Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle.

BACKGROUND: A rising isolation trend of drug-resistant M. bovis from human clinical cases is documented in the literature. Here we assessed Mycobacterium tuberculosis complex isolates from cattle for drug susceptibility by the gold standard agar proportion method and a simplified resazurin microtitre assay (d-REMA). A total of 38 M. tuberculosis complex strains, including M. bovis (n = 36) and M. caprae (n = 2) isolates, from cattle in Tunisia were tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and pyrazinamide. RESULTS: M. caprae isolates were found to be susceptible to all test drugs. All M. bovis strains were resistant to pyrazinamide, as expected. In addition, one M. bovis isolate showed high-level resistance to streptomycin (MIC > 500.0 µg/ml). Concordant results with the two methods were found. The most common target genes associated with streptomycin resistance, namely the rrs, rpsL and gidB genes, were DNA sequenced. A non-synonymous mutation at codon 43 (K43R) was found in the rpsL gene. To the best of our knowledge, this is the first report describing the isolation of a streptomycin-resistant M. bovis isolate from animal origin. CONCLUSIONS: Antitubercular drug susceptibility testing of M. bovis isolates from animals should be performed in settings where bTB is endemic in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.

Effects of natural treatments on the varroa mite infestation levels and overall health of honey bee (Apis mellifera) colonies.

The survival of the honey bee (Apis mellifera), which has a crucial role in pollination and ecosystem maintenance, is threatened by many pathogens, including parasites, bacteria, fungi and viruses. The ectoparasite Varroa destructor is considered the major cause of the worldwide decline in honey bee colony health. Although several synthetic acaricides are available to control Varroa infestations, resistant mites and side effects on bees have been documented. The development of natural alternatives for mite control is therefore encouraged. The study aims at exploring the effects of cinnamon and oregano essential oils (EOs) and of a mixed fruit cocktail juice on mite infestation levels and bee colony health. A multi-method study including hive inspection, mite count, molecular detection of fungal, bacterial and viral pathogens, analysis of defensin-1, hymenoptaecin and vitellogenin immune gene expression, colony density and honey production data, was conducted in a 20-hive experimental apiary. The colonies were divided into five groups: four treatment groups and one control group. The treatment groups were fed on a sugar syrup supplemented with cinnamon EO, oregano EO, a 1:1 mixture of both EOs, or a juice cocktail. An unsupplemented syrup was, instead, used to feed the control group. While V. destructor affected all the colonies throughout the study, no differences in mite infestation levels, population density and honey yield were observed between treatment and control groups. An overexpression of vitellogenin was instead found in all EO-treated groups, even though a significant difference was only found in the group treated with the 1:1 EO mixture. Viral (DWV, CBPV and BQCV), fungal (Nosema ceranae) and bacterial (Melissococcus plutonius) pathogens from both symptomatic and asymptomatic colonies were detected.

Geo-epidemiology of animal tuberculosis and Mycobacterium bovis genotypes in livestock in a small, high-incidence area in Sicily, Italy.

Introduction: The persistence of animal tuberculosis (TB) in livestock is a major concern in Sicily, Italy. The objective of this study was to elucidate the transmission dynamics of M. bovis infection in a highly circumscribed, and at the same time geographically diverse, high-risk area of the island through an in-depth geo-epidemiological investigation of TB in cattle and black pigs raised in small-scale extensive farms across the district of Caronia. Methods: We used genotype analysis coupled with geographic information system (GIS) technology and phylogenetic inference to characterize the spatial distribution of TB and M. bovis genotypes in livestock and the genetic relationships between M. bovis isolates. A total of 589 M. bovis isolates collected from slaughtered cattle (n = 527) and Sicilian black pigs (n = 62) over a 5-year period (2014-2018) were included in the study. Results: TB was widespread throughout the district and was most frequent in the north-central area of the district, especially along one of the district's streams. We identified a total of 62 M. bovis genotypes. Identical genetic profiles were isolated from both neighboring and non-neighburing herds. The 10 most frequent genotypes, accounting for 82% of M. bovis isolates, showed geographic specificities in that they tended to cluster in specific spatial niches. The landscape structure of these niches-i.e. steep slopes, rocky ridges, meadows and streams-is likely to have had a significant influence on the distribution of TB among livestock in Caronia. Higher concentrations of TB were observed along streams and in open meadows, while rocky ridges and slopes appeared to have hampered the spread of TB. Discussion: The geographical distribution of TB cases among livestock in Caronia is consistent with several epidemiological scenarios (e.g., high density of infected herds along the streams or in hilly plateau where livestock share pastures). Landscape structure is likely to play an important role in the transmission and persistence of M. bovis infection across the district. Additional potential risk factors, such as livestock trading and extensive breeding methods, are also discussed. Our results will contribute to the improvement of surveillance, control and eradication activities of TB in Sicily by the implementation of ad hoc TB control measures, especially in farms located along streams, sharing common pastures or with mixed animal species.

