Symptomatic macromastia causes physical and psychological problems that can lead to restrictions in the patients’ social and working lives and a reduced quality of life. Associated medical treatments also have a considerable impact on health-care costs. Several studies have assessed these costs, but the total disease costs of macromastia have never been evaluated on the basis of real-world data.The data for 76 patients who underwent reduction mammoplasty between 2008 and 2016 were collected using a two-part questionnaire (preoperative and postoperative), as well as the patient files. Topics surveyed, besides demographic data, included physician visits, medical imaging, medical procedures, medical treatments, rehabilitation and convalescent measures, drug intake, medical aids, exercise activity, and sick leave days before surgery, to calculate the costs per year of conservative treatment of symptomatic macromastia.The mean time from start of symptoms to surgery was 11.82 years. The data for this group of patients with symptomatic macromastia show that costs per patient amount to €1677.55 per year. These costs include medical consultation, radiological imaging, medical treatments and procedures, physical therapy and rehabilitation, medication, special brassieres, exercise classes costs for sick leave due to problems with macromastia, and travel expenses.These results show that considerable health-care costs arise due to macromastia with conservative treatment. Overall, macromastia costs €1677.55 per patient/year. In particular, lost productivity due to sick days and the costs of physiotherapy are factors driving the high costs. There is no unique definition of macromastia. There are several different synonyms for it, such as gigantomastia and hyperplasia of the mammary gland. Macromastia is usually defined as excessive growth of the mammary gland beyond the normal range . HoweverThe precise cause of macromastia is still as yet unknown. Several triggers or causes have been reported, such as hormone-related causes, anovulation, and mutations in oncogenes –6. As thBreast hypertrophy or macromastia in patients is often accompanied by several somatic problems such as chronic pain in the chest, shoulders, back, and neck, degenerative changes in the spine, and headaches , 7–20. NDue to the chronic pain, patients often reduce their leisure activities , 16, 25.Although the most effective and normally the only causal treatment is reduction mammoplasty, this type of surgery is often regarded as cosmetic surgery by patients, insurance companies, and even by medical professionals. In addition to weight reduction, conservative treatments are, therefore, often recommended by insurance companies, with approval for insurance coverage of surgical costs depending on previous implementation of such treatments. Treatments that are often recommended include physiotherapy, fango, massage, and acupuncture. Insurance companies may recommend nutritional counseling to promote weight loss, or psychotherapy to strengthen body perception , 28. SpeAll of these treatments create costs. Several studies have compared the costs of conservative treatment with surgical procedures, but, to the best of our knowledge, there has been no research analyzing the precise costs arising for insurance companies and in particular for society as a whole. More detailed information about these costs may be helpful in health-care decision-making. The aim of the present study was, therefore, to fully calculate the costs arising for insurance companies, and society for a patient with macromastia.This is a retrospective study including patients who underwent surgery at the University Breast Center for Franconia (Departments of Gynecology and Plastic Surgery) from 2008 to 2016. The patients were identified from the hospital information system using the operation and procedure (OPS) codes 5–884 ff. (reduction mammoplasty) and ICD-10 diagnosis N62 .Patients with breast cancer, adaptive one-sided reduction mammoplasty, anisomastia, gynecomastia, transsexualism, anomalies of the mammary gland, and previous surgery were excluded Fig. .Fig. 1PaPatient’s characteristics.Medical consultations.Medical imaging procedures.Treatment procedures carried out by physicians.Medical treatments such as medical exercise, rehabilitation, and sports.Medication (including pain relief).Work status and sick days per year.All of the patients were asked to complete a two-part questionnaire. In the first part, they were asked to report on the period before surgery. Part one comprised questions regarding:Information was also requested about reimbursement for these treatments by insurance companies and the availability and costs of objection procedures (when reimbursement is declined). The final item was about quality of life before surgery. All these items targeted known and often-recommended conservative treatments for symptomatic macromastia. The second part of the questionnaire was concerned with the postoperative period, including medical treatments as above, and also quality of life. The present study investigated part one as a complete, real-world disease cost analysis. Approval for the study was provided by the local ethics committee (ref. no. 34_16B). All data were anonymized. Data from the patients’ charts regarding surgery, pathology, and hospital stays were also retrieved.Costs were analyzed per patient and year. The patients were either asked about numbers of procedures per year, from which the annual costs were calculated ; or the patients reported the total number of treatments during the period before surgery and the annual costs were calculated by dividing the total by the mean period between the onset of symptomatic disease and the time of surgery . The figures were averaged for the complete cohort to calculate the mean frequency.Patients were asked if they had consulted their family physician due to symptoms of macromastia. The number of consultations per year, numbers of nonproductive work days, travel expenses, and the duration of the consultations were recorded. This complex of questions was repeated for orthopedics, dermatology, neurology, gynecology, plastic surgery, psychiatry, psychotherapy, and psychology. For consultations with psychotherapists, the type of therapy  had to be specified.Einheitlicher Bewertungsmassstab, EBM) [The basis used for cost accounting in the out-patient setting was the German 2015 Physicians’ Fee Schedule  . ReimburHochschulambulanzpauschale) for gynecology and plastic surgery in Bavaria (€103.30).Consultations at university hospitals were calculated on the basis of the 2015 university out-patient flat rate  and the frequency. Cost accounting was carried out on the basis of the 2015 EBM. The mean costs of the examinations were calculated. Preoperative imaging was not calculated, as this is covered by the disease-related group for in-patient treatment and is not associated with conservative treatment. The various costs for medical imaging are listed in Table The patients were asked to list medical procedures they had undergone and their frequency in relation to macromastia. The patients reported acupuncture, chiropractic, local anesthesia, and osteopathy. These costs were calculated on the basis of the 2015 EBM and are presented in Table Sozialgesetzbuch V) using the Bavarian statutory insurance company  as a reference point.The patients were asked to provide information about the different medical treatments they had received due to macromastia and to report their frequency. They were asked about physiotherapy, massage, fango, osteopathy, and special educational courses for back therapy or nutrition. The costs for medical modalities such as massage, fango, and physiotherapy were calculated on the basis of §125 of the German Social Security Code  and Association of Osteopaths in Germany  [Costs for osteopathy were mainly not covered by insurance. The mean costs for osteopathic treatment were taken as indicated by the Professional Association of German Osteopathic Medical Associations  , 32  [Gesundheitsberichterstattung des Bundes) [In addition, patients were asked about rehabilitation stays and treatments at health resorts associated with the symptoms of macrosomia. Frequencies and types were recorded, as well as travel expenses. The costs for these procedures were calculated on the basis of data from the German Pension Insurance Fund (cherung) . Costs f Bundes) , 35.Patients were asked about medication due to symptoms related to macromastia. The various substances used were divided into specific groups: analgesics, antidepressants/psychopharmaceuticals, relaxants, ointments/dermatologic medication, and other medication. The patients had to provide information about the drug name, dosage, frequency, and duration.Rote Liste 2015 (www.rote-liste.de). The least expensive drug and package size were always used. All prices were adjusted for inflation.Costs were calculated using drug prices from the German pharmacopeia, Frequency was classified into six categories . Days of medication intake were calculated for each drug. The total was multiplied by the daily drug costs for each drug. Costs for all the drugs in the above categories were calculated.Patients were asked if they had a special brassiere due to macromastia. The number of brassieres was reported. Patients were asked to state whether the brassiere was covered by insurance. Costs for the special brassieres were obtained from medical supply stores and were €70 on average. If they were covered by insurance, the patient had to pay 10% of the price.Patients were asked if they had attended a fitness center or gymnastics club due to the symptoms of macromastia. The costs for these activities were reported and monthly costs calculated.Patients were asked about their employment before reduction mammoplasty, stating whether they had missed work due to symptoms of macromastia. The number of working hours lost was reported, as well as the number of sick days. To calculate costs due to absence from work, the gross domestic product (GDP) per gainfully employed person in Germany in 2015 was used, divided by average working days minus 24 days’ vacation . The GDPMicrosoft Excel was used for data collection. Data analysis was