Detection of Influenza D-Specific Antibodies in Bulk Tank Milk from Swedish Dairy Farms.

Influenza D virus (IDV) has been detected in bovine respiratory disease (BRD) outbreaks, and experimental studies demonstrated this virus's capacity to cause lesions in the respiratory tract. In addition, IDV-specific antibodies were detected in human sera, which indicated that this virus plays a potential zoonotic role. The present study aimed to extend our knowledge about the epidemiologic situation of IDV in Swedish dairy farms, using bulk tank milk (BTM) samples for the detection of IDV antibodies. A total of 461 and 338 BTM samples collected during 2019 and 2020, respectively, were analyzed with an in-house indirect ELISA. In total, 147 (32%) and 135 (40%) samples were IDV-antibody-positive in 2019 and 2020, respectively. Overall, 2/125 (2%), 11/157 (7%) and 269/517 (52%) of the samples were IDV-antibody-positive in the northern, middle and southern regions of Sweden. The highest proportion of positive samples was repeatedly detected in the south, in the county of Halland, which is one of the counties with the highest cattle density in the country. In order to understand the epidemiology of IDV, further research in different cattle populations and in humans is required.

Associations between Bovine Coronavirus and Bovine Respiratory Syncytial Virus Infections and Productivity, Health Status and Occurrence of Antimicrobial Resistance in Swedish Dairy Herds.

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) affect dairy herds worldwide. In this study, effects on herd health, morbidity, and antimicrobial resistance (AMR) were assessed. Herds were considered free of infection (FREE), recently infected (RI) or past steadily infected (PSI) based on antibody testing of milk from primiparous cows. Data from farm records, national databases, and AMR of fecal Escherichia coli from calves were used as outcome variables. Compared to BRSV FREE herds: BRSV PSI herds had significantly higher odds of cough in young stock, a higher proportion of quinolone-resistant E. coli (QREC), but a lower proportion of cows with fever. BRSV RI herds had significantly higher odds of diarrhea in calves and young stock, a higher proportion of QREC and higher odds of multidrug-resistant E. coli. Compared to BCoV FREE herds: BCoV PSI herds had significantly higher odds of cough in all ages, and of diarrhea in young stock and cows, and a higher proportion of cows with fever. BCoV RI herds had significantly higher odds of diarrhea in young stock and cows and of cough in all ages. The results support previous research that freedom from BRSV and BCoV is beneficial for animal welfare and farm economy and possibly also mitigates AMR.

Exploring milk shipment data for their potential for disease monitoring and for assessing resilience in dairy farms.

The use of routinely recorded data for research purposes and disease surveillance is an attractive proposition. However, this requires that the validity and reliability of the data be evaluated for the purpose for which they are to be used. This manuscript reports an evaluation of milk shipment data for evaluating their usefulness in disease monitoring and the resilience of organic and conventional dairy herds in Sweden. A large number of inconsistencies were observed in the data, necessitating substantial efforts to "clean" the data. Given that the selection of rules used in the cleaning process was subjective in nature, a sensitivity analysis was carried out to determine if different cleaning routines produced substantially different results. Despite the cleaning efforts we observed far more large residuals at the shipment level than expected. Thus, it was concluded that the data were too "noisy" to be used for identification of short term impacts on milk production. Resilience was evaluated by examining the residual variance in milk shipped per cow per day under the assumption that herds with high resilience would have lower residual variance. The effects on residual variance of organic status or whether or not the herd used an automatic milking system were evaluated in models in which the residual variance was stratified or not by these factors. We did not find consistent evidence to suggest that organic herds had higher resilience than conventional herds, but this could be partly due to using residual variance as the measure indicating resilience.

FOXP3+ regulatory T cells in normal prostate tissue, postatrophic hyperplasia, prostatic intraepithelial neoplasia, and tumor histological lesions in men with and without prostate cancer.

