Mycobacterium Avium Spp. Paratuberculosis Research Articles

Association between major histocompatibility complex haplotypes and susceptibility of unvaccinated and vaccinated cattle to paratuberculosis

Bovine Johne’s disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Mycobacterium avium Subspecies paratuberculosis in Asymptomatic Zoo Herbivores in Poland

Mycobacterial infections are significant issues in zoo animals, influencing animal welfare, conservation efforts, and the zoonotic potential of pathogens. Although tuberculosis is recognised to be highly dangerous, paratuberculosis can also lead to animal losses and is potentially dangerous for humans. The aim of the current study was to confirm whether Mycobacterium avium spp. paratuberculosis (MAP) infections are currently present in zoos in Poland. Faeces samples (n = 131) were collected from different animal species from eight zoos in Poland. The faeces were decontaminated and inoculated into Herrold's Egg Yolk Media. The species was determined using commercial DNA testing. The IS900 was checked using RT-PCR. The culture was positive in seven samples: five with M. avium, one with Mycobacterium fortiatum, and one without any identified Mycobacterium species. RT-PCR confirmed MAP genetic material in nine animals. Our findings represent the first confirmation of MAP in bongo (Tragelaphus eurycerus), indicating that it is present in Polish zoological gardens. Fortunately, the disease can be monitored more easily due to recent legislation (the Animal Health Law).

Estimating the sensitivity and specificity of serum ELISA and pooled and individual fecal PCR for detecting Mycobacterium avium subspecies paratuberculosis in Canadian cow-calf herds using Bayesian latent class models.

While Johne's disease (JD) is less common in beef than in dairy herds, consolidation is increasing transmission risk. Estimates of Mycobacterium avium spp. paratuberculosis (MAP) prevalence and test performance in cow-calf herds are needed to inform control programs. Objectives of this study included describing the prevalence of MAP in Canadian cow-calf herds and comparing the relative performance of a serum ELISA, pooled fecal PCR and individual fecal PCR using Bayesian latent class models, and to investigate factors associated with positive MAP tests. Blood and fecal samples (n = 3,171) were collected from 159 Canadian cow-calf herds. All samples were analyzed using serum ELISA and fecal PCR (pools of five samples) and a subset of 913 fecal samples were also tested with individual PCR. Based on latent class analysis, MAP prevalence was higher in eastern compared to western Canada for both animals {East, 3% [95% Credible Interval (CrI) 1–7%]; West, 1% [95% CrI 0.2–2%]} and herds [East, 15% (95% CrI 2–35%); West, 10% (95% CrI 1–26%), based on one or more positive results]. Sensitivity (Se) and specificity (Sp) for animal level individual PCR were 96% (95% CrI 80–100%) and 98% (95% CrI 96–100%), respectively followed by pooled PCR [Se = 54% (95% CrI 36–72%), Sp > 99.9% (95% CrI 99.8–100%)] and ELISA [Se = 36% (95% CrI 22–52%), Sp = 98% (95% CrI 96–99%)]. Based on 20 samples per herd, the herd level Se of ELISA was 79% (95% CrI 47–100%) (at least one positive sample) compared to 43% (95% CrI 14–94%) for pooled PCR. Herd-level Sp was 99% (95% CrI 96–100%) for pooled PCR and 90% (95% CrI 83–100%) for ELISA. Cows from herds with dairy cattle on farm and cows with symptoms of JD in the past 3 years were more likely to be MAP positive. Herds that had animals with JD symptoms in the previous 3 years and those with more breeding females were most likely to test positive for MAP. While serum ELISA can be effective for herd screening, PCR performed better for animal testing. Pooled PCR testing could be a less costly option; however, determining the most cost-effective approach will require further economic analysis.

Seroprevalence of selected endemic infectious diseases in large-scale Estonian dairy herds and their associations with cow longevity and culling rates

