Edible insects are expected to become an important nutrient source for animals and humans in the Western world in the near future. Only a few studies on viruses in edible insects with potential for industrial rearing have been published and concern only some edible insect species. Viral pathogens that can infect insects could be non-pathogenic, or pathogenic to the insects themselves, or to humans and animals. The objective of this systematic review is to provide an overview of the viruses detected in edible insects currently considered for use in food and/or feed in the European Union or appropriate for mass rearing, and to collect information on clinical symptoms in insects and on the vector role of insects themselves. Many different virus species have been detected in edible insect species showing promise for mass production systems. These viruses could be a risk for mass insect rearing systems causing acute high mortality, a drastic decline in growth in juvenile stages and in the reproductive performance of adults. Furthermore, some viruses could pose a risk to human and animal health where insects are used for food and feed. Edible insects are expected to become an important nutrient source for animals and humans in the Western world in the near future and traditionally hold this status in many tropical countries ,2. The rBoth insects collected in Nature and those raised on farms may be infected with pathogenic microorganisms, including bacteria, viruses, fungi, protozoa and other organisms that can affect their safety as food ,29. InseThere is an abundant literature on the presence of viruses in insects of economic value or importance to public health, as in the case of silkworms and mosquitoes ,40,41. FThe focus of this study was on virus species detected to date in edible insect species currently considered for use in food and/or feed in the EU or with characteristics suited to mass rearing systems.For this systematic review, we performed a literature search to identify scientific articles reporting viruses in diverse edible insect species. The information retrieved from the literature conforms to the Preferred Reporting Items for Systematic reviews and Meta-analyses (PRISMA) statement .Bombyx mori: Musca domestica, Hermetia illucens, Tenebrio molitor, Zophobas morio, Alphitobus diaperinus, Galleria mellonella, Achroia grisella, Acheta domesticus, Gryllodes sigillatus, Locusta migratora and Schistocerca americana  have already been listed by Mleck et al. [G. assimilis and G. bimaculatus are listed by Weissmann et al. [In addition to the species considered promising by EFSA, we decided to also include: k et al.  as suitan et al.  as two oBombyx mori (Silkworm) and Apis mellifera (Western honeybee), respectively. Although the edible part of these insects (silkworm pupae and bee brood) is already consumed in many parts of the world and constitutes a promising edible resource, it is not specifically covered in this review because it is only a marginal part of the production system of these insect species [Silk and honey are the primary products of the  species ,54,55.Relevant studies were searched through two online database repositories, PubMed and Web of Science, using the keywords: “virus*” AND “scientific name of edible insect species” OR “common name of edible insect species”  have been considered separately [The edible insect species considered in this review are grouped below by order . A specific paragraph has been created for each insect species, describing the main nutritional and productive characteristics, potential for mass rearing systems, and associated viruses. Information on virus characteristics , insect stage involved during viral infection, type of viral infection , any detected symptoms or mortality, have been summarized in the tables  is used as synonym of invertebrate iridescent virus 6 (IIV-6), the type species of the genus oviridae ; Note 2:parately ,58,59,60Alphitobius diaperinus, is one of the most abundant insect pests in commercial poultry production facilities, with cosmopolitan occurrence [A. diaperinus has been listed as one of the seven insect species authorized to date for use in the large-scale production of processed animal protein (PAP) for aquaculture feeds, in accordance with EU Regulation (EC) No. 2017/893 [A. diaperinus belongs to the species with the greatest potential to be used as food and feed in the EU [The darkling beetle, currence ,62,63. S2017/893 . For thi2017/893 ,67,68,69n the EU , it couln the EU ,70,71. In the EU ,77,78,79n the EU ,76,77,79n the EU .A. diaperinus, which appears to act only as a vector and reservoir for honeybee viruses [Only two insect viruses have been found in the lesser mealworm that parasitize honeycombs: black queen cell virus (BQCV) and Israeli acute paralysis virus (IAPV) . These v viruses . At the  viruses  but sincTenebrio molitor is one of the largest stored-product beetles, which are widespread across the world [T. molitor, in the form of dried larvae, has recently been declared a safe novel food pursuant to Regulation (EU) No. 2015/2283 [T. molitor can be infected by or harbor parasites, entomopathogenic fungi, and viruses which reduce mealworm survival or reproductive success [Iridovirus and Densovirus genera, were isolated from or tested against T. molitor and in one paper this insect was used as an animal model. To date, no foodborne viruses have been detected in industrially reared mealworm larvae [The yellow mealworm he world . This behe world ,66,83. Fhe world . Besideshe world , T. moli success . To datem larvae .Iridovirus can cause disease leading to death in T. molitor: invertebrate iridescent virus 6 (IIV-6) and invertebrate iridescent virus 29 (IIV-29). IIV-6 causes paralysis in T. molitor larvae 3 days after virus infection [Iridovirus genus [T. molitor pupae [T. molitor larvae is Acheta domesticus densovirus (AdDNV) [T. molitor was used as a bioassay animal for Penaeus merguiensis densovirus (PmergDNV), which is particularly relevant for aquaculture since it causes disease in crustaceans [Two species of nfection  and thenus genus , rarely or pupae ,87 withi (AdDNV) . Mealwor (AdDNV) ,88. T. mstaceans , but it Zophobas morio, also known as the super worm, is a large beetle originating in South and Central America and introduced into other regions of Europe and Asia in recent years [Z. morio has been used as a protein source for small pets such as birds, reptiles, and small mammals [Z. morio as a nutrient source for food, livestock animal feed, and aquaculture has increased considerably [Z. morio is not listed in Regulation (EU) No 2017/893 and is therefore not officially authorized for inclusion in aquafeeds in the EU [T. molitor, density independent cannibalism [nt years ,90,91. Z mammals , but has mammals . Over thiderably ,93,94. Tn the EU . The mainibalism , increasnibalism , high denibalism ,98.Z. morio larvae [Z. morio colony in Hungary, where sudden large losses (80–90%) of Z. morio larvae were observed by a breeder in Budapest. Larvae displayed less activity, unusual behavior, and failed to pupate. Diagnostic methods detected densovirus in colony larvae but no further identification was performed [Z. morio larvae cultured in Russia; in one month, middle-aged larvae developed symptoms of acute disease affecting the locomotion system, inducing swirling, rolling and chaotic wandering of larvae, and within several days, up to 90–100% of the colony had died. Prior to death the larvae showed no morphological changes but after death the cadavers blackened quickly, were more intensively stained, and the inner content was partially liquefied. Through biomolecular techniques the virus was attributed to Densoviridae, with maximal similarity to two Blatella germanica densovirus-like isolates from the bat [Little information is available on viral diseases in the super mealworm, with only two reports available to date on virus infection in o larvae ,100. Theerformed . Tokarev the bat . Thus, tViruses detected in coleopteran edible species are listed in Acheta domesticus, is currently one of the most widely farmed insects, particularly in North America and Europe, and constitutes a thriving pet/reptile feeder insect market worldwide. This cricket also represents an emerging, vibrant insect-based food industry due to its high protein content (about 70% by dry weight), its short life cycle (around 5 weeks), and its prolificacy  [A. domesticus has been authorized for use as processed animal protein (PAP) for aquafeeds (EU Regulation 2017/893) [The European house cricket, 00 eggs) ,102. Nut00 eggs) ,105,106.017/893) .A. domesticus industry is Acheta domesticus densovirus, which is able to decimate commercial mass rearings in just a few days, leading to a fall in production and even extinction of local cricket populations [A. domesticus from a Swiss commercial mass rearing facility in 1977 [Viruses affecting this species of cricket have been reported in several papers ,35,42. Iulations ,109,110. in 1977 . In Nort in 1977  in a sma in 1977 . Symptom in 1977 ,108,109. in 1977 ,109. AdD in 1977 .Acheta domesticus mini ambidensovirus (AdMADV), a new ambisense densovirus, and Acheta domesticus volvovirus (AdVVV), a single-stranded, circular DNA virus; but no further information is currently available on these viruses [Acheta domesticus iflavirus (AdIV), has recently been isolated from both wild and commercially reared A. domesticus [A. domesticus is highly susceptible to both IIV-6 and its novel strain, cricket iridovirus (CrIV) [A. domesticus nymphs and adults from colonies of a commercial cricket producer in Europe. This virus, transmitted orally, led to unusual mortalities as well as greatly reduced fecundity and life span [Other viruses observed in cricket facilities are  viruses ,113,114.mesticus ,115. A. s (CrIV) . CrIV waife span . The fatife span .A. domesticus from dead crickets of a commercial cricket farm in the United States of America [A. domesticus is an omnivorous scavenger which could ingest virus-killed insects, thereby simply becoming a mechanical vector of other insect viruses, as Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and two recombinant strains (AcMNPV.AaIT and AcJHE.SG AcMNPV), bait-cricket virus (BCV), and Solenopsis invicta virus 3 (SINV-3) [Cricket paralysis virus (CrPV) was isolated from  America ,118. A. (SINV-3) ,121,122.