Molecular and serological prevalence of Coxiella burnetii in bovine dairy herds in southern Chile: A PCR and ELISA-based assessment of bulk tank milk samples.

Coxiella burnetii (C. burnetii) is a highly resilient zoonotic bacterium responsible for Q fever, a disease which occurs worldwide, with the exception of New Zealand. However, in Chile, the prevalence and impact of C. burnetii in cattle herds remain poorly understood due to limited research. This study aimed to assess the presence of C. burnetii in dairy cattle herds in southern Chile, using two diagnostic methods on bulk tank milk samples. The results of the study revealed a high prevalence of C. burnetii infection in the analyzed herds. Of the 271 milk tank samples tested, 76% (208/271, CI: 71.1-81.5) tested positive using ELISA, while 73% (200/271, CI: 68.0-78.8) tested positive using qPCR. These findings indicate a significant presence of C. burnetii in the cattle herds studied. Despite the high prevalence observed, no new Q fever outbreaks have been reported in the study area. This discrepancy highlights the need for further research to better understand the transmission dynamics, environmental factors, and livestock management practices associated with C. burnetii infection. These studies will contribute to the development of effective prevention and control strategies and promote public health regarding Q fever.

Effectiveness of copper ions against Mycobacterium avium subsp. paratuberculosis and bacterial communities in naturally contaminated raw cow's milk.

AIM: The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow's milk. METHODS AND RESULTS: A copper ions treatment was evaluated in naturally contaminated cow's milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical-chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively). CONCLUSION: With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow's milk. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow's milk, information that could be useful as a tool for MAP control.

More insights about the interfering effect of Mycobacterium avium subsp. paratuberculosis (MAP) infection on Mycobacterium bovis (M. bovis) detection in dairy cattle.

The estimated herd and within herd Mycobacterium bovis (M. bovis) infection prevalence in the southern Chile regions are 0.3 and 0.67%, respectively. However, higher rates of infection still remain in some herds. In parallel, it is well established that a big proportion of cattle herds are infected with Mycobacterium avium subsp. paratuberculosis (MAP), which has been also associated with a clear interference effect on M. bovis diagnosis. The present study aims to provide more insights about the diagnostic interference for Mycobacterium bovis detection due to co-infection with MAP. To better understand the dynamics of this identified interference, the effect of MAP genotype present, as well as MAP faecal shedding values (as proxy of the infection progression), for each of the CFT results was compared. No relationship was observed between MAP genotype with any type of differential response to the diagnostic tests of M. bovis infection. However, MAP shedding values in animals with positive CFT diagnostic results for M. bovis infection was significantly lower than animals with a negative CFT result, observing that as the MAP shedding load raises, the response to the bovine tuberculin test tends to be negative. The findings reported in this study allows to interpret that one of the causes of the prolonged elimination of M. bovis infection from some cattle herds may be due in part to the advanced MAP infection status in co-infected individuals affecting the outcome of screening in-vivo diagnostic techniques such as CFT. These false negative animals that show negative results to M. bovis detection tests, may maintain the infection at herd level and spread the pathogen to healthy individuals.

In vitro inactivation of Mycobacterium avium subsp. paratuberculosis (MAP) by use of copper ions.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. RESULTS: Moderate numbers (103 CFU ml-1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. CONCLUSIONS: The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

Effect of Mycobacterium avium subsp. paratuberculosis (MAP) infection on the diagnostic accuracy for Mycobacterium bovis (M. bovis) infection under field conditions in cattle belonging to low M. bovis prevalence herds.

Currently, the Chilean authority has implemented a National Eradication Program for bovine tuberculosis (bTB), aimed at controlling and eradicating the disease in Chile. The area under study has a low within-herd prevalence, has a relatively low number of infected herds, and is one of the major milk and beef producing areas in the country. However, so far, no attempts at eradicating the disease have been successful. It has been suggested that the diagnostic tests used were either not sensitive or specific enough. In addition, previous studies have shown that a great number of herds are infected with Mycobacterium avium subsp. paratuberculosis (MAP). The present study estimates the effect of MAP infection under field conditions, on the diagnostic sensitivity and specificity of routine tests in live animals for Mycobacterium bovis infection diagnosis in cattle. In general, the estimations of test accuracy observed an increase in the sensitivity and specificity on MAP-infected animals for tuberculin test but observed a decrease in the sensitivity of gamma interferon tests for MAP-infected cattle. These results are different from those of previous studies considering the role of MAP infection as an interfering infection. More research is needed in order to understand the complex interactions of the different mycobacteria that can be found infecting production cattle.

