The African swine fever (ASF) virus is one of the deadliest viruses plaguing the global swine industry. There are no effective treatments for pigs becoming infected with the ASF virus. Eliminating infected pigs is the only effective method to tackle an outbreak. Whole farm biosecurity is therefore critical to prevent the virus from entering in the first place. In recent years, feed and feed ingredients have been identified as potential vectors of viral diseases including ASF. The objective of this research was to simulate contamination of feed by ASF virus in a laboratory setting and use this simulation to test the efficacy of some select feed additives for reducing the amount and viability of this virus in feed. The main result shows that the inoculated virus in feed disappeared in a shorter time when the feed had been treated with the compounds. We conclude that these feed additives could be adopted as part of a comprehensive biosecurity system for pig holdings. Pigs might be protected from feed possibly contaminated by this virus, and the pork supply will thus be less likely to be impacted by this viral disease.African swine fever virus is one of the most highly contagious and lethal viruses for the global swine industry. Strengthening biosecurity is the only effective measure for preventing the spread of this viral disease. The virus can be transmitted through contaminated feedstuffs and, therefore, research has been conducted to explore corresponding mitigating measures. The purpose of the current study was to test a combination of pure benzoic acid and a blend of nature identical flavorings for their ability to reduce African swine fever viral survival in feed. This virus was inoculated to feed with or without the supplementation of the test compounds, and the viral presence and load were measured by a hemadsorption test and quantitative real time polymerase chain reaction. The main finding was that the combination of pure benzoic acid and nature identical flavorings could expedite the reduction in both viral load and survival in a swine feed. Therefore, this solution could be adopted as a preventive measure for mitigating the risk of contaminated feed by African swine fever virus. African swine fever (ASF) is a highly contagious and usually fatal disease for both domestic and feral pigs. This disease has been endemic for decades, initially in sub-Saharan Africa and then in some European regions. In 2018, China, a country holding half of the global population of pigs, reported its first outbreak of ASF, and it quickly spread to some other Asia-Pacific countries. According to the recent update by the World Organization for Animal Health (OIE) [The African swine fever virus is a large, enveloped virus with an average diameter of 200 nm and a genomic core consisting of a single molecule of linear, double-stranded deoxyribonucleic acid (DNA) . This viAn effective biosecurity program entails a comprehensive approach that addresses every potential threat enabling viral entry into pig holdings. According to a recent review by Jones et al. , ASF virAs part of a comprehensive biosecurity program, feed biosecurity should be included. Proactive mitigation through quarantining ingredients, thermal processing, or the use of feed additives as a final barrier should be considered. Additionally, feed additives may be more successful in terms of preventing cross-contamination . The traTherefore, the aim of this study was to simulate the contamination of feed with ASF virus in a laboratory setting and validate whether the combination of pure benzoic acid and nature identical flavorings could exert similar effects on the viability and load of ASF virus as previously reported for other viruses. This study was based on the hypothesis that the addition of nature identical flavorings to pure benzoic acid could decrease both the viral load and viability in complete feed when contamination of ASF virus occurred.The test products were either pure benzoic acid  Limited, Shanghai, China) or the combination of pure benzoic acid and a mixture of nature identical flavorings including thymol, eugenol, piperine, and curcumin as the main ingredients , a positive control with the inoculation of ASF virus (PC), and the NC with the inoculation of ASF virus and supplemented with either 0.5% pure benzoic acid or the combination of 0.5% pure benzoic acid and 0.02% nature identical flavorings. The assays were performed in 2 matrices, i.e., having the test products either dissolved in phosphate-buffered saline (PBS) or mixed in a pig diet. The assays in PBS were considered secondary and supportive for assays in feed matrix considering the poor solubility of pure benzoic acid and nature identical flavorings in water. All the operations with the ASF virus were carried out in a biosafety level (BSL)-3 laboratory at South China Agricultural University .2); a series of dilutions  of the supernatant or the aliquot were added to the wells and incubated for 3 h; 10 µL of CCK-8 solution was added to each well of the plate and incubated for 1 h; and finally, absorbance at 450 nm was measured using a microplate reader . Each dilution was tested in 3 replicates.The supernatant of the treatments in feed matrix and aliquots of the treatments in PBS were tested for their cytotoxicity according to the cell counting kit-8  assay. Briefly, 100 µL of cell suspension were dispensed in a 96-well plate and incubated for 3 h in an incubator /mL (ASFV/China/GZ201801), whereas 1 mL cell culture solution was added to the 20 mL solution of NC. The 4 test tubes of each treatment group corresponded to day 1, 3, 6, and 9 post inoculation as the sampling days. The test tubes were continuously agitated at 1200 rpm/min. On each of the pre-determined sampling days, one tube per treatment was taken for analysis. Aliquots of the solutions (100 µL) after 10-fold dilution were used to infect porcine alveolar macrophages in plate wells for 2 h before the removal of the supernatant and then the addition of RPMI 1640 medium . After further 24-h incubation, the real-time quantitative polymerase chain reaction (RT-PCR) and hemadsorption test were performed. The primary alveolar macrophages were collected from piglets tested negative for PRRS virus, porcine circovirus 2, and pseudorabies virus.Four treatment solutions were prepared with PBS according to the previously defined 4 treatments. Four 20-mL of the solution of PC or with test compounds were pipetted into 4 centrifuge tubes , then inoculated with 1 mL ASF virus with the titer of 105.3 HAD50/mL ASF virus (ASFV/China/GZ201801), whereas 1 mL cell culture solution was added to the tubes of NC group. The sample tubes were continuously agitated at 1200 rpm/min to prevent clumping. On each of the pre-determined sampling days, one sample tube per treatment was taken for analysis. Ten mL sterile PBS were added to each sample tube for collection of supernatant after centrifugation with aseptic techniques. One aliquot (100 µL) of the supernatant after 100-fold dilution was used to infect porcine alveolar macrophages in plate wells for 2 h before the removal of supernatant and then the addition of RPMI 1640 medium. After a further 24-h incubation, RT-PCR and hemadsorption test were performed.Four samples of commercial pig feed with corn and soybean meal as the main ingredients were mixed individually in a miniature blade mixer according to the previously defined treatments. These samples were gamma-irritated at 25 kGy. Four 10-g sub-samples of each treatment were weighed into 4 centrifuge tubes  to be sampled on day 1, 3, 6, and 9 post inoculation, respectively. Each sample tube of the PC and the treatments with supplements was inoculated with 1 mL 10For hemadsorption, 20 µL 1% red blood cells were added to each well for overnight incubation. On the following day, the agglutinated red blood cells with a ring appearance were observed under a microscope .The DNA was extracted with a commercial Axygen kit  and then stored at −20 °C before analysis by RT-PCR with a commercial kit for detecting ASF virus  according to the manufacturer’s instructions. This assay targets the VP72 gene of the ASF virus and uses the 5′-nuclease assay system to detect the amplicons. The analytical results were presented as cycle threshold (Ct) at which virus was detected. A greater Ct value indicates less genetic materials in the test samples. Samples were declared negative when Ct value is greater than 40 and positive when less than 38.p < 0.05.Data were analyzed with Fit model platform of JMP  to determine the main effects of sampling time, treatment, and their interaction. Tukey’s test were used to compare the means. The least square means were presented. Significance was declared at The survival rate of cells in the presence of 100- and 1000-fold dilutions of the supernatant of feed treated with pure benzoic acid or pure benzoic acid combined with nature identical flavorings reached 100% and thereby, 100-fold dilution was used in downstream hemadsorption tests to ensure no toxic effects on the porcine alveolar macrophage cells . With thThe NC samples tested negative for hemadsorption and were confirmed to be free of ASF virus DNA by RT-PCR on all sampling days, while the PC samples were all positive for hemadsorption and had an initial Ct value of 28.4 on day 1 post inoculation . This CtThe NC samples tested negative for hemadsorption and were confirmed to be free of ASF virus DNA by RT-PCR on all sampling days, while the PC samples were all positive and had an initial Ct value of 30.4 on day 1 post inoculation and then remained relatively stable from day 3 to 9 post inoculation . The incEscherichia coli F4 [Traditionally, both the pure benzoic acid and the nature identical flavorings evaluated in this study are supplemented to feed for improving the growth and health performance of pigs. Pure benzoic acid is a common preservative for foods and beverages and is an efficacious acidifier for weaned piglets and fattening pigs ,24. Pure coli F4 .The application of pure benzoic acid and nature identical flavorings for the control of viral transmission through feed is still novel, despite the broad recognition that feed is a valid vector for transmitting viruses. The first evidence that contaminated feed can serve as a vehicle for PED virus infection of naïve pigs was provided by Dee et al. , with coThis study has demonstrated two components of significance. Firstly, the ASF virus was successfully inoculated both to the PBS solution and a complete feed and survived for 9 days (the longest duration tested in this study) as shown by the PC in comparison to the NC. These procedures could be used as models for evaluating the efficacy of other potential compounds for mitigating ASF viral transmission in similar vectors. Secondly, both pure benzoic acid and its combination with nature identical flavorings showed efficacy for impairing the viability of live ASF virus and decreasing the viral load in the matrices, which provided supportive evidence for the hypothesis of the current study. The potential mechanisms could be related to the viral envelop being degraded by the acid, since its integrity is indispensable for accomplishing the cellular infection , while tThe results of the current study imply that both water and feed could harbor the ASF virus and then infect pigs, which calls attention to the need for mitigation strategies as part of a comprehensive biosecurity program for pig holdings. Feed might pose a greater threat in comparison to water sources because feed delivery is a high-frequency event and the likelihood of infection via contaminated feed increases dramatically with increasing exposures . The appWe acknowledge some limitations with this study. The inoculated dose of ASF virus might not represent all situations in reality. The mitigating effects of the test compounds were not confirmed with a bioassay study with the mitigated or non-mitigated vectors fed to live pigs. Further study is warranted to understand the mechanism by which adding nature identical flavorings to pure benzoic acid seems to provide a boosting effect for reducing the survival of virus in feed matrix. It is also worth emphasizing that mitigating the risk of viral spread via feed with feed additives can be an important component of a holistic biosecurity system, but does not reduce the importance of other preventative biosecurity practices.In conclusion, the current study developed laboratory models simulating the contamination of PBS and a complete swine feed with ASF virus and validated the mitigating effects of some feed additives. Under the conditions of this study, it was demonstrated that both pure benzoic acid and its mixture with nature identical flavorings can reduce the viral load and survivability of the ASF virus in these matrices, and the combinational solution appeared to be more advantageous when compared to pure benzoic acid alone in terms of preventing the survival of ASF virus in feed faster.