(SINV-3) ,123,124.Gryllodes sigillatus, is a cosmopolitan cricket that lives in close association with human dwellings [G. sigillatus is sold in both US and European pet food stores [Acheta domesticus densovirus (AdDNV) has been detected in G. sigillatus but it is less susceptible to AdDNV compared to other orthopteran species [The banded cricket , wellings . Little wellings ,126 but wellings . G. sigid stores  and its d stores ,127,128. species ,81.Gryllus assimilis, was first described from Jamaica and is widespread in the West Indies, Brazil, Central America, Mexico, and in five of the southernmost U.S. States [G. assimilis: cricket iridovirus (CrIV), Acheta domesticus densovirus (AdDNV), and a new volvolovirus named Acheta domesticus volvolovirus (AdVVV). G. assimilis, especially its first instars, is highly susceptible to CrIV [G. assimilis, reared to adulthood, were reported to produce large numbers of eggs that hatched and developed without displaying signs of virus infection [G. assimilis in Japan but little is known about it [The Jamaican field cricket, . States . This cr. States , but lim. States . To date to CrIV , but usu to CrIV . Furthernfection . The novnfection  has beenabout it .Gryllus bimaculatus is apparently the most widely distributed Gryllus species and is found at the tip of South Africa, in northern Europe, and as far east as Thailand [G. bimaculatus is commonly consumed as food in different parts of the world [Commonly called the two spotted cricket, Thailand . G. bimahe world ,131. Numhe world ,133, andhe world  and foodhe world ,136,137.G. bimaculatus [Iridovirus, Densovirus, Nudivirus and Cripavirus. G. bimaculatus seems to be highly susceptible to iridovirus but resistant to densovirus [Iridoviridae family, invertebrate iridescent virus (IIV-6) and two variants or novel strains of IIV-6—Gryllus bimaculatus iridescent virus (GbIV) and cricket iridovirus (CrIV) could seriously damage G. bimaculatus mass production, causing colony collapse in two weeks [Gryllus bimaculatus iridescent virus (GbIV) caused the death of all individuals of a colony by 14 days, showing clinically apparent behavior as apathy, ataxia, and disorientation [G. bimaculatus was tested, revealing mortality rates of between 20% and 35%, and the virus was then re-isolated from several fat-body samples of the cricket [To date seven different viral pathogens have been described in reared aculatus ,35,42 bensovirus ,88. Withwo weeks ,116. Invwo weeks . Cricketentation . The facentation  and coul cricket . These f cricket ,138,139. cricket .Gryllus bimaculatus nudivirus (GbNV) [G. bimaculatus nymphs but has a chronic course in adult specimens. Infections are reported to occur primarily during nymphal development, especially by cannibalistic feeding on moribund or dead specimens [Acheta dometicus densovirus (AdDNV), while testing positive at the lowest dilution, G. bimaculatus so far appears resistant [s (GbNV) ,141,142 pecimens . Affectepecimens . As regaesistant ,88. Cricesistant ,118,143.Locusta migratoria is one of the insects responsible for crop devastation in certain developing countries and among the most economically important locusts [L. migratoria belong to four families, i.e., Poxviridae, Baculoviridae, Iridoviridae, and Reoviridae [L. migratoria is susceptible, to varying degrees, to entomopoxviruses (EPVs), isolated from different orthopteran species [L. migratoria nymphs fed with entomopoxvirus occlusion bodies isolated from Melanoplus sanguinipes reached 90% in the 60 days after virus inoculation [The African migratory locust  locusts ,144. Viroviridae ,149,150. species ,146, and species ,146. Forculation . Arphia culation .L. migratoria can be infected with nucleolyhedrovirus of Spodoptera littoralis (SINPV-type B), a Lepidopteran species [L. migratoria, showing a disease pattern termed ‘dark cheeks’. However, the viral amounts in the infected locusts were very low and the role of the virus in generating the observed disease remained ambiguous [L. migratoria instars [L. migratoria was recorded during cytoplasmic polyhedrosis virus (CPV) infection [L. migratoria were heavily infected by the two virus isolates, invertebrate iridescent virus (IIV-6) and cricket iridovirus (CrIV), with mortality as high as 100% [L. migratoria, causing characteristic symptoms and fatal disease [ species ,149. Thimbiguous . A subsenfection ; to date as 100% . Iridovi disease .Schistocerca gregaria, due its high reproductive potential, represents another major pest in agriculture and, as other locust species, has been extensively studied in previous years [S. gregaria, showing signs of inactivity and a high death rate [Spodoptera littoralis nucleopolyhedrovirus (SINPV) was reported to be involved in a disease outbreak in immature S. gregaria [S. gregaria was detected by Purrini and Rohde [S. gregaria (L1–L3) could also be heavily infected by the two Iridovirus: invertebrate iridescent virus (IIV-6) and cricket iridovirus (CrIV) [The desert locust us years ,144. In ath rate . Spodoptgregaria . Entomopnd Rohde . Youngers (CrIV) .Schistocerca americana, commonly known as the American grasshopper [M. sanguinipes, and infected S. americana. The latter species proved only slightly susceptible: after a latent period of about 12 days, infected specimens exhibited general torpor, a decreased rate of development, and eventual death. The fat body—the only tissue invaded—became generally hypertrophic, changing from its normal glossy yellow to a fluffy gray. Examination of sectioned tissues revealed that most of the fat-body cells were filled with large numbers of polyhedral bodies [S. americana specimens, showing symptoms and signs of disease, were found to be infected with Melanoplus sunguinipes entomopoxvirus. Placed in a rearing facility, these specimens spread the virus to other grasshopper species through horizontal transmission, apparently by consumption of infected cadavers [S. americana 6 days after intrathoracic inoculation [Limited information exists on sshopper . In 1966sshopper  isolatedl bodies . Field-ccadavers ,155. Theculation ,157. Viruses detected in orthopteran edible species are listed in Hermetia illucens, is a saprophytic insect, which currently has a cosmopolitan distribution in tropical and temperate areas [H. illucens is one of the most promising insect species for food and feed production, efficient bioconversion of food waste, and biodiesel and fertilizer production [The Black Soldier Fly (BSF), te areas ,160. H. oduction ,164,165.oduction ; (ii) thoduction ; (iii) poduction .Salmonella spp., Orthoreovirus, Mastadenovirus, Teschovirus), but the analyses were performed only on the substrate and not on the larvae [In recent years, economic interest in this species has grown yielding abundant scientific literature on its behavior, rearing, nutritional value, and industrial applications ,172,173.e larvae .Musca domestica, distributed worldwide, is the most common species of fly, living in close association with humans and domestic animals, making it one of the most highly studied insect pests [M. domestica to reproduce quickly could be exploited to produce larvae as a source of protein for animal feed [H. illucens, larvae could grow in different substrates making M. domestica a promising insect for organic waste degradation [M. domestica [The housefly, ct pests ,177. Besct pests . The abimal feed ,181,182.radation ,185,186.omestica ,187.M. domestica can harbor several viruses pathogenic for both humans and livestock but, to date, only two insect viruses that are pathogenic for the fly itself have been detected [H. illucens, M. domestica adults are synanthropic and therefore any pathogens being carried could easily be transmitted to humans, animals, and other flies. Adult flies act only as mechanical carriers of human viruses after contamination by infected human fecal material [The housefly is known to carry more than 130 pathogens, including bacteria, viruses, fungi and parasites, some of which can cause serious, life-threatening diseases in humans and animals, or diseases in flies themselves ,177. M. detected ,190,191.detected ,190. Conmaterial ,193. Thematerial ,196,197.material .M. domestica is Musca domestica salivary gland hypertrophy virus (MdSGHV). It was isolated in 1993 [M. domestica (only adults) are naturally infected with MdSGHV but its incidence varies widely among farms and at different times of the year [M. domestica flies , in addition to suppressing ovarian development in infected females, inhibiting egg production and resulting in female sterility [The main virus affecting adult the year , with ththe year ,201. Theterility ,204,205.terility . Since sterility ,203,204.terility . Typicalterility  and the terility . The virterility ,209,210.M. domestica adults is a reovirus, detected by Moussa in 1978 and now named idnoreovirus 3 (Idno-3) [The second virus causing disease and mortality in (Idno-3) . This vi(Idno-3) ,211. No M. domestica has also been reported to mechanically transmit several types of viral pathogens to livestock including: avian influenza virus (AIV), both high and low pathogenic strains [ strains ,215,216, strains , Newcast strains ,221,222, strains , porcine strains ,227,228, strains , porcine strains , African strains ,232, Auj strains , senecav strains , Rift Va strains , Aleutia strains ,237, and strains ,239.ViruAchroia grisella, is a species closely related to Galleria mellonella [The lesser wax moth, llonella . As compllonella ,243. To G. mellonella, is a ubiquitous pest of field-based honeybee colonies and stored combs due to the destructive feeding habit of its larvae [G. mellonella has been available as pet food and bait for many years in several European countries and in the USA [G. mellonella is a reliable model organism to assay pathogenicity of human, animal and insect pathogens  as well as to test the effectiveness and toxicity of antimicrobial compounds [G. mellonella [G. mellonella infected with bovine herpes simplex virus-1 (BHSV-1) [G. mellonella larvae, but no mortality was detected [The greater wax moth (GWM), s larvae ,244. Rec the USA . This mo the USA ,248,249, the USA . G. mellompounds ,252,253.llonella ,250, but(BHSV-1) . In the detected .Nodamura virus, an insect picornavirus that can also infect vertebrates, is able to infect and kill greater wax moth larvae ,256. TheG. mellonella larvae. They belong to Densovirus, Iridovirus, Baculoviruses, Cripavirus and Triatovirus genera and can lead to patent and asymptomatic infections or severe symptoms and mass mortality. For example, Galleria mellonella densovirus (GmDV) can cause the death of the entire colony while Acheta domesticus densovirus (AdDNV) cannot even infect the colony [Galleria mellonella densovirus (GmDV) is the most highly studied and described densovirus in this moth species [G. mellonella specimens and other insect species . All said viral isolates have been shown to be pathogenic especially for G. mellonella larvae [Many insect viruses have been detected in e colony ,258. Gal species ,259. GmDa larvae ,264,265.Galleria mellonella nucleopolyhedrovirus (GmNPV) is highly pathogenic for G. mellonella, particularly in the preimaginal stages (third instars) [G. mellonella larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) ceased to feed and began to wilt after 17 days. Mortality occurred during between 19 and 27 days. Some infected insects did not die until entering the pupal stage [Malacosoma franconicum nucleopolyhedrovirus (ManeNPV-T3) caused 25% mortality in third instars of G. mellonella [instars) ,266,267.instars) . During instars) . G. mellal stage . The Malllonella .G. mellonelIa is highly permissive, with different levels of magnitude, to many Iridoviruses (IVs) isolated from insect species belonging to different orders [G. mellonella larvae [G. mellonella larvae [G. mellonella, with the average period to death occurring at about 14 days after injection [G. mellonella larvae [Sericesthis pruinosa, develops in G. mellonella plasmatocytes and adipohemocytes leading to visible alteration (hypertrophy) in these cells within 24 h of virus inoculation [G. mellonella larvae [t orders ,272,273.a larvae . Invertea larvae ,271. Thea larvae . Cricketa larvae ,116. Tipa larvae ,279,280.njection . Wax-motnjection . Apis cea larvae . Serisceculation ,284,285.a larvae ; specifia larvae . Honeybea larvae .A. fumigatus with A78 mycovirus caused a significant increase in radial growth and virulence in a moth model [G. mellonella concern a small spherical virus, Galleria free virus (GFV), isolated from G. mellonella [Galleria mellonella cell line virus (GmclV), apparently persistently infects the G. mellonella cell line (GmclV) and can be efficiently induced to replicate by the introduction of other insect viruses, causing complete death of the colony in 5 days [G. mellonella—Pariacoto virus and Heteronychus arator virus—show striking similarities to Nodamura virus [Spodoptera eridania), is able to multiply in G. mellonella larvae [G. mellonella larvae inactive, flaccid, unresponsive to touch, and paralyzed. Mortality reaches 50% in 20 days after infection [Mycoviruses are a specific group of viruses that naturally infect and replicate in fungi and are able to alter fungal growth. Accordingly, a lower initial concentration of spores can still become lethal post infection. Infection of th model . Other rllonella . The Galn 5 days . The lasra virus . Pariaoca larvae ,293. Founfection  and the Viruses detected in lepidopteran edible species are listed in At present there is no treatment for viral infections in edible insects and therefore prevention and control measures are pivotal for insect mass rearing systems. Mass rearing strategies must focus on defining and standardizing Good Farming Practices (GFP) . First oA. grisella and H. illucens), while reports on others are sometimes very limited in size or old. Among the insect orders considered, orthopteran seems to be the one most affected by viral pathogens belonging to seven families ; the most concerning viruses affecting Orthoptera are species belonging to Iridoviridae and Densoviridae families. The Lepidoptera order (represented by G. mellonella) seems heavily affected by virus species belonging to Baculoviridae and Iridoviridae families, while Densoviruses are rarely reported; other virus species reported to affect G. mellonella are member of Dicistoviridae, Parvoviridae and Picornaviridae families. Two iridoviruses and one densovirus are reported to cause mortality in the coleopteran order while only two insect viruses have been described as pathogenic for Diptera.The retrieved literature reveals that many viruses, belonging to 22 different families, have been observed in edible insect species . HoweverM. domestica). In the other two cases (viruses that multiplied in edible insect species), edible insects act as a biological vector. In the investigated edible insect species, different patterns of viral infection can occur ranging from asymptomatic to highly pathogenic, or even lethal [In the retrieved literature, viruses detected in edible insects can be divided into three major categories: (i) viruses that neither multiply nor cause disease in edible insect species; (ii) viruses that multiply and cause disease and mortality in edible insect species; (iii) viruses that multiply but do not cause disease or decrease performance in edible insect species (asymptomatic). In the first case, those viruses are casually associated with edible insects that act only as a mechanical vector without affecting their productivity or prolificacy  with no consequences for larvae, representing the valuable edible part, while other viruses affect only larvae . Greater attention must therefore be paid to these viruses in the production sector of insect facilities.Viruses that pose the highest risk for the collapse of mass insect rearing systems are those causing acute and high mortality because they can decimate commercial mass farms or entire colonies within a few days ,100,258.A. domesticus) [A. domesticus is highly susceptible to Acheta domesticus densovirus while G. assimilis usually seems to be infected with AdDNV to a much lower degree; G. sigillatus is less susceptible to AdDNV compared to other orthopteran species and G. bimaculatus so far appears resistant [G. assimilis, G. sigillatus, and G. bimaculatus have been proposed as the best replacement crickets to avoid heavy losses in commercial production [In heterometabolic insects, all stages can be infected by viruses but the juvenile stages are usually the most affected  ,116. Theesistant ,47,90. Foduction ,81. Besioduction ,63 and toduction .T. molitor, A. diaperinus and G. sigillatus) is considered low. Foodborne viruses could be introduced through rearing substrate or operator handling and transferred beyond primary production. This prompts the need to carry out more studies and experimental infections to produce more evidence of the low safety risk from foodborne viruses.To date, only one article has investigated the presence of foodborne viruses in three species of industrially reared insects for food , yieldinThe retrieved literature revealed that the number of viruses detected in edible insects is high, with more than 70 species listed and 36 able to cause disease and mortality. Viruses could be more or less species-specific and could infect edible insects at different life stages. Only insect-specific viruses could be a matter of concern in mass-rearing systems as they actively replicate and persist on the target species. Viral infection could have different consequences on mass rearing systems ranging from asymptomatic infection to the entire collapse of the colony. Since to date there is no cure for viral infections in edible insects, preventative measures are the only affordable strategy available. Thus, biosecurity is pivotal for insect mass rearing systems.To enable edible insects to become a safe nutrient source for animals and humans in the Western world, more investigations are warranted to better understand the effective impact of both insect and vertebrate viruses in industrialized rearing systems.


