1 (Babesia venatorum) using BLAST analysis. Real time Taqman PCR gave positive results for A. phagocytophilum in 23 out of 292 samples (7.9%), and for B. burgdorferi s.l. in 78 out of 292 samples (26.7%). I. ricinus was the only species found positive for pathogens by molecular analysis; 16 tick samples were co-infected with at least 2 pathogens. DISCUSSION The peak of nymph presence was in May, and the higher prevalence of pathogens occurred in April-June, most often in nymphs; therefore, spring season could represent the higher risk period for the transmission of pathogens. These data could provide guidelines for the preventions of tick-trasmitted diseases in this region.

Human exposure to piroplasms in Central and Northern Italy.

TA serosurvey has been conducted in Northern and Central Italy to investigate the presence in humans of antibodies against zoonotic Babesia and Theileria species. The study focused on a total of 432 volunteers, of which 290 were persistently exposed to tick bites because of their jobs (forester employees, livestock keepers, veterinary practitioners, farmers and hunters) and 142 resident in the same area less frequently exposed. An indirect fluorescent antibody test (IFAT) for humans was used to detect antibodies to Babesia microti, IFAT tests for veterinary use were modified to detect reactivity to Babesia bovis, Babesia canis and Theileria equi. A laboratory-derived ELISA was employed to detect antibodies to Babesia divergens. Both reactive and 10 negative sera were analysed against plasmodial antigens to evaluate possible aspecificity. A high reactivity to piroplasm antigens was found, showing significant difference between the sera of the two groups of volunteers (24% vs 7.%; p<0.001). No cross-reactivity was observed, while each professional group showed reactivity that would fit with the professional risk exposure. In particular, a high reactivity to B. microti and B. divergens antigens was observed in foresters and hunters (32% and 12%, respectively). This is the first report on the human seroreactivity to piroplasms in Italy; it also provides additional epidemiological information on these tick-borne zoonoses in Europe. Our findings suggest the possible occurrence of piroplasm infections in Italy and alert physicians to consider these otherwise neglected parasitic diseases when dealing with any febrile illness, especially in subjects exposed to tick bites.

Molecular evidence of Leishmania infantum in Ixodes ricinus ticks from dogs and cats, in Italy.

Leishmaniosis, caused by Leishmania infantum, is an endemic zoonosis in the Mediterranean basin. To date, phlebotomine sand flies are the only accepted biological vectors of Leishmania parasites to dogs and humans. The absence of the primary vector in autochthonous Leishmania outbreaks suggests a possible role of fleas or ticks as alternative vectors. In this study, 119 ticks were collected between August 2007-June 2008 and between March 2010-October 2010 from various animal species and humans living in Italian areas where canine leishmaniosis is endemic (i.e. rural areas of the North) and were tested for the presence of L. infantum DNA. Nine (7.5%) out of 119 ticks resulted PCR positive. All ticks were morphologically identified as Ixodes ricinus ticks, 3 from 1 cat, 6 from 4 dogs. To our knowledge, this is the first evidence of L. infantum DNA in ticks from cat, suggesting that the debate about the epidemiological role of ticks in canine leishmaniosis might be extended to feline leishmaniosis.

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

Chlamydiae in corvids

AVIAN chlamydiosis is primarily caused by the intracellular bacterium Chlamydia psittaci , belonging to the Chlamydiaceae family. Depending on the species and age of the bird and the virulence of the infectious bacterial strain, avian chlamydiosis can be subclinical or characterised by respiratory, digestive, or systemic disorders (Knittler and others 2014). Seven C. psittaci outer-membrane protein A ( omp A) genotypes (A-F and E/B), have been initially detected in birds. All these genotypes can be transmitted to humans by contact with contaminated faeces or feathers or by inhalation of an infectious aerosol, causing a mild flu-like illness or severe atypical pneumonia. Recently, six additional C. psittaci omp A genotypes, all occurring in wildlife birds, have been proposed (Sachse and others 2008). Recent studies suggested that more chlamydial agents, beyond C. psittaci, can be involved in avian chlamydiosis. In this respect, Chlamydia abortus , Chlamydia pecorum, Chlamydia trachomatis and Chlamydia pneumoniae have been detected in birds (Pantchev and others 2009, Sachse and others 2012, Frutos and others 2015). Recently, two new bacterial species belonging to the Chlamydiaceae family have been described: Chlamydia avium from pigeons and psittacine birds and Chlamydia gallinacea from poultry (Sachse and others 2014). In addition, a novel candidate species, named Chlamydia ibidis, …

Grains colonised by moulds: fungal identification and headspace analysis of produced volatile metabolites

Abstract The aim of this work was to verify if the headspace analysis of fungal volatile compounds produced by some species of Fusarium can be used as a marker of mould presence on maize. Eight samples of maize (four yellow maize from North Italy and four white maize from Hungary), naturally contaminated by Fusarium and positive for the presence of fumonisins, were analyzed to detect moisture content, Aw, volatile metabolites and an enumeration of viable moulds was performed by means of a colony count technique. Headspace samples were analysed using a gas-chromatograph equipped with a capillary column TR-WAX to detect volatile metabolites of moulds. Furthermore mac ro- and microscopic examination of the colonies was performed in order to distinguish, according to their morphology, the genera of the prevalent present moulds. Prevalent mould of eight samples was Fusarium, but other fungi, like Aspergillus, Penicillum and Mucora were observed. The metabo-ceae, lites produced by F. graminearum and F. moniliforme were Isobutyl-acetate, 3-Methyl-1-butanol and, only at 8 days, 3-Octanone. The incubation time can affect off flavour production in consequence of the presence of other moulds. Further studies on maize samples under different conditions are needed in order to establish the presence of moulds using the count technique and through the identification of volatile compounds.