Epidemiological significance of the domestic black pig (Sus scrofa) in maintenance of bovine tuberculosis in Sicily.

Bovine tuberculosis (bTB) is an emerging disease among wild animals in many parts of the world. Wildlife reservoir hosts may thus represent a potential source of infection for livestock and humans. We investigated the role played by the Sicilian black pig, an autochthonous free- or semi-free-ranging domestic pig breed, as a potential source of bTB infection in an area where bTB prevalence in cattle is high. We initially performed a preliminary field study to assess the occurrence of bTB in such animals. We sampled 119 pigs at abattoir and found 6.7% and 3.4% of them to be affected by gross tuberculous-like lesions (TBL) and Mycobacterium bovis culture positive, respectively. We then proceeded to investigate the dissemination and characteristics of lesions in a second field study performed on 100 animals sampled from infected herds. Here, tissues collected at the abattoir were examined macroscopically, microscopically, and by culture tests. Most pigs with TBL showed generalized lesions in both gross and histological examinations (53% and 65.5%, respectively). Head lymph nodes were the most frequently affected in both localized and generalized TB cases observed macroscopically and microscopically. M. bovis was the most frequently isolated etiologic agent. The molecular characterization of isolates from both field studies by spoligotyping and analysis of 12 mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR) loci, followed by their comparison to isolates of cattle origin, suggested a potential transmission of mycobacteria from domestic animals to black pigs and vice versa. Our findings, along with ethological, ecological, and management considerations, suggest that the black pig might act as a bTB reservoir in the ecosystem under study. However, additional studies will be necessary to establish the true epidemiological significance of the Sicilian black pig.

Natural Mineral Waters and Metabolic Syndrome: Insights From Obese Male and Female C57BL/6 Mice on Caloric Restriction.

Metabolic syndrome (MetS) represents one of the greatest challenges to public health given its serious consequences on cardiovascular diseases and type 2 diabetes. A carbohydrate-restricted, low-fat diet is the current therapy for MetS. Natural mineral waters (NMWs) are known to exert beneficial effects on human health. Our primary objective was to shed light on the potential therapeutic properties of NMWs in MetS. A total of 125 C57BL/6 male and female mice were included in the study. Of these, 10 were left untreated. They were fed a standard diet with tap water throughout the study period, and stayed healthy. The remaining 115 mice were initially fed a high-calorie diet (HCD) consisting of a high-fat feed (60% of energy from fat) with 10% fructose in tap water, served ad libitum over a period of 4 months to induce MetS (the MetS induction phase). Mice were then randomly divided into six treatment groups and a control group, all of which received a low-calorie diet (LCD), but with a different kind of drinking water, for 2 months (the treatment phase). Five groups were each treated with a different kind of NMW, one group by alternating the five NMWs, and one group - the control group - was given tap water. Body weight and blood biochemistry were monitored over the 6-month trial. After 4 months, male and female mice on HCD developed obesity, hypercholesterolaemia and hyperglycaemia, although gains in body weight, total cholesterol, and blood glucose in males were greater than those observed in females (P < 0.0001). When combined with an LCD, the NMWs rich in sulphate, magnesium and bicarbonate, and the minimally mineralised one were the most effective in reducing the blood levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, and glucose. Sex differences emerged during both the MetS induction phase and the treatment phase. These results suggest that NMWs rich in specific macronutrients, such as bicarbonate, sulphate and magnesium, and minimally mineralised water, in combination with an LCD, may contribute to controlling blood lipid and glucose levels in subjects with MetS. Further studies are needed to confirm these results and to extend them to humans.

Neurobrucellosis associated with syndrome of inappropriate antidiuretic hormone with resultant diabetes insipidus and hypothyroidism.

Neurological involvement of the central nervous system in brucellosis is uncommon. We describe a rare case of meningoencephalitis due to Brucella melitensis infection, associated with the syndrome of inappropriate antidiuretic hormone secretion and leading to diabetes insipidus and hypothyroidism. Neurobrucellosis, although rare, should be considered in cases of neurological disease of unknown etiology.