Candida auris provides a substantial global nosocomial threat clinically. With the recent emergence that the organism can readily colonize skin niches, it will likely continue to pose a risk in health care units, particularly to patients undergoing surgery. The purpose of this study was to investigate the efficacy of antifungal-loaded calcium sulfate (CS) beads in combatting C. auris infection. We demonstrate that the CS-packed beads have the potential to interfere with planktonic and sessile C. auris. Candida auris is a nosocomial pathogen that was first identified in 2009  as a biomaterial is not uncommon. CS has been developed as a vehicle for delivering antibiotics to infected surgical sites . The mecC. auris biofilm formation and associated pathogenicity. The efficacies of three antifungals, caspofungin (CSP), fluconazole (FLZ), and amphotericin B (AMB) incorporated into CS beads, were tested against planktonic and sessile C. auris NCPF 8978. The potency of the antifungal-loaded CS beads would also be assessed in a three-dimensional (3D) organotypic skin epidermis model.This study serves to investigate the potential use of antifungal-loaded CS beads in preventing C. auris was found to be susceptible to a released concentration of approximately 1.2 μg/mL for CSP across all days, varied from 40 to 80 μg/mL for FLZ, and maintained at 0.9 μg/mL for AMB over the full 7 days. Specifically, the concentration of FLZ was 80 μg/mL on days 1, 5, 6, and 7, while 40 μg/mL on days 2, 3, and 4.First, a broth microdilution test was used to assess drug release across the 7 days of suspension of CS beads in RPMI media. C. auris biofilms across all three time points   .For the biofilm studies, results of the XTT  and crystal violet CV) assays indicated that CSP and AMB were more effective than FLZ in reducing the metabolic activity and biomass of the V assays < 0.001) . Interes< 0.001)  and C bu< 0.001) . Biofilm< 0.001)  but sign< 0.001)  and F. CC. auris   . SEM imaC. auris fungal load within a skin epidermis coculture model. The experimental setup is highlighted in 3; ***, P < 0.001). CSP was effective in reducing the fungal load 2-fold from ∼1.2 × 104 colony forming equivalents (CFE)/tissue in controls to ∼0.6 × 104 CFE/tissue following treatment . FLZ treatment did not change the overall levels of C. auris colonized within the tissue  and drug efflux pumps  serve to increase FLZ tolerance . In brief, coverslips containing the biofilms were removed and initially fixed and then further processed for SEM imaging as described previously , 20. BioC. auris was inoculated on a wounded 3D skin epidermis model  with CS beads. The model set up was created as previously described , respectively. Fungal load was assessed using qPCR as above. For the host gene response, RNA was converted to cDNA before a custom RTlsewhere .P was <0.01.Statistical analyses and graph production were performed using GraphPad Prism . One-way analysis of variance (ANOVA) with Tukey’s post-test was used to assess differences in antifungal efficacies. For CFU and CFE counts, data was log transformed before analysis. Statistical significance was achieved if


We reconstructed the 2016–2017 Zika virus epidemic in Puerto Rico by using complete genomes to uncover the epidemic’s origin, spread, and evolutionary dynamics. Our study revealed that the epidemic was propelled by multiple introductions that spread across the island, intricate evolutionary patterns, and ≈10 months of cryptic transmission. Puerto Rico reported the first confirmed case of Zika virus (ZIKV) disease in November 2015 and subsequently experienced epidemic transmission that peaked by mid-August 2016 .In addition, we analyzed the temporal molecular evolutionary signal in our dataset by reconstructing time-calibrated phylogenies by using genomes annotated with date of sample collection based on year, month, and days for temporal precision. The correlation between date of sample collection and root-to-tip genetic distance supported the heterochronous nature of our dataset. The estimated divergence from the root  occurred in February 2013 (because 2013–2014 ZIKV genomes from French Polynesia were used as the root), and the within-epidemic evolutionary rate was 1.09 × 10−3 (95% HPD 1.13–2.10 × 10−3) substitutions/site/year. In addition, PR C1 was observed to diverge further into 2 subclades (SC1 and SC2) spreading across the island. The second clade, Puerto Rico clade 2 (PR C2), presents a smaller autochthonous monophyletic cluster that originated from viruses in Central America, including Nicaragua and Honduras (−3 (95% HPD 1.1–2.64 × 10−3). We compared the ZIKV epidemic history of Puerto Rico to the time-calibrated Bayesian phylogenies and observed that the tMRCA of PR C1 precedes the initial confirmation of ZIKV in the island through traditional surveillance methods by 3–10 months and that expansion of all PR lineages coincides with the peak of the epidemic curve (Bayesian reconstruction of Puerto Rico clade 1 (PR C1) presents the largest autochthonous monophyletic cluster that originated from viruses from South America and the Caribbean, including Brazil, Suriname, French Guyana, the US Virgin Islands, and Dominican Republic . tMRCA eHonduras . Our tMRic curve . We asseWe inferred past viral population dynamics by using Bayesian Skygrid plots, which show an increase in genomic diversity that coincides in time with the emergence of ZIKV in the Americas, followed by a series of fluctuations in the effective population size, characteristic of the virus spreading rapidly through the region . In Puerto Rico, we observed a similar sharp increase upon emergence and subsequent patterns that mirror the trends observed in the Americas.Our study revealed the origin and epidemic spread of ZIKV in the island after a period of cryptic transmission undetected by traditional surveillance. Similar cryptic transmission was reported in Brazil and Colombia (The integration of genomic epidemiology to arbovirus surveillance has proven to be central to the ascertainment of disease epidemiology, uncovering information otherwise concealed by the nature of the disease and limitations of surveillance systems. Fundamentally, integrated proactive genomic surveillance may help us to predict virus emergence and mitigate more effectively their regional or global expansion.Additional information about tracing the origin, spread, and molecular evolution of Zika virus, Puerto Rico, 2016–2017.