performed using IBM SPSS Statistics for Windows, version 24.0 . Normally distributed data are described as means and categorical data as percentage frequencies.n = 93 no answer; n = 3 missing consent). The patients’ mean age at the start of macromastia symptoms was 26.48 years, and their mean body mass index (BMI) was 27.93 kg/m2. The mean period between the start of symptoms and surgery was 11.82 years.A total of 172 patients were identified at the first step. Seventy-six out of 172 questionnaires were returned completed  of the patients had undergone imaging procedures due to symptoms of macromastia. The costs for imaging procedures were calculated by multiplying the number of procedures with the above-calculated prices. Costs for computed tomography (CT), magnetic resonance imaging (MRI), X-rays, and ultrasound are listed in Table .3% of thIn all, 28.9% of the patients underwent medical procedures such as acupuncture or local anesthesia  due to problems related to macromastia. The annual costs for medicine procedures were between €3.97 for acupuncture and €0.05 for chiropractic therapy. Total annual cost was €4.37 per patient. The frequency of procedures and the costs are listed in Table Over half of the patients 57.9%) stated that they had been treated with physiotherapy to obtain relief from chronic pain and musculoskeletal problems resulting from macromastia. A mean of 8.37 visits per year had taken place before surgery, resulting in annual costs of €132.66. Over 60% of the patients had received massage treatment for pain relief, resulting in annual costs of €98.15. Costs for these and other remedial methods are listed in Table 7.9% statOnly 5.3% of the patients reported that they had received orthopedic rehabilitation treatment before surgery, representing €17.86 per patient. Psychosomatic rehabilitation treatment was reported by 2.6%, representing costs of €323.40 per patient. Prevention treatment at a health resort or spa was used by 6.6% of the patients, with costs of €241.04 per patient . The total days of intake per patient between the start of symptoms and surgery were calculated as 1145.38 days. The total costs per patient were €276.67 for the period between the start of symptoms and surgery. The annual cost of analgesics per patient was €23.40.Figures for antacid agents, antidepressant drugs/psychopharmaceuticals, relaxants, and ointments are listed in Table Special brassieres were needed by 51.3% of the patients for pain relief before reduction mammoplasty. On average, each patient had 3.12 special brassieres during the period before surgery. The costs were covered by insurance in only a single case. Costs for brassieres were around €70 per item, resulting in total preoperative costs of €218.40 per patient, or €18.47 per year.More than a third of the patients (34.2%) had attended exercise classes for a mean of 10.37 months before reduction mammoplasty. The average costs were €16.23 per patient and month, representing annual costs of €14.23. Among the patients, 18.4% had been members of a gym for a mean of 14.06 months before surgery, with mean costs of €11.00 per patient and month, representing annual costs of €13.08 per patient.As the questionnaire included questions about travel to and from various relevant activities such as consultations, nutritional courses, and physiotherapy, it was possible to calculate travel expenses. On average, each patient had traveled 130.93 km during the period before surgery. Assuming a cost of €0.30 per kilometer—the standard amount for commuter tax relief in Germany—this represents €3.32 per patient and year.The main cost factor was loss of productivity due to sick leave resulting from symptoms of macromastia. Most of the patients were in employment before surgery (88.2%), with a mean of 28.73 work hours per week. One-third of the patients had a mean of 4.68 sick days per year before surgery. However, the symptoms had become worse over time and their ability to work had been restricted for a mean of 3.83 years before reduction mammoplasty. The total loss of GDP during the period from first symptoms to surgery was equivalent to €5555. Due to the variability of sick days per year in the preoperative period, the total costs for each patient had to be divided by the mean period of symptoms. The annual costs per patient were thus €470.Each stakeholder in the health-care system has a different view of the costs. Society as a whole is the stakeholder with the highest total costs, due to the German health-insurance system and employee protection legislation. Costs for initiating objection proceedings against the health-insurance funds’ medical service departments if they declined to cover the costs for reduction mammoplasty were not included in this calculation, as the aim was to present a cost analysis of conservative treatment for macromastia.The total annual cost per patient amounted to €1677.55. Figure Insurance companies and other cost-bearers cover the costs of medical consultations and imaging procedures. Acupuncture and chiropractic therapy also have to be covered by insurance. Costs for physical therapy, back therapy education, and nutritional education are subsidized at the level of €75 per course twice a year by the insurance funds. Rehabilitation measures and prevention treatments at health resorts, including travel expenses, are mainly covered by health insurance companies or pension insurance funds. Special brassieres were only covered by one health insurance company. Drug costs are covered by the health insurance companies, with the exception of the insurance excess amount that has to be paid by patients themselves.A total of €1161.18 per year and patient have to be covered by insurance companies and other cost-bearers before reduction mammoplasty, representing €13,725 over the period of 11.82 years.There have been several studies on the cost-effectiveness of surgical treatment for macromastia, but there is a lack of data on the complete costs of the condition. This questionnaire survey for the first time provided an opportunity to calculate the costs arising from conservative treatment for macromastia on the basis of real-world data. The limited data previously available are based on single cases and often do not include indirect and nonmedical costs—e.g., those due to loss of productivity . Scholz In contrast, the present study is based on real-world data, averaged for more than 70 patients with macromastia. The socioeconomic costs, including travel expenses and loss of productivity, are also taken into account. Although the costs calculated are lower than existing estimates, they appear to be reasonable. As the costs are averaged over a large cohort and a long period of time, their dynamic is flattened.The annual costs for conservative treatment of macromastia are calculated on the basis of the data from the questionnaires completed by the patients. The median age for the start of symptoms was 26.48 years. The period from the beginning of symptoms to surgery was 11.82 years. Assuming hypothetically that all patients were to be treated conservatively without surgery until the end of their lives, with an average life expectancy of 82 years, the costs have to be multiplied. In this example, the total lifetime costs for conservative treatment for macromastia would be 55.52 × €1677.59 = €93,140 per patient.However, it can be assumed that symptoms and problems due to macromastia will increase over time, leading to an increase in the annual costs, so that the total lifetime costs are likely to be even higher than calculated. On the other hand, costs due to loss of productivity will decrease after the age of 65–67 due to retirement.Assuming that 1–5% of the female population suffer from macromastia , the cosDecision-making on whether the costs of macromastia are to be covered by insurance is currently based on different criteria. It is mainly the nature of the surgery that is important whether it is only aesthetic or is medically indicated. Insurance companies often argue that reduction mammoplasty is a form of plastic surgery and decline to reimburse the costs. The evidence provided here of the total costs of macromastia may provide further arguments in support of insurance coverage.Regardless of the costs for each category, the usefulness of each form of treatment has to be assessed. Several studies have evaluated the effect of weight reduction, for example, and found that it did not have any positive effects on the symptoms of macromastia , 11, 20.In this study, costs were calculated from the start of symptoms to surgery with reduction mammoplasty, as the study cohort was recruited from this group of patients. There might, therefore, be some bias in the patient selection, since women with macromastia who do not have any symptoms will not seek medical treatment. It might be assumed that these patients do not need special treatment and so will not give rise to any additional costs. However, as one reasonable definition of macromastia makes the presence of chronic pain an obligatory element, the present cohort of patients is in fact representative of this diagnostic group.Recall bias: As the patients were recruited several years after surgery, it is possible that the information which they provided about the years before surgery is not complete. Moreover, no exact information is available about variability in costs during the years before surgery. For example, patients might have consulted their family physician several times at the start of symptoms, before the diagnosis and the cause of pain were identified, with no further consultations being needed after the diagnosis. On the other hand, it is quite possible that the symptoms increase over time, with the frequency of appointments with a physiotherapist, for example, also increasing.Since it is known from several studies that conservative treatments such as weight reduction and exercise  fail to On the other hand, quality assessment is nowadays becoming increasingly important in the calculation of health-care costs, and the loss of quality of life also needs to be taken into account during therapeutic decision-making. A cost–utility calculation would, therefore, be helpful, and this should be the focus of further research.


