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.

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.

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.

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.

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.

Genomic and antigenic characterization of viruses from the 1993 Italian foot-and-mouth disease outbreak.

The origin and evolution of the type O foot-and-mouth disease viruses (FMDV) that caused the outbreak occurrence in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on the viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that these Italian viruses were grouped in the ME-SA topotype, closely related to viruses that circulated previously in the Middle East. The analysis of the nucleotide sequences in VP1, VP2 and VP3 showed a co-circulation during the epizootic of genetic variants, including viruses with amino acid replacements in VP3. For some of the isolates analyzed, values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.5 to 5.1 x 10(-2). The use of a panel of new monoclonal antibodies raised against an isolate from this outbreak, as well as monoclonal antibodies to FMDV O1-Switzerland 1965, showed differences in the reactivity pattern among some of the Italian isolates analyzed, which were consistent with the co-circulation of antigenic variants. These results support the potential for FMDV diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented.

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples.

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.

Isolation of Verocytotoxin-producing Escherichia coli O157:H7 from cattle at slaughter in Italy.

Cattle arriving for slaughter at a large abattoir in northern Italy between April 1997 and January 1998 were examined for intestinal carriage of Verocytotoxin-producing Escherichia coli (VTEC) O157 using an immunomagnetic separation technique. Sixty sorbitol non-fermenting VTEC O157 strains were isolated from 59 (13.1%) of the 450 cattle examined. In particular, VTEC O157 was found in 37 (16.6%) of 223 feedlot cattle and in 22 (16.1%) of 137 dairy cull cows, but not in the 90 veal calves sampled. The isolation rate was higher during warm weather (17.5%), falling to an average of 2.9% during the winter months. VT-negative, O157 latex-agglutinating E. coli strains were isolated from 23 (5.1%) of the 450 animals. PCR analysis showed that all 60 VTEC O157 strains carried the VT2 gene and that 25 strains also carried the VT1 gene. In addition, four of the VT-negative, O157 latex-agglutinating E. coli strains carried the VT2 gene. Atypical biochemical features were observed in some VTEC O157: two strains (3.3%) showed beta-glucuronidase activity, and seven (11.7%) produced urease.

A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant.

Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.

Detection and preliminary characterization of a new rabbit calicivirus related to rabbit hemorrhagic disease virus but nonpathogenic.

A new rabbit calicivirus related to the rabbit hemorrhagic disease virus (RHDV) was identified. The new virus contains significant differences from the previously characterized RHDV isolates in terms of pathogenicity, viral titer, tropism, and primary sequence of the structural protein. Cross-protection experiments, antigenic data, and sequence comparisons demonstrate that the new virus is more closely related to RHDV than to the European brown hare syndrome virus, another member of the caliciviruses of the lagomorph group. The existence of a nonpathogenic calicivirus, which we propose to name rabbit calicivirus (RCV), provides an explanation for the early discrepancies found in the course of serological surveys of the rabbit population in European countries.

Leptospira interrogans serovar sejroe infection in a group of laboratory dogs.

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1 : 100 to 1 : 6400, against a serovar (hardjo) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA.

Primers for PCR were selected from a sequenced fragment of clone pL590, which contains a repetitive element present in the genome of Leptospira interrogans serovar hardjo type hardjoprajitno (M. L. Pacciarini, M. L. Savio, S. Tagliabue, and C. Rossi, J. Clin. Microbiol. 30:1243-1249, 1992). A specific DNA fragment was amplified from the genomic DNAs of serovar hardjo type hardjoprajitno and nine serovars also belonging to L. interrogans as a consequence of the spread of the same or a closely related repetitive element within this species (Pacciarini et al., J. Clin. Microbiol. 30:1243-1249, 1992). In addition, specific amplification was obtained from two Leptospira borgpetersenii serovars (tarassovi and hardjo type hardjobovis). Negative PCR results were observed with all of the other Leptospira serovars tested, including nonpathogenic ones (serovars patoc and andamana), another spirochete (Borrelia burgdorferi), bacteria commonly found in biological samples, and swine and bovine cell lines. Direct PCR on biological samples such as kidney samples demonstrated that preliminary isolation and culture of Leptospira cells are not required for efficient detection. Furthermore, digestion of the amplified DNA with the enzymes HinfI and DdeI yielded specific polymorphic patterns, allowing discrimination among the majority of the serovars. These methods were applied to 25 field isolates of serovar pomona, leading to the conclusion that they were suitable for the simple and rapid detection of L. interrogans and for serovar identification.

Identification of Leptospira interrogans strains by monoclonal antibodies and genomic analysis.

A recombinant probe derived from a genomic library of serovar hardjo strain Hardjoprajitno, and a panel of serovar specific Monoclonal Antibodies (MAbs) were used for the characterization of 31 Leptospira isolates from cattle and swine. The two methods performed equally well in serovar identification except for the distinction of the genotypes hardjoprajitno and hardjobovis within serovar hardjo which could only be obtained by genomic analysis. The combination of immunological and genetic information was also useful to evaluate the degree of variability of Leptospira strains. The quality of the patterns and the sensitivity provided by a digoxigenin labelled probe were comparable to those obtained with a radioactive reagent.

The search for improved methods for diagnosing leptospirosis: the approach of a laboratory in Brescia, Italy.

The authors describe work in progress at the laboratory in Brescia, Italy, on the application of molecular methods to the diagnosis of leptospirosis. This work includes the following: a) Development of polymerase chain reaction (PCR) assays capable of amplifying specific deoxyribonucleic acid fragments from most Leptospira interrogans strains. b) Development of a microtitre-based assay for rapid detection of PCR-positive samples. c) Characterisation of Leptospira strains through restriction endonuclease analysis of PCR products and amplified fragment length polymorphism.

Repetitive sequences cloned from Leptospira interrogans serovar hardjo genotype hardjoprajitno and their application to serovar identification.

We selected, from a genomic library of Leptospira interrogans serovar hardjo genotype hardjoprajitno, two probes containing repetitive sequences (pL1 and pL590). The hybridization patterns of these probes to DNA isolated from a variety of Leptospira serovars were examined and their ability to detect subtle differences at the genomic organization level was established. We identified the DNA fragments within pL1 and pL590 which are sufficient to yield polymorphic hybridization patterns; these results define the upper size limit of two novel repetitive elements in the Leptospira genome. The pattern and degree of hybridization observed for the serovars tested in this work were used to divide Leptospira spp. into groups which share genetic relatedness; our conclusions are consistent with previous classifications by other authors.