Herd culling rates and longevity represent herd health and welfare status as well as farm economic performance. The contribution of endemic circulation of the main cattle pathogens to herd performance has not been previously analysed. The aim of this study was to estimate the herd prevalence of selected endemic bovine pathogens among large commercial dairy herds and to analyse their associations with herd culling rates and longevity.Bulk tank milk (BTM) samples and 10 heifer serum samples were collected from 120 Estonian dairy herds with at least 100 cows, between August 2019 and July 2020. All samples were tested for antibodies against bovine herpesvirus 1 (BHV-1), bovine viral diarrhoea virus (BVDV), bovine respiratory syncytial virus (BRSV), Mycoplasma bovis, Mycobacterium avium spp. paratuberculosis (MAP) and Salmonella Dublin using commercial ELISA. Data on herd size, milk yield, culling rate (CR) and mean age of the culled cows (MAofCC) were collected from the Estonian Livestock Performance Recording Ltd. The apparent herd and animal prevalences were calculated, and linear regression models were used to identify associations between the herd status of six tested pathogens and CR and MAofCC.The herd seroprevalences for antibodies based on BTM and heifer serum sample testing were BHV-1 56.7 % (95 % CI 47.3; 65.7), Mycoplasma bovis 48.3 % (95 % CI 39.1; 57.6), MAP 2.5 % (95 % CI 0.5; 7.1) and S. Dublin 24.2 % (95 % CI 16.8; 32.8) in all tested herds. Excluding vaccinated herds, herds prevalence for BVDV was 27.0 % (95 % CI 19.0; 36.3) and for BRSV 94.7 % (95 % CI 88.1; 98.3).Herd seropositive status for BRSV was associated with lower MAofCC, and herds with BSRV-seropositive youngstock had increased CR. Herds with positive BTM test results for S. Dublin culled cows at an older average age (Coef = 3.79 months, 95 % CI 0.52; 7.07, p = 0.023). MAP-positive herds had somewhat lower herd MAofCC (Coef = −6.18 months, 95 % CI −12.98; 0.63, p = 0.075). There was also a tendency of BVDV-negative herds to have a lower CR than BVDV-positive herds (Coef = −3.03 %, 95 % CI −6.54; 0.49, p = 0.090), and vaccination against BVDV tended to be protective against high CR (Coef = −6.26 %, 95 % CI −12.61; 0.09, p = 0.053 compared to infected herds).This study shows that Estonian large-scale dairy herds are endemically infected with several important cattle pathogens. Most of the studied pathogens influence longevity and culling rates, thus entailing health and economic consequences.

29 Conditional GWAS using sequence-based genotypes for susceptibility to Mycobacterium avium subsp paratuberculosis infection in Canadian Holstein

Abstract Paratuberculosis is an incurable disease caused by Mycobacterium avium spp. paratuberculosis (MAP). Variability in the definition of a true infection could explain the lack of consensus in published results on host genetic susceptibility to MAP. The objective of this study was to identify quantitative trait loci (QTL) for susceptibility to paratuberculosis in Canadian Holstein with an accurately defined status. Cows with known parentage were recruited from 23 Canadian dairy herds and enrolled in a longitudinal study. Blood and feces were collected semi-annually over a 3-5-yr period. Serum samples were analyzed by ELISA (IDEXX) and feces by qPCR. Cows were labelled based on positive ELISA (&amp;gt; 45%) and on at least two consecutive fecal excretion of MAP (n = 382). Control cows (n = 669) did not excrete MAP nor have MAP antibodies (&amp;lt; 10%) over the 3–5 year period. Their genotypes (GGP Bovine 50K chip) were imputed to HD-SNP (within-breed), then to WGS using FImpute software and Run6 of 1,000 Bulls’ genome project as a multi-breed reference. Using GCTA software, heritability (h2) was estimated and conditional GWAS was performed to define multiple true causal mutations in QTL regions from those caused by linkage disequilibrium. The h2(se) was 0.52 (0.01) and after FDR correction, 1,357 variants presented significant effects (-log10 P &amp;gt; 6), grouped within 7 QTL located on chromosome BTA7, BTA16, BTA20, and BTA23. The most significant variant was on BTA20 (-log10 P =12.39). Functional annotation showed that 76% of all the variants of the QTL were intergenic, with the rest being mostly intronic. Of interest was variant located on BTA7 with effect size (se) of -0.77 (0.02). In conclusion, using accurate phenotypes, chromosomal regions and candidate genes that could be functionally related to MAP infection were uncovered. The significant variants found in this study could contribute to the enhancement of genomic evaluation for MAP resistance.