Evaluation of PMS-PCR technology for detection of Mycobacterium avium subsp. paratuberculosis directly from bovine fecal specimens.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with <100 MAP per 2g feces (30.06) (p<0.05). PMS-PCR technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection.

Comparative investigations on digestion in grazing (Ceratotherium simum) and browsing (Diceros bicornis) rhinoceroses.

Rhinoceroses represent the largest extant herbivores with extensive dietary specialization for plant groups like browse (black rhino Diceros bicornis) or grass (white rhino Ceratotherium simum). However, it is not clear to what extent such diet selection patterns are reflected in adaptations of digestive physiology of the respective feeding types. In this study, feeding trials with four black and five white rhinos were conducted in four zoos. The animals had ad libitum access to the same batch of grass hay (second cut; neutral detergent fiber (NDF) 63% dry matter (DM), crude protein 10.2% DM). Total intake, fecal N content, in vitro digestibility of NDF residues of feces, fecal particle size and mean retention time (MRT) of particles (Cr-mordanted fiber; 1-2mm) and fluid (Co-EDTA) were quantified. The average daily DM intake was 70+/-12 g/kg BW(0.75) for white and 73+/-10 g/kg BW(0.75) for black rhinos. In the in vitro fermentation test fecal NDF residues of black rhinos resulted in higher gas productions at fermentation times of 12 to 24h, indicating that white rhinos have a superior capacity to digest NDF. Average MRT for fluids and particles was 28+/-4h and 43+/-5h in white and 34+/-4h and 39+/-4h in black rhinos. The selectivity factor (SF=MRT(particle)/MRT(fluid)) was higher for white (1.5+/-0.2) than for black rhinos (1.2+/-0.1) (p=0.016). In a comparison of 12 ruminant and 3 rhino species, SF was correlated to percentage of grass in diet (R=0.75). Mean fecal particle size was higher in white (9.1+/-1.94 mm) than in black rhinos (6.1+/-0.79 mm) (p=0.016). The results demonstrate differences between white and black rhinos in terms of retention times and fiber digestibility. The more selective retention of particles by the white rhino corresponds with the higher digestion of fiber measured indirectly. Furthermore there is indication for a general pattern of high SF in grazing ruminants and rhinos. The difference in fecal particle size between both rhino species might be due to the considerable difference in body weight.

High-resolution electron-microscopic study of the relationship between human enamel and dentin crystals at the dentinoenamel junction.

The development of dentin and of enamel share a common starting locus: the dentinoenamel junction (DEJ). In this study the relationship between enamel and dentin crystals has been investigated in order to highlight the guiding or modulating role of the previously mineralized dentin layer during enamel formation. Observations were made with a high-resolution electron microscope and, after digitalization, image-analysis software was used to obtain digital diffractograms of individual crystals. In general no direct epitaxial growth of enamel crystals onto dentin crystals could be demonstrated. The absence of direct contact between the two kinds of crystals and the presence of amorphous areas within enamel particles at the junction with dentin crystals were always noted. Only in a few cases was the relationship between enamel and dentin crystals observed, which suggested a preorganization of the enamel matrix influenced by the dentin surface structure. This could be explained either by the existence of a proteinaceous continuum between enamel and dentin or by the orientation of enamel proteins by dentin crystals.

High-resolution electron microscopy study of synthetic carbonate and aluminum containing apatites.