Influenza virus (IV) defective interfering particles (DIPs) were first described by Preben Von Magnus in the 1940s as a noninfectious form of the virus that accumulated during infection . SubsequIV DIPs are characterized by large internal deletions within 1 or more genome segments that render them incapable of independent replication (hence the “defective” descriptor). The “interfering” comes from the well-characterized ability of DIPs to directly inhibit the replication of wild-type (WT) virus by outcompeting WT gene segments for replication resources and packaging. These phenotypic classifications have become more complicated over time. Replication-incompetent particles are not necessarily “defective” from the standpoint of a virus population as they can contribute to viral replication through collective interactions ,5. It isDelVGs and DIPs occur naturally during virus infection and can be easily generated and propagated in the lab. There are 2 separate processes involved in DIP generation: (1) the formation of DelVGs during genome replication and (2) the packaging and propagation of DelVGs within DIPs. For the sake of brevity, we will only discuss the formation of DelVGs here.Influenza DelVGs are likely formed by viral polymerase errors that occur during the replication process, rather than by ligation of viral RNA fragments or splicing ,6. This Two main models have been proposed for how this process might work at the molecular level. The first model, called “looping-out”, suggested that an RNA structure could bring 2 distant sites of a gene segment strand close to each other, forming a loop that the polymerase can skip without dissociating from the template or the nascent strand  6]. How. How6]. The specific molecular determinants that trigger the pausing of polymerization remain unclear. Potential candidates include the incorporation of base mismatches or local RNA structure. The downstream reinitiation of polymerization has been proposed to be guided by complementarity between the nascent strand and the site of reinitiation on the template . SupportAnalysis of DelVG sequences has provided contradictory information about the importance of complementarity at DelVG deletion junctions. Two recent studies reported the presence of overlapping sequences at the flanking regions of IV DelVG junction sites ,18. In cIn summary, the specific molecular determinants that govern the formation of DelVG remain a mystery. There are also several outstanding questions about the patterns of DelVG formation across the genome. For instance, we and others have shown that DelVGs form much more readily from some genome segments compared with others and that these patterns can differ between viral strains and types . One thiBeyond the mysteries of how DIPs form, the key question of why they form remains unresolved. For decades, the dogma was that DIPs, as indicated by their name, are defective products that interfere with WT virus replication through competition for resources and packaging . They arIn the past, this was explained by the idea that DIP production was simply an artifact of high multiplicity of infection  in vitro replication conditions, with minimal relevance for natural infection. Sustained high MOI infection creates conditions in which the emergence of DelVGs within viral populations might be inevitable . Recent If DIP production is as harmful to the virus as believed, then there should be a strong selective pressure to minimize DIP production. Based on this assumption, we would expect that viruses that produce fewer DIPs to outcompete and numerically dominate those with greater DIP production. This raises the question of why DIPs appear to be so common during natural infection.There are multiple possible explanations for why DIP production has not been eliminated by selection. For instance, DelVG production may be an unavoidable side-product of the influenza virus RNA replication process. The influenza virus polymerase may be unable to rapidly synthesize genomic RNAs without making occasional mistakes . A similar trade-off between replication speed and mutation rate has been proposed for poliovirus . ArguingAfter several decades of study, DIPs remain a weird and fascinating complication in our understanding of influenza biology, and numerous basic questions remain unanswered. First and foremost, how do DelVGs form, and what are the viral genetic determinants that govern their formation? Second, is DIP production as bad for the virus as has long been believed? How does the presence of DIPs alter the fitness and transmissibility of viral populations? Finally, how does DIP production influence that pathogenic potential of IV populations? The answers to these questions will greatly improve our fundamental understanding of influenza biology and are likely to reveal that DIPs are way more delightfully interesting than is commonly believed.


Ornithodoros soft ticks are the only known vector and reservoir of the African swine fever virus, a major lethal infectious disease of Suidae. The co-feeding event for virus transmission and maintenance among soft tick populations has been poorly documented. We infected Ornithodoros moubata, a known tick vector in Africa, with an African swine fever virus strain originated in Africa, to test its ability to infect O. moubata through co-feeding on domestic pigs. In our experimental conditions, tick-to-tick virus transmission through co-feeding failed, although pigs became infected through the infectious tick bite. Suidae caused by the African swine fever virus (ASFV). ASF is endemic in southern and eastern Africa, where ASFV circulates among different hosts, such as domestic pigs and warthogs, as well as soft tick vectors from the Ornithodoros moubata group . ThO. moubata were infected with the Liv13/33 strain by blood feeding on viremic pigs with a viremia ranging from 107,8 hemadsorbing dose 50%/mL (HAD50/mL) to 108,1HAD50/mL, as previously described )  , confirm–24.71]) , indicatO. moubata, which is known to acquire the Liv13/33 virus strain in salivary glands after infectious engorgement [The experimental design defined in the study was able to guarantee the reliability of the results obtained. Indeed, to maximize the success of transmission we used the African tick species orgement  and to torgement . To mimiorgement , the traorgement . Naive nO. moubata-Liv13/33 tick-virus pair. However, her experimental design was quite different, since she used adult stages for both the infected “source” and the uninfected “recipient” ticks and separated them on the pig with a cardboard barrier inside the feeding unit to distinguished them after blood feeding. Interestingly, in a second trial, Rennie (1998) carried out co-feeding on mice, which is a non-sensitive animal for ASFV, and that time transmission occurred [Our results completed those obtained on pigs by Rennie (1998) , who sugoccurred . In thisO. moubata-Liv13/33 tick-virus pair, but additional experiments are needed to confirm these results. Co-feeding studies with others tick development stages are also needed to take into account the impact of the difference in hypostome (mouthparts) length. This difference may have an impact on the depth of the bite, and the nymphs-1 could be unable to acquire the virus secreted by the infected adults during the blood meal. Warthogs, which are susceptible to ASFV, but which develop no or low viremia when they are infected, might mediate ASFV in the same manner as domestic pigs and may not be a major route for tick infection through co-feeding in the wild. Conversely, it is not possible to exclude the role of non-susceptible animals that may colonize ancient warthog burrows where soft ticks still settle, thereby contributing to tick infection through co-feeding and thus the persistence of the virus in tick populations. ASFV maintenance in soft ticks in the field is one cause of ASFV re-emergence [Taking our results and those of Rennie together, we can suggest that ASFV co-feeding cannot occur in domestic pigs using the mergence . However


Natural service is currently the predominant method of breeding in extensively managed beef herds. In these herds, the importance of the bull has been largely overlooked, focusing instead on female fertility. However, the bull might be the most important factor in determining the reproductive performance of the herd, because one subfertile or infertile bull could mean a decrease of 25 to 40 calves per year. Thus, bull management is critical to increase herd fertility. Infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) are infections associated with reduced conception rates. In this study, we analyzed the effect of IBR, BVD, and bull: cow ratio on seminal parameters of the bull and herd fertility, finding a significant negative effect of BVD on sperm concentration and motility, and a negative correlation between the number of cows per bull and herd fertility. Our data suggest that serological control of BVD and sperm parameters, as well as including less than 40 cows per bull, could improve the reproductive efficiency of the herd in extensively managed herds.Natural service remains the main breeding method in extensively managed beef herds. Although the bull might be the most important factor in determining herd fertility, its importance has been largely overlooked, focusing instead on female fertility. Management of the bull is critical to maximize the opportunities for cow conception. Infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) are infections associated with reduced conception rates. This study aimed to determine the effect of both IBR and BVD infection, and bull: cow ratio on seminal parameters in the bull and herd fertility. The presence of antibodies to IBR and BVD, seminal parameters , and herd fertility were analyzed in 158 extensively managed bulls. Sperm concentration and mass motility, as well as herd fertility, were significantly lower in BVD-positive bulls. No significant differences were found between IBR-positive and -negative bulls in any reproductive parameter. Sperm concentration was negatively affected by BVD infection in both Charolais and Limousin bulls, whereas mass motility and herd fertility were reduced in Limousin bulls only. No differences were observed in the cow: bull ratio between BVD+ and