Alessandro Mannelli

Ecology of Borrelia burgdorferi sensu lato in Europe: transmission dynamics in multi-host systems, influence of molecular processes and effects of climate change.

The analysis of different multi-host systems suggests that even hosts that are not capable of transmitting Borrelia burgdorferi sensu lato (s.l.) to the tick vector, Ixodes ricinus, or that are secondary reservoirs for these agents contribute to the intensity of transmission and to the overall risk of Lyme borreliosis, through the process of vector augmentation and pathogen amplification. On the other hand, above certain threshold densities, or in the presence of competition with primary reservoir hosts or low attachment rate of ticks to reservoir hosts, incompetent or less competent hosts may reduce transmission through dilution. The transmission of B. burgdorferi s.l. is affected by molecular processes at the tick-host interface including mechanisms for the protection of spirochaetes against the hosts immune response. Molecular biology also increasingly provides important identification tools for the study of tick-borne disease agents. Ixodes ricinus and B. burgdorferi s.l. are expanding their geographical range to northern latitudes and to higher altitudes through the effects of climate change on host populations and on tick development, survival and seasonal activity. The integration of quantitative ecology with molecular methodology is central to a better understanding of the factors that determine the main components of Lyme borreliosis eco-epidemiology and should result in more accurate predictions of the effects of climate change on the circulation of pathogens in nature.

Borrelia lusitaniae in Immature Ixodes ricinus (Acari: Ixodidae) Feeding on Common Wall Lizards in Tuscany, Central Italy

Abstract Lizards and small rodents were live captured in Tuscany, central Italy, from May through August 2005. Prevalence of infestation by larval Ixodes ricinus L. (Acari: Ixodidae) and mean numbers of larvae per host were not significantly different for common wall lizards, Podarcis muralis Laurenti, and Apodemus spp. mice, whereas infestation levels by nymphs were significantly greater on lizards. Borrelia lusitaniae, which was previously shown to be dominant in host-seeking I. ricinus in the same study area, was detected by polymerase chain reaction (PCR) in 19.8% (95% confidence interval: 14.4, 26.0) of larval ticks and in 52.9% (27.8, 77.0) of nymphs that were collected from lizards. Moreover, 18.8% (7.2, 36.4) and 25.0 (3.2, 65.1) of lizards’ tail biopsies and blood samples, respectively, were positive for B. lusitaniae. Conversely, attached ticks and ear biopsies from Apodemus spp. mice were PCR negative. Passerine birds belonging to 10 species were live captured in March 2005, and Borrelia valaisiana was detected in 57.1% (18.4, 90.1) of I. ricinus nymphs feeding on Eurasian blackbirds, Turdus merula L. Results of this study suggest that lizards play an important role as reservoirs for B. lusitanae and may affect the dominance of this genospecies in the Mediterranean area.

Rickettsia slovaca in Dermacentor marginatus and Tick-borne Lymphadenopathy, Tuscany, Italy

Of 263 patients in Tuscany, Italy, from whom ticks were removed during July 2005–May 2007, five showed signs of tick-borne encephalopathy. Of the ticks, 17 were Dermacentor marginatus; 6 (35.3%) of these were identified by sequence analysis as containing Rickettsia slovaca. Tick-borne lympadenopathy occurs in this area.

Prognostic Indicators for Dogs with Dilated Cardiomyopathy

The purpose of this study was to investigate the prognostic value of various clinical, ECG, echocardiographic, and Doppler echocardiographic variables in dogs with dilated cardiomyopathy. The relationship to survival of 11 variables was evaluated in 63 dogs. Studied variables were age at time of diagnosis, class of heart failure (HF), dyspnea, ascites, atrial fibrillation (AF), ejection fraction (EF), E-point septal separation, end-diastolic volume index, end-systolic volume index (ESV-I), and restrictive or nonrestrictive transmitral flow (TMF) pattern. Median survival time was 671 days (lower 95% confidence limit, 350 days). Survival curves showed that severity of HF, ascites, ESV-I greater than 140 mL/m2, EF less than 25%, and restrictive TMF pattern had a significant negative relation to survival time. Thirty-nine dogs with both sinus rhythm and AF presented adequate TMF recordings; in these dogs, after stratification by TMF pattern, the restrictive TMF pattern was the most important negative prognostic indicator. We conclude that in dogs with dilated cardiomyopathy the restrictive TMF pattern appears to represent a useful prognostic indicator. Class of HF, ascites, ESV-I, and EF are also useful indexes if an adequate TMF pattern is not recorded.

Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.

The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.

Diagnostic and prognostic value of serum lactate dehydrogenase (LDH) and LDH isoenzymes in canine lymphoma

In humans, serum lactate dehydrogenase (LDH) is used as a prognostic indicator in non-Hodgkin’s lymphoma (NHL). In particular, elevated values of LDH (Schneider et al., 1980) and increases of the isoenzymes LDH2 and LDH3 (Dumontet et al., 1999) have been correlated with shortened survival times. In another study, the same isoenzymes were elevated in patients affected by latent NHL (Rotemberg et al., 1984). However, in veterinary medicine only a very limited amount of information is available regarding the pattern of LDH isoenzymes related to lymphoma. An increase in the value of serum LDH has been reported in dogs affected by leukaemia (Leifer and Matus, 1986) and in cats with large granular lymphoproliferative disorder (Buracco et al., 1992). In canine lymphomas, however, similar correlations between high concentrations of serum LDH and clinical staging or survival times have not been found (Greenlee et al., 1990). Bezzecchi and colleagues (1979) found that an increase in LDH3 was a possible indicator of latent disease. In an earlier study by our group, we showed that LDH2 and LDH3 were increased in dogs with lymphoma (Abate et al., 1997). The present report evaluates the clinical utility of monitoring changes in total and isoenzymatic patterns of LDH at diagnosis and during the course of therapy.