Aluminum (Al)-containing calcium-deficient carbonated hydroxyapatites were produced by a precipitation method from aqueous solution with carbonate (0-6.1%) and aluminum (0.1-0.5%) concentrations close to those found in biological materials. Two series of apatites were prepared: one at pH 7.0 and another at pH 9. 0. High-resolution electron microscopy has shown that many of them possess structural defects such as screw dislocations, grain boundaries, and central defects. Samples with high carbonate content and high water and high Al(3+) content had a high amount of structural defects. Accordingly, a sample (7Al1) with a relatively high carbonate content (6.1%) and a sample (7Al6) without carbonate but with a relatively high water (2.0 mol) and Al(3+) content (0. 39%) presented the highest amount of structural defects, 54% and 47%, respectively. A sample (7Al13) with a low level of crystalline water (1 mol) and low carbonate (2.5%) showed a small amount of defects. The presence of water associated with Al(3+) induced a high number of crystals having a central defect with a great similarity to the so-called water layer of octacalcium phosphate (OCP). Observed images of all these crystals have shown good correspondence with the computer-simulated image based on the crystal structure of hydroxyapatite, indicating that the addition of Al(3+) and carbonate does not perturb the apatitic structure.

First experimental evidence for human dentine crystal formation involving conversion of octacalcium phosphate to hydroxyapatite.

Biological apatite-crystal formation is a complex process starting with heterogeneous nucleation of inorganic calcium phosphate on an organic extracellular matrix [Cuisinier et al. (1995), J. Cryst. Growth, 156, 443-453]. Further stages of crystal growth are also controlled by the organic matrix and both nucleation and growth processes are under cellular control [Mann (1993), Nature (London), 367, 499-505]. The final mineral in calcified tissue is constituted by poorly crystalline hydroxyapatite (HA) with a low Ca:P ratio, containing foreign ions such as carbonate and fluoride. This study reports the first observation of octacalcium phosphate (OCP) [Brown (1962), Nature (London), 196, 1048-1055] in a biological tissue; OCP was found in the central part and HA at the extremities of the same crystal of calcifying dentine. This observation is of key importance in understanding the first nucleation steps of biological mineralization. The presence of OCP in a forming human dentine crystal and the observation in the same tissue of nanometer-sized particles with a HA structure [Houllé et al. (1997), J. Dent. Res. 76, 895-904] clearly proves that two mechanisms, direct nucleation of non-stoichiometric HA crystals and nucleation of OCP, occur simultaneously in same area of mineralization. OCP is found to be a transient phase during the growth of biological crystals. In small crystals, OCP is completely transformed into HA by a hydrolysis reaction (Brown, 1962) and can only be detected in larger crystals because of its slow kinetics of transformation.

High resolution electron microscopy: structure and growth mechanisms of human dentin crystals.

Biological crystal formation was postulated to begin by a nucleation process. Such processes have been demonstrated for human amelogenesis and bone mineralization. The aim of this study was to confirm if such mechanisms occur during dentin crystal formation. The structure of human fetal dentin crystals and the earliest stages of mineral growth were followed by High Resolution Electron Microscopy (HREM) associated with digitalized image analysis. Micrographs of the mineralization front were first digitalized, and selected areas were transformed in the reciprocal space by Fast Fourier Transform. The resulting diffractograms were compared with computer-simulated diffractograms and used to determine the orientation of crystals. Dentin crystals, found close to the mineralization front, show a structure closely related to that of hydroxyapatite (HA), as determined by comparison of HREM images with simulated images. These crystals present numerous structural defects such as dislocations and grain boundaries. These defects appear to be present in dentin crystals at an early stage of growth. We have also observed nanometer-sized particles in mineralization areas. Calculated diffractograms of these areas show significant similarities with HA diffraction patterns, and in one case, their structure could be correlated to HA structure through an image simulation process. These nanometer-sized particles could be related to the nucleation process, and their growth, orientation, and formation appear to be mediated by extracellular matrix components.

Human amelogenesis: high resolution electron microscopy of nanometer-sized particles.