A case of generalized bovine tuberculosis in a sheep.

The present report describes a rare case of generalized bovine-type tuberculosis in a slaughtered 4-year-old ewe discovered during routine surveillance at an abattoir. A postmortem examination revealed lesions in the ewe's thoracic and abdominal cavities, ranging from encapsulated, mineralized foci to extensive, soft, caseous tissue. Lesions in the lungs, liver, and lymph nodes were consistent with mycobacterial infection. A histopathological examination detected granulomatous lesions in all tissue samples. The presence of Mycobacterium tuberculosis complex genome was confirmed through a polymerase chain reaction (PCR) analysis of tissues, using IS6110 primers, followed by a nucleotide sequence analysis of PCR products. Acid-fast bacteria, characterized as Mycobacterium bovis, were isolated from lesions following 38 days of incubation.

Isolation, Molecular Typing, and Antibiotic Susceptibility Testing of Mycobacterium avium Subspecies hominissuis From a Dog With Generalized Mycobacteriosis.

Mycobacterium avium-intracellulare complex infections are becoming an increasing concern in veterinary medicine because they affect livestock, wildlife, and companion animals. Here we describe the isolation, molecular typing, and antibiotic susceptibility testing of the causative agent of a rare case of generalized mycobacteriosis in a crossbred dog. Mycobacterial colonies were isolated from a popliteal lymph node aspirate sample and molecular typed by SNPs typing of the genes gyrB and rpsA, the 3' region of the hsp65 gene and the internal transcribed spacer (ITS), and MIRU-VNTR analysis. Colonies were also tested in vitro against the macrolide clarithromycin and other drugs, using a resazurin microdilution assay, in order to provide the most appropriate treatment for the dog. Results from SNPs typing of gyrB and ITS, as well as from MIRU-VNTR analysis suggested the isolation of a single strain of M. avium subsp. hominissuis (Mah). On the other hand, SNP typing of rpsA revealed DNA polymorphisms that led colonies to cluster into two groups. The presence of two distinct strains of Mah has been assumed. All colonies, regardless of the nucleotide sequence of rpsA, were found to be sensitive to all of the drugs tested except for ethambutol. Although the therapy administered was adequate, the dog's overall clinical status worsened progressively and the animal died 8 months later. In conclusion, we report on the isolation of Mah from a dog with generalized mycobacteriosis.

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis.

BACKGROUND: Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. RESULTS: Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. CONCLUSION: In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

Genotype diversity and distribution of Mycobacterium bovis from livestock in a small, high-risk area in northeastern Sicily, Italy.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important re-emerging disease affecting livestock, wildlife and humans. Epidemiological studies are crucial to identifying the source of bTB infection, and its transmission dynamics and host preference, and thus to the implementation of effective strategies to contain it. In this study, we typed M. bovis isolates from livestock, and investigated their genetic diversity and distribution. A total of 204 M. bovis isolates were collected from cattle (n = 164) and Sicilian black pigs (n = 40) reared in a limited area of the province of Messina, northeastern Sicily, an area that had previously been identified as having the highest incidence of bTB in livestock on the island. All M. bovis isolates were typed by both spoligotyping and 12-loci MIRU-VNTR analysis. Results from both methods were then combined in order to improve the discriminatory power of M. bovis typing. We identified 73 combined genetic profiles. Thirty-five point six percent of the profiles were common to at least two animals, whereas 64.4% of profiles occurred in only one animal. A number of genetic profiles were predominant in either cattle or black pigs. We identified common genetic patterns in M. bovis isolates originating not only from neighboring districts, but also from non-neighboring districts. Our findings suggest that bTB is widespread in our setting, and is caused by a large number of genetically diverse M. bovis strains. The ecology and farming practices characteristic of the area may explain the substantial M. bovis heterogeneity observed, and could represent obstacles to bTB eradication.

Rapid and safe one-step extraction method for the identification of Brucella strains at genus and species level by MALDI-TOF mass spectrometry.