BACKGROUND: The tumor promoting or counteracting effects of the immune response to cancer development are thought to be mediated to some extent by the infiltration of regulatory T cells (Tregs ). In the present study we evaluated the prevalence of Treg populations in stromal and epithelial compartments of normal, post atrophic hyperplasia (PAH), prostatic intraepithelial neoplasia (PIN), and tumor lesions in men with and without prostate cancer. METHODS: Study subjects were 102 men consecutively diagnosed with localized prostate cancer undergoing radical prostatectomy and 38 men diagnosed with bladder cancer undergoing cystoprostatectomy without prostate cancer at the pathological examination. Whole mount sections from all patients were evaluated for the epithelial and stromal expression of CD4+ Tregs and CD8+ Tregs in normal, PAH, PIN, and tumor lesions. A Friedmans test was used to investigate differences in the mean number of Tregs across histological lesions. Logistic regression was used to estimate crude and adjusted odds ratios (OR) for prostate cancer for each histological area. RESULTS: In men with prostate cancer, similarly high numbers of stromal CD4+ Tregs were identified in PAH and tumor, but CD4+ Tregs were less common in PIN. Greater numbers of epithelial CD4+ Tregs in normal prostatic tissue were positively associated with both Gleason score and pT-stage. We observed a fourfold increased risk of prostate cancer in men with epithelial CD4+ Tregs in the normal prostatic tissue counterpart. CONCLUSIONS: Our results may suggest a possible pathway through which PAH develops directly into prostate cancer in the presence of CD4+ Tregs and indicate that transformation of the anti-tumor immune response may be initiated even before the primary tumor is established.

Prevalence of thermophilic Campylobacter species in Swedish dogs and characterization of C. jejuni isolates.

BACKGROUND: The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping. RESULTS: Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3-12 months of age while dogs shedding C. upsaliensis were found in all ages. CONCLUSIONS: The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.

Bovine respiratory syncytial virus and bovine coronavirus in Swedish organic and conventional dairy herds.

BACKGROUND: Infections with bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BoCV) are endemic to the cattle populations in most countries, causing respiratory and/or enteric disease. It has been demonstrated that herds can remain free from these infections for several years also in high prevalence areas. Organically managed (OM) dairy herds have been shown to have lower seroprevalence of both viruses compared to conventionally managed (CM) herds. The objective of this study was to challenge the hypothesis of a lower occurrence of BRSV and BoCV in OM compared to CM dairy herds. In November 2011, May 2012 and May 2013 milk samples from four homebred primiparous cows were collected in 75 to 65 OM and 69 to 62 CM herds. The antibody status regarding BRSV and BoCV was analysed with commercial indirect ELISAs. Herds were classified as positive if at least one individual sample was positive. RESULTS: The prevalence of positive herds ranged from 73.4% to 82.3% for BRSV and from 76.8% to 85.3% for BoCV among OM and CM herds, over the three sampling occasions. There was no statistically significant difference between OM and CM herds at any sampling occasion. The incidence risk of newly infected herds did not differ statistically between OM and CM herds at any sampling occasion, neither for BRSV nor for BoCV. The incidence of herds turning sero-negative between samplings corresponded to the incidence of newly infected. Bulk tank milk (BTM) samples were also sampled in the herds and analysed. Several herds were negative on individual samples but positive in BTM. Herd-level data on production, health and reproduction were retrieved from VÄXA Sweden and the study herds were representative of the source population. CONCLUSION: There was no difference in prevalence of or incidence risk for BRSV or BoCV between Swedish OM and CM herds. Because the incidence of herds becoming seropositive was balanced by herds becoming seronegative it should be possible to lower the prevalence of these two infections among Swedish dairy cattle herds if biosecurity is improved.

Surveys on Coxiella burnetii infections in Swedish cattle, sheep, goats and moose.

BACKGROUND: Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey. RESULTS: The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%. CONCLUSIONS: The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.

A comparative study of ERG status assessment on DNA, mRNA, and protein levels using unique samples from a Swedish biopsy cohort.