BVD- bulls. A significant negative correlation was detected between the number of cows per bull and herd fertility, which was negatively affected when herds had more than 40 cows per bull. In conclusion, BVD and bull overuse negatively affect the reproductive performance of the herd. Extensive cattle production generates enormous wealth, yielding economic benefits, employment, and environmental sustainability . FertiliIn contrast to intensively managed cattle, in which artificial insemination was introduced in the 1950s, displacing natural service as the selected breeding method, logistical difficulties have limited its use in extensively managed herds . Moreover, infectious diseases, such as bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR), and infection by Besnoitia, Trichomonas, Neospora, and Campylobacter, have also been linked to fertility and reproductive disorders in this species, causing an important economic impact in the livestock industry ,11. PartOn the other hand, IBR is another widely recognized cause of abortion, infertility, and suboptimal productivity in the herd ,22. InfeThe number of cows per bull represents another important parameter to evaluate in extensively managed herds in order to obtain better reproductive performance. The percentage of bulls per cow is very variable between herds. Some studies have estimated that it ranges from 1.5% to 10% of bulls in different herds and that 2% might be enough to obtain successful pregnancy rates ,24. ThesThe study was conducted between May and August 2014 and 2015 and involved 158 bulls  and 4299 cows that were randomly selected in two adjacent areas in Spain (Cáceres (Extremadura) and Salamanca (Castilla y León)). No fertility evaluations had been performed on the bulls prior to the start of the study. All procedures were approved by the Spanish Ethics Committee of Animal Welfare (RD 53/2013). Twenty-one farms that did not vaccinate the animals for IBR and BVD were included in the study to avoid interferences in the analyses. The bulls included in the study tested negative for Trichomonas and Campylobacter and their age ranged from 1 to 10 years old (mean of 4.06 years old). The average size of the cow herd was 30.49 ± 0.68 (mean ± SEM), ranging from 15 to 51 cows per bull. No differences were observed in bull: cow ratio between different farms (One-way ANOVA).Animals were fed on natural pastures supplemented with water, feed additives, and good quality straw in the summer season. Only cows in good physical condition were considered to analyze cow: bull ratio and herd fertility. Fertility was evaluated based on calving rate, which was assessed as the percentage of calving cows within the herd per year. Data were collected by both veterinarians and farm owners.Bulls included in the study were kept in sexual rest for 2 weeks. They were then held for semen collection in a handling sleeve. A transrectal massage was performed with a flattened hand. Feces were manually removed and electroejaculation was performed using a 75 mm diameter rectal probe with three ventrally oriented longitudinal electrodes . During semen collection, the pre-seminal fraction was discarded. The electroejaculator was set on automatic mode; one circuit contained a series of 40 cycles, with each cycle delivering a slightly higher intensity voltage. Each cycle lasted for 2 s followed by 2 s pause. Once a minimum sample was obtained (3 mL), the stimulation stopped. This procedure was carried out by a veterinarian, following the same protocol in all bulls included in the experiment.Immediately after collection, semen samples were analyzed. Each sample was processed for volume, concentration, mass motility, and progressive motility. The volume of the sample was determined using a graduate micropipette . The sperm concentration was determined using a photometer . Mass and progressive motility were subjectively evaluated by the same researcher for all analyses included in this study. Mass and progressive motile sperm were subjectively determined under phase-contrast microscopy at 400× magnification (B-500 TpH OPTIKA), using a scale ranging from 1 to 4, in which 1 = 0–39%, 2 = 40–59%, 3 = 60–79%, and 4 = ≥80% spermatozoa showed total or progressive motility. g for 10 min, and frozen at −20 °C until use. Serum samples were analyzed for antibodies against IBR virus and BVDv p80  by ELISA as previously described with some modifications [Peripheral blood samples were obtained from the bulls included in this study. Samples were then allowed to clot, and the serum was separated by centrifugation at 1500 ications . t-test. The differences in BVD and IBR prevalence between breeds were analyzed by Chi-square test. The differences in seminal parameters  and herd fertility between breeds were analyzed by One-way ANOVA. To exclude bull age as a possible variant affecting the results, t-test was performed between infected and non-infected animals. To exclude a farm effect on bull: cow ratio, One-way ANOVA was performed. Linear regression and correlation analyses were performed between cow: bull ratio and reproductive parameters. All statistical analyses were performed with Prism GraphPad software  and a value of p < 0.05 was considered significant.The differences in seminal parameters  and herd fertility between infected and non-infected animals were analyzed by t-test).Bull age was excluded as a variable that could affect the results because no statistically significant differences were found between BVD+ (3.35 ± 0.32 years) and BVD- bulls (4.06 ± 0.18 years); or between IBR+ (3.67 ± 0.26 years) and IBR- bulls (3.99 ± 0.19 years) (p = 0.002) and mass motility (p = 0.044) compared to their non-infected counterparts. Although volume and progressive motility were slightly lower in bulls infected with BDV, no significant differences were found between BVD-positive and -negative animals. However, fertility was significantly lower in those herds in which the bull was infected  (t-test), which excludes a possible interaction of cow: bull ratio in these results. Forty-two out of 157 bulls (26.58%) tested positive for BVD. Bulls infected with BVD showed a significantly reduced sperm concentration ( = 0.01) . No diffp = 0.004), mass motility (p = 0.02) and herd fertility (p = 0.047) were significantly reduced only in Limousin bulls. No differences were found between BVD-positive and -negative bulls from autochthonous breeds for any of the parameters analyzed, although this could be due to the reduced number of animals in this group , Limousin , and bulls from autochthonous breeds . When we analyzed whether BVD affected reproductive parameters differently in the main breeds, we found that while sperm concentration was negatively affected by BVD infection in Charolais bulls  were positive for IBR. In this study, no correlation was found between the presence of IBR infection in the bull and the reproductive parameters evaluated .p = 0.001), and mass motility was significantly lower in Charolais bulls (p = 0.047) , Limousin , and bulls from autochthonous breeds . Similarly, no differences were found in reproductive parameters between IBR-infected and non-infected bulls in the main breeds . When we= 0.047) .p = 0.0102, p = 0.034 and p = 0.046, respectively). However, there were no statistically significant differences between animals infected with both BVD and IBR, and animals infected with only BVD or IBR  were positive for both IBR and BVD, 19 (12.02%) were only positive for BVD, 44 (27.85%) were only positive for IBR, and 72 (45.57%) were negative for both. Higher sperm concentration, mass motility, and herd fertility were found in bulls free of both IBR and BVD virus compared to animals positive for both diseases (D or IBR .p > 0.05). However, there was a statistically significant negative correlation between herd fertility (r = −0.2853) and the number of cows per bull. Interestingly, when herds had more than 40 cows per bull, the negative effect on fertility was statistically significant (p = 0.002), excluding a possible effect of BVD infection on this analysis.A significant negative correlation (r = −0.1833) between progressive motility and the number of cows per bull (ranging from 15 to 51) was found. Although sperm concentration and mass motility were lower when the number of cows per bull increased, no differences were found between sperm volume, concentration, and mass motility and the cow: bull ratio  and BVD (26.58%) in extensively managed bulls in Spain and points out the negative effect of BVD on the reproductive parameters of the bull. Furthermore, we report that bull overuse decreases the reproductive efficiency of extensively managed herds, which is significantly reduced when more than 40 cows are allocated per bull. BVDv transmission within a herd or between herds depends primarily on contact between susceptible individuals and PI animals . These iIBR infection is another widely recognized cause of abortion, infertility, and suboptimal productivity in the herd ,22. MoreInterestingly, in our study, a high number of cows per bull appeared to directly affect reproductive parameters. A negative correlation (r = −0.2853) was found between the number of cows per bull and fertility. Moreover, fertility was significantly affected when herds were held with more than 40 cows per bull. Other studies have reported that pregnancy rates can be successful with ratios around 50 cows per bull; however, the authors point out that the bulls need to be of high reproductive performance ,24. MoreThis study provides an extensive analysis of the prevalence of BVD and IBR and of seminal parameters of extensively managed bulls in Spain. In the present study, no correlation was found between the presence of IBR infection in the bull and the reproductive parameters evaluated. Sperm concentration, mass motility, and herd fertility were negatively affected by BVD infection and by the overuse of bulls, decreasing the fertility of the herd. In order to improve the reproductive parameters of the herd, it seems mandatory to perform serological analyses for BVD and sperm analyses, as well as restricting the number of cows per bull to less than 40. However, further studies focusing on the number of cows per bull are necessary to narrow down the adequate ratio to improve the reproductive parameters of the herd.