Growth of inorganic crystals of enamel is described as a two-stage process with growth of ribbon-like crystals in length and width, followed by their development in thickness. In early stages of crystal growth during human amelogenesis nanometer-sized particles with a mean diameter of 1.1 nm were described between ribbon-like crystals. These small particles had a crystalline structure but their lattice parameters did not seem to be directly related to those of calcium phosphates. The nanometer-sized particles appear to correspond to initial stages of apatite crystal growth. Their localization close to ribbon-like crystals and their progressive increase in size and number may indicate that they represent a precursor phase for these crystals. Nucleation areas at both extremities, of elongated ribbon-like crystals could be involved in the two-directional growth of ribbons and/or in nanometer-sized particle nucleation.

Observation of the loss of the hydroxyapatite sixfold symmetry in a human fetal tooth enamel crystal.

A deviation from the hydroxyapatite hexagonal symmetry of a human tooth enamel crystal observed by high-resolution electron microscopy is reported. This symmetry deviation is characterized by: (1) 'preferential' planes that can be indexed as (100) with an intensity that differs from the (300) and the other (100) hexagonal equivalent planes; and (2) streaking of higher order reflections in the optical diffractogram of the image of the crystal. Computer simulations show that similar 'preferential' planes can also be observed at specific crystal tilt angles (and/or beam tilt and/or objective aperture misalignment) and at crystal thickness/microscope defocus values in images of hydroxyapatite crystals observed along the [0001] or [2243] zone axes. The streaking of higher order reflections in the optical diffractogram is related to a deformation of the crystal itself and does indeed show a symmetry deviation of the crystal under observation.

Human amelogenesis. I: High resolution electron microscopy study of ribbon-like crystals.

Ribbon-like crystals, from developing enamel of human fetuses, were studied by high resolution electron microscopy. These crystals were classically described as the first organized mineral formed during amelogenesis. They were characterized by a mean width-to-thickness ratio (W.T-1) of 9.5, and 40% were bent. On lattice images we noted the presence of the central dark line (CDL) associated with white spots. Both structures were found in crystals with a minimum thickness of 8-10 nm. CDLs were localized in the center of the crystals and seemed to be linked to the initial growth process, but their exact structure and function were not fully determined. We were able to study the structure of the ribbon-like crystals with a Scherzer resolution close to 0.2 nm. The good correspondence between experimental and computed images showed that their structure was related to hydroxyapatite (HA). In addition, the presence of ionic substitutions and deficiencies were also compatible with HA. In this study, about 50% of the crystals showed structural defects. Screw dislocations were the most often noted defects and were observed within crystals aligned along five different zone axes. Low- and high-angle boundaries were also detected. Low-angle boundaries, found in the center of the crystals, could thus be related to CDLs and be implicated in the nucleation step of crystal formation, whereas high-angle boundaries could result from the fusion of ribbon-like crystals. Such mechanisms could induce an acceleration of the growth in thickness of the crystal observed during the maturation stage of amelogenesis.

High resolution electron microscopy of young apatite crystals in human fetal enamel.

Apatite crystal growth in enamel has been followed during the secretory stage in 5 month-old human fetuses. The crystals were observed in a high resolution transmission electron microscope operating at 300 kV. This microscope has a Scherzer resolution of 0.19 nm and for each micrograph, the absence of astigmatism and drift was checked with optical diffractograms. Crystals in various stages of growth from approximately 3 to 10 unit cells in thickness could be observed along [0001] and mean value of 1010 zone axes. For growing crystals, a highly ordered atomic structure with an ionic distribution consistent with hydroxyapatite was noted as demonstrated by the correspondence of computer simulated and observed images. Moreover, most of the crystals showed structural defects such as edge dislocations and periodically repeated strain fields created by screw dislocations. The observed dislocations were described by Burgers vectors running parallel to mean value of 1120 axes.

Ultrastructural study on human root caries.