Percutaneous Ultrasound Guided Cholecystocentesis and Microbiological Analysis of Bile in Chronic Diarrhoea of Cattle

Background: Chronic diarrhoea is a frequently presenting complaint by the cattle owners and some presents as non-responsive to routine treatment. Such cases are often a diagnostic challenge because of wide ranging etiologies. In Indian context few data is available on prevalent causes of chronic diarrhoea in bovines. Salmonellosis is one of the important cause of chronic diarrhoea in crossbred cattle. Therefore, the study was conducted to investigate the role of Salmonella in chronic diarrhoea of crossbred cattle and to determine microbiological profile of bile in cattle with chronic idiopathic diarrhoea. Methods: Twenty (n=20) adult dairy cattle presented with the history of chronic diarrhoea at Large Animal Clinics, GADVASU, Ludhiana from September 2015 to June 2016 were selected for the study. Case histories were recorded, faecal, blood and bile samples were collected from 20 animals. Bile was collected through ultrasound guided cholecystocentesis (PUC) from 20 cattle and five healthy cattle. The faecal and bile samples were cultured for isolation and identification of bacterial pathogens using selective and non-selective media with special emphasis on isolation of Salmonella spp. Microscopic examination of faeces were done to rule out parasitic cause of diarrhoea and for acid-fast bacilli (AFB). The serum samples were employed to detect antibodies against Mycobacterium avium spp paratuberculosis using serum ELISA. Result: Investigations have showed culture of bile and faecal did not yield Salmonella spp; however E.coli was isolated from 6 cows (30%), Klebsiella spp in 9 cows (45%) and Pseudomonas spp from 1 cow (5%) from bile culture of diarrhoeic cattle. PCR results of E.coli culture from bile showed hlyA virulence genes which expected band at 534 bp however these isolates were negative for other virulence gene of E.coli. It was observed in this study that bactibilia was associated with chronic diarrhoea in three fourth of the diarrhoeic cattle.

Association of SNP markers in immune response genes with resistance or susceptibility to Mycobacteriun avium paratuberculosis in Cattle

Background: Bovine paratuberculosis, also known as Johne's disease (JD), is a chronic, granulomatous inflammatory disease of the intestines of ruminants and some wild-type species caused by Mycobacterium avium spp. paratuberculosis (MAP). The present study was conducted to find the association between SNPs in TLR 1 gene and occurrence of paratuberculosis in cattle. Methods & Materials: A total of 178 cattle were subjected to Johnin PPD, ELISA test (indigenous as well as Parachek kit method), faecal microscopy and faecal culture for detection of presence of bovine paratuberculosis infection. Based on the results of diagnostic tests, 45 animals each were assigned to case and control population. Two SNPs viz. rs135207279 and rs437316199 in TLR 1 gene were validated using PCR-RFLP with restriction enzymes TspRI. For SNP rs135207279, three genotypes i.e. AA, AC and CC were observed with genotype frequencies in case as 24, 54 and 22% respectively while in control population as 33, 24 and 43% respectively. The association study was carried out by PROC LOGISTIC procedure of SAS 9.3. Results: AA, AC and CC genotypes significantly different (p < 0.05) between case and control cattle. The ODDs of AA and AC genotypes verses CC genotype were 1.00 (0.0301-3.321; 95% CI) and 3.66 (1.2-11.17; 95%CI) respectively. For rs437316199, the genotype frequencies of GG, GT and TT in case and control population were 36, 18, 46%; and 40, 16and 44% respectively. AA, AC and CC genotypes were not significantly (P > 0.05) different in case than control population. Conclusion: Genotypes in SNP rs135207279 showed significant association with occurrence of paratuberculosis in the analyzed cattle population. There is further need to confirm the association of TLR 1 gene with bovine tuberculosis infection status in a larger population of cattle.

Assessment of yeast Saccharomyces cerevisiae component binding to Mycobacterium avium subspecies paratuberculosis using bovine epithelial cells.

BackgroundSince yeast Saccharomyces cerevisiae and its components are being used for the prevention and treatment of enteric diseases in different species, they may also be useful for preventing Johne’s disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium spp. paratuberculosis (MAP). This study aimed to identify potential yeast derivatives that may be used to help prevent MAP infection. The adherence of mCherry-labeled MAP to bovine mammary epithelial cell line (MAC-T cells) and bovine primary epithelial cells (BECs) co-cultured with yeast cell wall components (CWCs) from four different yeast strains (A, B, C and D) and two forms of dead yeast from strain A was investigated.ResultsThe CWCs from all four yeast strains and the other two forms of dead yeast from strain A reduced MAP adhesion to MAC-T cells and BECs in a concentration-dependent manner after 6-h of exposure, with the dead yeast having the greatest effect.ConclusionsThe following in vitro binding studies suggest that dead yeast and its’ CWCs may be useful for reducing risk of MAP infection.

Evaluating contribution of the cellular and humoral immune responses to the control of shedding of Mycobacterium avium spp. paratuberculosis in cattle.