Initially, every animal was tested for PI within the first year, followed by testing for the presence of virus in all newborn calves for the next four years. Prevalence of calves being born PI thus diminished substantially from around 1.4% to <0.02%, which enabled broad testing for the virus to be abandoned and switching to economically more favourable serological surveillance with vaccination being prohibited. By the end of 2020, more than 99.5% of all cattle farms in Switzerland were free of BVDV but eliminating the last remaining PI animals turned out to be a tougher nut to crack. In this review, we describe the Swiss BVD eradication scheme and the hurdles that were encountered and still remain during the implementation of the programme. The main challenge is to rapidly identify the source of infection in case of a positive result during antibody surveillance, and to efficiently protect the cattle population from re-infection, particularly in light of the endemic presence of the related pestivirus border disease virus (BDV) in sheep. As a consequence of these measures, complete eradication will (hopefully) soon be achieved, and the final step will then be the continuous documentation of freedom of disease.Bovine viral diarrhoea virus (BVDV) and related ruminant pestiviruses occur worldwide and cause considerable economic losses in livestock and severely impair animal welfare. Switzerland started a national mandatory control programme in 2008 aiming to eradicate BVD from the Swiss cattle population. The peculiar biology of pestiviruses with the birth of persistently infected (PI) animals upon  Pestivirus in the family Flaviviridae comprised the four species bovine viral diarrhoea virus (BVDV)-1 and−2, classical swine fever virus (CSFV) and border disease virus (BDV) from sheep  strategy indicates that the host is chronically or even lifelong infected, which mostly requires that the virus evolved sophisticated means to evade the host's immune system , 20. WelThe successful worldwide survival of BVDV  and otheIn addition to this persistence, acute infection of adult, naïve cattle with either biotype of BVDV results in transient viremia that is often asymptomatic or accompanied by only mild diarrhoea or respiratory symptoms, but in rare cases, severe thrombocytopenia and haemorrhages might be observed . During Ruminant pestiviruses have probably circulated for hundreds of years in their hosts , 35 causThe entire cattle population in Switzerland comprises ~1.5–1.6 million animals, and annually, 600,000–700,00 calves are born . The disall bovines were virus-positive, and 20.0% of all herds had at least one virus positive animal  , 62. TheAll dairy herds should be tested twice yearly and all non-dairy herds once every three year. The spot test should include at least five animals not <6 months of age and born after September 2009, or 10% of the stock in larger herds. Additional requirements for animals to be included in a spot test were (i) that they were born at least 1 month after the elimination of the last PI animal in the herd; (ii) that they stayed in the herd to be tested for at least 6 months, (iii) that they were not part of a herd containing a PI animal during and after its stay in that herd, and (iv) that the animals were not previously tested seropositive. Spot tests were also performed in dairy herds with a “non-negative” bulk milk result. The classification of a bulk milk test result as “non-negative” was adapted at the end of 2014, as the interpretation of the progression of the antibody titre in herds categorised as class 2 by the PP value between two samples was too complex. To simplify the interpretation, all samples in class ≥2 were defined as non-negative, despite this leading to an increase in positive results and a higher workload for the regional veterinary services. In the first 3 years of surveillance, samples for spot tests were taken on the farm by the cantonal veterinary services. This resulted in only about 80% of the non-dairy herds being sampled at least once in the 3 years. In addition, these herds were not tested uniformly in this period but most of them were tested in the last year. Thus, the increase in the case count 2013–2015  could beIn 2015, the sampling frequency for dairy herds was reduced to once per year, with the sampling in spring 2015 accounting for the same year, whereas the samples taken in autumn 2015 were counted for 2016. This led to an elongated period without testing dairy farms from autumn 2015 to autumn 2016. At the time this decision was taken, the prevailing opinion was that the virus had almost been eradicated, so surveillance could be considerably reduced. Unfortunately, this turned out not to be the case at all. The eradication scheme suffered a severe setback, with yearly case numbers doubling from 2015 to 2017 . As a coThe regional veterinary services responsible for sampling on-site estimated the workload as being too high if all non-dairy herds were to be sampled yearly. To overcome this limitation, the project RiBeS  was initiated in 2016 to take blood samples for surveillance in cattle during meat inspection at large abattoirs, as similarly proposed later in Japan . SamplinA spot test is considered positive if at least one animal is serological positive. For small herds, the size can be reduced from five to two animals as it was sometimes impossible to find more animals fulfilling all the requirements. In situations where only one animal is positive, the regional veterinary services perform a risk analysis to determine whether a suspected case is established and whether measures should be imposed on the herd. Spot tests in dairy herds are always taken from animals living on the farm by a veterinarian on a single day. Since 2018, spot tests from non-dairy herds are mostly taken during meat inspection at the abattoirs. The two big differences compared to the sampling in dairy herds by the classical spot test is that (i) the sampling takes place over a prolonged period and (ii) no second sample can be taken from the tested animals. Consequently, mistaken sample identification or false-positive test results are more difficult to verify. Evaluation of the results of the spot tests and the bulk milk samples indicated that in 2018, in about 89% of the positive screening results, no PI animal could be identified. Reasons might be that the animal might already has left the herd, or the spot tests were false-positive in both herd types. This provides evidence that serologically positive animals are still not restricted to animals that had contact with known PI animals. As a consequence, these seropositive animals are a major problem for effectively targeting the control efforts only to the herds where active transmission is indeed occurring.As a result of these constantly high surveillance efforts, case numbers have dropped again from 258 infections in new herds in 2017 to 121 in 2020 . ExperieThe conceptual layout of the Swiss computerised data management has been previously described . In shorThe applications RiBeS and RiBeS-App, used to indicate which animals the meat inspectors need to sample at the abattoirs, are completely independent from the other software applications, but they provide an interface with the resource-planning software of the enterprise of the large and small abattoirs, respectively. Taking samples according to RiBeS is now well-established in the meat inspection process. Nevertheless, food safety clearly remains the priority in the process of meat inspection, and the additional effort needs to be financially compensated. To profit from possible synergisms, a more integrated system from management to integration of laboratory results for all cattle surveillance programmes is planned in the future. A more detailed data structure would be of great value especially for the spot tests, as with the current systems, differentiation of the results of the spot test from the ones of other serological assays in dairy herds proved to be difficult.In the last 3 years, the use of a data warehouse combining information from different sources allowed the production of useful reports for epidemiological investigations and contact tracing of animals and herds with positive results in serological surveillance. These possibilities are increasingly used by the regional veterinary services. The format of flexible reports combining the information from the AMD, the laboratory database, ISVet, and RiBeS proved to be an important improvement. The animal movement database is the most important source for tracing of animals, and current efforts are directed toward specifically transforming data available from the AMD into information useful for BVD control, such as the calculation of calving periods and the proportion of twins and stillbirths per herd. The data management systems used offer great flexibility, but as a disadvantage, retrospective evaluations are rather difficult as the previous status of animals and herds are not available, e.g., in contrast to data management systems used in Germany .As virus is shed from all secretions, e.g., saliva, semen, tears, milk, and to a lesser extent faeces, direct contact of susceptible animals to persistently or transiently infected cattle is the most prominent way of horizontal transmission. Summer grazing on one of the 6,740 communal alpine pastures (as of 2019) is very common in Switzerland , with ap0), which indicates the expected number of new infections generated by one case in a completely susceptible herd, might be well above 30 for the transmission by PI animals  animals starts at around 2–3 days post-infection (p.i.), and last up to 1–2 weeks until seroconversion of the infected host occurs. This indicates that the presence of infectious virus in secretions from acutely infected animals can be expected. Indeed, infectious virus could be isolated from nasal swabs from 5 out of 6 experimentally infected animals between day 5 and 10 p.i. , whereasThis rather short time window of virus secretion together with a reduced amount of virus shed by acutely infected compared to PI animals leads to strongly reduced efficiency of virus transmission. Thus, none of the 14 sentinel animals were infected by nose-to-nose contact with 5 TI calves . This waIn rare cases, the persistence of BVDV in testicles of postpubertal bulls was described despite these bulls seroconverting after transient infection and being free of virus in serum thereafter. In these animals, infectious virus could be detected in semen for months –91, even0 were rarely done, and the results were quite diverse, but in most cases, R0 for TI animals was below 1. Thus, R0 was reported to be around 0.25 for BVDV-1 and−2 being transmitted by experimentally generated TI animals, whereas the introduction of PI animal led to an unlimited increase of R0 [“R0 = ∞”  can be estimated. Of course, herd size, cattle management, general health status etc. will influence the efficiency of transmission, but TI animals appear to be an even smaller risk than surface or fomite contamination by secretions of PI animals, especially during the birth of PI animals. Therefore, the detection of TI animals is not the main risk factor to maintain a chain of infection, but rather represents an important indicator of the presence of a source of infection, e.g., a PI calf. In later stages of BVDV eradication with a highly susceptible cattle population and intense surveillance, transient infections might nevertheless be observed and might lead to either costly investigations or, in rare cases, to the transmission to a pregnant heifer and the birth of new PI calf.Summarising these studies , transmiSince the first description of BVDV in 1946 , 102, a rns  is highly sensitive, and was and still is the gold standard, but the requirement of cell cultures limits its use to more specialised laboratories. Thus, agar gel immunodiffusion test and enzyme-linked immunosorbent assay (ELISA) were rather routinely applied , 104, wirns , 107. Byrns .In countries where HoBi-like pestiviruses (Pestivirus H) are circulating, specific assays need to be developed as the test routinely applied for BVDV and BDV appear to unreliably detect these types of antibodies –112. IndCoxiella burnetii in addition to BVDV  or HoBi-like viruses , are not specified by the animal disease regulation . As routinely applied diagnostic tests do not differentiate between BVDV and BDV, any positive result is defined as a positive case with all its consequences defined in the TSV for BVDV. However, if antibody monitoring or further investigations on virus-positive cattle indicate that, e.g., a PI animal is infected with BDV, the TSV does not apply, and any further actions rely only on a general act regulating animal disease control measures in the animal disease law (“Tierseuchengesetz TSG”). In light of new pestiviruses that were recently described and might be discovered in future, a broader definition for pestivirus infection in cattle might be advantageous and legally assured in an animal disease regulation in countries with scheduled or ongoing eradication programmes. Similar problems were faced with the eradication of caprine arthritis encephalitis virus (CAEV) in Swiss goats, where sheep infected with Visna-Maedi virus (VMV) or CAEV play a significant role as a reservoir for such small ruminant lentiviruses (SRLV) and, therefore, as a risk factor in the control of CAEV in the Swiss goat population –166.As stated above (chapter Data management systems), the enormous logistical tasks encountered in the eradication programme could not have been achieved without appropriate data management systems, including the animal movement database (AMD) . In the The very strict and ambitious BVDV eradication in Switzerland led to a quick initial success [Currently, there appears to be around 10 chains of infection remaining, with some of them circulating for several years. The largest cluster we observed, i.e., isolates with identical sequences in the short stretch of the 5′-UTR, contains samples from around 1,000 animals collected since 2011. With pestiviruses being RNA viruses with a considerable mutation rate, it is, however, not plausible that all these isolates represent identical viruses, albeit a common origin cannot be excluded. Thus, the rather low resolution in the 5′-UTR is clearly sufficient to allocate the sequences to a specific (sub-)genotype, but it is insufficient to differentiate individual virus isolates. To enhance the resolution in sequencing to be of help for molecular epidemiology, we established a pilot scheme where we sequenced fragments of 800–1,000 nucleotides in length of selected clusters with identical sequences in the 5′-UTR by classical Sanger sequencing. This study confirmed that BVD viral strains can be further differentiated using these larger fragments, as exemplified in Based on the rather high number of BVD antibody positive animals prior to 2008 , SwitzerInitially, it was assumed that it would take around 10 years for eradication to be completed, as was described for other countries . HoweverFor the final achievement of BVD eradication in Switzerland, the following factors and measures are important for the programme to be successful, most of them already being in place:- Consistent completion of the animal movement database by every user without any gaps, possibly applying more severe consequences for fraudulent entries.- Continued strict application of biosecurity measures, incl. cattle trade and summer pasturing.- Enhanced biosecurity measures and strict supervision and surveillance during calving of possible “Trojan dams”.- Nationwide standardised procedure following positive results in antibody surveillance to achieve faster response across cantonal borders.- Immediate start of investigations upon a positive result through antibody surveillance, if appropriate with coordination across cantonal borders. During these assessments, the role of transient infections and the fact that no test is 100% sensitive and specific must be taken into consideration.- Shorten the time interval between active surveillance on the “farms of concern,” i.e., the few farms repetitively harbouring PI animals in recent years.- Transfer of every virus positive sample to the reference laboratory for sequencing. Molecular epidemiology is a great tool to track chains of infection, but this is only of help if all sequence data are available nationwide.- Separation of cattle and sheep. Where this is not feasible, voluntary sanitation of the sheep population concerned should be envisioned to avoid costly investigations over and over again.The aforementioned measures should enable the identification if any remaining source of infection as quickly as possible, and to reduce the risk of further transmission of the virus to naïve pregnant animals within this time interval. A final, more rigorous effort for a rather short time might be required to achieve the final aim of eradicating BVDV from the cattle population in Switzerland. Nevertheless, even after successful completion of this task, continued surveillance needs to be implemented (i) as ruminant pestiviruses might be re-introduced into the highly susceptible cattle population, e.g., by animal import or contaminated semen or vaccines, and (ii) as pestiviruses remain endemic in small ruminants in Switzerland, mainly in sheep, and pose a constant risk for re-introduction. This surveillance scheme will also be a necessity for federal and European regulations to continuously report the freedom of disease , which wMS conceptualised the manuscript. MS and HSch wrote the first draught of the manuscript with helpful advice and input by AH, EDL, HSt, and MG. HSch, HSt, and MS prepared the figures. All authors edited the manuscript and have read and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Greenland sled dogs (GSD) are a unique, genetically isolated population of dogs living under exceptional environmental conditions. Metabolism, and thereby thyroid hormones are affected by multiple factors. Among other activity, energy balance and environmental conditions are important. A breed-specific reference interval (RI) can be useful for diagnostics of potential thyroid-related pathologies. The aim of this study was to establish RIs of the thyroid hormones thyroxin (T4), free thyroxin (fT4), and thyroid stimulating hormone (TSH) in GSD. In addition to evaluate the effect of sex, age, season, management, and body condition score (BCS) in GSD. Physical exams and cephalic venous blood sampling were performed in the period of 2018–2019 from 265 GSD managed either privately or by the Danish navy. Serum biochemical analyses, including C-reactive protein, were performed and RIs were determined for TSH, T4 and fT4 in only healthy dogs. The RIs were determined using American Society for Veterinary Clinical Pathology guidelines and the effect of varying factors were evaluated by linear regression and further tested by Mann–Whitney test.144 GSD were included in the reference group resulting in RIs: T4: 6.44–48.65 nmol/L; fT4: 3.91–18.51 pmol/L; and TSH: 0.04–0.55 ng/mL. Female GSD had significantly higher concentrations of T4 (P = 0.039) and fT4 (P = 0.015) compared to males; a positive correlation between TSH and aging was found; T4 concentrations were significantly higher (P = 0.003) during summer; and TSH concentrations were lower in GSD managed by the navy (P < 0.0001). BCS was higher (P < 0.0001) in Sirius GSD compared to civilian GSD, and BCS was positively correlated with T4 and negatively correlated with TSH.Reference intervals for T4, fT4 and TSH in GSD were established. The RI for T4 and fT4 was lower compared to other breeds. In addition, sex, age, season, management and BCS demonstrated variable effects on thyroid hormones. Our results can be used as a foundation for improving management and further research of GSD.The online version contains supplementary material available at 10.1186/s13028-021-00617-8. Greenland sled dogs (GSD) is an original landrace of the Greenlandic Inuit. The Inuit of modern-day Greenland originate from an aboriginal Inuit culture that migrated to- and settled in Greenland about 900 years ago . With th[…] without their help [“Eskimo dogs”], success could never have crowned the efforts of the exhibition. They are sturdy, magnificent animals. There may be larger dogs than these, there may be handsomer dogs; but I doubt it. Other dogs may work as well or travel as fast and far when fully fed; but there is no dog in the World that can work so long in the lowest temperatures on practically nothing to eat.”The purpose of the GSD has evolved over time. Historically, the GSD were used only for subsistence hunting, and while this is still important for some GSD owners, the use now includes other activities such as racing, transportation, fishing, and tourism . GSD in Greenland are however typically inactive during summer, and active during late autumn, winter, and spring, when temperatures fall, and snow covers the landscape. The dogs exhibit great stamina and the ability to transport heavy loads across long distances at extreme low temperatures and with minimal sustenance . Their aThis early quote indicates a dog breed that possesses a unique metabolism. Several studies have examined how metabolism is affected by prolonged endurance exercise, intense physical training as well as extreme environmental conditions –14. ThyrThis study aims: (i) to determine the concentration of thyroid hormones  in healthy GSD and establish their reference intervals, (ii) to examine the behaviour of thyroid hormones in GSD under different circumstances , and, finally (iii) to describe the relationship between thyroid hormone concentrations and body condition score.Greenland sled dogs (> 6 months old) located in Greenland were included in the study. Information regarding age, sex, management, body condition score (BCS) , muscle g in 10 min) within 4 h of collection. Whole blood, serum, and EDTA plasma were stored in cryotubes and frozen upright at − 20 °C until further analyses at Veterinary Diagnostic Laboratory, University of Copenhagen, Denmark.Blood was collected by cephalic venepuncture into serum and EDTA plastic vacutainers, respectively . Fasting prior to blood sampling was not imperative. Analysis of T4, fT4, and TSH do not require dogs to be fasted prior to sample collection, according to the manufacturer. Blood samples were kept at 0–10 °C and was centrifuged  were performed using an Immulite 2000 . Concentrations of T4 and fT4 were determined using a solid-phase, chemiluminescent competitive immunoassay  and TSH was determined by a solid-phase, enzyme-labelled chemiluminescent immunometric assay . Biochemical analysis including C-reactive protein (CRP) were performed using an Advia 1800 . All analyses were performed following the manufacturer’s instructions. For all analytes, the analyses were performed within one analytical run.http://stat.ethz.ch/CRAN/). Following the American Society for Veterinary Clinical Pathology (ASVCP) guidelines , fT4, and TSH  . One outlier for TSH was excluded. According to histograms and the D’Agostino & Pearson omnibus normality test, the distribution of the reference sample group was skewed for T4 and TSH, but not for fT4.Lower detection limits of the Immulite 2000, were 6.44 nmol/L for T4 and 3.86 pmol/L for fT4. 15 dogs  presented a concentration below the analytical detection limit for T4, and three dogs (2% of the reference sample group) were below the detection limit for fT4..014 ng/mL/year, 95% CI ]  during summer (median = 18.80 nmol/L) vs. winter (median = 13.25 nmol/L). There were no significant differences in concentrations of fT4 and TSH across season  in Sirius dogs (median = 0.096 ng/mL) vs. civilian (median = 0.206 ng/mL) Fig. .Significant differences for summer observations were found between Sirius and civilian GSD for T4 , fT4 , and TSH . Furthermore, significant differences were observed for winter observations between Sirius and civilian GSD for T4  and TSH , but not for fT4 ], and negatively correlated with TSH, with a slope of − 0.043 ng/mL per BCS-point [95% CI ] , was observed for Sirius GSD. This finding was significant at a < 0.1 significance level, but not at a conventional < 0.05 significance level. Surprisingly, a negative correlation with a slope of − 0.038 nmol/L TSH per BCS-point (P = 0.03) was found among civilian GSD , alkaline phosphatase (ALP), alanine aminotransferase (ALT)], high creatinine kinase (CK) and changes in electrolytes or a mix of these changes.Reference intervals were established for serum concentrations of T4, fT4, and TSH in healthy GSD. Lower concentrations of T4 and fT4 were demonstrated in GSD compared to standard reference intervals based on other dog breeds. For all thyroid hormones, the width of 90% CI of lower reference limits were smaller than 20% of the width of respective reference intervals, as is recommended . HoweverA previous study by Hegstad-Davies et al.  of sevenboth corresponding clinical signs and thyroid levels. Typical clinical signs of hypothyroidism may nevertheless be difficult to detect in GSD; for instance, obesity is unlikely to occur, despite pathological hypothyroidism, due to generally restricted feeding regimens. Typical clinical signs of hypothyroidism  were likewise rarely observed or reported by owners among GSD in this study. Our physical examinations did detect clinical signs attributable to hypothyroidism, i.e., poor hair coat, seizures, and dermal hyperpigmentation, but these are not specific to hypothyroidism. Only a few dogs, displayed these symptoms accompanied by low T4 levels . Altogether, evaluation of clinical signs in GSD is challenging, largely because of the type of management and the extreme environment in Greenland, and not least because of lack of veterinary assistance.In case of thyroid gland disease, it is important to be able to evaluate hormone levels against reference intervals, but a clinical diagnosis of hypothyroidism is based on Relatively speaking, generally low thyroid levels have also been reported in other sled dog breeds , 13. ThiThe need for partitioning of dogs into distinct groups was assessed according to the ASVCP reference interval guidelines , i.e. paThe sex-associated variation in T4 and fT4 concentrations is consistent with a previous study including a variety of breeds . FurtherA positive correlation between age and concentrations of TSH, T4 and fT4 have previously been reported in dogs , in our Median BCS was 3/9 and 5/9 for civilian and Sirius GSD, respectively. This difference is most likely due to different feeding regimens, de-worming regimens and not least diverging views on optimal BCS for GSD . Furthermore, Sirius GSD have access to veterinary consultancy and care, unlike the majority of civilian GSD. BCS was obtained by different people throughout the study but general protocols were followed to minimize potential biases .Overall, a positive correlation was found between BCS and T4, and a negative correlation was found between BCS and TSH. No such relationship has been identified in the literature before, and it is argued that the results should be interpreted with care as there may be other factors affecting BCS and thyroid hormone levels simultaneously. Importantly, type of management was shown to affect levels of TSH considerably, i.e., focusing on Sirius GSD, an increasing trend (P < 0.1) for TSH with increasing BCS was demonstrated, which is opposite that of the overall significant result. A significantly higher BCS in Sirius GSD compared to civilian GSD possibly contributes to this discrepancy, but this assumption cannot be confirmed from our available data alone. Further work examining why civilian GSD differ from Sirius GSD regarding conflicting correlations between BCS and TSH is prudent and may help us understand specific coping mechanisms of these dogs, as well as the role and behaviour of thyroid hormones in general.Overall, this study showed significantly higher concentration levels of T4 in samples collected during summer compared to winter samples. Previous studies have observed fluctuations in thyroid hormone concentration levels throughout the year for other breeds , 30. No en route 6 h or 30 km per day). This makes it difficult to partition groups of dogs into different activity levels and the strong connection between season and activity level makes it challenging to conclude whether the estimated correlation is due to variation in season , activity levels, or a combination of both. Optimally, effect of activity level should be measured for samples obtained in the same season.Estimating effect of season is challenging for GSD in Greenland as their activity level is also highly dependent on season. GSD are largely confined to a chain during summer and work (and otherwise chained) during winter. Comparisons of activity levels during winter were difficult due to variable types and intensity of activities among civilian dogs . Activity level of Sirius GSD is generally very homogenous. They are used for long-range patrols up to 4500 km, pulling 300–550 kg, over 6 months  test was not conducted due to financial limitations. Elevated TgAA concentrations are rare (< 1%) but occur. TgAA artificially elevates T4 measurements even when the dog may in fact be hypothyroid . MoreoveReference intervals for the thyroid hormones T4, fT4 and TSH have been established and the effect of sex, age, season, management, and BCS have been assessed. These new data can be used for evaluating thyroid hormone profiles from e.g., clinically diseased GSD in the future. Throughout this study, results showed a difference between civilian and Sirius GSD but separate RI were not established based on guideline recommendations , moreoveOur results can inform veterinarians, caregivers, and owners on improved management of GSD, including assessment of thyroid-related diseases and/or conditions. Further studies are however needed focusing on susceptibility or potential resistance, including potential breed-specific clinical signs, of GSD to thyroid-related disease, hypothyroidism in particular. Overall, our results also shed light upon the function and behaviour of thyroid hormones in a mammalian species, in an extreme environment, and under multiple stressors.Additional file 1. Correlation between body condition score (BCS) and T4, fT4, and TSH concentrations in healthy Greenland sled dogs. Lines represent linear correlation between age and thyroid hormones. Shaded areas denote 95% confidence intervals. T4 and TSH were found to be correlated with BCS with slopes of 1.46 nmol/L per BCS-point and − 0.043 ng/mL per BCS-point, respectively. There was no correlation between BCS and fT4. a) T4, b) fT4, c) TSH.Additional file 2. Correlation between body condition score (BCS) and thyroid stimulating hormone (TSH) concentrations in healthy Greenland sled dogs measured by type of management (Sirius (a), civilian (b)). Lines represent linear correlation between age and thyroid hormones. Shaded areas denote 95% confidence intervals. Civilian, referring to dogs owned by civilian Greenlanders; Sirius, referring to dogs owned by the special Danish military unit: Sled dog Patrol Sirius. TSH was found to be correlated with BCS with slopes of 0.015 nmol/L per BCS-point and − 0.038 ng/mL per BCS-point for Sirius GSD and civilian GSD, respectively.


Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration . In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV. Bromoviridae family with tripartite genome. RNA1 and RNA2 are associated with virus replication and encode 1a and 2a proteins, respectively while 5′ part of RNA3 encode movement protein (MP). Coding sequence of coat protein (CP) is located on subgenomic RNA4 [Brome mosaic virus (BMV) is typical member of the Bromovirus genus in the mic RNA4 . Howevermic RNA4 . Nonethemic RNA4 . The resmic RNA4 .Solemoviridae family. The virus has positive single-stranded polycistronic RNA genome of 4082 nt in size, encapsidated in a spherical particles of about 30 nm in diameter [ulema melanopus and O. gallaeciana) in non-persistent manner. CfMV induces lethal, severe or mild leaf mottle and stunting of infected plants [Dactylis glomerata but it was revealed also in wheat and it could be artificial transmitted to other cereals [The second virus, a new potential threat for cereal crops, is cocksfoot mottle virus (CfMV). This is single stranded RNA virus belonging to the Sobemovirus genus in the diameter . The 5′ diameter . The virdiameter . In Poladiameter . The vird plants . It has  cereals , 13. As  cereals –19. A mu cereals , 21. Forhttp://bioinfo.ut.ee/primer3-0.4.0/) [2 to 4.5 × 10–5 ng/µL of total RNA) of BMV-ML1, sample co-infected with BMV and CfMV, were used as template for reactions. The detection limit was verified by electrophoresis as described above.The studies were conducted with total RNA isolated from barley cv. Bażant infected with several BMV and CfMV-P1 isolates collected in 2013–2020 from different locations in Wielkopolska, Dolny Śląsk and Małopolska regions of Poland (Table -0.4.0/)  based on-0.4.0/)  and CfMV-0.4.0/)  primer p-0.4.0/)  and thenBased on published results , 13 BMV


Geminiviruses are circular single-stranded DNA plant viruses encapsidated into geminate virion particles, which infect many crops and vegetables and, hence, represent significant agricultural constraints worldwide. To maintain their broad-range host spectrum and establish productive infection, the geminiviruses must circumvent a potent plant antiviral immune system, which consists of a multilayered perception system represented by RNA interference sensors and effectors, pattern recognition receptors (PRR), and resistance (R) proteins. This recognition system leads to the activation of conserved defense responses that protect plants against different co-existing viral and nonviral pathogens in nature. Furthermore, a specific antiviral cell surface receptor signaling is activated at the onset of geminivirus infection to suppress global translation. This review highlighted these layers of virus perception and host defenses and the mechanisms developed by geminiviruses to overcome the plant antiviral immunity mechanisms. Geminiviridae family) . Wh. Wh1 (ME pathway . LikewisL1) gene .S-adenosyl methionine (SAM), the universal donor of methyl groups in methylation reactions [N. benthamiana [C4/AC4 is also part of the virus arsenal against antiviral RNA silencing by interacting and sequestering dsRNA precursors from DCL cleavage and siRNA from RISC loading ,72. C4 ieactions . Therefoeactions . Additioeactions ,75. The eactions . Mungbeathamiana .AV2 and V2 also suppress antiviral RNA silencing in the amplification phase and interfere with host methylation activities, downstream events of TGS ,78,79. TS-adenosyl-L-homocysteine hydrolase (SAHH) activity, a methyl cycle enzyme required for TGS, interfering with the host methylation–mediated virus defense pathway [Geminiviruses are often associated with alpha- and betasatellites, which encode efficient suppressors of viral genome methylation in infected plants and PTGS ,85. The  pathway ,87. ThisIn plant–virus interactions, the host immune system often recognizes viral components or effectors to activate defenses . The plaN-myristoylated C4 protein is de-myristoylated and translocated to the chloroplast. Inside the chloroplast, non-myristoylated C4 interacts with the thylakoid membrane-bound plant calcium-sensing receptor (CAS) and hampers SA biosynthesis and mediated defenses [Arabidopsis thaliana and N. benthamiana , and inhibits early PTI responses  Figure . TYLCV Cdefenses . Finallythamiana  [96,97,9The second level of microbe perception by the host plant is mediated by intracellular immune receptors (R proteins), which recognize pathogen avirulent effectors to activate ETI ,93,99. EArabidopsis, CabLCV infection induces HR- and senescence-related genes without developing a visible cell death phenotype [Compelling evidence that plants deploy ETI against geminiviruses results from infectivity assays demonstrating that geminiviruses induce and suppress HR and ETI-like responses. In henotype . More sphenotype ,103,104.henotype ,106. Likhenotype . In conthenotype . C4 inteArabidopsis, this subfamily of RLKs encompasses14 proteins that harbor four complete LRRs (with 24 residues) and a fifth incomplete LRR (with 16 residues) arranged in a single continuous block within the N-terminal extracellular domain, a single-pass transmembrane segment, and a highly conserved serine/threonine kinase domain at the C-terminal cytosolic side [As obligate intracellular parasites, viruses interact extensively with the host cell functions to complete their life cycle. Independent of the repertoire of viral genome–encoded proteins, all viruses are dependent on the host protein synthesis machinery to translate viral messenger RNAs (mRNAs) ,109,110.lic side . As a colic side . Accordilic side . The pholic side ,118. Liklic side . Like BABegomovirus-derived nucleic acids act as viral a PAMP to activate NIK1 via phosphorylation on Thr-474  and tomato severe rugose virus (ToSRV) [Arabidopsis causes stunted growth, although, in tomato plants, the transgenic lines are phenotypically indistinguishable from the untransformed lines under greenhouse standard conditions [The NSP binding site on NIK1 was mapped to an 80-amino acid stretch within the activation loop, overlapping the essential Thr-474 for activation . Therefo (ToSRV) ,113. Thi (ToSRV) . Howevernditions ,113. ThiPseudomonas infection, the bacterial PAMP flagellin induces FLS2 and BAK1 oligomerization and transphosphorylation, forming an active immune complex to initiate PTI signaling. The active FLS2–BAK1 immune complex, in turn, phosphorylates NIK1 on Thr-474, strengthening the NIK1 interaction with FLS2–BAK1 and concurrently activating the NIK1-mediated antiviral signaling. Significantly, bacterial flagellin (PAMP)-induced phosphorylation of NIK1 requires both FLS2 PRR and BAK1 coreceptor, indicating that NIK1 acts downstream of receptor signaling [Recent studies have shown that NIK1 not only plays an essential role in the translational control of antiviral immunity but regulates bacterial PTI negatively . NIK1 inignaling . These rignaling . FurtherIn RNA-silencing-mediated antiviral immunity, two mechanisms are fundamental to protect plants against invading nucleic acids from viruses, PTGS and TGS. PTGS is used as a defense mechanism against RNA and DNA viruses, whereas TGS targets virus DNA. PTGS acts at the post-transcriptional level by directing mRNA targets to degradation or translational suppression. In contrast, TGS represses transcription of target loci by controlling the chromatin methylation status and heterochromatin formation. Geminiviruses have evolved different suppressing strategies of host RNA-silencing-derived antiviral defenses. The geminiviral suppressors of RNA silencing can interfere with PTGS, TGS, and downstream events of TGS. They also act by activating or inducing the expression of endogenous suppressors. The immune-suppressing activities of geminiviruses may account for their broad host range and compromise the success of siRNA-based strategies for engineering host resistance.The interactions of the plant’s innate immune system with geminiviruses are far less understood. Nevertheless, growing evidence has demonstrated that the classical plant PTI limits geminivirus infection similarly to nonviral pathogens. Geminiviral proteins have been shown to target PTI components to both induce and suppress PTI-like responses. Furthermore, begomoviruses-derived nucleic acids act as viral PAMPs and activate the transmembrane receptor NIK1, which shares with PTI coreceptors conserved regulatory mechanisms for activation. However, geminiviral PAMPs and their cognate PRRs have yet to be identified, and hence, the mechanism of geminiviral PTI activation remains unknown.Likewise, plants may deploy ETI as part of the defense arsenal to combat geminiviruses. Accordingly, geminivirus infection induces ETI-like responses, including cell death, HR, ROS, SA accumulation in resistant genotypes, and at least one isolated resistant gene to TYLCV encodes an NLR protein, although the cognate geminiviral effector (Avr gene) is missing. The current studies designed to uncover geminiviral PTI- and ETI-suppressing functions are limited as they mostly target conserved features between the antiviral and anti-nonviral pathogen immunity. The discovery of geminiviral PAMPs and their cognate PRRs, intracellular R proteins, and the matching geminiviral effectors will potentially shed light on the mechanism of antiviral PTI and ETI activation uncovering specific and conserved features and their specific roles in resistance against viruses.