The ERG rearrangement is identified in approximately 50% of prostate cancer screened cohorts and is known to be highly specific. This genetic aberration, most commonly leading to the TMPRSS2-ERG fusion, but also SLC45A3-ERG or NDRG1-ERG fusions, all leading to an overexpression of a truncated ERG protein. Most studies have applied in situ hybridization (FISH) methods or mRNA-based assays to investigate the ERG status. Recently, studies showed that ERG protein levels assessed by ERG antibodies can be used as a surrogate marker for ERG rearrangement. In the current study, we investigate ERG status on a series of diagnostic biopsies using DNA-based, mRNA-based, and protein-based assays. We formally compared 3 assay results (ie, FISH, fusion mRNA, and immunohistochemistry) to identify which method could be most appropriate to use when having limited amount of tissue. ERG rearrangement was found in 56% of the cases. Comparing ERG rearrangement status by FISH with ERG overexpression and TMPRSS2-ERG fusion transcript we found 95.1% (154/162, Fisher exact test 9.50E-36) and 85.2% (138/162, Fisher exact test 7.26E-22) concordance, respectively. We show that the ERG antibody highly correlates with the ERG rearrangement with high sensitivity and specificity. We also identified the most common TMPRSS2-ERG isoform in the majority of ERG rearranged cases. These results provide compelling evidence that the ERG antibody can be used to further investigate the role of ERG in prostate cancer.

Comparison of Bovine coronavirus-specific and Bovine respiratory syncytial virus-specific antibodies in serum versus milk samples detected by enzyme-linked immunosorbent assay.

Bovine coronavirus (BCV; Betacoronavirus 1) and Bovine respiratory syncytial virus (BRSV) are significant causes of enteric and respiratory disease in beef and dairy cattle throughout the world. Indirect enzyme-linked immunosorbent assays are widely used to detect serum antibodies for herd monitoring and prevalence studies. In dairy herds, milk is more readily collected than serum. Hence, in order to investigate the test agreement between serum and milk, both serum and milk samples from 105 cows in 27 dairy herds were analyzed in parallel for presence of immunoglobulin G antibodies to BCV and BRSV. The Bland-Altman analyses of data demonstrated good agreement between serum and milk antibody titers for both viruses. The results indicate milk samples are sufficient for surveillance of antibodies to BCV and BRSV.

CD4 helper T cells, CD8 cytotoxic T cells, and FOXP3(+) regulatory T cells with respect to lethal prostate cancer.

Prostate cancer represents a major contributor to cancer mortality, but the majority of men with prostate cancer will die of other causes. Thus, a challenge is identifying potentially lethal disease at diagnosis. Conflicting results have been reported when investigating the relationship between infiltration of lymphocytes and survival in prostate cancer. One of the mechanisms suggested is the recruitment of regulatory T cells (T(regs)), a subpopulation of T cells that have a role in promoting tumor growth. T(regs) counteract tumor rejection through suppressive functions on the anti-immune response but their prognostic significance is still unknown. We report here the results of a conducted case-control study nested in a cohort of men treated with transurethral resection of the prostate and diagnosed incidentally with prostate cancer. Cases are men who died of prostate cancer (n=261) and controls are men who survived >10 years after their diagnosis (n=474). Infiltration of both T(helper) and T(cytotoxic) cells was frequently observed and the majority of the T(regs) were CD4(+). T(helper) or T(cytotoxic) cells were not associated with lethal prostate cancer. However, we found a nearly twofold increased risk of lethal prostate cancer when comparing the highest with the lowest quartile of CD4(+) T(regs) cells (95% confidence interval: 1.3-2.9). Our conclusion is that men with greater numbers of CD4(+) T(regs) in their prostate tumor environment have an increased risk of dying of prostate cancer. Identification of CD4(+) T(regs) in tumor tissue may predict clinically relevant disease at time of diagnosis independently of other clinical factors.

Tracing the transmission of bovine coronavirus infections in cattle herds based on S gene diversity.

Bovine coronavirus (BCoV) is found worldwide and causes respiratory infections and diarrhoea in calves and adult cattle. In order to investigate the molecular epidemiology of BCoV, 27 reverse transcription polymerase chain reaction (RT-PCR) positive samples from 25 cattle herds in different parts of Sweden were analysed. A 1038-nucleotide fragment was PCR amplified and directly sequenced. The analysed BCoV strains showed a high sequence identity, regardless of whether they were obtained from outbreaks of respiratory disease or diarrhoea or from calves or adult cattle. Circulation of an identical BCoV strain during a 4-month period was demonstrated in calves in one dairy herd. In a regional epizootic of winter dysentery in Northern Sweden, highly similar BCoV strains were detected. In the Southern and Central regions, several genotypes of BCoV circulated contemporaneously, indicating that in these regions, which had a higher density of cattle than the Northern regions, more extensive transmission of the virus was occurring. Identical BCoV sequences supported the epidemiological data that inter-herd contact through purchased calves was important. Swedish BCoV strains unexpectedly showed a high homology with recently detected Italian strains. This study shows that molecular analysis of the spike (S) glycoprotein gene of BCoV can be a useful tool to support or rule out suspected transmission routes.