Several viruses have the capacity to cause serious infections of the nervous system in patients who are immunosuppressed. Individuals may be immunosuppressed because of primary inherited immunodeficiency, secondary immunodeficiency due to particular diseases such as malignancy, administration of immunosuppressant drugs or organ or bone marrow transplantation. The viruses capable of such opportunistic infection of the nervous system include herpes simplex virus (HSV), Varicella-Zoster virus (VZV), Cytomegalovirus (CMV), Epstein –Barr virus (EBV), Human Herpes virus type 6 (HHV-6), JC virus (JCV), enterovirus, measles virus and Covid-19. In most cases it seems likely that immunological defence mechanisms in the immunosuppressed are deficient which creates a suitable environment for certain viruses to become opportunistic in the nervous and other systems. Further research is required both to understand these opportunistic mechanisms in more detail and also to determine how many virus infections are modified by specific inborn errors of immunological responses. An individual may become immunocompromised as a result of multiple factors. These include the administration of immunosuppressant drugs given for various medical conditions, an underlying disease such as Human Immunodeficiency Virus (HIV) infection or malignancy such as lymphoma which affects the immune system producing secondary immunodeficiency, various primary immunodeficiency syndromes such as severe combined immunodeficiency (SCID) with defects in T cells, B cells, and NK cells , or fromViruses may reactivate under conditions of immunosuppression from various causes primarily because the immune mechanisms that normal suppress or limit viral replication become disrupted. In the situation of latent virus infection which is kept in check by the immune system the virus may reactivate and cause neurological disease. For example, in the case of Varicella Zoster (VZV) infection, following primary infection (chicken pox) the virus becomes latent in neurons in peripheral ganglia . It may Here I give an overview of the several viruses which have a propensity to replicate and/or reactivate under conditions of immunosuppression causing neurological disease. These include Herpes viruses including Herpes Simplex Virus type 1 and 2 , VZV, Cytomegalovirus (CMV), Epstein Barr virus (EBV) and Human Herpes Virus type 6 (HHV-6). Also included here is JC Virus, and, in brief, enteroviruses, measles and the recently recognised Covid-19 (SARS-CoV2) infection. In many cases there is included an indication of the Class of Evidence and Level of Recommendation for a therapeutic intervention or an investigative procedure, and these are based on previously published criteria which are also used for official European Academy of Neurology guidelines , 8, 9.While HSV-1 is classically associated with herpes simplex encephalitis (HSE) , HSV-2 azoster sine herpete where VZV reactivates in the absence of the typical zosteriform rash [2 for 7–10 days) (Class IV). The role of acylcovir in imunocompetent individuals below 50 years is less clear. In patients with VZV-induced myelitis and vasculopathy it has been suggested that treatment should be with intravenous acyclovir for 14 days in combination with oral prednisolone for 5–7 days but this is based on experience with only a small number of patients [zoster sine herpete can be obtained using PCR to detect VZV DNA in the CSF , though detection of anti-VZV IgG in the CSF has a higher sensitivity for correctly diagnosing VZV vasculitis than does PCR [Reactivation of latent VZV from human ganglia is a well-recognised complication of immunsuppression resulting in a variety of neurological complications, due, as indicated above, to symptomatic impairment of cell-mediated immunity to the virus. Indeed VZV infections are among the most important of all viral infections that affect the central nervous system (CNS) and peripheral nervous system (PNS) in immunocompromised individuals. The protean manifestations of VZV-induced neurological disease have been described in detail elsewhere , but theorm rash , 17, 18.orm rash . Treatmepatients   is a demyelinating disease associated mainly with immunosuppression, especially AIDS (5% at post-mortem), organ transplants, leukaemia, and the Multiple Sclerosis drug Natalizumab (Tysabri) , 28, 29.While enteroviruses such as poliovirus, echoviruses and coxsackie viruses are known to infect the immunocompetent human host causing a range of diseases including those of the CNS such as encephalitis, they may also be significant in the immunocomprised individual. Diseases such as meningitis, myocarditis and myopathy can be more common in the immunocompromised or neonatal host . Of partWhile measles virus infection, normally a fairly mild though certainly uncomfortable condition, has the capacity to be serious in the immunocompetent, it is typically a far more dangerous infection in the immunosuppressed. Measles may rarely be followed by an acute post-measles encephalitis, which is the most common CNS complication in the immunocompetent as well as a rare primary measles encephalitis. Immunodeficient children are susceptible to measles inclusion body encephalitis where the measles virus persists in the brain. The latter is a very serious infection with a mortality of 75% . In a reIn the author’s view, it is relevant to mention this recently recognised infection in the current context. It is known that malignant disease predisposes to Covid-19 infection with 7.2% of 138 patients having malignant disease as a co-morbidity in 1 major study . Neurolo


Canine distemper virus (CDV) is a highly contagious pathogen transmissible to a broad range of terrestrial and aquatic carnivores. Despite the availability of attenuated vaccines against CDV, the virus remains responsible for outbreaks of canine distemper (CD) with significant morbidity and mortality in domesticated and wild carnivores worldwide. CDV uses the signaling lymphocytic activation molecule  and nectin-4 (PVRL4) as entry receptors, well-known tumor-associated markers for several lymphadenomas and adenocarcinomas, which are also responsible for the lysis of tumor cells and apparent tumor regression. Thus, CDV vaccine strains have emerged as a promising platform of oncolytic viruses for use in animal cancer therapy. Recent advances have revealed that use of the CDV reverse genetic system (RGS) has helped increase the understanding of viral pathogenesis and explore the development of recombinant CDV vaccines. In addition, genetic engineering of CDV based on RGS approaches also has the potential of enhancing oncolytic activity and selectively targeting tumors. Here, we reviewed the host tropism and pathogenesis of CDV, and current development of recombinant CDV-based vaccines as well as their use as oncolytic viruses against cancers. Morbilivirus genus within the Paramyxoviridae family [Lycaon pictus), about 10,000 Caspian seals (Phoca caspica), around 35% of Serengeti lions (Panthera leo), and 30% of rhesus monkeys (Macaca mulatta) [Canine distemper virus (CDV) is an enveloped negative-RNA virus, which, along with measles virus (MeV), Rinderpest virus (RPV), peste des petits ruminants virus (PPRV), phocine distemper virus (PDV), Cetacean morbillivirus (CeMV), and recently discovered feline morbillivirus (FeMV), belongs to the e family ,2. Notede family . Both vie family ,5,6,7. Hmulatta) ,9,10.Currently, attenuated CDV strains are routinely used worldwide as live vaccines against CDV infection in dogs, minks, and other carnivores. However, these vaccines have often been reported as insufficiently attenuated for ferrets and other wildlife species and can cause symptomatic and sometimes fatal infections after administration, resulting in an increased interest for a safe and efficient CDV vaccine for highly susceptible species ,13,14,15This review highlights the potential use of the CDV genome as a viral vector for the development of novel vaccines and oncolytic virotherapy against cancers. Specifically, the development of a CDV reverse genetics system (RGS) and identification of the viral cellular receptors over the past decades, contributed to the understanding of viral pathogenesis and the generation of recombinant CDV-based vaccines, as well as the exploitation of the oncolytic potential of the virus against cancers by targeting tumor markers as viral receptors.The development of the RGS of RNA viruses can be used to produce modified viruses that have specific properties with potential applications in vaccine development, gene therapy, and basic science. Concomitantly, foreign genes were inserted to (1) allow localization of viral replication in vivo through the expression of marker genes, (2) develop candidate multivalent vaccines against vector viruses and other pathogens, and (3) create candidate oncolytic viruses. The vector use of RNA viruses was experimentally encouraged by the pronounced and unexpected genetic stability of the recombinants, and by the high load of insertable genetic material .Mononegavirales, cannot function as mRNA. Thus, to generate the viral RNP, the minimal infectious unit of negative-strand RNA viruses, simultaneous expression of viral protein components forming the polymerase complex, and full-length viral antigenomic RNA (cRNA) from cDNA are required [The CDV genome is 15,690 