D. Salvatore

Canine leishmaniasis surveillance in a northern Italy kennel

An epidemiological survey on canine leishmaniasis (CanL) was performed during a 3-year period (2007-2009) in a public kennel of the Bologna province. The presence of the disease was shown in the canine population for the first time in 2007 by indirect fluorescent antibody test (IFAT). The parasite circulation was confirmed also by direct diagnostic tools, as PCR, cytology and cultural method, performed on different bioptic materials. The parasite was isolated and identified as Leishmania infantum zymodeme MON 1. The serological monitoring was performed also in 2008 and 2009 on animals that previously showed negative or uncertain results. The incidence values calculated by significant seroconversions in IFAT titre ≥ 1/160, ranged between 4.9% and 6.6%, indicating a stable focus of leishmaniasis. The entomological survey, performed by sticky and CO(2)-baited traps in 2008, showed the presence of the vector Phlebotomus perfiliewi. This study allowed us to identify a stable focus of CanL in an area that was not considered eco-compatible with the presence of the vector and infection. Our results confirm the northward spread of CanL towards areas not previously affected by autochthonous foci.

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.

Seroepidemiologic Survey for Chlamydia suis in Wild Boar (Sus scrofa) Populations in Italy

We used serology to estimate the prevalence of exposure to chlamydiae in Italian populations of wild boars (Sus scrofa). Sera from 173 hunter-killed wild boars harvested during the 2006–2009 hunting seasons in three Italian regions were tested for antibodies to Chlamydia suis, Chlamydophila pecorum, Chlamydophila abortus, and Chlamydophila psittaci by the microimmunofluorescence test. Antibody titers to chlamydiae ≥1:32 were detected in 110 of the 173 samples tested (63.6%). Specific reactivity could be assessed only in 44 sera with antibody titers to C. suis that were two- to threefold higher than antibody titers against the other chlamydial species; the other 66 sera had similar reactivity against all the chlamydia species tested. Antibody to C. suis was detected in sera from wild boar populations with rare or no known contact with domestic pigs. These results suggest that the wild boar could be a chlamydia reservoir and may acquire chlamydiae independent of contacts with the domestic pig.

Molecular characterization of Leishmania infantum strains by kinetoplast DNA RFLP-PCR.

Multilocus enzyme electrophoresis is the tool most frequently used to classify Leishmania spp., although it is time consuming and, sometimes, a not enough discriminative method. In the present study a kinetoplast DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to characterize 16 zymodeme MON-1 Leishmania infantum strains: 15 were from dogs housed in public kennels of 7 geographical areas in the Emilia-Romagna region, Northern Italy, 1 was the L. infantum reference strain MHOM/TN/1980/IPT1. Six enzymatic patterns were observed. Kinetoplast DNA RFLP-PCR confirmed to have a good discriminatory power within the same zymodeme and proved to be useful for comparing few strains or discriminating between relapse and reinfection in the same host. We therefore recommend it use for discriminating between relapse and reinfection in the same host rather than supporting large-scale epidemiological studies.

Chlamydiosis: Seroepidemiologic Survey in a Red Deer (Cervus elaphus) Population in Italy

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.

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.

Canine leishmaniasis surveillance program in a San Marino Republic kennel.

The Republic of San Marino is an autonomous State that, in view of its geographical and environmental features, can be considered a part of the Northern Italian territory, where the canine leishmaniasis (CanL) is endemic. In the past, a CanL focus in the Republics kennel was described. As a consequence of this epidemiological situation, a surveillance program was carried-out covering a 6-year period (2006-2012). A total of 1,094 sera were collected from 420 kennel dogs and examined for antibodies to Leishmania infantum by the indirect fluorescent antibody test (IFAT). Eighty-eight (21%) dogs resulted IFAT positive (antibody titre ≥1/40). The overall seroprevalence increased in the first 4 years (2006-2010), going from 5.5% to 26.8% and then decreased in the 2 following years going to 17.9%(2011) and 3.9% (2012). The cumulative incidence constantly increased from 0.6% to 2.6%. This trend could be attributed to a changed infection pressure due to the dog turnover in the kennels. According to the observed incidence values, the CanL focus seems to be stable, supported by autochthonous transmission, new case introduction and Leishmania spp. circulation in owned dogs in the same area.

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, …

Multilocus microsatellite typing (MLMT) reveals host-related population structure in Leishmania infantum from northeastern Italy

Background Visceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic. Methodology/Principal findings Multilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs. Conclusions/Significance A peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities.