Denosumab for craniofacial fibrous dysplasia: duration of efficacy and post-treatment effects.

Denosumab has been advocated as a potential treatment for the rare skeletal disorder fibrous dysplasia (FD); however, there is limited data to support safety and efficacy, particularly after drug discontinuation. We report a case of successful treatment of aggressive craniofacial FD with denosumab, highlighting novel insights into the duration of efficacy, surrogate treatment markers, and discontinuation effects. A 13-year-old girl presented with persistent pain and expansion of a maxillary FD lesion, which was not responsive to repeated surgical procedures or bisphosphonates. Pre-treatment biopsy showed high RANKL expression and localization with proliferation markers. Denosumab therapy was associated with improved pain, decreased bone turnover markers, and increased lesion density on computed tomography scan. During 3.5 years of treatment, the patient developed increased non-lesional bone density, and after denosumab discontinuation, she developed hypercalcemia managed with bisphosphonates. Pain relief and lesion stability continued for 2 years following treatment, and symptom recurrence coincided with increased bone turnover markers and decreased lesion density back to pre-treatment levels. This case highlights the importance of considering the duration of efficacy when treating patients with FD and other nonresectable skeletal neoplasms that require long-term management.

Clinical and molecular characterization of Chilean patients with X-linked hypophosphatemia.

We report the most comprehensive clinical and molecular characterization of XLH patients performed in Chile. We show high prevalence of musculoskeletal burden and pain, associated with significantly impaired physical capacity and quality of life, with many relevant complications presenting more frequently than previously reported in cohorts from developed countries. INTRODUCTION: Our current understanding of the clinical presentation and natural history of X-linked hypophosphatemia (XLH) comes mainly from cohorts from developed countries, with limited data on the clinical and genetic abnormalities of XLH patients in South America. OBJECTIVE: To describe the clinical, biochemical, and molecular presentation of patients with XLH in Chile. METHODS: Patients with XLH referred by endocrinologist throughout Chile were included. Demographic data and clinical presentation were obtained from a clinical interview. Surveys were applied for quality of life (QoL), pain, and functionality. FGF23 was measured by ELISA, and genetic testing was performed. Imaging studies were conducted to assess skeletal and renal involvement. RESULTS: We included 26 patients, aged 2-64 years, from 17 unrelated Chilean families. All pediatric patients but only 40% of adults were receiving conventional therapy, while 65% of all patients had elevated alkaline phosphatase. All patients had mutations in PHEX, including 5 novel variants. Radiographic skeletal events (RSE) and enthesopathies in adults were frequent (34% and 85%, respectively). The duration of treatment was associated with fewer RSE (p < 0.05). Most adults reported pain and impaired QoL, and 50% had impaired physical capacity. The number of enthesopathies was associated with worse pain and stiffness scores (p < 0.05). CONCLUSION: Chilean patients with XLH have a high prevalence of musculoskeletal burden associated with pain and impaired physical capacity and QoL, especially in adults who were generally undertreated. These data identify a significant unmet need, inform our understanding of the current status of patients, and can guide care for XLH patients in similarly socioeconomically defined countries.

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.

Fate of Mycobacterium avium subsp. paratuberculosis and changes in bacterial diversity populations in dairy slurry after chemical treatments.

AIMS: A major drawback of using dairy slurry as fertilizer is that it may contains pathogens such as Mycobacterium avium subsp. paratuberculosis (MAP), and it could represent a risk to animal and public health. Thus, the aim of this study was to evaluate the fate of MAP and bacterial communities in dairy slurry after chemical treatments. METHODS AND RESULTS: Cattle slurry, naturally contaminated with MAP, was collected from a dairy herd and divided into 32 glass bottles which were assigned to eight different treatments (control, 3·0% CaO, 0·5% NaOH; 0·087%, 0·11% and 0·14% H2 SO4 ; and 1·0 and 2·5% KMnO4 ). Treated dairy slurry samples were evaluated at 0, 1, 3, 7, 15, 30 and 60-days following treatment application for viable MAP and dairy slurry pH, and in addition temperature in this material was monitored continuously. Bacterial counts were estimated at each sampling time. A Bayesian zero-inflated Poisson mixed model was fitted to assess the effect of each treatment on the count of MAP cells. Model results indicated that only the 3·0% CaO treatment had a statistically important negative effect on MAP counts during the study period. For most treatments, MAP was undetectable immediately after chemical treatment but re-appeared over time, in some replicates at low concentrations. However, in those cases MAP counts were not statistically different than the control treatment. Regarding the fate of the other bacterial populations, the Firmicutes phylum was the dominant population in the un-treated slurry while Clostridia class members were among the most prevalent bacteria after the application of most chemical treatments. CONCLUSION: Only 3% CaO treatment had a statistically important negative effect on MAP viability in cattle slurry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence of MAP partial control in dairy slurry. This information should be considered as a best management practice to reduce MAP and other pathogens for slurry management on dairy farms.

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.

Bone marrow failure and extramedullary hematopoiesis in McCune-Albright syndrome.

In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), bone and bone marrow are, to varying degrees, replaced by fibro-osseous tissue typically devoid of hematopoietic marrow. Despite the extensive marrow replacement in severely affected patients, bone marrow failure is not commonly associated with FD/MAS. We present a 14-year-old girl with FD/MAS, who developed pancytopenia and extramedullary hematopoiesis (EMH) with no identified cause, in the setting of iatrogenic thyrotoxicosis and hyperparathyroidism. Pancytopenia, requiring monthly blood transfusions, persisted despite multiple strategies to correct these endocrinopathies. Due to worsening painful splenomegaly, likely as a result of sequestration, splenectomy was performed. Following splenectomy, pancytopenia resolved and patient has since been transfusion-independent. We report the first detailed case of bone marrow failure and EMH in FD/MAS. The etiology of marrow failure is likely multifactorial and related to the loss of marrow reserve due to extensive polyostotic FD, exacerbated by iatrogenic thyrotoxicosis and hyperparathyroidism. Mini Abstract: A patient with fibrous dysplasia developed bone marrow failure and extramedullary hematopoiesis. The etiology likely involved loss of hematopoetic marrow space and uncontrolled endocrinopathies. Splenectomy was therapeutic.

Tumor-induced osteomalacia in association with PTEN-negative Cowden syndrome.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic condition in which phosphaturic mesenchymal tumors (PMTs) secrete high levels of fibroblast growth factor 23 (FGF23) into the circulation. This results in renal phosphate wasting, hypophosphatemia, muscle weakness, bone pain, and pathological fractures. Recent studies suggest that fibronectin-fibroblast growth factor receptor 1 (FN1-FGFR1) translocations may be a driver of tumorigenesis. We present a patient with TIO who also exhibited clinical findings suggestive of Cowden syndrome (CS), a rare autosomal dominant disorder characterized by numerous benign hamartomas, as well as an increased risk for multiple malignancies, such as thyroid cancer. While CS is a clinical diagnosis, most, but not all, harbor a mutation in the tumor suppressor gene PTEN. Genetic testing revealed a somatic FN1-FGFR1 translocation in the FGF23-producing tumor causing TIO; however, a germline PTEN mutation was not identified. To our knowledge, this is the first reported case of concurrent TIO and CS.

Fibrous dysplasia of bone: craniofacial and dental implications.

Fibrous dysplasia (FD) is a rare bone disease caused by postzygotic somatic activating mutations in the GNAS gene, which lead to constitutive activation of adenylyl cyclase and elevated levels of cyclic AMP, which act on downstream signaling pathways and cause normal bone to be replaced with fibrous tissue and abnormal (woven) bone. The bone disease may occur in one bone (monostotic), multiple bones (polyostotic), or in combination with hyperfunctioning endocrinopathies and hyperpigmented skin lesions (in the setting of McCune-Albright Syndrome). FD is common in the craniofacial skeleton, causing significant dysmorphic features, bone pain, and dental anomalies. This review summarizes the pathophysiology, clinical findings, and treatment of FD, with an emphasis on the craniofacial and oral manifestations of the disease.

Stability and degradation of fibroblast growth factor 23 (FGF23): the effect of time and temperature and assay type.

UNLABELLED: There is growing need for a reliable assay for measuring fibroblast growth factor 23 (FGF23), a regulator of phosphorus and vitamin D. In this work, we analyze and compare the performance of three available assays, including the effect of temperature and time. This knowledge will allow for better understanding of FGF23 in the future. INTRODUCTION: Intact and C-terminal FGF23 (iFGF23 and cFGF23) concentrations are important in the diagnosis of hypo- and hyperphosphatemic diseases. The effects of temperature, storage, and specimen handling on FGF23 levels are not well known. We investigated the effects of various factors on plasma and serum measurement of FGF23 using three different assays. METHODS: Serum and plasma FGF23 were measured using three commercially available ELISA assays-two measuring iFGF23 and one measuring cFGF23. Samples from subjects with known FGF23 disorders were stored at 4, 22, and 37 °C and analyzed at different intervals up to 48 hours (h). A subset of samples underwent repeated freeze-thaw cycles, and samples frozen at -80 °C for up to 60 months were reanalyzed. The effect of adding a furin convertase inhibitor on FGF23 degradation was investigated using samples stored at 37 °C for 48 h. Intact FGF23 levels were measured from plasma samples of four different groups to test the correlation of the two assays. RESULTS: Plasma FGF23 levels were stable when stored at 4 and 22 °C for 48 h. Both plasma and serum FGF23 levels demonstrated relative stability after five freeze-thaw cycles. Long-term storage at -80 °C for 40 months induced some variability in FGF23 levels. The addition of a furin inhibitor did not affect FGF23 degradation. Intact FGF23 levels showed good correlation only at the upper limit of the assay range when comparing the two assays. CONCLUSIONS: Sample type, handling, and choice of assay are factors that affect FGF23 levels and should be considered when measuring this hormone.

Cutaneous skeletal hypophosphatemia syndrome: clinical spectrum, natural history, and treatment.

Cutaneous skeletal hypophosphatemia syndrome (CSHS), caused by somatic RAS mutations, features excess fibroblast growth factor-23 (FGF23) and skeletal dysplasia. Records from 56 individuals were reviewed and demonstrated fractures, scoliosis, and non-congenital hypophosphatemia that in some cases were resolved. Phosphate and calcitriol, but not skin lesion removal, were effective at controlling hypophosphatemia. No skeletal malignancies were found. PURPOSE: CSHS is a disorder defined by the association of epidermal and/or melanocytic nevi, a mosaic skeletal dysplasia, and an FGF23-mediated hypophosphatemia. To date, somatic RAS mutations have been identified in all patients whose affected tissue has undergone DNA sequencing. However, the clinical spectrum and treatment are poorly defined in CSHS. The purpose of this study is to determine the spectrum of the phenotype, natural history of the disease, and response to treatment of hypophosphatemia. METHODS: Five CSHS subjects underwent prospective data collection at clinical research centers. A review of the literature identified 45 reports that included a total of 51 additional patients, in whom the findings were compatible with CSHS. Data on nevi subtypes, bone histology, mineral and skeletal disorders, abnormalities in other tissues, and response to treatment of hypophosphatemia were analyzed. RESULTS: Fractures, limb deformities, and scoliosis affected most CSHS subjects. Hypophosphatemia was not present at birth. Histology revealed severe osteomalacia but no other abnormalities. Skeletal dysplasia was reported in all anatomical compartments, though less frequently in the spine; there was no clear correlation between the location of nevi and the skeletal lesions. Phosphate and calcitriol supplementation was the most effective therapy for rickets. Convincing data that nevi removal improved blood phosphate levels was lacking. An age-dependent improvement in mineral abnormalities was observed. A spectrum of extra-osseous/extra-cutaneous manifestations that included both benign and malignant neoplasms was present in many subjects, though osteosarcoma remains unreported. CONCLUSION: An understanding of the spectrum, natural history, and efficacy of treatment of hypophosphatemia in CSHS may improve the care of these patients.

Short communication: Heritability estimates for susceptibility to Mycobacterium avium subspecies paratuberculosis infection defined by ELISA and fecal culture test results in Jersey cattle.

Paratuberculosis (Johne's disease), an enteric disorder in ruminants caused by Mycobacterium avium ssp. paratuberculosis, causes economic losses in excess of $200 million annually to the US dairy industry. Costly diagnostic testing, cumbersome control programs, incurability, and ineffective vaccination all make M. avium ssp. paratuberculosis susceptibility a good candidate for genetic studies and genetic selection a potentially useful adjunct to management-based control programs. No report has been published for heritability of susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle. The objective of this study was to estimate variance components and heritability for susceptibility to M. avium ssp. paratuberculosis infection in US Jersey cattle. Data consisted of complete serum ELISA and partial fecal culture results on a total of 2,861 Jersey cows from 23 commercial herds throughout the United States after editing. Four M. avium ssp. paratuberculosis susceptibility phenotypes were defined using (1) ELISA sample-to-positive ratios as a continuous trait, (2) ELISA results as a binary trait (positive=1, negative=0), (3) ELISA results as an ordered categorical trait, and (4) a combined test in which ELISA and fecal culture results were both taken into account in a binary analysis. Three statistical models, including linear, binary threshold, and ordered threshold sire models, were used to analyze the data. All analyses were executed using the restricted maximum likelihood method in ASReml 3 software. The heritability estimates were low to moderate and ranged from 0.08 (±0.03) to 0.27 (±0.11) based on different trait definitions. The nonzero heritability indicates that susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle is influenced by genetic factors. Therefore, selection of the least susceptible animals could decrease genetic predisposition to M. avium ssp. paratuberculosis infection in Jersey populations in future generations.

Effect of soil slope on the appearance of Mycobacterium avium subsp. paratuberculosis in water running off grassland soil after application of contaminated slurry.

The study assessed the effect of soil slope on Mycobacterium avium subsp. paratuberculosis transport into rainwater runoff from agricultural soil after application of M. avium subsp. paratuberculosis-contaminated slurry. Under field conditions, 24 plots of undisturbed loamy soil 1 by 2 m(2) were placed on platforms. Twelve plots were used for water runoff: 6 plots at a 3% slope and 6 plots at a 15% slope. Half of the plots of each slope were treated with M. avium subsp. paratuberculosis-contaminated slurry, and half were not treated. Using the same experimental design, 12 plots were established for soil sampling on a monthly basis using the same spiked slurry application and soil slopes. Runoff following natural rainfall was collected and analyzed for M. avium subsp. paratuberculosis, coliforms, and turbidity. M. avium subsp. paratuberculosis was detected in runoff from all plots treated with contaminated slurry and one control plot. A higher slope (15%) increased the likelihood of M. avium subsp. paratuberculosis detection but did not affect the likelihood of finding coliforms. Daily rainfall increased the likelihood that runoff would have coliforms and the coliform concentration, but it decreased the M. avium subsp. paratuberculosis concentration in the runoff. When there was no runoff, rain was associated with increased M. avium subsp. paratuberculosis concentrations. Coliform counts in runoff were related to runoff turbidity. M. avium subsp. paratuberculosis presence/absence, however, was related to turbidity. Study duration decreased bacterial detection and concentration. These findings demonstrate the high likelihood that M. avium subsp. paratuberculosis in slurry spread on pastures will contaminate water runoff, particularly during seasons with high rainfall. M. avium subsp. paratuberculosis contamination of water has potential consequences for both animal and human health.

Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome.

UNLABELLED: The Hajdu-Cheney syndrome is a very rare disease that affects several organ system, leading to severe osteoporosis and other abnormalities. We describe clinical and genetic findings of nine patients with this disease. INTRODUCTION: The Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts, and other abnormalities. Recently, heterozygous mutations in NOTCH2 were identified as the cause of HCS. METHODS: Nine patients with typical presentations of HCS took part in this study: five affected patients from two small families and four sporadic cases. Peripheral blood DNA was obtained and exome sequencing performed in one affected individual per family and in all four sporadic cases. Sanger sequencing confirmed mutations in all patients. RESULTS: One of the identified mutations was introduced in a plasmid encoding NOTCH2. Wild-type and mutant NOTCH2 were transiently expressed in HEK293 cells to assess intracellular localization after ligand activation. Deleterious heterozygous mutations in the last NOTCH2 exon were identified in all patients; five of the six mutations were novel. CONCLUSION: Consistent with previous reports, all mutations are predicted to result in a loss of the proline/glutamic acid/serine/threonine sequence, which harbors signals for degradation, therefore suggesting activating mutations. One of the six mutations furthermore predicted disruption of the second nuclear localization signal of NOTCH2, but the mutant revealed normal nuclear localization after transfection, which is consistent with the proposed gain-of-function mechanism as the cause of this autosomal dominant disease. Our findings confirm that heterozygous NOTCH2 mutations are the cause of HCS and expand the mutational spectrum of this disorder.

Cold-activated brown adipose tissue is an independent predictor of higher bone mineral density in women.

UNLABELLED: In animals, defective brown adipogenesis leads to bone loss. Whether brown adipose tissue (BAT) mass relates to bone mineral density (BMD) in humans is unclear. We determined the relationship between BAT mass and BMD by cold-stimulated positron-emission tomography (PET) and dual-energy X-ray absorptiometry (DXA) in healthy volunteers. Higher BAT mass was associated with higher BMD in healthy women, but not in men, independent of age and body composition. INTRODUCTION: Contrary to the traditional belief that BAT is present only in infants, recent studies revealed significant depots of BAT present in adult humans. In animals, defective brown adipogenesis leads to bone loss. While white adipose tissue mass is a known determinant of BMD in humans, the relationship between BAT and BMD in humans is unclear. We thus examined the relationship between BAT and BMD in healthy adults. METHODS: BAT volume (ml) and activity (standard uptake value) were determined by 18F-fluorodeoxyglucose PET after overnight mild cold exposure at 19 °C, and BMD was determined by DXA. RESULTS: Among 24 healthy adults (age 28±1 years, F=10), BAT volumes were 82.4±99.5 ml in women and 49.7±54.5 ml in men. Women manifested significantly higher BAT activity, by 9.4±8.1% (p=0.03), than men. BAT volume correlated positively with total and spine BMD (r2=0.40 and 0.49, respectively, p<0.02) in women and remained a significant predictor after adjustment for age, fat, and lean body mass (p<0.05). Total and spine BMD were higher in women who harbored visually detectable BAT on PET images than those without by 11±2% (p=0.02) and 22±2% (p<0.01), respectively. No associations were observed between BAT parameters and BMD in men. CONCLUSIONS: This study demonstrated higher BMD among healthy women with more abundant BAT, independent of age and other body compositional parameters. This was not observed in men. The data suggest that brown adipogenesis may be physiologically related to modulation of bone density.

A meta-analysis of the effect of dose and age at exposure on shedding of Mycobacterium avium subspecies paratuberculosis (MAP) in experimentally infected calves and cows.

A meta-analysis was performed using all published and one unpublished long-term infection-challenge experiments to quantify the age- and dose-dependence of early and late shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in cattle. There were 194 animals from 17 studies that fulfilled the inclusion criteria, of which 173 received a known dose of MAP and 21 were exposed naturally. Results from parametric time-to-event models indicated that challenging older calves or using multiple-exposure experimental systems resulted in a smaller proportion and shorter duration of early shedding as well as slower transition to late shedding from latent compartments. Calves exposed naturally showed variable infection progression rates, not dissimilar to other infection routes. The log-normal distribution was most appropriate for modelling infection-progression events. The infection pattern revealed by the modelling allowed better understanding of low-grade endemicity of MAP in cattle, and the parameter estimates are the basis for future transmission dynamics modelling.

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils.

Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.

Whole-Genome association analysis of susceptibility to paratuberculosis in Holstein cattle.

The objective of this study was to identify genetic markers and genomic regions associated with susceptibility to Mycobacterium avium ssp. paratuberculosis (MAP) infection in Holstein cattle. Associated single nucleotide polymorphisms (SNPs) were identified by genotyping 521 MAP-infected Holstein cows and comparing SNP allele frequencies of these infected cows with allele frequencies estimated from specific reference populations. Reference population allele frequency estimates used Holstein sire genotype data and were weighted estimates based on sire usage within the population in question. The 521 infected cows were 233 and 288 cows from two resource populations of approximately 5000 cows each, collected independently. Population 1 was comprised primarily of daughters of twelve Holstein artificial insemination sires used heavily within the US dairy cattle population. Samples were obtained from 300 co-operating commercial dairy herds throughout the US and were tested by both MAP faecal culture and blood-enzyme-linked immunosorbent assay (ELISA). Population 2 consisted of dairy cattle from six co-operating dairy herds in Wisconsin, with all animals in the herds tested by blood enzyme-linked immunosorbent assay (ELISA) for MAP infection. Genotyping was performed with the Illumina Bovine SNP50 Bead Chip, providing genotypes for 35,772 informative SNPs. Data from the two resource populations were analysed both in separate and combined analyses. The most significant autosomal markers from the individual and combined analyses (n=197, nominal P<0.001) were used in a stepwise logistic regression analysis to identify a set of 51 SNPs that could be used as a predictor of genetics for Holstein cattle susceptibility to MAP infection.

