Transmission patterns of a Mycobacterium avium subsp. paratuberculosis clone within a single heard investigated by Whole Genome Sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale.

Evaluation of Mycobacterium avium subsp. paratuberculosis survival during the manufacturing process of Italian raw milk hard cheeses (Parmigiano Reggiano and Grana Padano).

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis in ruminants, is suspected to be involved in the aetiology of some human diseases. Notably, the consumption of milk and dairy products is considered to be the main route of human exposure to MAP because of its ability to survive during pasteurization and manufacturing processes. The aim of this study was to investigate, through a microbiological challenge test, the survival of MAP during the manufacturing and ripening period of two Italian hard cheeses, Parmigiano Reggiano and Grana Padano, made from raw bovine milk. The challenge test was performed in two different phases: the creaming phase and the manufacturing phase. The creaming phase, which is the first step of cheese production, was reproduced in the laboratory employing raw cow's milk spiked with a MAP reference strain at a final concentration of 5.58 log10 CFU/mL. After the creaming at 18 °C and 27 °C for 12 h, a decrease of 0.80 log10 and 0.77 log10 was observed in partially skimmed milk, respectively. In the second phase, two batches of raw cow's milk (1000 L each) were inoculated with MAP reference and wild strains, respectively. Then, the entire manufacturing process for Parmigiano Reggiano and Grana Padano, both of Protective Designation of Origin (PDO), was reproduced in an experimental cheese factory, starting from a concentration in milk of 5.19 ±â€¯0.01 and 5.28 ±â€¯0.08 log10 CFU/mL of MAP reference and wild strains, respectively. Heating the curd at 53 °C for 20 min did not affect MAP survival, however a significant decrease (p < 0.05) in MAP viability was observed during the moulding phase and after salting in brine, regarding the wild strains and the reference strain, respectively. In addition, a significant decrease was observed during the ripening period, at which time the MAP concentration dropped below the limit of detection from the second and the third month of ripening, for the wild and reference strains, respectively. Taking into account the poor data availability about MAP survival in hard cheeses, this study may improve the knowledge regarding the effect of the cheese manufacturing process on the MAP dynamics, supporting also the safety of traditional raw milk hard cheeses.

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing.

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2-1.3 × 105 somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 102 cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.

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.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant.

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013-March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170-230 MAP cells/g (62-115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

First outbreak of bovine mastitis caused by Prototheca blaschkeae.

The most important animal disease caused by yeast-like algae belonging to the genus Prototheca is bovine mastitis. Although the infection can be caused by both Prototheca zopfii genotype 2 and Prototheca blaschkeae, the bulk of prevalence of bovine protothecal mastitis has been so far attributed to the former, being P. blaschkeae only sporadically isolated. However, we report here the first outbreak of bovine mastitis caused by P. blaschkeae in an Italian dairy herd. One hundred and four individual milk samples, three bulk tank milk and 16 environmental samples within the herd were screened for the presence of Prototheca: five, one and four positive samples, were respectively observed. Molecular analysis revealed that, with the sole exception of one environmental isolate belonging to P. zopfii genotype 2, all Prototheca strains were identified as P. blaschkeae. Our results might suggest that even P. blaschkeae can induce mastitis outbreaks, while it is not clear if the higher incidence of P. zopfii genotype 2 as causative agent of protothecal mastitis could reflect an intrinsic higher pathogenicity or it could be simply the consequence of its, so far observed, higher diffusion in worldwide dairy herd ecosystems.

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species.

AIM: The study describes the development of a simple and rapid tool to identify yeast-like microalgae belonging to the genus Prototheca. METHODS AND RESULTS: The method, based on two-step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non-pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype-specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. CONCLUSIONS: The assay two-step Real Time PCR is accurate, robust, cost-effective and faster than auxonographical, biochemical or conventional molecular biology methods. SIGNIFICANCE AND IMPACT OF THE STUDY: the rapid and high throughout two-step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.

Molecular characterization of Prototheca strains isolated from Italian dairy herds.

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.

Screening for cognitive toxicity of anticholinergic drugs.

Impairment of memory by muscarinic anticholinergic drugs has been demonstrated with a variety of agents using both acute administration in normal volunteers and chronic treatment in patients. Cognitive toxicity must be considered in the evaluation of the risks versus the benefits of psychopharmacologic treatment. The relationship between serum levels of anticholinergic drugs measured by radioreceptor assay and performance on the Buschke Selective Reminding Test were investigated to develop strategies for identifying patients with drug-related cognitive impairment. The radioreceptor assay may be of value in screening for patients at high risk for cognitive toxicity. However, there appears to be no alternative to careful longitudinal evaluation of learning and memory in patients after appropriate trial modifications of their medication regimens to identify patients with drug-related impairment.