A stretching intervention program is performed to maintain and improve range of motion (ROM) in sports and rehabilitation settings. However, there is no consensus on the effects of stretching programs on muscle stiffness, likely due to short stretching durations used in each session. Therefore, a longer stretching exercise session may be required to decrease muscle stiffness in the long-term. Moreover, until now, the retention effect (detraining) of such an intervention program is not clear yet. The purpose of this study was to investigate the training (5-week) and detraining effects (5-week) of a high-volume stretching intervention on ankle dorsiflexion ROM (DF ROM) and medial gastrocnemius muscle stiffness. Fifteen males participated in this study and the plantarflexors of the dominant limb were evaluated. Static stretching intervention was performed using a stretching board for 1,800 s at 2 days per week for 5 weeks. DF ROM was assessed, and muscle stiffness was calculated from passive torque and muscle elongation during passive dorsiflexion test. The results showed significant changes in DF ROM and muscle stiffness after the stretching intervention program, but the values returned to baseline after the detraining period. Our results indicate that high-volume stretching intervention  may be beneficial for DF ROM and muscle stiffness, but the training effects are dismissed after a detraining period with the same duration of the intervention. Joint flexibility, known as range of motion (ROM), and muscle stiffness could be contributors to movements of daily living and sports performance. Previous studies have pointed out that a poor ROM  and incrno statistical changes were observed for the muscle-tendon mechanical properties (including the muscle stiffness) after a chronic stretching intervention. However, both the short duration of interventions and the low volumes of stretching performed per week may be responsible for not allowing sufficient stimuli for muscle adaptations. To the best of our knowledge, the study that implemented the longest stretching duration per week was conducted by Many previous studies showed that ROM and muscle stiffness were significantly improved immediately after static stretching intervention . On the Another interesting issue could be to explore to what extent there is any retention effect after a stretching program in sports and rehabilitation settings. According to literature, also concerning the effect of detraining, findings are controversial. Therefore, this study aimed to investigate the effects of a stretching intervention on muscle stiffness using a longer stretching duration per week than in any other studies . Interestingly, two mechanisms have been proposed to explain the changes in ROM after a stretching intervention program: the mechanical theory, which is based on changes in the mechanical properties of the muscle-tendon unit, such as a decrease in muscle stiffness; and the sensory theory, which is related to changes in tolerated passive torque, also known as stretch tolerance . In ordeIn this study, the participants visited the laboratory >72 h prior to the baseline measurement for a familiarization session. The ankle dorsiflexion ROM (DF ROM), passive torque, and ultrasound measurements were determined before (PRE) and after (POST) the 5-week of static stretching (SS) program in both the dominant and non-dominant legs. In addition, all variables were measured after a 5-week detraining period (De-Tr). After PRE assessment, we defined the dominant side  as the intervention side (SS side) and the non-dominant side as the control side (CON side) to minimize between-group variability due to personal factors such as exercise and activity patterns . To contFifteen males participated in this study . Inclusion criteria were as follows: no regular resistance training within the past 6 months, no neuromuscular disease, and no history of orthopedic disease. None of the participants was competitive athletes or engaged in regular resistance training or stretching programs for the lower limbs. All participants were fully informed about the procedures and purpose of this study, and they provided written informed consent. This study was approved by the Ethics Committee of the Niigata University of Health and Welfare (#18305), Niigata, Japan.The participants sat on a dynamometer chair with a 0° knee angle  and adjustable belts fixed over the trunk and pelvis . The participants were reclined  to prevent tension at the back of the knee. The footplate of the dynamometer was passively and isokinetically dorsiflexed at a speed of 5°/second from the neutral anatomical position to the dorsiflexion angle just before subjects started to feel discomfort or pain . Before After familiarization trials, the subjects stopped the dynamometer by activating a hand-held safety remote button when they started to feel discomfort or pain, and the angle just before this point was defined as DF ROM. The measurement was performed twice, and the average value was used for future analysis. In addition, passive torque at DF ROM was defined as the stretch tolerance .Throughout the passive dorsiflexion test, participants were requested to relax completely and not offer any voluntary contraction. We confirmed that there was no voluntary contraction of the medial gastrocnemius (MG) by monitoring muscle activity on the surface electromyography . Surface electrodes  were placed on the muscle belly of the MG. After the passive dorsiflexion assessment, muscle activity of the MG was measured during a 3 s maximal voluntary isometric contraction (MVIC), and we confirmed that all data were collected during a relaxed state, i.e., did not show muscle activity exceeding 5% of MVIC . The musA B-mode ultrasound imaging device  and an 8-MHz linear array probe were used to assess elongation of the MG during the passive dorsiflexion test. We obtained the longitudinal ultrasound images of the MG, which were synchronized to the passive torque and joint angle outputs. The ultrasound probe was placed on the distal MG, near to the muscle-tendon junction (MTJ). The ultrasound probe was secured with a standard orthopedic stocking to prevent movement of the probe during the passive dorsiflexion test. In POST and De-Tr measurements, ultrasound images were obtained and compared to images acquired at PRE to assure the same position of measurement. An acoustically reflective marker was placed on the skin under the ultrasound probe proximal to the MTJ of MG to verify that the probe remained stable during the measurement . UltrasoThe muscle force of the MG was estimated by multiplying the measured passive torque by the relative contribution of the physiological cross-sectional area (18%) of the MG within the plantarflexor muscles  and thenThe SS program was performed only on the intervention side with a stretching board . Participants stood erect with one foot on the stretching board and the other foot on its edge and both arms against a wall in front of the body for balance . StretchThe test-retest reliability of the measurement for DF ROM, passive torque at DF ROM, and muscle stiffness was determined by a coefficient variation (CV) and the intraclass correlation coefficient (ICC) using seven healthy males . The CVs of the measurements for DF ROM, passive torque at DF ROM, and muscle stiffness were 4.7 ± 5.2, 7.2 ± 6.3, and 3.6 ± 3.1%, respectively, and the ICCs of the measurements were 0.973, 0.868, and 0.881, respectively.t-tests. For all variables, a two-way repeated measure ANOVA was performed using the factors of time (PRE vs. POST vs. De-Tr) and side (SS side vs. CON side) to determine the interaction and main effects. As a post hoc test, we used the Bonferroni multiple comparison test to determine significant differences in time on each side. The Spearman’s rank correlation coefficients (rs) were computed to quantify the relationship between PRE and POST measurements of DF ROM, passive torque at DF ROM, and muscle stiffness on the SS side. Statistical significance was defined as p < 0.05 and the descriptive data were reported as means ± SD.IBM SPSS Statistics version 24.0  was used to conduct statistical analyses. Between-side differences in DF ROM, passive torque at DF ROM, and muscle stiffness at PRE assessment values were determined using paired t-test showed that there were no significant differences in any variables between the SS and CON sides in PRE assessments. The repeated two-way ANOVA indicated significant interaction effects for DF ROM , passive torque at DF ROM , and muscle stiffness . On the SS side, the DF ROM and passive torque at DF ROM measurements at POST were significantly increased compared to PRE measurements (p < 0.05), whereas there were no significant differences between PRE and De-Tr or POST and De-Tr measurements. Similarly, on SS side, the muscle stiffness value at POST was significantly decreased from the PRE value, and there were no significant differences between PRE and De-Tr or POST and De-Tr values. There were no significant changes in any of the variables in CON side.The results of all variables were presented in rs = 0.792, p < 0.01; rs = −0.366, p = 0.179; The relationships between changes in DF ROM, passive torque at DF ROM, and muscle stiffness were shown in In this study, we investigated the effect of a 5-week high-volume stretching intervention program with 3,600 s of SS per week as well as the sustained effects after a 5-week detraining period. To the best of our knowledge, our study not only implemented the longest stretching duration per week of any study to date, but it was also the first to examine the detraining effect on passive muscle stiffness. Our results showed that after the 5-week intervention, DF ROM and passive torque at DF ROM were significantly increased, and muscle stiffness was significantly decreased. Contrary to our hypothesis, the increased DF ROM and decreased muscle stiffness returned to their baseline values after the 5-week detraining period.Our results showing that DF ROM and passive torque at DF ROM increased after our 5-week stretching intervention program were consistent with findings in previous studies . HoweverThe results of this study also revealed that muscle stiffness was decreased after the 5-week high-volume stretching intervention program. While the meta-analysis conducted by Contrary to our hypothesis, the decrease in muscle stiffness after the stretching intervention program was returned to baseline value after a 5-week detraining period, which was consistent with the results of rs = 0.792, p < 0.01; rs = −0.366, p = 0.179; As stated previously, the two mechanisms proposed to explain the changes in ROM after a stretching intervention program are the mechanical theory and the sensory theory . Our resBased on previous studies that have pointed out that poor ROM  and incrOur study showed that DF ROM and passive torque at DF ROM were significantly increased after a 5-week high-volume stretching intervention program but returned to baseline after a 5-week detraining period. Similarly, our results showed that muscle stiffness was significantly decreased after the stretching intervention program, but also returned to baseline after a 5-week detraining period. Our results suggest that the increase in ROM after the stretching intervention program could be attributed to the change in passive torque at DF ROM, i.e., the stretch tolerance, which supports the sensory theory.