Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis.

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.

Molecular identification of cryptic cysticercosis: Taenia ovis krabbei in wild intermediate and domestic definitive hosts.

The complex life cycle of taeniids represents an ideal model of a multi-host system. The complexity of these parasites can therefore cover the epidemiological issues of the interface between wild and domestic animals, especially once spatial overlap between wild and domestic definitive and intermediate hosts occurs. Here we use the occurrence of Taenia ovis krabbei in two model areas as an example of this epidemiological complexity. In two contiguous areas in the Italian northern Apennines, two hunted roe deer (Capreolus capreolus) showed numerous cysticerci in the muscles of their whole body and an adult tapeworm was recorded in a semi-stray dog (Canis lupus familiaris). Through molecular typing of the mitochondrial cytochrome c oxidase I (cox1) gene, cysticerci and the adult tapeworm of T. krabbei were identified. Taenia krabbei cysticercosis was recorded for the first time in Italy. Although the role of dogs in the parasite's life cycle emerges, the overlap between wild and domestic definitive hosts and the increase of wild population densities raise concerns about the temporal (old or new) introduction and the spread of this parasite by one of these canid species (wolf (Canis lupus) or dog). Although T. krabbei is not a public health issue, economic concerns emerged for hunters and meat producers, related to the damage of carcasses by cysticerci. Therefore, there is a need to evaluate the spread of T. krabbei in the intermediate and definitive host populations, and to ensure the relevant sanitary education for hunters in order to avoid practices that could favour the spread and maintenance of its life cycle.

Five-year surveillance of human tuberculosis caused by Mycobacterium bovis in Bologna, Italy: an underestimated problem.

Human tuberculosis (TB) caused by Mycobacterium bovis surveillance is affected by a lack of data. The aims of the present study were: (i) to estimate the proportion of human TB caused by M. bovis over a period of 5 years in Bologna, Northern Italy, which, like most Western European countries, has been declared bovine TB-free; (ii) to compare the genetic profiles of M. bovis strains identified in humans with those circulating in cattle in the last 15 years in Italy. Among 511 TB patients, the proportion of human TB caused by M. bovis was 1·76%, significantly associated to extra-pulmonary localization (P = 0·004) and to being elderly (P < 0·001) and Italy-born (P = 0·036). The molecular epidemiology analysis by spoligotyping and Multilocus Variable Tandem Repeat Analysis confirmed that most M. bovis strains from Italy-born patients matched those circulating in cattle herds in Italy between 2001 and 2016. Two cases of Mycobacterium bovis BCG infection were also characterized. In conclusion, the rate of human TB caused by M. bovis was not negligible, highlighting the relevance of molecular typing in evaluating the effectiveness of programmes designed to eradicate TB in cattle in Italy.

Characterization of Brachyspira hyodysenteriae isolates from Italy by multilocus sequence typing and multiple locus variable number tandem repeat analysis.

AIMS: To evaluate and compare the capabilities of multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) techniques to characterize Brachyspira hyodysenteriae isolates and to investigate the relationship between pleuromutilin resistance and genetic variability. METHODS AND RESULTS: MLST genotyping was performed on 180 B. hyodysenteriae isolates, and the results were evaluated considering profiles from 108 other strains previously reported in the database. In total, 37 sequence types were obtained. The MLVA approach completely characterized 172 strains and grouped the isolates into 22 different profiles. The combination of MLST and MLVA showed a slight increase in the discriminatory power, identifying 33 joint profiles. An antibiotic resistance analysis showed a reduction in the susceptibility to pleuromutilins over time, and a weak association between susceptibility to valnemulin and inclusion in clonal complex 4. CONCLUSION: MLST and MLVA are reliable methods for characterizing B. hyodysenteriae strains and they have comparable discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping of B. hyodysenteriae isolates and a database of all the genetic profiles collected during the diagnostic activities could support traditional epidemiological investigations in identifying infection sources and routes of transmission among herds, and in developing more effective control measures.

Spatiotemporal and Ecological Patterns of Mycobacterium microti Infection in Wild Boar (Sus scrofa).

Mycobacterium microti has recently been described as the causative agent of tuberculosis-like lesions in wild boar (Sus scrofa), a reservoir specie of Mycobacterium tuberculosis complex (MTBC) in some European Mediterranean ecosystem. Through a five-year survey on tuberculosis in free-living wild boars, the epidemiological trend of M. microti infections and the host and population risk factors linked with its occurrence were described. Retropharyngeal and mandibular lymph nodes of 3041 hunted wild boars from six different districts were macroscopically inspected. The sex and age of each animal were registered, as well as the animal abundance in each district. Lesions compatible with tuberculosis (190) were collected and analysed using a gyrB PCR-RFLP assay. M. microti was identified directly in 99 tissue samples (Prev = 3.26%; 95% CI: 2.67-3.97%), while neither Mycobacterium bovis, nor other members of the MTBC were detected. The probability of being M. microti positive showed spatio-temporal variability, with 26% of increase of risk of being infected for each year. Moreover, a positive effect of wild boar abundance and age on the prevalence was detected. The generalized increase in the European wild boar population, coupled with its sensitivity to M. microti infection, poses a future concern for the identification and management of MTBC members in wild boar.

Tuberculosis in domestic animal species.

M. bovis and M. caprae, members of the Mycobacterium tuberculosis complex (MTC), are the major causative agents of tuberculosis in domestic animals. Notably, M. bovis exhibits a wide host range; the infection has been reported in many domesticated animals and free or captive wildlife. Despite most of them acting as spill-over hosts in particular epidemiological scenarios, some domesticated species as pigs, camelids and goats may display high rates of infection and possibly play a role in the inter-species transmission of the disease. The aim of this review is to make an updated overview of the susceptibility and the role in the transmission of the disease of the most common domesticated animals species such as small ruminants, pigs, horses, camelids, dogs and cats. An overview of the diagnostic approaches to detect the infection in each of the species included in the review is also presented.

Isolation of Mycobacterium caprae (Lechtal genotype) from red deer (Cervus elaphus) in Italy.

During tuberculosis (TB) surveillance, 53 hunted red deer (Cervus elaphus) were collected to determine whether TB was present in free-ranging animals from an Italian alpine area. Samples (lungs, liver, intestine, and lymph nodes) were cultured and analyzed by real-time PCR assay carried out directly on tissue. Mycobacterium caprae was isolated from small granulomatous, tuberculosis-like lesions in the liver of a 12-yr-old female. Identification of suspect colonies was done by PCR restriction fragment length polymorphism analysis of the gyrb gene, and genotyping was performed by spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat analysis. The isolated strain was genetically identical to strains isolated in the study area in 2001 from dairy cows imported from Austria and in 2010 from an indigenous cow. The genotype, called "Lechtal," is the most frequently detected in the TB outbreaks in Austria and Germany. The possibility that red deer act as a maintenance host of M. caprae between TB outbreaks could be not excluded. Despite the high red deer population density, the detection of only one infected red deer could suggest that the wildlife management measures applied in the study area (prohibition of artificial feeding and secure removal of offal from hunted animals) may reduce the risk of TB spreading.

Identification of Mammalian orthoreovirus type 3 in Italian bats.

Summary This study describes the isolation and molecular characterization of Mammalian orthoreovirus (MRV) in microbats. Faecal samples and dead individuals available from rehabilitation centres or collected from known roost sites were virologically tested. In total, 112 carcasses of bats found dead, and 44 faecal samples were analysed. Nineteen viral strains were isolated by in vitro cell culture from faecal and tissue samples of different bat species (Pipistrellus khulii, Tadarida teniotis, Rhinolophus hipposideros and Vespertilio murinus), and they were morphologically identified as reoviruses by negative staining electron microscopy observation. The definitive assignment of all isolates to MRV was confirmed by RT-PCR assays targeting the L1 gene. Through a multiplex RT-PCR assay targeting the S1 gene, we typed 15 of 19 isolates as MRV type 3. Partial L1 (416 bp) and complete S1 (1416 bp) sequences of the isolates were analysed and compared with those of reference strains obtained from GenBank, belonging to the three serotypes. Molecular analysis of the S1 gene revealed that the amino acid residues associated with neurotropism (198-204NLAIRLP, 249I, 350D and 419E) were highly conserved among the Italian bat strains. These results suggest that potentially neurotropic MRV type 3 strains are widespread among Italian bats. Furthermore, the identification of MRV type 3 in bat species such as Pipistrellus Khulii, which is common in urban areas and known for its close contact with humans, underlines the need for vigilance.

Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs.

Mycobacterium bovis recognizes as hosts a wide spectrum of animal species. In particular epidemiological situations, high prevalence of infection is found also in pigs. In the present study, we evaluated the capability of the interferon-gamma (IFN-γ) assay to identify pigs infected with M. bovis. The results of the immune-diagnosis were correlated to the findings of the post mortem inspection and the bacterial culture of lymph nodes. Blood samples of 146 pigs, belonging to a local breed of Sicily reared in free or semi-free roaming conditions, were collected to assess the specificity and the sensibility of the IFN-γ assay. Thirty-one pigs, from M. bovis free herds, did not react to the IFN-γ assay, yielding a specificity of 100%. The IFN-γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculous lesions, with a sensibility of 78.9-84.6%, respectively. Out of 26 reactors to the test, 15 pigs (57.7%) confirmed to be infected after the bacterial culture and 22 (84.6%) had tuberculous lesions. The IFN-γ assay was able to reveal 4 animals with no visible lesions (NVL). Together, these findings support the feasible use of the IFN-γ assay as an intra vitam tool for the surveillance and management of M. bovis infection in swine populations.

A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex.

The activity of cyclin-dependent kinases (CDK) is crucial for cell-cycle transitions. Here, we report the identification of a CDK activity that phosphorylates the retinoblastoma-related (RBR) protein. A CDK/cyclin complex that binds to and phosphorylates RBR may be isolated from various plant sources, e.g. wheat, maize, Arabidopsis thaliana and tobacco, and from cells growing under various conditions. The presence of an RBR-associated CDK activity correlates with the proliferative activity, suggesting that phosphorylation of RBR is a major event in actively proliferating tissues. In A. thaliana, this activity comprises a PSTAIRE CDKA and at least cyclin D2. Furthermore, this CDK activity is cell-cycle-regulated, as revealed by studies with highly synchronized tobacco BY-2 cells where it is maximal in late G1 and early S phase cells and progressively decreases until G2 phase. Aphidicolin-arrested but not roscovitine-arrested cells contain a PSTAIRE-type CDK that binds to and phosphorylates RBR. Thus, association with a D-type cyclin is a likely mechanism leading to CDK activation late in G1. Our studies constitute the first report measuring the activity of CDK/cyclin complexes formed in vivo on RBR, an activity that fluctuates in a cell-cycle-dependent manner. This work provides the basis for further studies on the impact of phosphorylation of RBR on its function during the cell cycle and development.

The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G(1)/S regulators.

Association of the retinoblastoma (Rb) protein with E2F transcription factors is central to cell cycle-specific gene expression and growth in animal cells. Whether Rb-E2F complexes are also involved in plant cell growth and differentiation is still unknown since E2F proteins have not yet been identified in plants. Here we report the isolation and characterisation of a wheat E2F (TmE2F) cDNA clone. Interestingly, the overall domain organisation of plant E2F is related to the human E2F-1/2/3 subset but its primary sequence is slightly more related to the E2F-4/5 subset. TmE2F-Rb binding depends on residues, located at the C-terminus, which are different from those of animal E2Fs. However, the acidic or hydrophobic nature of certain residues is maintained, strongly suggesting that they may have a crucial role in E2F activities. Plant E2F is expressed in proliferating cultured cells and in differentiated tissues and is up-regulated early in S phase. Our studies reinforce the idea that G(1)/S regulators in plants are unrelated to those of yeast cells but similar to those of animal cells and provide new tools to analyse the links between cell cycle regulators, plant growth and developmental signals.

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.

Two independent pathways of expression lead to self-assembly of the rabbit hemorrhagic disease virus capsid protein.

The rabbit hemorrhagic disease virus capsid protein was expressed in insect cells either as an individual protein species, from a mRNA analogous to the viral subgenomic RNA, or as part of a polyprotein that included the viral 3C-like protease and the RNA polymerase. Both pathways of expression led to the assembly of viruslike particles morphologically and antigenically similar to purified virus.