Meta-analysis of two genome-wide association studies of bovine paratuberculosis

Background

Bovine paratuberculosis (ParaTB) also known as Johne''s disease, is a contagious fatal disease resulting from infection by Mycobacterium avium subspecies paratuberculosis (MAP). Previous studies have identified loci associated with ParaTB using different measurements to define cases and controls. The objective of this study was to combine the data from two recent studies to identify genetic loci associated with MAP tissue infection and humoral immune response, defined by MAP ELISA-positive cattle, by comparing cases and control animals for one or both measures of infection.

Methodology/Principal Findings

The two populations used for the association analyses were a cohort of MAP tissue infected animals and control Holstein cows from the USA and the second cohort composed of ELISA-positive and ELISA-negative Holstein cows from Italy. Altogether 1190 cattle were genotyped with the Illumina BovineSNP50 BeadChip. SNP markers were removed if the minor allele frequency <0.01 or genotyping failure was >5%. Animals were removed with >5% genotyping failure. Whole genome association analyses were conducted with the GRAMMAR-CG method using two different definitions of control populations.

Conclusion/Significance

The analyses identified several loci (P<5 e-05) associated with ParaTB, defined by positive ELISA and presence of bacteria in tissue compared to ELISA and tissue negative animals, on chromosomes 1, 12 and 15 and one unassigned SNP. These results confirmed associations on chromosome 12 and the unassigned SNP with ParaTB which had been found in the Italian population alone. Furthermore, several additional genomic regions were found associated with ParaTB when ELISA and tissue positive animals were compared with tissue negative samples. These loci were on chromosomes 1, 6, 7, 13, 16, 21,23 and 25 (P<5 e-05). The results clearly indicate the importance of the phenotype definition when seeking to identify markers associated with different disease responses.     

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.     

Genome-wide association study of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">Paratuberculosis</Emphasis> infection in Chinese Holstein

Background

Paratuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle.

Results

Using Illumina Bovine 50?K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50?K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P?<?5?×?10^??5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1?Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5?Mb on BTA23, where the TDP2 gene was anchored.

Conclusions

In conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

     

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH₂ (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.     

Genetic Loci Involved in Antibody Response to Mycobacterium avium ssp. paratuberculosis in Cattle

Background

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne''s disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision.

Methodology/Principal Findings

The objective of the current study was to localize genes having an impact on Johne''s disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip) to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal.

Conclusion/Significance

The analysis of the genotype data identified several chromosomal regions associated with disease status: a region on chromosome 12 with high significance (P<5×10⁻⁶), while regions on chromosome 9, 11, and 12 had moderate significance (P<5×10⁻⁵). These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information into breeding programmes for the improvement of health status.     

Relationship between Presence of Cows with Milk Positive for Mycobacterium avium subsp. paratuberculosis-Specific Antibody by Enzyme-Linked Immunosorbent Assay and Viable M. avium subsp. paratuberculosis in Dust in Cattle Barns

Paratuberculosis, or Johne''s disease, in cattle is caused by Mycobacterium avium subsp. paratuberculosis, which has recently been suspected to be transmitted through dust. This longitudinal study on eight commercial M. avium subsp. paratuberculosis-positive dairy farms studied the relationship between the number of cows with M. avium subsp. paratuberculosis antibody-positive milk and the presence of viable M. avium subsp. paratuberculosis in settled-dust samples, including their temporal relationship. Milk and dust samples were collected in parallel monthly for 2 years. M. avium subsp. paratuberculosis antibodies in milk were measured by enzyme-linked immunosorbent assay (ELISA) and used as a proxy for M. avium subsp. paratuberculosis shedding. Settled-dust samples were collected by using electrostatic dust collectors (EDCs) at six locations in housing for dairy cattle and young stock. The presence of viable M. avium subsp. paratuberculosis was identified by liquid culture and PCR. The results showed a positive relationship (odds ratio [OR], 1.2) between the number of cows with ELISA-positive milk and the odds of having positive EDCs in the same airspace as the adult dairy cattle. Moreover, the total number of lactating cows also showed an OR slightly above 1. This relationship remained the same for settled-dust samples collected up to 2 months before or after the time of milk sampling. The results suggest that removal of adult cows with milk positive for M. avium subsp. paratuberculosis-specific antibody by ELISA might result in a decrease in the presence of viable M. avium subsp. paratuberculosis in dust and therefore in the environment. However, this decrease is likely delayed by several weeks at least. In addition, the data support the notion that M. avium subsp. paratuberculosis exposure of young stock is reduced by separate housing.     

