Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

Surveillance of the source of poultry infections with Enterococcus hirae and Enterococcus cecorum in Slovenia and E. hirae antibiotic resistance patterns.

Enterococcus cecorum and Enterococcus hirae can cause locomotor problems, septicaemia, and endocarditis in broiler chickens. Understanding transmission routes and resistance patterns are essential for effective treatment. The aim of this study was to follow the same animals from the breeder flock to the hatchery and up to 14-day-old broiler chickens on the farm to find the source of E. cecorum and E. hirae. During the production cycle, only faeces and organs of broilers were E. hirae positive in all three sampled farms in which recurrent enterococcal infections were previously confirmed. None of the isolates possessed virulence genes. Based on resistance profiles, a variety of different strains were present in faeces and organs of different broilers' ages. Samples from the breeder flock and hatchery were negative. Faecal shedding on the farm and tolerance of enterococci to the environmental conditions enable persistence of pathogenic enterococci in farm dust; therefore, adequate cleaning and disinfection after depopulation of the farms could prevent disease recurrence in the new cycle. Susceptibility testing of E. hirae isolates showed no resistance to the drugs of choice for the treatment of enterococcal infections in poultry.

Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov., isolated from gastric mucosa of red foxes (Vulpes vulpes).

Six Helicobacter-like isolates were recovered from 15 gastric mucosa samples of red foxes (Vulpes vulpes) shot by hunters in the surroundings of Ljubljana, Slovenia. Gram-negative, tightly coiled, intensely motile, 7-15 µm long and ≤1 µm wide bacteria grew on the biphasic blood agar plates. By using a genus-specific polymerase chain reaction (PCR), all isolates were confirmed as Helicobacter sp. and subsequently subjected to whole-genome sequencing (WGS). Five isolates showed a genome-wide average nucleotide identity (ANI) value of <95 % to the previously described Helicobacter species and one isolate was classified as Helicobacter felis. In the five unidentified isolates, the 16S rRNA gene sequence similarity to the type strains of all Helicobacter species ranged from 98.6 to 98.9 %. Their taxonomic status was established using a polyphasic taxonomic approach comprising the core genome-based phylogeny, morphological and phenotypic characteristics, including an analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. Phylogeny revealed the existence of three novel and well-supported clusters, with Helicobacter bizzozeronii and Helicobacter baculiformis being the most closely related species. The isolates also differed from the previously described species in their MALDI-TOF profiles and some biochemical characteristics. In conclusion, the data presented herein indicate that the obtained isolates, excluding H. felis isolate, represent three novel Helicobacter species, for which the names Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov. are proposed, with isolates L9T (=DSM 108823T=CRBIP 111719T), L15T (=DSM 108730T=CCUG 72910T) and L2T (=DSM 108727T=CCUG 72909T) as type strains, respectively.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

Quantification of Clostridioides (Clostridium) difficile in feces of calves of different age and determination of predominant Clostridioides difficile ribotype 033 relatedness and transmission between family dairy farms using multilocus variable-number tandem-repeat analysis.

BACKGROUND: Community acquired Clostridioides (Clostridium) difficile infection (CA-CDI) is a significant health problem in human and veterinary medicine. Animals are often considered as potential reservoirs for CA-CDI. In Europe, family farming is the most predominant farming operation, with a complex interaction between animals and the community. Therefore, it is pertinent to evaluate transmission patterns of C. difficile on such prominent European farming model. Fecal samples from calves (n = 2442) were collected biweekly over a period of one year on 20 mid-size family dairy farms. Environmental samples (n = 475) were collected in a three month interval. Clostridioides difficile was detected using qPCR in 243 fecal samples (243/2442); positive samples were then quantified. Association between prevalence/load of C. difficile and age of the calves was estimated with logistic regression model. Most common C. difficile isolate from calves (n = 76) and the environment (n = 14) was C. difficile ribotype 033, which was further analyzed using multilocus variable-number tandem-repeat analysis (MLVA) to assess intra- and between-farm relatedness. RESULTS: Clostridioides difficile was detected in feces of calves less than 24 h old. Results showed a non-linear statistically significant decrease in shedding load of C. difficile with age (P < 0.0001). A nonlinear relationship was also established between the number of calves and the farm C. difficile prevalence, whereas the prevalence of C. difficile ribotype 033 increased linearly with the number of calves. MLVA revealed close intra-farm relatedness among C. difficile ribotypes 033. It also revealed that the between-farms close relatedness of C. difficile ribotypes 033 can be a direct result of farm to farm trade of calves. CONCLUSIONS: Implementation of better hygiene and management measures on farms may help decrease the risk of spreading CA-CDI between animals and the community. Trading calves older than 3 weeks would decrease the possibility C. difficile dissemination in the community because of lower prevalence and lower load of C. difficile in feces.