To determine the extent of exposure to Zika virus (ZIKV) and chikungunya virus (CHIKV) in Jamaica, we collected serum from 584 pregnant women during 2017–2019. We found that 15.6% had antibodies against ZIKV and 83.6% against CHIKV. These results indicate potential recirculation of ZIKV but not CHIKV in the near future.  The recent introductions of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas led to widespread epidemics with substantial health and economic effects. Small island developing states in the Caribbean are particularly affected by emerging mosquitoborne disease, primarily because of year-round climatic conditions favorable for mosquito breeding, high levels of poverty, and extensive migration.In Jamaica, introductions of CHIKV in 2014 and ZIKV in 2016 led to epidemics that overwhelmed the healthcare system  from June 28, 2017, through April 15, 2019. Pregnant women https://www.euroimmun.com) and BLACKBOX Zika virus ELISA . Because serologic cross-reactivity with related flaviviruses can be problematic for accurately identifying past ZIKV infections, it is imperative that ZIKV serologic assays account for cross-reactive antibodies. The BLACKBOX Zika virus ELISA is a ZIKV immune complex–binding IgG ELISA that is highly specific and does not show cross-reactivity with dengue virus (DENV)  were positive for CHIKV IgG , and 15.https://www.moh.gov). All 89 samples were negative for CHIKV IgG and ZIKV IgG , indicating that false-positive results were unlikely to affect the reported CHIKV and ZIKV seroprevalence for 2017–2019.To ensure that the seroprevalence results were not inflated by false positives, we also examined 89 archived serum samples from pregnant women who had attended 1 of the 5 clinics examined (clinic D) during June–December 2013. This period predates CHIKV and ZIKV introductions into Jamaica and was a period of low DENV circulation, similar to 2017–2019 (Ministry of Health Epidemiology Bulletin, Our study indicates a high level of past CHIKV infections and a low level of ZIKV exposure among pregnant women in Jamaica receiving antenatal care during 2017–2019. We offer several possible explanations for the higher seroprevalence of CHIKV compared with ZIKV. CHIKV infections result in substantially greater viremia, which could lead to increased transmission rates for CHIKV. In addition to the lower viremia associated with ZIKV, preexisting antibodies to DENV can be cross-protective against ZIKV infection  Figure cCases of CHIKV and ZIKV infection in Jamaica have been extremely limited since their initial epidemic years. Only 21 CHIKV cases were reported in Jamaica during 2015–June 2020, and only 1 ZIKV case was reported during 2017–June 2020. CHIKV cases will probably remain limited in Jamaica until a more substantial portion of the population lacks immunity. In contrast, ZIKV could possibly circulate again in Jamaica in the near future because of low population immunity and waning cross-reactive DENV antibodies.Additional methods and results for study of antenatal seroprevalence of Zika and chikungunya viruses, Jamaica, 2017–2019.


Cost–benefit analysis (CBA), as a common instrument in the decision making process on how to allocate financial resources, has been widely used in various research areas and in almost all of countries over the world. However, the origin and the historical development of CBA has long been subject to neglect. We attempt to fill this gap and clarify the origin and the early development of CBA.A comparative analysis is used to investigate the origin and the early development of CBA in France and the USA. The comparison is focused on two questions: (1) which criteria should be applied to decide whether or not a project should be carried out, and (2) with which procedure these criteria can be used for real projects.The origin of CBA can be dated back to the work of Saint-Pierre in France in 1708. Dupuit introduces the concept of consumer’s surplus that founds the economic basis of CBA. These works are not taken seriously in France and do not draw attention from other countries. Hence, until the 1930s, the principle of CBA is newly proposed in the US and the Green Book marks the mature of CBA.The early development of CBA in France and the US is independent from the aspects of historical background, personnel, approaches and standardization. This study could help researchers of various disciplines be sure about the history of CBA when they perform this analysis in their research areas. Cost–benefit analysis (CBA) is defined as a systematic cataloguing of impacts as benefits (pros) and costs (cons), valuing in dollars with assigned weights, and then determining the proposal relative to the status quo by the net benefits (benefits minus costs) or the benefit–cost ratio (divide benefits by costs) . CBA is By 2020, CBA has been widely applied to various research areas in almost all countries over the world. Based on the Web of Science Core Collection, the search of cost–benefit analysis in the topic during the period 1900–2020  results in a total of 54,445 publications. The first application of CBA was published in 1951, and studies using CBA have seen significant increase since the 1990s . Ha. Ha28]).Therefore, in this paper, we attempt to fill this gap and clarify this confusion by tracing out the origin of CBA and the early development of calculation methods in France and the US, respectively. This study could help researchers of various disciplines be sure about the history of CBA when they perform this analysis in their research areas.The comparative analysis method is used to investigate the origin of CBA. The comparison is focused on two questions: (1) which criteria should be applied to decide whether or not a project should be carried out, and (2) with which procedure these criteria can be used for real projects. Ekelund and Hébert  and HufsThe idea of a national transportation network in France began in the seventeenth century, since when French engineers worked consciously at introducing a decision rule to guide the construction of public works. The earliest demands depended on French engineers by military considerations, the most prominent engineer of this era was Sébastien Vauban, whose key role was associated to his formative influence on the Corpes des Ingénieurs des Ponts et Chaussées, which was instituted in 1716. A central office within the Corps was established in 1747 for the purpose of training more men and improving their effectiveness, it gradually evolved into the École des Ponts et Chaussées in 1775 and was renamed the École Nationale des Ponts et Chaussées (ENPC) after the Revolution. At the ENPC mathematics was highly appreciated, courses were also given in the engineering subjects of road, bridge and canal building, flood control, harbour improvement, and railroad construction, and economic studies were incorporated into coursework early on. In the first half of the nineteenth century the graduates of the ENPC have been a well-trained group, referred to as Ponts engineers, who developed some fundamental principles and analytical tools to solve cost–benefit calculations of the construction of public works. Jules Dupuit was the culmination among those brilliant engineers in this field .The first formal cost–benefit study was undertaken in 1708 by the Abbé de Saint-Pierre in measuring the incremental benefits of road improvements. He theorized that incremental benefits would result from increased trade and reduced transport costs. The benefit of increased trade was calculated in two steps. First, the annual value of agricultural output in each province from the annual tax revenue collected was estimated. Then a loss factor, percentage of annual output not produced due to the impossibility of transport, was added to this magnitude. The resulting figure indicated the benefit of increased trade because this loss would be restored by improving the roads. The benefit of reduced transport costs was calculated by the savings per horse and driver. Assuming that the roads were equally passable in all seasons, horses would be able to carry 20 percent more weight, and more trips could be made, only 80 percent as many horses would be required as before the road improvements, resulting in annual savings of 20 percent fewer horses and drivers. On the cost side, the annual additional expenses consisted of costs for administration, repair and continuing maintenance .From the very beginning of CBA, Saint-Pierre was sensitive to the use of incremental analysis in evaluating public goods. He was also alert to the indirect of secondary benefits by observing that better roads could attract industry and trade, which in turn could increase employment. Additionally, he was well aware that this kind of analytical techniques could be fruitfully applied to all public works. Saint-Pierre framed the important issues that were confronted by generations of Ponts engineers, and his argument was accepted by the engineers of the newly formed Corps .The era of canal construction in pre-revolutionary France gave the engineers the possibility to turn their cost–benefit calculations to greater effect because the benefits and costs of canals are more precisely economic and more susceptible to measurement. However, due to technical and financial difficulties, the wave of canal construction failed to stimulate much analytical progress. A project for constructing a canal must be certified by engineers according to the public utility of the canal, but the engineers at that time did not understand the concept of demand. Benefits could neither be calculated by increased trade nor by reductions in transport costs, because no trade had existed before. Therefore, benefits were identified chiefly in terms of value of time saved in the shipment of goods, which was notoriously difficult to deal with. Two decision rules emerged eventually to solve these difficulties. First, a canal produced net utility when the resulting savings in transport costs were greater than its construction costs, and second, a canal adds utility when, treating construction costs as sunk, its toll revenues exceed its maintenance costs. But the appropriate level of tolls posed another vexing problem because any levy reduced the public utility of canals .The revolutionary spirit made all past institutions suspect, including the Corps and the École, so that the old regulations in relate to public works were in part abolished, in part abandoned, and altogether dependent on local conditions. After ascending to power Napoleon Bonaparte helped restore the order to the administration of the