nucleotides in length and consists of a short 3′ leader region and 6 genes encoding the nucleocapsid (N), phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins, which are separated by intergenic regions of 3 nucleotides and followed by a short 5′ trailer region. The V and C nonstructural proteins are encoded within the P gene. The V protein is expressed by cotranscriptional RNA editing, and C is expressed from an overlapping reading frame . The N, required .Paramyxoviridae family. The T7 RNA polymerase in this RGS was delivered by MVA-T7, a derivative of the modified vaccinia Ankara (MVA) virus, which can drive high-level expression of T7 RNA polymerase in the cytoplasm. However, the use of a vaccine virus has several disadvantages, primarily high poxvirus-mediated cytotoxicity, challenges associated with separating vaccinia virus from the rescued viruses, and increasing restrictions on the use of vaccinia virus, especially for vaccine development. To avoid these possible problems, cells lines stably expressing T7 RNA polymerase has been developed as alternatives to the use of helper T7-vaccinia virus. The most commonly used of which are BSR-T7 cells, a baby hamster kidney (BHK)-derived cell line stably expressing T7 RNA polymerase [Paramyxoviridae genera [In standard RNA virus RGSs, an exact 3′-end of the antigenome is generated by autocatalytic cleavage from a hepatitis delta virus ribozyme (HDV-Rbz) sequence positioned directly downstream of the antigenome. Accordingly, the first established CDV RGS was the Vero cell-adapted Onderstepoort vaccine strain, which was recovered from Vero cells overlaying vaccinia-T7 virus-infected HeLa or 293 cells after cotransfection with a plasmid encoding the full-length cDNA  nucleotides at the 3′-end), and supporting plasmids encoding the N, P, and L proteins ,26. The lymerase ,29,30,31lymerase , and as lymerase . By incoe genera . Insteade genera ,35; the e genera .Recently, several generated RGSs have been reported for the rescue of CDV wild-type strains from marmoset lymphoblastoid cells (B95a) or Vero cells expressing the signaling lymphocytic activation molecule (SLAM) receptor (Vero-SLAM) that allows for propagation of wild-type CDVs and maintenance of their virulence in vivo . ResultsIdentification of cellular receptors has vastly improved our understanding of CDV viral tropism and pathogenesis. Two cellular receptors, the SLAM/CD150, and nectin cell adhesion molecule 4 (nectin-4/PVRL4), involved in the entry of morbillivirus in the cell, and subsequent infection, have been identified for both the vaccine and wild-type CDV strains. Noted, SLAM is reported to be expressed on the surface of activated T-lymphocytes, and B-lymphocytes, macrophages, and dendritic cells, but not in epithelial cells, whereas nectin-4 is known to be expressed on the apical and basolateral surface of polarized epithelial cells, as well as in endothelial cells in the central nervous system (CNS) of dogs ,40,41,42The CDV RGS has also been used to identify the key residues in the H protein interacting with cellular receptors and evaluate their contribution to the viral entry. Recently, the P493S/Y539A double mutations in the H protein were introduced in an unable to recognize nectin-4 (nectin-4-blind) recombinant CDV strain, which was recovered and used to infect ferrets. Although the infected ferrets showed no clinical signs or viral shedding, there was a rapid and efficient spreading of immune cells observed, producing the same levels of leukopenia and inhibiting lymphocyte proliferation activities as in the case of wild-type strain infected animals. This finding highlights the fact that the SLAM-expressing immune cells appear to be necessary for CDV entry, dissemination, and initial infection, as well as immunosuppression, whereas infection of nectin-4 mediated epithelial cells seems to be required for viral shedding and clinical disease, as well as viral transmission . AdditioIn addition to the identification of key residues in the H and F proteins sustaining the SLAM or nectin-4 receptor dependent cell entry and fusion in vitro ,49,50, sUrocyon cinereoargenteus) and ferrets (Mustela nigripes) [Mustela lutreola) and ferrets [Both live-attenuated and inactivated CDV vaccines have been used to reduce the number of CD outbreaks in ferret, mink, and dog populations . Howeverigripes) ,60, whil ferrets ,14. Prob ferrets ,62,63,64 ferrets . Recentl ferrets ,67,68. ADue to the availability of an RGS for manipulating CDV genomes and generating respective recombinant viruses, a number of recombinant viruses based on attenuated CDV strains has been generated that present antigens of foreign pathogens during CDV replication. These recombinant vaccines have an excellent safety record in animal models and elicit humoral as well as cellular immune responses in vaccinated animals, which are responsible for long-lasting protection . Leishmania antigens (leishmania homolog of receptors for activated C-kinase (LACK), thiol-specific antioxidant (TSA), or protein antigen LmSTI1 (LmSTI1), was also reported suitable for use as a polyvalent vaccine vector for Leishmania, providing protection against both CDV and major infections of Leishmania in dogs [N-linked glycosylation sites in the H protein of the virus produced a recombinant CDV with an attenuated phenotype, which was no longer able to cause disease in ferrets [CDV vaccine strains have recently been used as a vector to stably express the glycoprotein (G) of the rabies virus (RABV) within an additional transcription unit of a CDV cDNA clone. The RABV-G can be incorporated in the viral envelope of the chimeric virus to improve immunological recognition. The chimeric virus was shown to be safe for both mice and dogs and resulted in the production of long-lasting neutralizing antibodies against both CDV and rabies, and even protected mice from a lethal dose of rabies, demonstrating potential for the production of multivalent vaccines against both pathogens ,35. A re in dogs . In addi in dogs ,71. Thes in dogs . The res in dogs . Similar ferrets . In general, as a nonsegmented negative-strand RNA virus, the lack of a significant nuclear phase in CDV is a favorable aspect with respect to vector development because it insulates these infectious agents from extensive interference of genetic and cellular processes. Thus, the fact that the CDV genome does not recombine is also of relevance for viral-vector development. In addition, most of CDV vaccine strains showed a good safety record in the context of canines, and diseases arising from Rockborn strain infection such as post-vaccination encephalitis have been stopped by vaccinations . AdditioCancer could be defined as the formation of a malignant tumor due to genetic mutations in normal cells leading to their indefinite growth and abnormal proliferation in the body and causing damage to the normal functions of the body. Many cancers remain incurable to modern therapy despite recent antitumor pharmacological regimens in both humans and animals. Roughly 1 to 2% of canines over the age of 10 die from cancer ,77,78, wThere are several possible mechanisms through which an oncolytic virus could mediate oncolysis . One of Taken together, oncolytic viruses have been shown to prevent neoangiogenesis either by direct infection and destruction of the tumor vasculature, concurrently enhancing innate immunity mediated antitumor effects or by exerting antiangiogenic abilities in tumor tissues. However, before oncolytic viruses can be widely used as therapeutic agents for the clinical treatment of human or animal cancer patients, major obstacles, including potential viral toxicity, ineffective delivery of virus to the tumor, and spread of the virus throughout the tumor mass should first be overcome. To resolve these problems, viral RGS approaches have been recently employed to generate attenuated tumor-selective vaccine strains, as well as selectivity- and efficacy-enhanced, oncolytic virus strains. For instance, MeV was first mentioned as a potential oncolytic agent in anecdotal reports regarding the regression of a Hodgkin lymphoma following natural MeV infection in humans . AttenuaIn the veterinary field, several viruses have been reported as oncolytic agents, including adenovirus, reovirus, vaccinia virus, and CDV. One advantage of CDV as an oncolytic virus is the relatively small size of CDV in comparison with DNA viruses. Only a few proteins of the virus are expressed in addition to those expressed from the artificial inserts, thus minimizing superfluous immune responses in vivo. Second, as members of the morbilliviruses, CDV shares many similarities with MeV, and represents an interesting candidate for its employment in oncolytic therapy, as naturally attenuated vaccine strains have been shown to be able to infect and induce apoptosis in many kinds of cancer cells . InfectiSafety is of paramount importance. CDV has a non-segmented genome rendering it stable, with a low risk of mutation, thus it is highly unlikely to revert to the pathogenic phenotype. As an effective oncolytic virus for canine cancer cells in vitro, live attenuated CDV strains also maintain the ability to enter the immune and epithelial cells in vivo via the SLAM and nectin-4 receptors. Thus, safety issues with respect to possible virus dissemination and therapeutic effects in animals and humans