Successful control of Johne's disease in nine dairy herds: results of a six-year field trial.

The objective was to evaluate if a standardized Johne's disease control program significantly reduced the prevalence of cattle infected with Mycobacterium avium ssp. paratuberculosis in dairy herds with a moderate to high initial infection prevalence of >or=10% ELISA-positive adult cattle. Nine Wisconsin dairy herds of diverse sizes and management styles completed the 6-yr study. The control program involved changes to heifer rearing practices in combination with a routine testing program. For heifers, the program specifically required 1) segregated maternity pens for ELISA-positive and ELISA-negative cattle; 2) removal of calves from the maternity pen in <2h; 3) use of colostrum only from individual ELISA-negative cows (no colostrum pooling); 4) hygienic collection of colostrum; 5) feeding of pasteurized milk as milk replacer or on-farm pasteurized milk until weaning; and 6) minimizing contact with manure from the adult cattle until weaning. The testing program was designed to detect the most infectious cattle by using a commercial ELISA once on every adult during each lactation. Producers were required to cull cows with strong-positive ELISA results before the next calving and to label cows with low- to medium-level ELISA results and manage them to limit infection transmission. Outcomes were measured by comparing the apparent prevalence based on ELISA or fecal culture in the whole herd and in first-lactation cohorts at 2 time points: before implementation of the control program and at the end of the trial. The combined results from the 9 herds showed a significant reduction in ELISA-positive cows, from 11.6% at the start of the trial to 5.6% at conclusion of the trial. The apparent prevalence decline among first-lactation cows was greater and was evident by ELISA (10.4 vs. 3.0%) and by fecal culture (17.0 vs. 9.5%). Although variations among farms were observed, the collective results demonstrated that bovine paratuberculosis can be controlled in dairy herds through effective heifer husbandry practices in combination with diagnostic testing to identify, for culling or management, cows most likely infectious.

A case of familial tumoral calcinosis/hyperostosis-hyperphosphatemia syndrome due to a compound heterozygous mutation in GALNT3 demonstrating new phenotypic features.

SUMMARY: A new case of familial tumoral calcinosis (FTC)/hyperostosis-hyperphosphatemia syndrome (HHS) due to a novel compound heterozygous mutation in N-acetylgalactosaminyltransferase 3 (GALNT3) and with new phenotypic findings is presented. The response in serum phosphate and fibroblast growth factor 23 (FGF23) to medical treatment is detailed. This case expands the genotype and phenotype of FTC/HHS and gives insight into its treatment and pathophysiology. INTRODUCTION: FTC and HHS are caused by mutations in FGF23, GALNT3, or KLOTHO. They are characterized by hyperphosphatemia, increased phosphate reabsorption, and elevated or inappropriately normal serum 1,25-dihydroxyvitamin D(3) (1,25-D(3)); FTC is associated with calcific masses, and HHS with diaphyseal hyperostosis. METHODS: A 36-year-old woman presented with abnormal dental X-rays at age 12 and was hyperphosphatemic at 22. She underwent radiographic, biochemical and genetic testing, and medical treatment. RESULTS: Serum phosphorus was 7.3 mg/dL (2.5-4.8), TmP/GFR 6.99 mg/100 mL (2.97-4.45), 1,25-D(3) 35 pg/mL (22-67). Radiographs revealed tooth anomalies, thyroid cartilage calcification, calcific masses in vertebral spaces, calcification of the interstitial septa of the soft tissue in the lower extremities, and cortical thickening of the long bones. Her total hip Z score was 1.9. C-terminus serum FGF23 was 1,210 RU/mL (20-108), but intact FGF23 was 7.4 pg/mL (10-50). DNA sequencing determined she was a compound heterozygote for mutations in GALNT3. Treatment with niacinamide and acetazolamide decreased TmP/GFR and serum phosphate, which was paralleled by a decrease in serum C-terminus FGF23. CONCLUSIONS: This case broadens the spectrum of phenotypic and genotypic features of FTC/HHS and suggests treatments to decrease renal phosphate reabsorption in the setting of a low intact FGF23.