Corps and began to assign many new projects, most of which were driven by political or military considerations. He was mainly concerned with cost and speed of project construction, therefore, the engineers’ attention was focused on cheapness and expediency rather than on calculating expected benefits. The problem of minimizing transport cost had been solved mathematically by Gaspard Monge in 1776, but this was not a complete solution to the problem at hand because it shed no light on the issue of benefits. It is just on the benefit side that real progress should be made .The engineers were recentralized in the Ministry of the Interior during the Restoration, making a fertile period in economic analysis from the 1820s to 1840s. The stimulus given to canal construction prompted a number of minor advances in the formulation of CBA.In 1822, focusing on the value of time saved in transport and the amortized costs of building and maintaining a canal, Pierre-Simon Girard tried to measure the benefit of the canal in physical terms by employing a curious combination of hydraulics and economics. The shortcomings of his method were demonstrated by Louis-Joseph Favier in 1824, who established the principle that a public work could be justified when it conveyed positive net utility, that is, the amount of net revenue from the public work must be greater than the cost of (re)construction, disregarding how the revenues and costs were assigned. To emphasize the choice between alternative public investments Favier derived a rule stating that a public work is to be preferred if its net utility exceeds that of another, taking into account the life of the respective constructions and amortized costs .C), the price of goods transported by road (r), the price of goods transported by canal (c), cost savings to consumers attributable to the canal (S = r − c), and the annual amount of goods transported on the new canal (n). Since n is a function of S, there must be some n′ and S′ such that n′S′ = C, so n′ is the minimum demand being sought. If n > n′, the state would gain annually a net amount equal to S times (n − n′), while if n < n′, the state would lose annually a similar amount [As early as 1830, a prominent Ponts engineer named Henri Navier set up a cost–benefit principle that public works should be provided only if the total benefits exceeds the total costs by measuring the benefits of new transport facilities based on an estimate of cost savings. His effort produced a decision rule that allowed for calculating minimum demand for new public works, below which the construction would be against the interests of the state. Algebraically, Navier defined the annual recurrent costs related to a new canal , the transport costs per ton and kilometer (p), the distance of the route (l), a sum of outlay (A), and the construction and maintenance costs (C). His analysis distinguished between communications already existed and new ones to be established. In the first case, he argued that the most preferable project should be the one over which, for a given outlay A, the greatest amount of goods could be carried. Since A = nlp, he took n = A/lp as the typical form of the solution and concluded that to the least product of l and p would correspond the greatest value of n, and consequently the most preferable project. In the second case, the construction and maintenance costs should be taken into account. Courtois introduced the ratio of the amount of goods carried per unit of construction and the maintenance costs as the benefit criterion which he called “the measure of advantages”. He took n/C = A/Clp as the typical form to determine the character of the project with the maximum of advantages [In 1833, Charlemagne Courtois developed a single principle for the selection of the most preferable transport project linking two cities. That was to choose the project that, given the costs, provided the greatest benefit. To identify the benefits of a project, Courtois considered as relevant variables the amount of goods in tons , the annual maintenance costs (d) normalized according to average costs of labor and materials, the appropriate discount rate (r), and the annual savings in maintenance costs on the old road (S) owing to less traffic after the new road is built. Thus, the annual expense of the new road (C) is equal to cr + d − S. On the benefit side, he defined the average transport cost on the new road (p) calculated as a function of weight, the average transport cost on the old road (q), and the estimated amount of goods to be transported on the new road (n). Thus, the total benefit of the new road (B) is equal to n(q − p). For the value n was not given, Mondot proposed the measure of “the real utility per unit of expenditure”, which is equal to (q − p)/C, as “the administrative value” of a project. What he called a “normal project” was the one with the highest “administrative value”. Mondot’s work typified the simple definition of CBA by proposing a criterion that compared disadvantages (C) with advantages (B). So long as a large number of projects were desirable, rigorous estimate of transport demand (n) on new routes was not always a pressing problem, for one could simply reject all projects for which B − C was not sufficiently positive [On the cost side, Mondot defined the construction costs  and water system in a town (public works), he came to the conclusion that “each consumer himself attaches a different utility to the same thing according to the quantity which he can consume”. Then he distinguished between the absolute utility and the relative utility. “In general, the relative utility of a product is expressed by the difference between the sacrifice which the purchaser would be willing to make in order to get it, and the purchase price he has to pay in exchange”, supposing that the market price of the product is more or less equivalent to the costs of production. Although Dupuit did not identify the exact concept of marginal utility, he did illustrate the idea from an empirical consideration that Ponts engineers typically confronted, and concluded that “in general every rise of fall in price decreases or increases utility by an amount equal to this variation for those who are consumers in both situations; for those who disappear or who appear, the utility lost or acquired is equal to the old or to the new relative utility yielded to them by the product” , 14, 36.q = f(p), thus placing the independent variable (price) on the x axis and the dependent variable (quantity) on the y axis. He showed that the absolute utility of Oq′ articles is equal to the area Oq′n′P under the consumption curve, and derived the relative utility, what is now called consumer surplus, by subtracting the costs of production shown as Oq′n′p′, which leaves the area n′p′P. Suppose the price decreases from p′ to p due to a reduction in costs of production, so that the quantity consumed increases from q′ to q. This raises the absolute utility to OqnP, subtracting costs of production Oqnp from this amount yields the relative utility of npP, so the net gain in relative utility is measured by pnn′p′ [By pointing out the error in Navier’s calculation of the utility of a canal, Dupuit proposed his method, arguing that the measure of utility for products already being consumed should be based on reduction in costs of production rather than reduction in costs of transportation, while in the case of new commodities being transported the measure of utility would be the lowest tax which would prevent their being carried by the new route. To operationally calculate the utility of public works, Dupuit derived his “consumption curve”, which was actually the marginal utility curve Fig. . He defiy pnn′p′ . In thisWe summarize the contributions to CBA made by French engineers in Table The American water resources development, including navigation, flood control, irrigation and water power, initiated from the beginning of the nineteenth century , 49. TheAs the first major construction agency with the requisite technical abilities, the Army Corps of Engineers established officially in 1802 on the model of the Corpes des Ingénieurs des Ponts et Chaussées, was given the responsibility for planning river and harbour improvements in 1824. The establishment of the Mississippi River Commission involved the Corps in flood control in 1879. The 1902 River and Harbor Act created a national-level Board of Engineers within the Corps to evaluate construction and maintenance costs, commercial benefits and necessity of river and harbour improvements. The 1917 Flood Control Act introduced the principle of local financial contributions to flood control, and authorized the Corps to undertake comprehensive studies of watersheds regarding the relationship of flood control to navigation, water power and other uses. The 1920 River and Harbor Act further required the reporting of local benefits for the recommendations of appropriate local cost sharing , 47. AmeThe modern economic analysis of project value began during the New Deal. The most important agency in relation to water resources in this period were four successive national resource planning organizations operating between 1933 and 1943, namely National Planning Board , National Resources Board , National Resources Committee , and National Resources Planning Board  . With thThe 1938 report of the NRC suggested that social and economic benefits, general and special benefits, potential and existing benefits should be taken into account in deciding whether or not large water projects should be undertaken as well as in distributing the costs of projects among the beneficiaries .The Water Resources Committee of the NRPB and its predecessors contributed greatly to the development of CBA. Although much of its contribution was not highly technical and far less than complete , the comBesides the Corps, there were many other agencies involved in water resources development, however, each agency adopted different and inconsistent methods of estimating costs and benefits. The 1941 report drew attention to these inconsistencies and advocated cooperative studies to develop uniform methods. After the NRPB was abolished in 1943, a new pattern of coordination arose with the establishment of the Federal Interagency River Basin Committee (FIARBC). In 1946 a subcommittee on benefits and costs was appointed “for the purpose of formulating mutually acceptable principles and procedures for determining benefits and costs for water resources projects”. This subcommittee issued a final report entitled “Proposed Practices for Economic Analysis