should be addressed. CDV is thought to be the result of a spillover transmission into dogs from MeV during human epidemics in the New World . The recUntil now, although there have been few studies on the use of CDV, particularly genetic engineering CDV strains, as an oncolytic virus for canine cancer patients compared with the advanced progress of human virotherapy in clinical trials, the high similarity between human and canine cancers, including colorectal cancer, fibrosarcoma, osteosarcoma, and soft tissue sarcoma ,85,86,87Mononegavirales, vesicular stomatitis virus (VSV) and MeV-mediated oncolysis, the strategies used to increase their oncolytic activity employed the use of VSV/MeV hybrid viruses generated by RNA virus RGS as a platform for oncolytic virotherapy [Today, several limitations remain in the development of an ideal oncolytic virus, including (1) selective targeting of oncolytic viruses to tumor tissue, (2) relatively poor viral spread throughout solid tumor tissues, (3) inefficient viral replication in immunocompetent hosts, and (4) disadvantageous ratio between the anti-viral and anti-tumoral immunity. As far as CDV is concerned, virotherapeutic studies have been primarily performed using CDV vaccine strains rather than wild-type strains, due to the exceptional genetic stability and safety record in canine populations ,18,19,20otherapy . This hyotherapy . AccordiRecently, “immunovirotherapy” strategies to enhance the oncolytic efficacy of MeV have been developed with the introduction of immunomodulatory transgenes  and antibodies against immune checkpoint inhibitors  that stimulate the native anti-tumor immune response. These strategies were used for MeV-susceptible cancer mouse models and showed significantly-senhanced tumor regression or prolonged overall survival, which were correlated with an influx of host neutrophils and tumor-infiltrating cytotoxic T cells ,120,121.This review summarized the important roles that CDV RGS approaches have played in (1) the construction of recombinant CDV-based vaccines; (2) understanding the mechanisms of the cellular tropism and pathogenesis of CDV from a molecular perspective, comprising not only the interactions of the viral proteins with host cellular receptors but also the influence of host factors on viral virulence; and (3) the potential ability of CDV, particularly vaccine strains, to be used as an oncolytic virus against cancers in humans and animals by targeting tumor markers as viral receptors. However, the action of CDV as an oncolytic virus in human and canine cancers has been primarily evaluated in tumor cells in in vitro systems only. Detailed in vivo studies are highly required for a better understanding of the mechanisms of interactions between the virus and tumor cells, as well as of the impact on the microenvironment and antitumor immune reactions.


The total mean cost of IPD increased in the PCV13 period by 31.4% (€3016.1 vs. €3963.9), mainly due to ICU stay . During the PCV13 period, direct IPD costs decreased due to a reduction in the number of cases, but cases were more severe and had a higher mean cost. During 2015, IPD costs increased due to an increase in the costs associated with non-PCV13 serotypes and serotype 3 and this requires further investigation.The lack of invasive pneumococcal disease (IPD) cost studies may underestimate the effect of pneumococcal polysaccharide conjugated vaccines (PCV). The objective of this study was to estimate the direct costs of hospitalized IPD cases. A prospective study was made in children aged <5 years diagnosed with IPD in two high-tech hospitals in Catalonia (Spain) between 2007–2009 (PCV7 period) and 2012–2015 (PCV13 period). Costs were calculated according to 2014 Catalan Health Service rates using diagnostic-related groups. In total, 319 and 154 cases were collected, respectively. Pneumonia had the highest cost , followed by meningitis . During 2007–2015, the costs associated with PCV7 serotypes (Pearson coefficient (Pc) = −0.79;  Streptococcus pneumoniae in 2015 accounted for 11% of deaths in children aged < 5 years not infected with HIV. Incidence and mortality rates are higher in developing countries and most deaths occur in the African and Asian regions [Invasive pneumococcal disease (IPD) is a major cause of morbidity and mortality in children aged < 5 years and occurs mostly as pneumonia or other serious forms such as meningitis, bacteremia or osteoarticular infections. In 2000, 14.5 million cases of IPD were estimated to have occurred in children aged < 5 years, causing 7,350,000 deaths [ regions , respectStudies show that, after the introduction of PCV10 and PCV13, there was a decrease in IPD incidence in children and adults ,13,14,15While the licensing of PCVs has led to numerous changes in the epidemiology of IPD, which have been extensively studied microbiologically and epidemiologically, there are few studies of the costs of IPD , which mThe costs of IPD vary greatly depending on the geographical scope and design of the study. Each region has its own health policies, its own health system, and its epidemiologic reality in terms of circulating serotypes . This maThe objective of this study was to estimate the costs of IPD cases requiring hospital admission in children aged < 5 years in high-tech pediatric hospitals in Catalonia (Spain) following the licensing of the PCV7 and PCV13 and to compare and identify the differences between the two periods and the main factors that may involve variations in the costs of IPD, both globally and in the mean cost per case.A prospective study was conducted in children aged < 5 years diagnosed with IPD in two periods. The first period was between 1 January 2007 and 31 December 2009 and corresponded to the period in which the vaccine licensed was PCV7. The second, from 1 January 2012 to 31 December 2015, corresponded to the period in which the vaccine licensed was PCV13. In both periods, two high-tech pediatric hospitals in Catalonia participated, namely the Sant Joan de Déu Hospital and the Vall d’Hebron Mother-Child Hospital, which account for 31.9% of hospital discharges in Catalonia of children aged < 5 years during the first period and 28.9% during the second period, according to data provided by the Minimum Basic Data Set of Hospital Discharges . The preS. pneumoniae by real-time PCR in a normally sterile sample according to the methodology described [Patients aged < 5 years hospitalized with a diagnosis of IPD during the study periods in the two participating centers were recruited. IPD was defined as clinical findings of infection together with isolation and/or detection of DNA of the neumolisin gene (ply) and a wzg capsular gene (cpsA) of escribed ,26.S. pneumoniae isolated by culture were serotyped using the Quellung reaction or dot blot by the National Center for Microbiology, Majadahonda, Madrid [S. pneumoniae serotype in samples with negative culture and positive PCR was made by PCR in accordance with the previously reported methodology [The strains of , Madrid . Identifhodology ,28.The following demographic, clinical and epidemiological variables were recorded for each case: age, sex, date of birth, date of symptom onset, previous care in other hospitals, hospitalization date, clinical form of IPD , complications, intensive care unit (ICU) admission and length of stay, and pre- and post-admission antibiotic therapy (days of treatment and antibiotics administered).p < 0.05 were considered statistically significant. The analyses were conducted using the Statistical Package for Social Sciences (SPSS 19.0).Differences between the two study periods in the demographic, clinical, and epidemiological variables were analyzed using Pearson’s chi-square test for categorical variables and the Student’s t-test for continuous variables. The annual evolution of the costs associated with IPD was analyzed by simple linear regression. The linear relationship was checked by ANOVA tests, and Pearson’s correlation coefficients (Pc) were obtained. The 95% confidence intervals (CIs) were calculated, and values of The costs of IPD cases were calculated taking into account the following health service resource use: hospitalization , interhospital transfer, follow-up medical visits, and outpatient treatment with antibiotics. The unit costs of each resource were the rates published by the Catalan Health Service for the provision of health services through the network of public hospitals and other suppliers.The cost of hospitalization was estimated using the diagnostic-related group (DRG) methodology. This method is based on two concepts: the relative resource intensity (RRI), which takes into account the complexity of the cases attended, and the intensity relative hospital intensity (RHI), which measures the structure that hospital has to address the disease. Both concepts are regulated annually by the Catalan Health Service .The mean cost per hospital discharge for each facility was calculated using the following formula .Mean HoSant Joan de Déu and Vall d’Hebron are classified as high technology hospitals (Group 4). The mean costs per hospital discharge and the number of cases treated in each hospital were considered, and the weighted average was obtained so that the same unit cost was allocated to the hospital discharge regardless of the period in which the patient entered. The mean costs per