Application of network analysis parameters in risk-based surveillance - examples based on cattle trade data and bovine infections in Sweden.

Financial resources may limit the number of samples that can be collected and analysed in disease surveillance programmes. When the aim of surveillance is disease detection and identification of case herds, a risk-based approach can increase the sensitivity of the surveillance system. In this paper, the association between two network analysis measures, i.e. 'in-degree' and 'ingoing infection chain', and signs of infection is investigated. It is shown that based on regression analysis of combined data from a recent cross-sectional study for endemic viral infections and network analysis of animal movements, a positive serological result for bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) is significantly associated with the purchase of animals. For BCV, this association was significant also when accounting for herd size and regional cattle density, but not for BRSV. Examples are given for different approaches to include cattle movement data in risk-based surveillance by selecting herds based on network analysis measures. Results show that compared to completely random sampling these approaches increase the number of detected positives, both for BCV and BRSV in our study population. It is concluded that network measures for the relevant time period based on updated databases of animal movements can provide a simple and straight forward tool for risk-based sampling.

Phylogenetic analysis of bovine respiratory syncytial viruses from recent outbreaks in feedlot and dairy cattle herds.

Bovine respiratory syncytial virus (BRSV) is one of the major causes of bovine respiratory disease worldwide. In order to study the molecular epidemiology of the virus, samples from 30 BRSV outbreaks in cattle herds located in different parts of Sweden were collected from 2007 to 2011. The samples were analyzed by PCR, and the glycoprotein (G) gene was sequenced. BRSV was detected in outbreaks of respiratory disease in both dairy and feedlot herds most often during the winter period but also during the summer months (May to August). This indicates that circulation of the virus between herds occurs throughout the year. Comparative sequence analysis revealed a high degree (more than 94.5%) of sequence identity among the collected strains. Phylogenetic analysis showed that 29 out of the 30 strains formed a unique clade. Identical sequences found in herds sampled within a few months' time suggested that these herds were part of a common transmission chain. One strain from a single outbreak in a herd in southern Sweden clustered with Danish strains and showed a distant relationship to the rest of the Swedish strains. Further studies are highly warranted to clarify the inter-herd transmission routes of BRSV. Such knowledge is essential for the control of the spread of this virus between herds, regions and even countries.

The relationship between antibody status to bovine corona virus and bovine respiratory syncytial virus and disease incidence, reproduction and herd characteristics in dairy herds.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) and bovine corona virus (BCV) affects cattle worldwide. Our objective was to evaluate the effects of these infections on general health and reproduction parameters measurable on herd level and to explore the association between antibody status and some herd characteristics. METHODS: We collected a pooled milk sample from five primiparous cows from 79 Swedish dairy herds in September 2006. The samples were analysed for immunoglobulin G antibodies to BCV and BRSV with indirect enzyme-linked immunosorbent assays. Herd level data from 1 September 2005 to 30 August 2006 were accessed retrospectively. The location of the herds was mapped using a geographical information system. RESULTS: Ten herds were antibody negative to both viruses and were compared with 69 herds positive to BCV or BRSV or both. Positive herds had a higher (P=0.001) bulk tank milk somatic cell count (BMSCC) compared with negative herds. The medians for all other analyzed health and reproductive parameters were consistently in favour of the herds negative to both viruses although the differences were not statistically significant. A higher proportion (P=0.01) of herds used professional technicians for artificial insemination, rather than farm personnel, amongst the 33 herds negative to BCV compared with the 46 positive herds. CONCLUSIONS: Our result shows that herds that were antibody positive to BCV and/or BRSV had a higher BMSCC compared with herds negative to BCV and BRSV. There was also tendency that negative herds had a better general herd health compared with positive. A higher proportion amongst the BCV negative herds used external technicians for AI instead of farm personnel, indicating that it is possible to avoid infection although having regular visits. Negative herds were located in close proximity to positive herds, indicating that local spread and airborne transmission between herds might not be of great importance and that herds can stay free from these infection transmission although virus is circulating in the area.