of River Basin Projects” in 1950, which became known as the Green Book , 42.The Green Book is recognized as the first landmark in the history of CBA in the United States , 28. CBAAnother advantage of the Green Book is to apply these principles to develop operational procedures for quantifying benefits of various project purposes, such as irrigation, flood control, navigation, electric power, and recreation, although not in sufficient detail to serve as a manual. For example, the primary benefits of flood control should either be measured in terms of the estimated costs that would be avoided with flood control but would be incurred without it, or be evaluated as the costs of repairing or rehabilitating the affected property. Measuring primary benefits from water power was based on the costs of equivalent power from alternative sources that would most likely be utilized in the absence of the water power .Here, all of cost-based methods that are used widely today, namely avoided cost method, restoration cost method, and replacement cost method, have their rudiments. Unfortunately, the Green Book argued that the benefits of navigation improvements were measured by savings in transportation costs rather than reduction of production costs, which was Dupuit’s main point before one hundred years. For the purpose of evaluating recreational benefits, the Green Book mentioned two approaches being used at that time : recreatAlthough the Green Book had considerable influence, it failed utterly to reconcile the cost–benefit practices of relevant agencies, especially the Bureau of Reclamation (BOR). The BOR, established by the Reclamation Act in 1902 for the purposes of making examinations and constructing irrigation works, was the most important rival agency against the Corps in this field. Since the BOR was the specialist on irrigation, it created a set of discrepant methods for quantifying the benefits of irrigation, which contained extravagant measures of indirect benefits. Possibly because the BOR was not involved in preparing the Green Book, the report took gentle but clear position against the Bureau on the issue of secondary benefits, stating that secondary benefits should only be considered under certain strict conditions. It was no surprise that the BOR did not accept these restrictions , 42. In Beginning in 1943, the Bureau of the Budget (BOB) was required to review and consolidate all public works including water resources projects. Attempting to supervise the economic justification of projects, the BOB in 1952 issued Budget Circular A-47, which was in many respects similar to but more restrictive than the Green book, to set forth uniform standards and procedures in reviewing proposed water resources projects , 42. TheThe early work on water resources evaluation until the early 1950s was undertaken by professionals from federal agencies Table . Many ofAlong with the consolidation of welfare economics in the 1950s , CBA becIn the 1960s, the applications of CBA had widened from water resource projects to almost all kinds of government activity, such as public health , transpoThe origin of CBA can be dated back to the work of Saint-Pierre in 1708. Through the efforts by a number of French engineers, Dupuit introduces the concept of consumer’s surplus in 1844 that founds the economic basis of CBA and measures benefits in terms of the reduction of production costs. However, these works are not taken seriously in France, and do not draw attention from other countries. Hence, the principle of CBA is newly proposed by the 1936 Flood Control Act in the US, and the Green Book in 1950 marks the mature of CBA by establishing cost-based methods for measuring benefits.The comparative analysis shows that the early development of CBA in France and the US is independent from four aspects. First of all, there is no considerable evidence suggesting that the American experts are familiar with the early works of French engineers. Although Charles Ellet Jr., an American civil engineer, indeed traveled to Paris in 1830 to study as an external student at the École des Ponts, his contributions to the economic thought lie mainly in the practical problem of monopoly profit maximization of a railroad rather than CBA , 42. TheSecond, the backgrounds for introducing CBA in France and America are clearly different. The French tradition rooted in the field of transport economics, whereas the American tradition was related to water resources projects. Third, the personnel who made efforts to the development of CBA are also distinct. French engineers have formal academic background, but American professionals work in federal agencies. Both facts result in that French engineers follow a theoretical approach and attached great importance to mathematical calculation, while American professionals adopt an empirical approach and pay more attention to practical application . This diFinally, the most crucial difference lies in the attempt for standardizing this approach in America, but such a progress does not take place in France. The political rival situation among agencies with overlapping responsibility is the major driving force for the standardization in the US , but the


Though the disease burden of varicella in Europe has been reported previously, the economic burden is still unknown. This study estimated the economic burden of varicella in Europe in the absence of Universal Varicella Vaccination (UVV) in 2018 Euros from both payer (direct costs) and societal (direct and indirect costs) perspectives.We estimated the country specific and overall annual costs of varicella in absence of UVV in 31 European countries . To obtain country specific unit costs and associated healthcare utilization, we conducted a systematic literature review, searching in PubMed, EMBASE, NEED, DARE, REPEC, Open Grey, and public heath websites (1/1/1999–10/15/2019). The number of annual varicella cases, deaths, outpatient visits and hospitalizations were calculated (without UVV) based on age-specific incidence rates  and 2018 population data by country. Unit cost per varicella case and disease burden data were combined using stochastic modeling to estimate 2018 costs stratified by country, age and healthcare resource.Overall annual total costs associated with varicella were estimated to be €662,592,061  in Europe in absence of UVV. Direct and indirect costs were estimated at €229,076,206  and €433,515,855 , respectively. Total cost per case was €121.45 . Almost half of the costs were attributed to cases in children under 5 years, owing mainly to caregiver work loss. The distribution of costs by healthcare resource was similar across countries. France and Germany accounted for 49.28% of total annual costs, most likely due to a combination of high numbers of cases and unit costs in these countries.The economic burden of varicella across Europe in the absence of UVV is substantial (over 600 M€), primarily driven by caregiver burden including work productivity losses.The online version contains supplementary material available at 10.1186/s12889-021-12343-x. Varicella-zoster virus, from the α-herpesvirus family, causes varicella (or chickenpox) on primary infection and herpes zoster (HZ) (or shingles) upon reactivation. Varicella is a highly communicable disease, typically affecting children 2–8 years of age , 2. VariVaricella vaccines are safe and efficacious in preventing varicella, particularly severe varicella . In the In 2014, The World Health Organization (WHO) recommended that countries which are able to sustain a vaccine coverage of at least 80% should consider introducing varicella into routine childhood immunization programs . Yet, asThis study was conducted in three successive steps: 1. A systematic literature review (SLR) was conducted to obtain unit cost and healthcare resource utilization parameters; 2. Disease burden was updated using published incidence rates and 2018 population estimates; and 3. Economic burden was estimated by combining burden of disease and unit cost/utilization parameters. All costs were estimated for the 2018 European population ) and were reported in Euros (year 2018).Unit cost items per varicella case: including outpatient physician visits, emergency room (ER) visits, hospital visits , hospitalizations , intensive care unit (ICU) stay, other healthcare professionals visits , over-the-counter (OTC) medications, prescription medications in outpatients , tests and procedures in outpatients.Per case resource utilization: including length of hospitalization, work days lost by caregiver for outpatient/inpatient cases, work days lost by adult outpatient/inpatient cases, utilization rate for ER visits, hospital visits, ICU stays, OTC medications, other healthcare professional visits, prescription medications and tests and procedures.We conducted a SLR for peer-reviewed studies published between 1 January 1999 and 15 October 2019 in any language to obtain country-specific unit costs per varicella case and healthcare resource utilization parameters. The database search was conducted in MEDLINE (via PubMed), EMBASE, NHS Economic Evaluation Database (NEED), Database of Abstracts of Reviews of Effectiveness (DARE), Research Papers in Economics (REPEC) and Open Grey. Data were also obtained from the WHO, European Center for Disease Prevention and Control (ECDC) and national public health institutes websites. Two types of outcomes were of interest:The SLR was conducted in accordance with the standards of the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) guidelines and a flowchart was prepared to describe the process . Titles The extracted cost and utilization outcomes were summarized for each country. For countries with multiple entries per outcome, a range (min-max) was generated based on all values extracted. Values expressed in currency prior to 2018 were adjusted for inflation using the 2018 price index (Gross Domestic Product (GDP) price deflator) . For couWe calculated the mean (range: min-max) annual number of varicella cases and deaths, outpatient visits and hospitalizations in 2018 using previously reported data on varicella incidence rates in Europe before UVV . We updaVariables to be included in the model were chosen based on data availability, and information from at least five countries was required to include an outcome. The following outcomes were included: unit cost of outpatient visit/hospitalization/prescription medication/OTC medication and utilization (proportion) of prescription medication/OTC medication, length of hospital stay, and number of work days lost by caregiver/adult case. To account