The folate in human milk.

In previous studies of the folate content of human milk, samples were prepared for assay by a method that resulted in a turbid solution that was then assayed by a turbidimetric microbiological method. We have used an improved microbiological assay in which the milks were treated with rennin to precipitate casein and heated in a buffered ascorbate to coagulate lactalbumin and lactoglobulin. Milks were obtained serially from nursing mothers for periods ranging from 1 day to 6 months postpartum. The results showed that the folate in human milk has few glutamate residues since treatment with a purified folate conjugase preparation release no additional folate activity for Lactobacillus casei. Colostrum is relatively low in folate, but milk folate increases as lactation proceeds. During each stage of lactation there was great variation in milk folate content among the women. In the case of a folate-deficient woman, supplementation with folic acid resulted in a prompt increase in milk folate level.

H-2 homology requirements for secondary cell-mediated lympholysis and miced lymphocyte reactions to TNP-modified syngeneic lymphocytes.

Secondary cell-mediated lympholysis (CML) and mixed lymphocyte reactions (MLR) were generated in a tissue culture system against trinitrophenyl (TNP)-modified murine syngeneic spleen cells. H-2 homology between primary and secondary TNP-modified stimulating cells was required in order to restimulate in the secondary CML. Strong proliferative responses (MLR) were detected only in the secondary cultures, for which H-2 homology was also required between TNP-modified primary and secondary immunogens. Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were I, D, and K and/or I-A. Homology throughout the entire major histocompatibility complex or at K plus I-A gave stronger MLR than did cultures in which there was homology between the primary and secondary phases at I or D only.

In vitro induction of F1 hybrid anti-parent cell-mediated cytotoxicity.

Spleen cells from normal (C57BL/6 X DBA/2)F1 mice were sensitized in vitro for 5 days with irradiated C57BL/6 or DBA/2 parental stimulating cells. Effector cells were generated which specifically lysed 51Cr-labeled targets (leukemia or mitogen-stimulated lymphoid cells) H-2-matched with the parental genotype used for sensitization. The response of F1 spleen cells to the C57BL/6 parent was stronger and more reproducible than that to the DBA/2 parent. The kinetics of generation of effector cells were similar for the F1 anti-parent and an F1 anti-allogeneic response. However, the magnitude of the F1 anti-C57BL/6 cytotoxic response was considerably lower than the F1 response to allogeneic cells. The ratio of responder to stimulator cells in the cultures was more critical for the former than for the latter response. Several lots of fetal bovine serum were found to be adequate for supplementing the medium in the induction of J1 hybrid anti-parent and anti-allogeneic cytotoxic effector cells. Based on these and other studies, it would appear that the F1 hybrid anti-parent cytotoxic response provides an in vitro model of murine hemopoietic graft rejection in vivo. This response may be elicited by a mechanism distinct from T cell-mediated cytotoxicity and involve different subpopulations of spleen cells.

Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by H-2K and H-2D serological regions of the murine major histocompatibility complex.

Splenic lymphocytes from four C57BL/10 congenic resistant mouse strains were sensitized in vitro with trinitrophenyl (TNP)-modified autologous spleen cellsmthe effector cells generated were incubated with 51-Cr-labeled unmodified or TNP-modified spleen or tumor target cells, and the percentage of specific lympholysis determined. The results obtained using syngeneic-, congenic-, recombinante, and allogeneic-modified target cells indicated that TNP modification of the target cells was a necessary but insufficient requirement for lympholysis. Intra-H-2 homology either between modified stimulating cells and modified target cells or between responding lymphocytes and modified target cells was also important in the specificity for lysis. Homology at the K serological region or at K plus I-A in the B10.A and B10BR strains, and at either the D serological region or at some other region (possibly K) in the B10.D2 and C57BL/10 strains were shown to be necessary in order to detect lympholysis. Experiments using (B10itimes C57BL/10)F1 responding lymphocytes sensitized and assayed with TNP-modified parental cells indicated that the homology required for lympholysis was between modified stimulating and modified target cellsmthe possibility is raised that histocompatibility antigens may serve in the autologous system as cell surface components which are modified by viruses or autoimmune complexes to form cell-bound modified-self antigens, which are particularly suited for cell-mediated immune reactions. Evidence is presented suggesting that H-2-linked Ir genes are expressed in the TNP-modified autologous cytotoxic system. These findings imply that the major histocompatibility complex can be functionally involved both in the response potential to and in the formation of new antigenic determinants involving modified-self components.