Brucellosis is essentially a disease of domesticated livestock; however, humans can also be infected via the consumption of contaminated meat or dairy products, underlying the need for rapid and accurate identification methods. Procedures for microbiological identification and typing of Brucella spp. are expensive, time-consuming, and must be conducted in biohazard containment facilities to minimize operator risk. The development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based assay has reduced the processing time while maintaining performance standards. In this study, to improve the identification accuracy and suitability of the MALDI-TOF-based assay for routine diagnosis, we developed a new protein extraction protocol and generated a custom reference database containing Brucella strains representative of the most widespread species. The reference library was then challenged with blind-coded field samples isolated from infected animals. The results indicated that the database could be used to correctly identify 99.5% and 97% of Brucella strains at the genus and species level, respectively, indicating that the performance of the assay was not affected by the different culture conditions used for microbial isolation. Moreover, the inactivated samples were stored and shipped to reference laboratories with no ill effect on protein stability, thus confirming the reliability of our method for routine diagnosis. Finally, we evaluated the epidemiological value of the protocol by comparing the clustering analysis results of Brucella melitensis strains obtained via multiple locus variable-number tandem repeat analysis or MALDI-TOF MS. The results showed that the MALDI-TOF assay could not decipher the true phylogenetic tree, suggesting that the protein profile did not correspond with the genetic evolution of Brucella.

In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens.

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions.

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

Purpose. Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing.Methodology. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient.Results. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here.Conclusion. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1ß and TNF-α levels were also found in L. lactis-inoculated glands. The above findings seem to suggest that food-grade L. lactis at a high-inoculum dose such as an overnight culture may elicit a suppurative inflammatory response in the mammary gland, thus becoming a potential mastitis-causing pathogen. Because of the unpredictable potential of L. lactis in acting as a potential mastitis pathogen, this organism cannot be considered a safe treatment for bovine mastitis.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

PURPOSE: Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. METHODOLOGY: In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. CONCLUSION: Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping.

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap".

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection.

Mycobacterium avium-intracellulare complex (MAC) infections have been described in many mammalian species, including humans and pets. We isolated and molecularly typed the causative agent of a rare case of disseminated mycobacteriosis in a dog. We identified the pathogen as M. avium subspecies avium by sequencing the partial genes gyrB and rpsA. Considering the zoonotic potential of this infection, and in an attempt to ensure the most effective treatment for the animal, we also determined the drug susceptibility profile of the isolate to the most common drugs used to treat MAC disease in humans. The pathogen was tested in vitro against the macrolide clarithromycin, as well as against amikacin, ciprofloxacin, rifampicin, ethambutol and linezolid, by the resazurin microdilution assay. It was found to be sensitive to all tested drugs apart from ethambutol. Despite the fact that the pathogen was sensitive to the therapies administered, the dog's overall clinical status worsened and the animal died shortly after antimicrobial susceptibility results became available. Nucleotide sequencing of the embB gene, the target gene most commonly associated with ethambutol resistance, showed new missense mutations when compared to sequences available in public databases. In conclusion, we molecularly identified the MAC pathogen and determined its drug susceptibility profile in a relatively short period of time (7 days). We also characterized new genetic mutations likely to have been involved in the observed ethambutol resistance. Our results confirmed the usefulness of both the gyrB and the rpsA genes as biomarkers for an accurate identification and differentiation of MAC pathogens.

In vitro stimulation of murine peritoneal monocytes induced by alginates.

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.

Multiple drug-susceptibility screening in Mycobacterium bovis: new nucleotide polymorphisms in the embB gene among ethambutol susceptible strains.

OBJECTIVES: Pyrazinamide-resistant Mycobacterium bovis isolates of animal origin were assessed for drug susceptibility to five antituberculosis drugs by the agar based Middlebrook 7H11 method as gold standard as well as by a simplified, dichotomous resazurin microtitre assay (d-REMA). METHODS: A total of 53 M. bovis isolates were typed and tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and the control drug pyrazinamide. On the basis of the results obtained, pncA and embB genes were PCR-amplified and DNA-sequenced for all isolates. RESULTS: All M. bovis isolates, classified into 21 spoligotype/MIRU-VNTR profiles, were resistant to pyrazinamide by both methods, as expected. The pncA gene sequencing confirmed the presence of the resistance-conferring H57D mutation. All strains were found to be susceptible to the other five drugs by the agar based gold standard method. The d-REMA was in agreement with these results for all five drugs, with the exception of 12 isolates, which showed ambiguous and therefore inconclusive results in ethambutol testing. Mutations in the embB gene were observed in all 53 isolates: four new single-nucleotide polymorphisms were identified. No association was found between embB genetic profiles and ethambutol resistance results by the gold standard. CONCLUSION: All M. bovis isolates were sensitive to the most common antituberculosis drugs used for treatment. There was a good agreement between the d-REMA assay and the agar based reference method. Among ethambutol susceptible isolates, four new embB mutations were found.