Superficial root caries lesions of incisors and premolars of 24 patients aged 52-60 years were studied by scanning electron microscopy and transmission electron microscopy. In the predominantly gram-positive bacteria of the root dental plaque, a great number of corncob configurations were observed. Bacterial infiltration into cementum occurred without any gradient of demineralization and mainly followed the borders between calcified cementum bundles as well as incremental lines. The narrow channels which were filled initially by a single row of gram-positive bacteria broadened progressively with simultaneous destruction of the apatite minerals and the collagenous matrix. Root dentin invasion showed great similarities to coronal dentin with an important gradient of demineralization. In the deep layers, sclerosed tubules contained sometimes rhombohedral crystals. In more superficial layers, bacterial invasion occurred initially in still partially sclerosed tubules. Tubular lumens as well as lateral side branches which had lost their peritubular dentin were filled with gram-positive microorganisms. An important diffuse destruction of the apatite minerals was observed at this stage in the intertubular dentin which still presented typical cross-banded collagen fibrils. The confluence of enlarged dentinal tubules filled by numerous microorganisms led to large bacterial zones with complete destruction of root dentin.

Transmission electron microscopy of the human odontoblast process in peripheral root dentine.

Various methods of fixation were tried on dentine of 45 premolars from children. Two methods based on immediate or delayed use of liquid nitrogen, as well as a cryoprotector (10 per cent dimethylsulphoxid), gave the best results in transmission electron microscopy. Using these methods, the presence of an odontoblast process could be demonstrated in the inner, middle and peripheral human root dentine. The presence of unmyelinated nerve fibril in outer root dentine supports the hypothesis of direct neural stimulation in peripheral dentine.

Ultrastructural development of dated plaque in case of denture stomatitis.

Using special acrylic discs glued into the palatal surface of a denture the development of denture plaque in a case of denture stomatitis as well as the relationships between the denture surface, pellicle, plaque and palatal epithelium were studied with transmission electron microscopy after 30 mn, 1 h, 4 h, 8 h, 24 h, 48 h, 9 days and 29 days. A thin pellicle which increased in thickness from 9 hours to 28 days was visible at the surface of the acrylic denture. It appeared that bacteria retained in the palatal epithelial intercellular spaces were the source of the plaque which developed at the surface of the acrylic denture. Initially loosely packed cocci-like and a few rod-shaped Gram positive bacteria appeared in 8 hours samples at the denture-epithelium interface. An important increase in denture plaque thickness was noted between 24 and 48 hours. In 9 and 28 days samples, coccoid and rod-shaped Gram positive and Gram negative bacteria were present and filamentous bacteria began to be apparent. In all the samples studied, Candida was rare.

The extent of the human odontoblast process as determined by transmission electron microscopy: the hypothesis of a retractable suspensor system.

After fixation of fully formed human permanent teeth in liquid nitrogen the extent of the odontoblast process has been studied in transmission electron microscopy. The odontoblast process, limited by a trilaminar plasma membrane, was found just under the dentine-enamel junction. In cross section, the cytoplasm contained a granular mass with light and/or dense core granules. Bare unmyelinated nerve-like fibrils were seen in close connection with the odontoblast process. In the periodontoblastic space non calcified collagen fibrils were occasionally present. Cytoplasmic globules and granules limited by a trilaminar membrane, with occasional myelinic figures were also noted in the peripheral tubular lumens. The hypothesis of a retractable suspensor system is advanced to explain why in normal fixation conditions the odontoblast processes associated with nerve fibrils have not been observed in the outer layers of dentine.

Transmission electron microscopy of plaque accumulations in denture stomatitis.

Denture pellicle in denture stomatitis has been studied with transmission electron microscopy after embedding the denture base in a water-miscible resin in seven patients with heavy plaque deposits on their dentures and in five patients with no apparent plaque accumulation. In the first group, the denture surface was covered by a well differentiated granular pellicle. A cell-free zone was interposed between the pellicle and the plaque, which consisted predominantly of rounded, rod-shaped, and filamentous microorganisms with a loose distribution, separated by an electron-lucent amorphous and gel-like matrix. C. albicans were scattered among the bacteria and often presented with degenerated cytoplasm. In the second group, a structurally heterogeneous pellicle was seen adjacent to the denture surface. A thin plaque that consisted mainly of dense accumulations of C. albicans, a narrow dense matrix, and few bacteria was found. Calculus accumulations on the dentures consisted of amicrobial calcifications in the deeper layers, whereas the superficial parts showed bacterial calcifications.