Mycobacterium avium spp. paratuberculosis (MAP) causes a persistent infection and chronic inflammation of the gut in ruminants leading to bacterial shedding in feces in many infected animals. Although there are often strong MAP-specific immune responses in infected animals, immunological correlates of protection against progression to disease remain poorly defined. Analysis of cross-sectional data has suggested that the cellular immune response observed early in infection is effective at containing bacterial growth and shedding, in contrast to humoral immune responses. In this study, 20 MAP-infected calves were followed for nearly 5 years during which MAP shedding, antigen-specific cellular (LPT) and humoral (ELISA) immune responses were measured. We found that MAP-specific cellular immune response developed slowly, with the peak of the immune response occurring one year post infection. MAP-specific humoral immunity expanded only in a subset of animals. Only in a subset of animals there was a statistically significant negative correlation between the amount of MAP shedding and magnitude of the MAP-specific cellular immune response. Direct fitting of simple mechanistic mathematical models to the shedding data suggested that MAP-specific immune responses contributed significantly to the kinetics of MAP shedding in most animals. However, whereas the MAP-specific cellular immune response was predicted to suppress shedding in some animals, in other animals it was predicted to increase shedding. In contrast, MAP-specific humoral response was always predicted to increase shedding. Our results illustrate the use of mathematical methods to understand relationships between mycobacteria and immunity in vivo but also highlight problems with establishing cause-effect links from observational data.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0204-1) contains supplementary material, which is available to authorized users.

Detection of Mycobacterium avium subspecies paratuberculosis in environmental samples by faecal culture and real-time PCR in relation to apparent within-herd prevalence as determined by individual faecal culture.

SUMMARY Environmental samples are a cost-saving and easy-to-use approach to diagnose paratuberculosis at the herd level. Detailed knowledge concerning its uncertainties in herds with a low prevalence of Mycobacterium avium spp. paratuberculosis (MAP) is required to design sampling strategies in control programmes. This study aimed to calculate a threshold level of the apparent within-herd prevalence (WHPapp) as determined by individual mycobacterial cultivation (faecal culture; FC) of all cows thus allowing the detection of a herd as MAP-positive at a certain probability level (P d). Out of 200 environmental samples taken twice from five predefined locations in a barn, 25 were positive by FC and 60 were positive by a quantitative real-time PCR method (qPCR). A logistic regression model was used to calculate the WHPapp threshold of detection. For 50% P d, a WHPapp threshold of 2.9% was calculated for the combination of three samples (milking area, main cow alleyways, holding pen) tested simultaneously both by FC and qPCR. The threshold increased to 6.2% for 90% P d. Repeated environmental sampling did not reduce the WHPapp threshold. Depending on the particular needs for prevalence estimation or in control programmes (single or repeated sampling) the provided WHPapp thresholds at different P d will enable decisions to be made about various sampling strategies.

Prevalence of Mycobacterium avium subsp paratuberculosis (MAP) ELISA-positive cows and assessment of MAP transmission risk on organic dairy farms in Ontario, Canada

Johne's disease (JD) is caused by Mycobacterium avium spp paratuberculosis (MAP). It is an incurable, chronic inflammation of the gastrointestinal tract of cattle that causes diarrhea, weight loss, and reduced production. Commonly recognized risk factors for MAP transmission include purchase of subclinically infected cows and the infection of calves through ingestion of manure, colostrum or milk containing MAP shed by infected cattle. To limit the negative effects of JD on the dairy industry in Ontario, Canada, the voluntary Ontario Johne's Education and Management Assistance Program (OJEMAP) was implemented in January 2010. Producers can participate in MAP antibody ELISA testing and an on-farm risk assessment and management plan (RAMP) with their herd veterinarian. High RAMP scores are associated with high risk for MAP transmission.&#x0D; Organic farming is one of the fastest growing agricultural sectors worldwide. In Ontario, approximately 80 dairy farms (2% of all dairy farms) are certified organic. While most organic farmers tend to maintain closed herds, which would reduce the risk for JD introduction, they also often support prolonged contact between newborn calves and their dams. These and other factors could potentially influence the risk of MAP transmission as well as JD prevalence and prevention efforts on organic dairy farms. The aim of this cross-sectional study was to assess the Johne's ELISA prevalence and Johne's disease transmission risk on organic dairy farms in Ontario.

Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.