Modeling the spread of vector-borne diseases on bipartite networks.

Background Vector-borne diseases for which transmission occurs exclusively between vectors and hosts can be modeled as spreading on a bipartite network. Methodology/Principal Findings In such models the spreading of the disease strongly depends on the degree distribution of the two classes of nodes. It is sufficient for one of the classes to have a scale-free degree distribution with a slow enough decay for the network to have asymptotically vanishing epidemic threshold. Data on the distribution of Ixodes ricinus ticks on mice and lizards from two independent studies are well described by a scale-free distribution compatible with an asymptotically vanishing epidemic threshold. The commonly used negative binomial, instead, cannot describe the right tail of the empirical distribution. Conclusions/Significance The extreme aggregation of vectors on hosts, described by the power-law decay of the degree distribution, makes the epidemic threshold decrease with the size of the network and vanish asymptotically.

Prevalence of cryptosporidian infection in cats in Turin and analysis of risk factors.

An epidemiological study was carried out to identify factors associated with the risk of cryptosporidian infection in cats. Faecal samples from 200 domestic cats were collected in the small animal clinic at the Faculty of Veterinary Medicine in Turin (north-west Italy). The faecal samples were analysed for the presence of Cryptosporidium species oocysts using a centrifugation concentration floatation method. For each cat, age, sex, breed, indoor/outdoor status, diet, diarrhoea and presence of other enteric parasites were analysed for association with Cryptosporidium species infection. Cryptosporidia oocysts were identified in 49 cats (24.5%) aged from 2 months to 18 years. Statistical analysis revealed that four variables are significantly associated with an increased risk of infection: less than 1 year of age (χ 2=6.5, P=0.01), feeding home-cooked diet (χ 2=6.92, P<0.01), presence of diarrhoea (χ 2=4.34, P<0.037), and presence of other enteric parasites (χ 2=10.31, P<0.01). No statistical differences were found for sex (χ 2=1.56, P=0.21), breed (χ 2=0.78, P=0.38) and outdoor/indoor status (χ 2=1.49, P=0.22). Cryptosporidium species was the parasite most frequently detected in the cats surveyed.

Rickettsia slovaca in immature Dermacentor marginatus and tissues from Apodemus spp. in the northern Apennines, Italy

Immature Dermacentor marginatus ticks and tissues from small rodents were tested for infection with Rickettsia slovaca in the northern Apennines, Lucca Province, where tick-borne lymphadenopathy (TIBOLA) was previously reported in people. Prevalence of infestation with D. marginatus was 30.5% (n=131, 95% CI: 22.8-39.2%) in Apodemus spp. and 26.5% (n=34, 95% CI: 12.9-44.4%) in Myodes glareolus, which were captured during 1980 trap nights in 2009 and 2010. Rickettsia slovaca was identified by polymerase chain reaction, targeting the gltA and OmpA genes, in ear biopsies from 8 out of 37 tested Apodemus (22%, 95% CI: 9.8-38.2%), but not from 9 M. glareolus. The prevalence of R. slovaca in D. marginatus feeding on Apodemus spp. was 53% in larvae (n=51, 95% CI: 38.5-67.1%) and 47.5% in nymphs (n=59, 95% CI: 34.3-60.9%). No larvae (0.0%, 95% CI: 0-36.9%), but one nymph removed from M. glareolus was positive (10%, 95% CI: 0.3-44.5%). Prevalence of R. slovaca in host-seeking D. marginatus larvae, collected in the same area, was 42% (n=38; 95% CI: 26.3-59.2%). Prevalence of R. slovaca was greater in larvae feeding on PCR-positive Apodemus than in those feeding on negative mice (78.6% vs. 37.1%). Furthermore, levels of infestation with D. marginatus larvae were greater for R. slovaca-positive mice. The infection of Apodemus spp. was probably the result of repeated bites by transovarially infected larvae. On the other hand, the finding of R. slovaca in mice tissues would be compatible with transmission from these hosts to feeding D. marginatus. Based on such a hypothesis, the most heavily infested Apodemus might play a role as amplifiers of the infection.

Detection of Invasive Borrelia burgdorferi Strains in North-Eastern Piedmont, Italy

Following reports of human cases of Lyme borreliosis from the Ossola Valley, a mountainous area of Piemonte, north‐western Italy, the abundance and altitudinal distribution of ticks, and infection of these vectors with Borrelia burgdorferi sensu lato were evaluated. A total of 1662 host‐seeking Ixodes ricinus were collected by dragging from April to September 2011 at locations between 400 and 1450 m above sea level. Additional 104 I. ricinus were collected from 35 hunted wild animals (4 chamois, 8 roe deer, 23 red deer). Tick density, expressed as the number of ticks per 100 m2, resulted highly variable among different areas, ranging from 0 to 105 larvae and from 0 to 22 nymphs. A sample of 352 ticks (327 from dragging and 25 from wild animals) was screened by a PCR assay targeting a fragment of the 16S rRNA gene of B. burgdorferi s.l. Positive samples were confirmed with a PCR assay specific for the 5S‐23S rRNA intergenic spacer region and sequenced. Four genospecies were found: B. afzelii (prevalence 4.0%), B. lusitaniae (4.0%), B. garinii (1.5%) and B. valaisiana (0.3%). Phylogenetic analysis based on the ospC gene showed that most of the Borrelia strains from pathogenic genospecies had the potential for human infection and for invasion of secondary body sites.