Identification of risk factors influencing Clostridium difficile prevalence in middle-size dairy farms.

Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence.

Mycobacterium angelicum sp. nov., a non-chromogenic, slow-growing species isolated from fish and related to Mycobacterium szulgai.

The name 'Mycobacterium angelicum' dates back to 2003 when it was suggested for a slowly growing mycobacterium isolated from freshwater angelfish. This name is revived here and the novel species is proposed on the basis of the polyphasic characterization of four strains including the original one. The four strains presented 100 % 16S rRNA gene sequence similarity with Mycobacterium szulgai but clearly differed from M. szulgai for the milky white aspect of the colonies. The sequence similarity with the type strain of M. szulgai ranged, in eight additionally investigated genetic targets, from 78.9 to 94.3 %, an evident contrast with the close relatedness that emerged at the level of 16S rRNA gene. The average nucleotide identity between the genomes of M. szulgai DSM 44166T and strain 126/5/03T (type strain of the novel species) was 92.92 %, and supported the status of independent species. The confirmation of the name Mycobacterium angelicum sp. nov. is proposed, with strain 126/5/03T ( = CIP 109313T = DSM 45057T) as the type strain.

Campylobacter jejuni contamination of broiler carcasses: Population dynamics and genetic profiles at slaughterhouse level.

Six slaughter batches deriving from six typical industrial broiler flocks were examined for the presence, quantity and genetic characteristics of contaminating Campylobacter jejuni (C. jejuni) during various stages of slaughtering and carcass processing. To assess the contamination dynamics of the carcasses, the analyses were always conducted on neck-skin samples from the same pre-selected and carefully marked carcasses in each batch. The skin samples were taken sequentially at three successive slaughter-line locations in the evisceration room, after three-day refrigeration and after three-day freezing procedure. Caecal samples from the same animals were also tested, as well as samples from the slaughterhouse environment before and after slaughtering. The samples were analysed by the ISO10272 isolation method; campylobacters from neck-skin samples were also quantified. Isolates were species-identified and genotyped by pulsed-field gel electrophoresis (PFGE). On average, the highest C. jejuni skin contamination was detected at the first sampling point (post-plucking), suggesting that the majority of Campylobacter contamination actually occurs before the entrance to the eviscerating room, probably during the preceding plucking stage. In two out of five positive batches, an additional increase in contamination was recorded after the evisceration step. An evident trend of increasing contamination level was detected when successive batches were compared at each of two initial sampling sites in the evisceration room, indicating an accumulation of contaminating C. jejuni at some point before the evisceration room. Three-day refrigeration and three-day freezing caused a 4.5- and 142-fold drop in mean C. jejuni CFU counts, respectively. All pre-slaughtering samples from the slaughterhouse environment were negative and all post-slaughtering samples, except water from the scalding tank, were positive. Pulsotypes were limited: altogether five different types were detected, typically one type per batch. The PFGE results from the slaughterhouse environment isolates indicate that cross-contamination is possible (multiple pulsotypes detected in e.g. eviscerating machine). Nevertheless, this was not confirmed in carcasses: analyses of neck-skin isolates suggest that carcasses are contaminated by their own caecal/farm/flock pulsotype.

International Clostridium difficile animal strain collection and large diversity of animal associated strains.

BACKGROUND: Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. RESULTS: Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). CONCLUSIONS: This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

Prevalence and molecular characterization of Clostridium difficile isolated from European Barn Swallows (Hirundo rustica) during migration.

BACKGROUND: Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species. Birds, especially migratory passerine species, can play a role in the spread of many pathogens, including Clostridium difficile. Barn Swallows (Hirundo rustica) nest in close proximity to human habitats and their biology is closely associated with cattle farming. Therefore, we hypothesized that Barn Swallows can be the reservoir of Clostridium difficile. RESULTS: Barn Swallows (n = 175) were captured on their autumn migration across Europe to sub-Saharan Africa. Droppings were collected from juvenile (n = 152) and adult birds (n = 23). Overall prevalence of Clostridium difficile was 4% (7/175); 4.6% (7/152) in juvenile birds and 0/23 in adults. Clostridium difficile ribotypes 078, 002 and 014 were identified, which are commonly found in farm animals and humans. Three new Clostridium difficile ribotypes were also identified: SB3, SB159 and SB166, one of which was toxigenic, harbouring genes for toxins A and B. CONCLUSIONS: Results of this study indicate that Barn Swallows might play a role in national and international dissemination of Clostridium difficile and could serve as a source for human and animal infection. Clostridium difficile ribotype 078 was identified, which has been reported as an emerging cause of community-associated Clostridium difficile infection in humans. Based on this and other studies, however, it is more likely that Barn Swallows have a more indicative than perpetuating role in Clostridium difficile epidemiology.