Detection of Mycobacterium avium ssp. paratuberculosis in ileocaecal lymph nodes collected from elderly slaughter cows using a semi-nested IS900 polymerase chain reaction

The aim of this study was to investigate the occurrence of subclinical Mycobacterium avium spp. paratuberculosis (MAP) infections at slaughter by testing ileocaecal lymph nodes with a semi-nested IS900 PCR. Tissue samples were available within the framework of a parallel study investigating BSE-susceptibility factors in members of BSE-cohorts in the German Federal State of Lower Saxony. Ileocaecal lymph nodes were collected over a 2-year sampling period from 99 slaughter cattle of a mean age of 6.5 years (5.5–7.5 years). A recently developed IS900 semi-nested polymerase chain reaction (snPCR) assay offering a sensitivity of 1 genome equivalent was used for the detection of MAP-DNA. Based on this snPCR, 17 out of the 99 samples gave positive results, indicating a MAP occurrence of 17.17% in the random sample. All PCR products were sequenced for screening of polymorphisms. Nucleotide homologies of 98.5–100% were found with respect to the MAP K10 reference sequence IS900 (GenBank: AE16958). PCR analysis of ileocaecal lymph nodes collected from slaughter cattle proved to be a suitable technique to determine MAP occurrence in the local cattle population.

A principal component regression based genome wide analysis approach reveals the presence of a novel QTL on BTA7 for MAP resistance in holstein cattle

Bovine Johne's disease (JD), caused by Mycobacterium avium spp. paratuberculosis (MAP), causes significant losses to the dairy and beef cattle industries. Effective vaccination or therapeutic strategies against this disease are currently unavailable and infected animals either get culled or die due to clinical disease. An alternative strategy to manage the disease is to selectively breed animals with enhanced resistance to MAP infection. Therefore, the objective of this study was to identify genetic loci putatively associated with MAP infection in a resource population consisting of Holstein cattle using a genome-wide association approach. The BovineSNP50 BeadChip, containing 54,001 single nucleotide polymorphisms (SNPs), was used to genotype 232 animals with known MAP infection status. Since, traditional case-control analytical techniques are based on single-marker analysis and do not account for the existence of linkage disequilibrium (LD) between markers, we used a novel principal component regression approach, where each SNP was fit in a logistic regression model, along with principal components of other SNPs on the same chromosome showing association with the trait, as covariates. Such an approach allowed us to account for the LD that exists between multiple markers showing an association on the same chromosome. Our analysis revealed the presence of at least 12 genomic regions on BTA1, 5, 6, 7, 10, 11 and 14 that were associated with the MAP infection status of our resource population. A brief description of these genomic regions, and a discussion of the analysis used in this study, have been presented.

Mycobacterium aviumssp.paratuberculosisHigh Shedding in an Adult Female Alpaca, and its Implications for the Rest of the Herd