Metabolomic Profiling in Cattle Experimentally Infected with Mycobacterium avium subsp. paratuberculosis

The sensitivity of current diagnostics for Johne''s disease, a slow, progressing enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), is too low to reliably detect all infected animals in the subclinical stage. The objective was to identify individual metabolites or metabolite profiles that could be used as biomarkers of early MAP infection in ruminants. In a monthly follow-up for 17 months, calves infected at 2 weeks of age were compared with aged-matched controls. Sera from all animals were analyzed by ¹H nuclear magnetic resonance spectrometry. Spectra were acquired, processed, and quantified for analysis. The concentration of many metabolites changed over time in all calves, but some metabolites only changed over time in either infected or non-infected groups and the change in others was impacted by the infection. Hierarchical multivariate statistical analysis achieved best separation between groups between 300 and 400 days after infection. Therefore, a cross-sectional comparison between 1-year-old calves experimentally infected at various ages with either a high- or a low-dose and age-matched non-infected controls was performed. Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS DA) yielded distinct separation of non-infected from infected cattle, regardless of dose and time (3, 6, 9 or 12 months) after infection. Receiver Operating Curves demonstrated that constructed models were high quality. Increased isobutyrate in the infected cattle was the most important agreement between the longitudinal and cross-sectional analysis. In general, high- and low-dose cattle responded similarly to infection. Differences in acetone, citrate, glycerol and iso-butyrate concentrations indicated energy shortages and increased fat metabolism in infected cattle, whereas changes in urea and several amino acids (AA), including the branched chain AA, indicated increased protein turnover. In conclusion, metabolomics was a sensitive method for detecting MAP infection much sooner than with current diagnostic methods, with individual metabolites significantly distinguishing infected from non-infected cattle.     

Phenotype definition is a main point in genome-wide association studies for bovine Mycobacterium avium ssp. paratuberculosis infection status

Paratuberculosis caused by Mycobacterium avium ssp. paratuberculosis (MAP) causes economic losses and is present in dairy herds worldwide. Different studies used different diagnostic tests to detect infection status and are the basis of genome-wide association (GWA) studies with inconsistent results. Therefore, the aim of this study was to identify and compare genomic regions associated with MAP susceptibility in the same cohort of cattle using different diagnostic tests. The GWA study was performed in German Holsteins within a case-control assay using 305 cows tested for MAP by fecal culture and additional with four different commercial ELISA-tests. Genotyping was performed with the Illumina Bovine SNP50 BeadChip. The results using fecal culture or ELISA test led to the identification of different genetic loci. Two single-nucleotide polymorphisms showed significant association with the ELISA-status. However, no significant association for MAP infection could be confirmed. Our results show that the definition of the MAP-phenotype has an important impact on the outcome of GWA studies for paratuberculosis.     

Genetic association between NOD2 polymorphism and infection status by Mycobacterium avium ssp. paratuberculosis in German Holstein cattle

The aim of the study was the analysis of the nucleotide‐binding oligomerization domain containing 2 (NOD2, formerly CARD15) as a candidate gene for Mycobacterium avium ssp. paratuberculosis infection in cattle. Eleven SNPs in the NOD2 gene were identified, and finally, four SNPs were included in a case–control study using 324 German Holstein cows tested for paratuberculosis using fecal culture and ELISA. The SNP (GenBank) AY518738S04:g.521G>A in exon 4 showed a significant association between the fecal culture status of the animals and NOD2 allele variants. The other three SNPs showed no associations in German Holstein cows.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.     

Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Fecal and Molecular Survey of Neospora caninum in Farm and Household Dogs in Mashhad Area, Khorasan Province, Iran

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dog is the definitive host for N. caninum and can infect dairy cattle. The aim of this study is to determine the prevalence of Neospora oocysts in feces of dogs from dairy farms. A total of 174 fecal samples was collected from 89 farm dogs and 85 household dogs during 2006 and 2008. Fecal samples of dogs were microscopically examined for detecting Hammondia Neospora-like oocysts (HNLO) by Mini Parasep®SF fecal parasite concentrator. HNLO were microscopically detected in 4 fecal samples (2.2%). The fecal samples with HNLO were examined by N. caninum-specific PCR. Two of the samples were positive for N. caninum. The 2 positive fecal samples were selected for inoculation to calves. Two inoculated calves were seronegative by ELISA for 4 months post-infection. This is the first report of finding N. caninum DNA in feces of farm dogs in Mashhad area, Iran.     