hospital discharge related to prior hospitalization were calculated using their own RHI and RRI values.The 2014 RRI and RHI indices were applied, and costs were expressed as 2014 euros . Days ofThe unit cost of follow-up visits after hospital discharge was considered according to the structural complexity of the hospital center , while tTo establish the unit cost of inter-hospital transfer, we differentiated between scheduled and urgent services, defined as those that accounted for direct entry into the hospital ICU. Since the amount of an urgent inter-hospital transfer is not published in the official documents, it was estimated to be twice the amount of a non-urgent transfer ,33.The unit cost of administering outpatient antibiotics was calculated from the public sale price of the prescribed generic antibiotic and only the portion financed by the Catalan Health Service was charged as a cost.No diagnostic tests were made or samples taken from any participant in addition to those required by routine care. The study complies with the principles of the Declaration of Helsinki and the legal structure according to international human rights and biomedicine and personal data protection legislation.The Ethics Committee of Hospital Sant Joan de Déu approved the study . Informed consent signed by parents or legal guardians was given for all participants. All data were treated as confidential and records were accessed anonymously.During the two study periods, 473 cases were collected: 319 (67.4%) during the first period  and 154 (32.6%) during the second . The first half of Of the 319 IPD cases in the first period, 300 (94.0%) were serotyped. Of these, 27 (9%) were PCV7 serotypes, 182 (60.7%) were serotypes added to the PCV13, and 91 (30.3%) were non-PCV13 serotypes. During the second period, 149 (96.7%) cases were serotyped; 19 (12.7%) were PCV7 serotypes, 70 (47.0%) were serotypes added to the PCV13, and 60 (40.3%) were non-PCV13 serotypes.p = 0.028), especially for meningitis . The proportion of urgent transfers was higher during the second study period both overall , and for pneumonia  .The second half of p = 0.006) and for cases of pneumonia . The mean number of ICU days per patient during the first period was 0.8 days and peaked in 2009 (1.2 days). In 2007 and 2008, the mean ICU stay per patient was 0.5 days. During the second period, the mean number of ICU days per patient was 1.6. The mean days of ICU stay in 2012, 2013, 2014, and 2015 were 1.3, 2.1, 0.8, and 2.4 days, respectively.More cases were admitted to the ICU during the second study period overall . There were no differences in hospital and primary care follow-up visits.No significant differences were found between the two study periods in the mean duration of hospitalization and outpatient treatment according to the clinical presentation, except for meningitis, where the days of hospital antibiotic treatment were greater during the second study period , followed by meningitis , other forms of IPD , and non-focal bacteremia .During the first period, the greatest overall costs were due to hospitalization in study hospitals (56.6%), days of ICU admission (34.9%), and days of previous hospitalization (4.3%). During the second study period, the greatest overall costs were days of ICU admission (47.1%), hospitalization in study hospitals (46.6%), and days of previous hospitalization (2.8%) .For meningitis, in both study periods, the ICU stay generated the most costs  followed by hospitalization in study hospitals , and hospital follow-up visits (1.5%) during the first study period, and days of previous hospitalization (3%) in the second period.For pneumonia, the ICU stay went from second to first position as the service that generated the most costs (20.2% during the first study period and 46.7% during the second).For non-focal bacteremia, days of hospitalization (96.4%) were the costliest service during the first study period, followed by primary care follow-up visits (2.8%) and days of outpatient antibiotic treatment (0.5%). During the second study period, days of hospitalization (78.4%) were the costliest service, days of ICU stay (19.2%) occupied the second place, followed by primary care follow-up visits (1.8%).The evolution of the total annual costs associated with IPD during the two study periods is shown in p = 0.005), hospitalization in study hospitals , days of outpatient antibiotic treatment , non-urgent hospital transfers , and hospital follow-up visits . There was a significant decrease in the direct costs associated with PCV7 serotypes  but not in those associated with the serotypes added to the PCV13  or in the total annual direct costs of IPD . The direct costs associated with non-PCV13 serotypes increased   A,B.The total mean cost of IPD increased in the second study period by 31.4% (€3016.1 vs. €3963.9) . The greThe greatest increases in costs were for urgent intra-hospital transfers (276.6%) and days of ICU stay (77.4%), due to the increase in these resources in treating pneumonia . The costs of days of hospital stay in study hospitals increased by 8.1%, while those of hospitalization in pre-transfer hospitals decreased by 13.9% .During the two study periods, the proportion of IPD costs caused by PCV7 serotypes decreased slightly , while there was an important decrease in the proportion of the costs associated with serotypes added to the PCV13 between the two periods . The proportion of the cost of IPD caused by non-PCV13 serotypes increased in the second period  A.Changes in the proportion of costs associated with serotypes added to the PCV13 were observed between the two study periods. In the first period, the serotypes that produced the highest costs were 19A and 3  followed by serotype 1 . In the second period, serotype 3 accounted for 37.5% of the total costs associated with serotypes added to the PCV13 followed by serotype 19A (28.5%) and serotype 7F (20.5%) B.Invasive pneumococcal disease entails a high cost for the health system. Our results show that, after the introduction of PCV13, the direct costs associated with IPD in children aged < 5 years in Catalonia (Spain) decreased globally and for all clinical presentations due to a reduction in the number of cases. These results are comparable to the decrease in hospitalizations observed by Baldo et al.  in childThe health resources that generated the most costs varied from one study period to the other. During the first period, the days of hospitalization in study hospitals generated the highest costs. Lagos et al.  calculatThe use of major health resources increased during the second study period, resulting in increased mean costs in general, as well as for meningitis, pneumonia, and non-focal bacteremia. Comparison of the two study periods showed that the factors that increased the most in the second period were the days of ICU admission and urgent health transfers, indicating that the severity of IPD, and therefore the costs, increased during the second study period. In addition, while the overall costs of IPD decreased between the first and second periods due to the reduction in the number of cases, the cases that occurred in the second period were more severe, consumed more health resources, and had a 31.4% higher mean cost. Although some authors had already warned of a possible increase in the severity of IPD after the introduction of PCV , to the Brotons et al.  and SongPossible confounding variables were taken into account in the interpretation of the results. Regarding the medical environment, in both study periods, the same hospitals participated; both centers were reference hospitals, a similar account of hospital discharges has been maintained, and medical care circuits did not vary and neither were other pediatric hospitals in the region growing or contracting during the study periods. The same research group participated in both study periods and there were no changes in the management of IPD pediatric patients. As our study was carried out only in high-tech hospitals, there may have been a bias to a higher cost of IPD, since the cost of hospitalization is higher than in other hospitals due to the resources available in these centers and there could have been a selection of the most serious cases of IPD. However, the costs associated with IPD in the form of pneumonia found (€3639.9) were similar to the €3909 found by Baldo et al. in a very similar region . The posA limitation of the study is that, to determine the impact of PCV13 on the total direct costs of pneumococcal disease, we had no information on outpatient cases which, although having much lower unit costs, have a high prevalence that implies a large burden on the health system. However, the USA study by Huang et al.  found thFollowing the introduction of the PCV13, the direct costs associated with IPD in children aged < 5 years in Catalonia (Spain) decreased globally and for all clinical presentations due to a decrease in the number of cases. IPD cases during the PCV13 period were more severe, consumed more health resources, and had a higher mean cost. There was a significant decrease in the direct cost associated with IPD cases produced by PCV7 serotypes and the serotypes added to the PCV13, but not in non-PCV13 serotypes. The increase in costs during 2015 could be explained by the increase in the costs associated with non-PCV13 serotypes and serotype 3.