A 6-year-old female alpaca (case 1) was examined at the George D. Widener Hospital for Large Animals of the University of Pennsylvania for evaluation of lethargy, diarrhea, and weight loss of 3 months’ duration. A diagnosis of Mycoplasma haemolamae had initially been made by the referring veterinarian based on blood smear examination findings. The animal was treated with a long-acting form of oxytetracyline (10mg/kg SQ q24h) for 10 days but its clinical signs failed to improve. Serial fecal floatations performed before referral showed no parasite ova. Additional treatments received before referral included ivermectin, fenbendazole, B vitamins, injectable iron, procaine penicillin, oral sulfadimethoxine, and oral electrolytes. As the diarrhea and weight loss persisted, the alpaca was referred for further evaluation. The animal was pregnant (approximately 155 days gestation) at the time of presentation. The animal was from a herd of 78 alpacas, and was pastured with a group of pregnant females and young animals. Routine vaccinations on the farm included rabies, Clostridium perfringens Types C and D, and tetanus. On presentation, the alpaca was quiet but responsive and in poor body condition (body condition score 3/10), weighing 60 kg. Rectal temperature, heart rate, and respiratory rate were 98.21F, 56 beats per minute, and 20 breaths per minute, respectively. The alpaca appeared well hydrated with pale mucous membranes. It had green, watery feces, and a poor appetite. Initial laboratory abnormalities included mild nonregenerative anemia (PCV, 23%; reference interval, 25–46%), hypoproteinemia (total protein, 4.2 g/dL; reference interval, 4.6–6.9 g/dL), hypoalbuminemia (albumin, 1.40 g/dL; reference interval, 2.50–4.20 g/dL), and azotemia (serum creatinine concentration, 2.28mg/dL; reference interval, 0.6–1.80mg/dL). Blood smear examination was negative for Mycoplasma haemolamae organisms. Feces were submitted for fecal floatation, Mycobacterium avium spp. paratuberculosis (MAP) RT-PCR, acid-fast staining for Cryptosporidium, fecal occult blood, and bacterial cultures. Blood was submitted for MAP antibody detection with ELISA.No parasite ova were observed on fecal floatation, and the acid-fast staining was negative forCryptosporidium organisms. The occult blood test result was negative. Fecal bacterial cultures were negative for Salmonella spp. and Clostridium spp. Fetal and abdominal ultrasound examinations were performed. The sonographic appearance of the visible gastrointestinal structures was within normal limits. Fetal sonographic evaluation detected an irregular heart rate, suggestive of fetal distress. Additional diagnostic tests performed during the hospital stay included determination of serum trace mineral concentrations (Cu, Fe, Se, and Zn), fecal coronavirus detection with electron microscopy, and bovine viral diarrhea (BVD) testing by PCR on the buffy coat. Serum trace mineral concentrations were within the reference range. Results of coronavirus and BVD tests later returned negative. The patient remained hospitalized for 5 days while awaiting diagnostic test results. During that time, supportive care in the form of IV fluids (Normosol-R, 1.5mL/kg/h) and partial parenteral nutrition (1.5mL/ kg/h) were administered. Ranitidine (1.5mg/kg IV q8h) was also administered for prevention of 3rd compartment ulceration. RT-PCR results for MAP detection identified a strong positive result with duplicate values of 17.43 and 17.79 cycles to positive threshold (CT). Because of the animal’s advanced clinical signs and potential risk to the rest of the herd, permission was obtained for euthanasia. The alpaca was euthanized by administration of a barbiturate overdose and a postmortem examination performed. From the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA (Fecteau, Ross, Tennent-Brown, Habecker, Sweeney, Whitlock); and the Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St Paul, MN (Sreevatsan). Dr Ross is presently affiliated with the Department for Environment, Food and Rural Affairs, Nobel House, 17 Smith Square, London SW1P 3JR, UK. Dr Tennent-Brown is presently affiliated with the Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA. This work was performed at the Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA. Corresponding author: Dr Marie-Eve Fecteau, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348; e-mail: mfecteau@vet.upenn.edu. Submitted June 16, 2009; Revised August 19, 2009; Accepted August 19, 2009. Copyright r 2009 by the American College of Veterinary Internal Medicine 10.1111/j.1939-1676.2009.0401.x Abbreviations:

M1118 Presence of Serum Antibodies Against Secreted Proteins from Mycobacterium avium Spp Paratuberculosis in Patients with Crohn's Disease

M1118 Presence of Serum Antibodies Against Secreted Proteins from Mycobacterium avium Spp Paratuberculosis in Patients with Crohn's Disease

One-Step, Nanoparticle-Mediated Bacterial Detection with Magnetic Relaxation

Acknowledging the need for the development of fast and sensitive bacterial detection methods, we functionalized superparamagnetic iron oxide nanoparticles to identify Mycobacterium avium spp. paratuberculosis (MAP), through magnetic relaxation. Our results indicate that the MAP nanoprobes (1) bind specifically to MAP, (2) can quantify the bacterial target quickly in milk and blood with high sensitivity, and (3) are not susceptible to interferences caused by other bacteria. The use of the described magnetic nanosensors in the identification and quantification of bacteria in clinical and environmental samples is anticipated.

Attachment of Mycobacterium avium subspecies paratuberculosis to bovine intestinal organ cultures: Method development and strain differences

Mycobacterium avium spp. paratuberculosis (MAP) causes chronic granulomatous inflammation of the intestinal tract in many species of animals, but the mechanisms of disease are poorly understood. Attachment of bacteria to epithelial cells is a critical step in pathogenesis of many mucosal diseases. The goal of these studies was to develop an in vitro method to study attachment of MAP to bovine intestinal epithelial cells. Short-term, bovine intestinal organ cultures were used to show a significant difference in the ability of radiolabelled MAP strains to attach to intestinal epithelium. We found significant differences in the ability of different strains of MAP to attach, but there were no differences in attachment among different regions of the intestinal tract. Examination of acid fast stained tissue sections of organ cultures demonstrated that organisms were located adjacent to mucosal epithelium or within goblet cells. Coating of the organisms with fibronectin, which has been shown to be involved in attachment of many mycobacteria, including MAP, affected the attachment of the MAP strains in different ways, but did not affect the overall attachment of the organisms to different regions of the gastrointestinal tract. This organ culture method should also prove useful for defining the molecular mechanisms of attachment and interactions of MAP with intestinal epithelium.