Clostridium difficile in goats and sheep in Slovenia: characterisation of strains and evidence of age-related shedding.

Diversity of Clostridium difficile in different age groups of goats (n = 109) and sheep (n = 105) was investigated. C. difficile was detected in 9.2% of goats and 5.7% of sheep. None of the adult animals were positive. Isolates belonged to four toxinotypes (0, V, XIa, XII), six PCR-ribotypes (010, 014/020, 045, 056, SLO 061, SLO 151) and six pulsotypes. PCR-ribotypes 010, 014/020, 045 and 056 were found previously in other animal species and humans in Slovenia. Additionally, three pulsotypes were indistinguishable from restriction patterns in our PFGE database of animal isolates. All strains were susceptible to metronidazol, vancomycin, moxifloxacin, and with the exception of a single non-toxigenic strain also to clindamycin and erythromycin. While all strains were resistant to ciprofloxacin and levofloxacin, oxacillin-resistance was observed only in strains of PCR-ribotype 045. This first study on C. difficile in small ruminants in Slovenia revealed the evidence of age-related shedding as the highest was demonstrated in neonatal goats and sheep aged up to 16 days.

Bovine Tuberculosis in Wild Boar (Sus scrofa) in Slovenia.

Mycobacteria of the Mycobacterium tuberculosis complex (MTC) are capable of infecting a wide variety of animals. Wild boar (Sus scrofa) has been recognized as an important wildlife reservoir for bovine tuberculosis. We screened wild boar in Slovenia for the presence of (1) Mycobacterium bovis in tissues and (2) antibodies to M. bovis in blood samples. In 2016 and 2017, 1284 tissue samples from 676 wild boar were subjected to cultivation. In 2018 and 2019, blood samples from 132 wild boar were examined using an ELISA kit. None of the MTC species were isolated from the tissue samples, and no antibodies to M. bovis were detected in the blood samples. Several nontuberculous mycobacteria (NTM), identified by 16S rRNA sequencing and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry, were found in the tissues of 9.8% of the wild boar: Mycobacterium nonchromogenicum, Mycobacterium peregrinum/Mycobacterium septicum, Mycobacterium avium, Mycobacterium engbaekii, Mycobacterium arupense, Mycobacterium algericum, Mycobacterium bohemicum, Mycobacterium confluentis, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium thermoresistibile, and Mycobacterium vaccae. Species-level identification was not possible for 21.2% of the isolates. At the time of the study, wild boar in Slovenia were not at risk from bTB; the significance of the presence of NTM in wild boar remains to be clarified and evaluated from a One Health perspective.

A scoping review on associations between paratuberculosis and productivity in cattle.

Paratuberculosis (PTB), or Johne's disease, is a disease with worldwide distribution caused by Mycobacterium avium subsp. paratuberculosis (MAP) that leads to chronic enteritis, primarily in ruminants. Even subclinical infection significantly reduces the animals' performance, and consequences of the disease lead to high economic losses for the cattle industry. To estimate the economic burden of bovine PTB and to evaluate the benefits of a potential control program, accurate estimates of the production effects associated with the disease are required. Therefore, the aim of this scoping review was to provide a comprehensive overview of associations between MAP infection and production parameters in cattle. The studies were collected from three electronic databases. Of the total 1,605 identified studies, 1,432 did not meet the set criteria in the title and abstract screening and a further 106 were excluded during full-text review. Finally, data on 34 different production parameters were extracted from 67 publications. Results show that the magnitude of reported performance losses varies depending on several factors, such as the type of diagnostic test applied, disease status or number of lactations. Studies reported a reduction in milk yield, changes in milk quality (e.g., higher somatic cell count, lower amount of produced milk fat and protein), reduced fertility (e.g., prolonged calving interval and service period, higher abortion rate and calving difficulties), reduced weaning weight, slaughter weight and slaughter value, or a higher risk for mastitis. Results from the studies included in our review show a median decrease of milk yield per infected cow of -452 kg/lactation for raw and -405 kg/lactation for modeled data. Similarly, the amount of produced milk protein fell by a median of -14.41 kg/lactation for modeled data and the amount of produced milk fat by a median of -13.13 kg/lactation. The reviewed studies revealed a prolonged calving interval by around 30 days and a 1.5 to 3 times higher likeliness of culling per lactation in PTB positive animals. Results from this scoping review provide evidence-based inputs for the development of economic models aiming at the estimation of the costs and benefits associated with different disease control scenarios for PTB.