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Niigata University of Health and Welfare, Niigata, Japan. The patients/participants provided their written informed consent to participate in this study.MN contributed to the study design and data collection and drafted and made the critical revisions to the manuscript. KY, SS, RK, and RY contributed to the data collection and made the critical revisions to the manuscript. TF, JN, and AK contributed to the study design and data analysis and made the critical revisions to the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A higher alpha angle has been proposed to correlate with lower hip range of motion, but the association in people with longstanding hip and groin pain is currently unclear. The aims were to: (1) assess the association between range of motion and alpha angle in patients with longstanding hip and groin pain; (2) examine if a cut-off value in range of motion variables could identify patients with an alpha angle above or below 60°.Seventy-two participants were consecutively recruited from an orthopaedic department after referral for hip- and groin-related pain. Passive hip range of motion was measured in flexion, internal rotation with 90° hip flexion, internal rotation in neutral hip position, external rotation with 90° hip flexion, and abduction. The alpha angle was calculated from a frog-leg lateral radiograph. Linear regression examined the association between range of motion and alpha angle, and an ROC-curve analysis was performed to identify the sensitivity and specificity of range of motion cut-offs.Lower range of motion in internal rotation in flexion, external rotation, and abduction were associated with higher alpha angle. Internal rotation of 27° or less displayed good sensitivity (81%) and specificity (85%) to detect an alpha angle above 60°, while a cut-off of 41° in external rotation and 27° in abduction showed a sensitivity of 72% and specificity of 50% and 60%, respectively.Less internal rotation in flexion, external rotation, and abduction are associated with a greater alpha angle in a cohort of people with longstanding hip and groin pain. A cut-off of 27° in internal rotation has good sensitivity and specificity to identify people with an alpha angle above or below 60° and have the potential to be used in the clinical setting to identify patients that require further imaging, or that are unlikely to have cam morphology.II. Longstanding hip and groin pain is a common problem in young to middle-aged adults, leading to reduced participation in sports and activities of daily living, and reduced quality of life , 33. SocThe hip joint is recognised as a source of groin pain, and femoroacetabular impingement syndrome (FAIS) is a common source of hip-related pain in a younger and physically active population . Cam-typCam morphology has been proposed to affect hip ROM, primarily in positions of hip flexion and internal rotation . SeveralThe diagnosis of FAIS is based on clinical findings, patient-reported symptoms and radiographic imaging . HoweverThe aim of this study was to examine the association between passive hip range of motion and alpha angle in patients with longstanding hip and groin pain. A secondary aim was to examine whether a cut-off value in ROM variables could identify patients with alpha angles above or below the clinical cut-off of 60°. The hypothesis was that lower range of motion would be associated with a higher alpha angle.http://www.strobe-statement.org). Approval for the study was granted by the Regional Ethical Review Board in Lund (reg. no 2014/12) and the participants signed an informed consent form.This cross-sectional study was reported in accordance with the Strengthening of Observational Studies in Epidemiology (STROBE) statement (n = 156) were screened for eligibility by the orthopaedic surgeon in charge of hip arthroscopy. Inclusion criteria were: age 18–55 years; unilateral or bilateral hip and/or groin pain > 3 months. Patients with verified moderate or severe osteoarthritis or any other musculoskeletal comorbidities overriding the hip and groin pain, or limitations preventing testing of ROM, were excluded as described  showed excellent intra-rater reliability (i.e. ICC > 0.8) for FLEX [ICC 0.839 (95% CI 0.643–0.933)], IRF [ICC 0.868 (95% CI 0.685–0.947)], IRN [ICC 0.941 (95% CI 0.859–0.976)], and ERF [ICC 0.842 (96% CI 0.641–0.934)]. ABD showed moderate intra-rater reliability (ICC > 0.6) [ICC 0.635 (95% CI 0.193–0.848)]. Passive extension was not included in the analyses due to poor intra-rater reliability (ICC < 0.2) [ICC 0.177 (95% CI − 0.293 to 0.571)]. The SEM was calculated for FLEX (4.3°), IRN (2.6°), IRF (3.6°), ERF (2.0°), and ABD (3.1°).Excellent inter- and intra-rater reliability have previously been reported for hip ROM using digital inclinometry in healthy populations  and in pSPSS version 25 (IBM Corp) was used for all statistical analysis. A linear regression model was used with alpha angle as the dependent variable and ROM as independent variables. An initial univariate linear regression was conducted for each ROM variable, and sex was later added to the model due to the uneven distribution of men in the high and low alpha angle groups. BMI also differed in the two groups, and age can influence hip ROM [t tests and Fisher’s exact test were performed for numerical and categorical data, respectively, to determine between group differences in age, BMI, sex, and HAGOS scores.Student’s Sensitivity and specificity were calculated by performing a ROC-curve analysis with the clinical cut-off at a 60° alpha angle and the ROM variables considered clinically and statistically relevant. The area under the curve (AUC) was determined to establish whether any ROM variables were suitable for predicting the alpha angle, with a value > 0.05 signifying predictive ability better than chance and a value of 1 indicating perfect predictive ability. The ROM threshold with the highest sensitivity and specificity to detect participants above or below the alpha angle cut-off was selected.n = 72 was considered sufficient, using the general rule of thumb of a minimum of 10 subjects per variable. Alpha was set at 0.05.As this was a secondary analysis of the data sample, no a priori power calculation was conducted. As only two predictors (ROM variable and sex) was used in the models, the sample of There were significant differences in ROM in people with alpha angles above and below 60°, with the patients with higher alpha angles presenting with less ROM Table . End ranAll ROM variables entered in the initial regression analysis were statistically significant. When adjusted for sex, FLEX and IRN displayed no significant predictive ability. IRF, ERF, and ABD predicted a higher alpha angle, with a 10° lower ROM predicting a higher alpha angle by 5.6° (95% CI 2.1°–9.2°) in IRF, 4.5° (95% CI 0.9°–8.0°) in ERF, and 5.4° (95% CI 0.6°–10.1°) in ABD, when adjusted for sex Table .Table 3RDue to their predictive ability in the linear regression, IRF, ERF, and ABD were further analysed with a ROC-curve analysis to explore their ability to detect an alpha angle > 60°. IRF had an AUC of 0.896 Fig. , ERF 0.6The most important findings of the present study were that (1) less ROM in IRF was associated with a greater alpha angle in a sample of patients with longstanding hip and groin pain, and (2) that a 27° cut-off of IRF had good sensitivity and specificity to classify participants above or below the alpha angle threshold of 60°. Also, less IRF, ERF, and ABD were associated with a greater alpha angle, where 10° less ROM corresponded to an approximately 5° higher alpha angle.p values to describe the strength of the association, did not report correlation coefficients or beta-values, and the correlation analysis was not adjusted for sex [A clinical assessment tool to help identify the presence of cam morphology could be of benefit in the diagnosis of FAIS, and ROM assessment is a low-cost and low-risk assessment that is routinely performed in this patient population. The association of ROM and the alpha angle found in the current study is in line with previous research with similar methodology. In one study, which used computed tomography to model and simulate ROM in people with FAIS, a 1° greater alpha angle corresponded to 0.46° less internal rotation in 90° hip flexion . The aut for sex . In the  for sex , 22, 27.In the ROC-curve analysis, IRF displayed large AUC and good sensitivity and specificity to identify patients above and below the alpha angle threshold. A study by Kapron et al. performed a ROC-curve analysis between ROM and cam morphology on asymptomatic male American football players, defining a cam as > 50° alpha angle . The autThere may be several reasons for limited ROM in an individual with longstanding hip and groin pain, such as sex, morphological variations, and perceived pain. Females generally have greater ROM than males , and malThe ROM assessment method used in the current study showed good to excellent intra-rater reliability and an SEM of 2.0°–4.3° for the different directions. These findings are similar to previous research , 38. BotIn clinical practice, ROM assessment is often conducted by visual estimation and perception of joint end feel. A recent study by Pålsson et al. reported moderate to high specificity for identifying patients with FAIS using a dichotomous  evaluation of hip ROM in flexion, rotation, and abduction . The finIn the current study, a frog-leg lateral projection and an alpha angle cut-off of 60° were used. Heterogeneity in the imaging projections and cut-offs in studies reporting on ROM in FAIS limit the possibility to compare the results of the present study to the results of previous research. The two published systematic reviews on physical impairments related to FAIS included studies of people with both cam and pincer FAIS, as well as people with isolated chondrolabral pathology , 10. AlsTo our knowledge, this is the first study exploring the association of cam morphology and hip ROM in patients with longstanding hip and groin pain. It is also the first to examine whether a cut-off in ROM has merit as a test to identify an alpha angle above 60°. However, this study is not without limitations. The results should be interpreted with caution due to the exploratory nature of this study and the wide confidence intervals in the regression models. Although there was a significant association between ROM and alpha angles in this study, other factors that may contribute to ROM limitations, such as femoral torsion, acetabular version, pincer morphology, and capsular thickness, were not measured. Another limitation is the disproportionate number of men compared to women in the high alpha angle group. While steps were taken to minimise the effect of this imbalance by checking for multicollinearity and adjusting for sex in the linear regressions, the differences in sex may affect the results of our ROC-curve analysis by shifting the cut-off and over- or underestimating the diagnostic accuracy.In the clinical setting, differential diagnosis of hip and groin pain can be a challenge. So far, ROM in patients with FAIS have primarily been compared to that of asymptomatic controls. The finding of the current study that greater cam morphology is associated with less ROM in a cohort of people with symptoms indicates the potential value of ROM testing to identify underlying morphology and informing differential diagnosis of hip and groin pain, especially in situations where imaging is not readily available.Specifically, passive internal rotation of 27° or less may identify a patient likely to have cam morphology, and can help physical therapists or general practitioners deciding who to refer for further investigation and/or orthopaedic consultation.In the current study, less internal rotation, hip external rotation, and abduction were associated with a higher alpha angle in a population with longstanding hip and groin pain. IRF had the best sensitivity and specificity to identify cam morphology.


Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses  in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus  and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.Phleboviruses (genus  Phlebovirus, family Phenuiviridae, include mosquito-, sand fly-, and tick-borne pathogens, and these viruses have three negative sense RNA segments (4). The first phlebovirus identified in the United States, Rio Grande virus (RGV), was isolated from southern plains woodrats (Neotoma micropus) in Texas [Lutzomyia anthophora, in the laboratory [Equus cabalus) and sheep (Ovis aries), were found to be antibody-positive, indicating prior infection. No antibodies were found in 278 human serum samples tested, and sera from native wild ruminants were not tested [Neotoma micropus, thought to be the natural host, ranges across North America from southeastern Colorado, east to central Kansas, west to the Arizona border, and south to the eastern coast of Mexico in northern Veracruz [L. anthophora, a sand fly found in or near rodent and lagomorph nests in southern Texas and northern Mexico [Viruses within the genus in Texas ,2, and wboratory . Some dot tested . Rio Grasegments . The firVeracruz . The pron Mexico .Odocoileus virginianus) were found to be susceptible to RVFV infection, potentially serving as a reservoir and amplification host [Rift Valley fever virus (RVFV), an African mosquito-borne phlebovirus, causes new born fatalities and abortions in ruminants, and potentially fatal hemorrhagic disease in humans. This zoonotic virus can be transmitted to humans by direct contact with meat or fluids from infected animals, or from an infected mosquito, and has been identified as a top concern by the CDC and the USDA . Diagnosion host . Due to Phlebovirus was previously reported [Identification of newly discovered viruses was previously based on virus serum neutralization assays which take advantage of cross-reacting antibodies between closely related viruses . It is ireported . The assreported .Misinterpretation of false positive results in diagnostics has the potential for significant consequences. Falsely identifying a foreign animal disease, such as RVFV, is likely to lead to regulatory responses including farm and ranch quarantines, trade restrictions, and elevated public health concern. Our research looked for potential for cross-reactions between RGV and RVFV based on diagnostic reagents used to detect virus antigens or antibodies.Viruses. RGV isolate TBM3-204 was provided by Dr. R. McClain, USDA-APHIS Wildlife Service, National Wildlife Research Center. The attenuated RVFV strain, MP-12, was obtained from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), as previously published [6.41, 106.50, 106.29, and 107.14 tissue culture infectious dosage 50% (TCID50) for Anhanga, Punta Toro, RGV, and MP-12, respectively.Antibodies. Primary anti-RVFV antibodies used in this study included sera from sheep vaccinated with MP-12 [th MP-12 . SecondaImmunocytochemistry. Indirect immunofluorescence testing was applied to noninfected V76 cells, or cells infected with Anhanga virus, Punta Toro virus, RGV, or MP-12 grown on microwell slides at a multiplicity of infection of 0.5. Cells were fixed with acetone when cytopathic effects were first observed, which ranged from 2 days post-infection for MP-12, to 4–5 days post-infection for Anhanga virus, Punta Toro virus, and RGV. Fixed cells were incubated with rabbit anti-RVFV NP protein or sera from MP-12-vaccinated sheep, diluted 1:50 in PBS. Secondary fluorescent labeled antibodies, diluted 1:1000 in PBS, were used to detect infected cells. Nuclei were stained with DAPI  and actin filaments were detected with rhodamine-conjugated phalloidin . Images were captured using an EVOSfl fluorescence microscope .Protein detection by Western blotting. Standard SDS-PAGE methods were used for protein detection [etection ,15. BrieWhole-Genome Sequencing. Whole-genome sequencing (WGS) was performed to verify the identity of the viruses used in this study, and to obtain the WGS of RGV. Briefly, the viruses were grown in 75 cm flasks and frozen at −80 °C when >80% CPE was observed. Cultures were thawed and clarified by centrifugation at 5000× g for 30 min, and virus particles were semi-purified by centrifugation through a 20% sucrose cushion at 105,000× g for 12 h. The pellet was resuspended in 400 μL of PBS and RNA extracted with Trizol reagent (Life Technologies), following the manufacturer’s recommendation. Sequencing was performed at the University of Illinois Keck Center using the Illumina MiSeq  platform. Sequencing reads were assembled using Ray de novo assembly [assembly , and conMeasurement of Fluorescence Intensity. The fluorescent intensity of granules from MP-12- and RGV-infected cells was measured using ImageJ software [t-test, with significance set at p < 0.05.software . Four arImmunofluorescent assays using anti-RVFV antibody reagents were used to determine their potential to also detect RGV-infected cells. Cells infected with the RVFV-attenuated strain, MP-12, stained with rabbit anti-NP primary antibody, exhibited strong fluorescence staining of abundant cytoplasmic granules, as seen in Western blots were used to identify proteins from virus-infected cells. When probed with rabbit anti-NP, bands of the expected size of 27.4–27.8 kD were identified in protein extracts from cells infected with MP-12, RGV, and Anhanga virus . WesternWhole-genome sequencing was performed to verify the identity of viruses used in this study, and to obtain WGS for RGV. Complete segments were obtained for RGV and were placed in GenBank with accession numbers MK503253 (L), MK503254 (M), and MK503255 (S). The identity of MP-12, Anhanga virus, and Punta Toro virus were verified by NCBI nucleotide Blast of complete sequences of all segments, with 99.2–100% identity.Rio Grande virus is the only known phlebovirus endemic to the United States, and the potential for cross-reactivity with RVFV has not previously been reported. The USDA has identified RVFV as the most significant arthropod-borne animal disease threat to U.S. livestock. Due to this concern, the development of diagnostic assays to detect RVFV or anti-RVFV antibodies has been a priority of the USDA, and APHIS National Veterinary Stockpile committee. The risk of RVFV introduction to the Western hemisphere is not limited to the United States. Numerous native phleboviruses are endemic to Central and South America ,19, and We tested the ability of antibodies developed to detect RVFV proteins to also detect proteins from RGV, Anhanga virus, and Punto Toro virus. Cross-reactions with RGV were identified by immunocytochemistry and Western blotting, which also detected Anhanga virus. This is a preliminary study, and further research is needed to verify and determine the extent of antigenic cross-reactions. Limitations of the study include the need to test using methods commonly used for high-throughput diagnostics, such as ELISA and fluorescence microsphere immunoassays. Such assays have been developed for diagnoses of RVFV ,22,23,24The results of this study suggest that some diagnostic tests designed to detect RVFV proteins in U.S. animals could give false positive results in RGV-endemic regions. Rio Grande virus is currently only known to occur in Texas, but the range of the southern plains woodrat includes Colorado, Kansas, New Mexico, and much of Mexico, and the sand fly vector species ranges from Texas to Alberta, Canada . The lik


BioMed Research International has retracted the article titled “Construction and Immunogenicity of DNA Vaccines Encoding Fusion Protein of Porcine IFN-λ 1 and GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus” [e Virus” , the artThe authors responded to explain that different shapes in the same lane may be due to the machine and electrophoresis, however, it was determined that this was not a satisfactory response, and the article is therefore being retracted with the agreement of the Editorial Board.The authors agree to the retraction and the notice.