Evaluation of bioelectronics sensor compared to other diagnostic test in diagnosis of Johne's disease in goats

A bioelectronics sensor has been developed and it is evaluated for the diagnosis of paratuberculosis in goats. Initially hematite nanoparticles were prepared and using this nanoparticles as core, electrically active polyaniline coated magnetic (EAPM) nanoparticles are synthesized from aniline monomer (made electrically active by acid doping). These EAPM nanoparticles were fabricated with rabbit anti-goat IgG for the detection of goat antibodies on the capture pad. The protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) immobilized onto the capture pad will detect the antibody against MAP in the goat sera samples. This bound goat antibody will be detected by the anti-goat IgG previously bound to EAPM. Upon detection the EAPM nanoparticles bridges an electric circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by EAPM, was measured as direct charge transfer between the electrodes. Testing of the biosensor with known Johne's disease (JD) positive and negative serum samples gave significant difference in the electrical conductance value. Further the efficacy of this biosensor was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA using field sera. Out of 265 goat sera tested, positive results recorded were; AGID 36 (13.59%), bioelectronics sensor 49 (19.14%), and absorbed ELISA 51 (19.25%). This biosensor was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals. Of which, positive results recorded in animals were; Johnin test 21 (32%), biosensor 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though the nanobioelectronics sensor was slightly less sensitive (not statistically significant) compared to absorbed ELISA and fecal nested PCR for mycobacterial DNA but it was simple to perform in field conditions and requires less time. The speed of detection and the equipment involved would support its application toward the various point-of-care opportunities aimed at control and management of Johne's disease in goats.     

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.     

Gastrointestinal parasitic infections in intensive dairy cattle breeding: Update on the epidemiology and associated risk factors in northern Italy

Parasitic infections such as Strongylida and Eimeria still represent a major health problem of dairy cattle impacting their health, welfare, and productivity. In view of the scarcity of data on risk factors contributing to the spread of parasitic infections in cattle breeding, an epidemiological study in intensive dairy farms in northern Italy was planned. 495 animals (lactating and dry cows, heifers, and calves) from 19 farms were enrolled in the study. Individual fecal samples were analyzed by a quantitative copromicroscopic analysis to detect the number of Strongylida eggs or Eimeria oocysts per gram of faeces (EPG/OPG). Data concerning management, sanitary and biosecurity measures were collected using a questionnaire; a management measures score (MMS) was also calculated. The possible influence of risk factors on Strongylida and Eimeria was thus assessed by statistical analysis using generalized linear mixed models (GLMMs).Eimeria spp. was the most frequently detected parasitic taxon (herd and individual prevalence: 89.5% and 46.2%, respectively), followed by Strongylida (herd and individual prevalence: 63.1% and 16.6%, respectively). The presence of Strongylida resulted associated to the productive category (p-value = 0.028), with heifers and dry cows at higher risk of infection than lactating cows and calves, and to the MMS (p-value = 0.007). Higher prevalence values were recorded in farms with intermediate or low MMS compared to those with optimal MMS. As regard Eimeria infection, a greater effect of MMS on OPG was recorded in calves when compared to those recorded in heifers (OR = 0.228, p-value = 0.003) and dry cows (OR = 0.241, p-value = 0.009).Gastrointestinal parasitic infections still remain an underestimated problem in intensive dairy cattle breeding. MMS may help in the choice of strategies aimed at minimizing the impact of parasites on animal health, thus improving the productivity of the entire herd.     

Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10¹⁰ CFU/animal) made resistant to nalidixic acid (Nal^r). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal^r E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal^r E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

Background

During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection.

Methods

Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture.

Results

Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele ''Q'' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele ''q''. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways ''dendritic cell maturation'' and ''acute phase response signaling'', whereas cell culture affected biological processes involved in ''cellular development''.

Conclusions

The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.     

Comparison of Rectoanal Mucosal Swab Cultures and Fecal Cultures for Determining Prevalence of Escherichia coli O157:H7 in Feedlot Cattle

We compared fecal samples with samples collected with rectoanal mucosa swabs (RAMS) to determine the prevalence of Escherichia coli O157 in feedlot cattle (n = 747). Escherichia coli O157 was detected in 9.5% of samples collected with RAMS and 4.7% of samples tested by fecal culture. Pulsed-field gel electrophoresis analysis of isolates suggested that the strains colonizing the rectoanal junction were the same as those from the feces. Mucosal swab sampling was more sensitive than fecal sampling for determining the prevalence of E.coli O157 in feedlot cattle.