Report on overcoming the poor quality of Apai pulsotypes with a short review on PFGE for listeria monocytogenes.

Since listeriosis, caused by Listeria monocytogenes, is one of the important concerns of public health in Europe related to foodborne zoonoses, an efficient protocol for isolate typing is necessary when performing epidemiological studies. Three standardized PFGE protocols available for L. monocytogenes were briefly reviewed. Since observing a poor-quality of ApaI pulsotypes in our laboratory, enzymes from three different manufacturers were compared. The obtained pulsotypes showed that restriction digestion with ApaI from New England BioLabs should be complemented with a subsequent overnight incubation of PFGE plugs in TE buffer for better performance.

Phylogeography of Brucella suis biovar 2 with focus on Slovenian wildlife.

Brucella suis commonly infects swine but occasionally also other animal species and humans. Wild boars are the most important reservoir of B. suis biovar 2, continually infecting susceptible hosts through close contact. Nevertheless, the genetic diversity of B. suis in wildlife remains understudied. Here, we typed 17 Slovenian B. suis biovar 2 isolates obtained in 2017-2019 from wild boars (n = 16) and a hare (n = 1) using whole-genome sequencing (WGS). To assess the global phylogenetic diversity of B. suis, we compared them to 126 publicly available B. suis genomes. All Slovenian isolates fell within the biovar 2 lineage, confirming the previous multiplex PCR typing results. According to MLST-21, the wild boar isolates were of sequence types (STs) ST16 (n = 8) and ST153 (n = 8); the maximum genetic distance between isolates of the same ST was 28 wgMLST alleles. The ST153 isolates were restricted to the Slovenian-Croatian border and clustered together with the Croatian ST153 isolates from swine, indicating cross-border transmission of B. suis ST153 strain. The hare isolate was of ST40 and was genetically distant (≥ 489 alleles) from the wild boar isolates. The genome-wide phylogeny clearly separated different B. suis biovars. The present study is the first report on the population structure of B. suis in wildlife in Slovenia and shows that the Slovenian B. suis population is genetically heterogeneous. At the species level, B. suis biovars are clearly separated in the WGS-based phylogenetic tree and can therefore be reliably predicted using WGS.

Assessment of Tuberculosis Biomarkers in Paratuberculosis-infected Cattle.

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and Methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.

Clostridium difficile genotypes other than ribotype 078 that are prevalent among human, animal and environmental isolates.

BACKGROUND: Characterising the overlap of C. difficile genotypes in different reservoirs can improve our understanding of possible transmission routes of this pathogen. Most of the studies have focused on a comparison of the PCR ribotype 078 isolated from humans and animals. Here we describe for the first time a comparison of C. difficile genotypes isolated during longer time intervals from different sources including humans, animals and the non-hospital environment. RESULTS: Altogether 786 isolates from time interval 2008-2010 were grouped into 90 PCR ribotypes and eleven of them were shared among all host types and the environment. Ribotypes that were most common in humans were also present in water and different animals (014/020, 002, 029). Interestingly, non-toxigenic isolates were very common in the environment (30.8%) in comparison to humans (6.5%) and animals (7.7%). A high degree of similarity was observed for human and animal isolates with PFGE. In human isolates resistance to erithromycin, clindamycin and moxifloxacin was detected, while all animal isolates were susceptible to all antibiotics tested. CONCLUSION: Our results show that many other types in addition to PCR Ribotype 078 are shared between humans and animals and that the most prevalent genotypes in humans have the ability to survive also in the environment and several animal hosts. The genetic relatedness observed with PFGE suggests that transmission of given genotype from one reservoir to the other is likely to occur.

Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA.

BACKGROUND: Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle. RESULTS: The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/µl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher's exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant. CONCLUSIONS: This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.

Coagulase-negative staphylococci from non-mastitic bovine mammary gland: characterization of Staphylococcus chromogenes and Staphylococcus haemolyticus by antibiotic susceptibility testing and pulsed-field gel electrophoresis.

During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (28·8%), followed by Staph. simulans (11·2%), Staph. xylosus (11·2%), Staph. epidermidis (7·5%), Staph. hyicus (6·3%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation.

Evidence of Anaplasma phagocytophilum in game animals from Slovenia.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.