for the uncertainty of the input parameters, assuming that all values falling within the min-max range were equally probable, a uniform distribution (min-max) was assigned to each parameter in our model. The parameter distributions were then combined through stochastic modeling; a total of 10,000 iterations were ran, and the mean and min-max output of the output values were used to estimate the population-level direct costs  and indirect costs (work loss caregivers/patients/deaths) were estimated for the 31 European countries. The distribution of costs by country, age group and healthcare resource was described. Finally, the mean  costs per varicella case were calculated by dividing the total annual costs by the number of annual varicella cases in Europe. In addition, sensitivity analysis was conducted by: 1. considering only countries without general recommendation for publicly funded varicella vaccination as of 2021 , 2. adjuAs described in the PRISMA diagram Fig. , 120 fulThe outcomes with sufficient data (i.e. > 5 countries with at least one data source) are described in Table Overall annual total costs associated with varicella in Europe were €662,592,061  . Adjusting the unit costs of the tradeable health care costs (OTC and prescription medication) using exchange rates instead of PPP resulted in a reduction of total costs of 662,375 EUR .The estimated economic burden of varicella in the absence of UVV across 31 European countries would be substantial, amounting to over €660 million annually in 2018 Euros. Of the total cost, 65% were attributed to work loss suggesting a significant societal burden and impact on productivity. To our knowledge, this study is the first study to systematically estimate the economic burden of varicella for individual European countries. The substantial costs associated with productivity losses estimated in our study are in line with findings from previous systematic literature reviews on varicella burden . Banz etPrimary care visits were the key driver among direct costs associated with varicella in almost all countries (46% of direct costs on average). It was previously shown that most varicella cases in Europe lead to a physician consultation (54%), whereas only a minority of cases (0.3%) are hospitalized [The greatest share of costs was associated with cases in children younger than 5 years, reflecting that varicella incidence is highest in this age group , whereasThe distribution of varicella costs by healthcare resource appeared to be generally similar across European countries. In the absence of UVV, more than three-quarters (77.40%) of the total varicella costs would be attributable to five countries, namely France, Germany, Italy, Spain and the UK. These countries represent the top-five European countries in terms of population size and annual varicella case counts. Annual varicella costs estimated in 2002 for France (148€ million) and Germany (144€ million) before introduction of routine immunization were in line with our findings . FurtherWhile varicella is commonly perceived as a mild disease, our study shows that it can pose significant economic and caregiver burden. It is important to consider strategies to reduce the clinical and economic burden of varicella across Europe. As of January 2021, nine European countries had UVV . CountriThe main limitation of our study concerns the lack of availability of several unit cost and utilization input parameters, such as the cost of tests or procedures, which were not included in the final model. Moreover, the extrapolation of data from countries with available information to other countries lacking such data might have introduced a bias due to heterogeneity of health resource costs and resource utilization rates that occurs across countries. Health-economic results are usually not directly comparable from one country to the other and need specific adjustments , 86, 101It is important to state that we have not investigated in our study the burden due to the loss in quality of life (QoL) nor the quality-adjusted life-year (QALY) although the latter is widely used as measure of the incremental effect in economic evaluations of vaccination. On the basis of Health Utilities Index mark 2 (HUI2) ratings, Brisson et al.  found 0.Varicella has a significant public health impact. Its economic burden in absence of UVV is considerable in Europe, mainly owing to high disease incidence and associated health care resource use and caregiver burden including work productivity losses. Assessing the economic burden of a disease is essential for prioritizing healthcare interventions among competing vaccine-preventable diseases, and this analysis underscores the need for more country-specific evaluations to allow informed decision making. These country specific economic data could be used for potential country-specific cost-effectiveness evaluations that would be valuable to support national immunization policy decisions.Additional file 1. Search strategy Systematic literature review.Additional file 2. Data management.Additional file 3. Estimated disease burden in absence of UVV: annual varicella cases, deaths and resource utilization by country and age group.Additional file 4. Cost model.Additional file 5. Varicella unit cost and utilization outcomes (SLR outcomes).


Giardia spp., Cryptosporidium spp. and Microsporidia are unicellular parasites spread by the fecal-oral route by environmentally resistant stages and can infect humans, livestock, and wildlife including non-human primates. Using immunoassay diagnostic kits and amplification/sequencing of the region of the triosephosphate isomerase, small ribosomal subunit rRNA and the internal transcribed spacer genes, we investigated Giardia, Cryptosporidium, and microsporidia infections, respectively, among humans, domesticated animals , and wild nonhuman primates (eastern chimpanzees and black and white colobus monkeys) in Bulindi, Uganda, an area of remarkably high human–animal contact and spatial overlap. We analyzed 137 fecal samples and revealed the presence of G. intestinalis assemblage B in two human isolates, G. intestinalis assemblage E in one cow isolate, and Encephalitozoon cuniculi genotype II in two humans and one goat isolate. None of the chimpanzee and colobus monkey samples were positive for any of the screened parasites. Regular distribution of antiparasitic treatment in both humans and domestic animals in Bulindi could have reduced the occurrence of the screened parasites and decreased potential circulation of these pathogens among host species.Zoonotic pathogen transmission is considered a leading threat to the survival of non-human primates and public health in shared landscapes.  Emerging zoonotic diseases are a serious threat to both public health and animal conservation. While emerging epidemics such as Ebola and more recently Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most likely resulting from zoonotic transmission, can be deadly among humans ,2, lethaHuman activities, including logging, forest clearance, and farming, have meant that NHP increasingly share anthropogenically modified landscapes with humans and livestock ,12,13. TGiardia intestinalis, Cryptosporidium spp., and microsporidia of genera Encephalitozoon and Enterocytozoon are common intestinal protists infecting humans and domesticated animals, including livestock, dogs, and cats [G. intestinalis have been reported, but infections were asymptomatic [Cryptosporidium infections in great apes is limited to a few studies. To date, several Cryptosporidium species have been described in great apes, namely C. parvum in mountain gorillas (Gorilla beringei beringei) and Bornean orangutans (Pongo pygmaeus), C. muris in western lowland gorillas (G. gorilla gorilla), Bornean orangutans and Sumatran orangutans (Pongo abelii), C. bovis and C. meleagridis in western lowland gorillas, and C. hominis, C. suis, and C. ubiquitum in eastern chimpanzees (Pan troglodytes schweinfurthii) [C. parvum, C. hominis, C. hominis “monkey”, C. felis, and C. cuniculus have been reported from other NHPs such as macaques, langurs, colobus, and baboons [Encephalitozoon spp. and E. bieneusi in humans and NHP are characterized by a variety of pathologies ranging from asymptomatic to lethal infections, mainly in immunodeficient hosts [and cats ,20,21,22and cats ,25,26,27and cats ,29. In fptomatic ,30,31,32furthii) ,34,35,36 baboons ,39,40,41 baboons . Microspnt hosts . In contnt hosts ,45,46, cGiardia, Cryptosporidium, and microsporidia infections result from fecal-oral transmission through ingestion of contaminated water and food, transmission might occur between humans, domestic animals, and wildlife sharing environments [G. intestinalis, Cryptosporidium spp., and microsporidia in wild great apes revealed potential for transmission of those parasites among humans, domestic animals, and mountain gorillas in Uganda [As ronments ,48. Prevn Uganda ,42,49,50n Uganda ,51, and n Uganda , and witn Uganda .Giardia, Cryptosporidium, and microsporidia infections for their potential zoonotic transmission among humans, domesticated animals , and wild NHP  in Bulindi, Uganda [The aim of this explorative study was to investigate , Uganda ,53,54 , and a goat, were identical to E. cuniculi genotype II , previously detected in a wide spectrum of hosts , causes human giardiosis only rarely [G. intestinalis assemblage B in this study was identical to isolates that have been previously reported from water samples and humans in Canada, humans in Australia (GenBank acc. no. EF688026), and from captive white-faced saki monkeys (Pithecia pithecia) in Japan [Encephalitozoon cuniculi has a broad host range, mainly among mammals, but also infects birds, NHP, and humans [Encephalitozoon cuniculi genotype II, the most common genotype, has been reported in numerous birds and mammals, including livestock, NHP, and humans [A very low occurrence of y rarely . The seqin Japan ,57. Enced humans . Four did humans . Encephad humans .P. anubis [Macaca fascicularis [G. b. beringei [E. cuniculi, E. bieneusi, or Cryptosporidium spp. in NHP [The results of our study contrast with several previous studies that reported a higher occurrence of the studied parasites in various primates with frequent contact with humans and livestock . Rather. in NHP ,36 in arG. intestinalis, E. bieneusi, and E. cuniculi, are often excreted intermittently. Thus, repeated sampling of the same individuals over several consecutive days is recommended [Giardia cysts (1.6%). These findings agree with the results of the present study and suggest a very low G. intestinalis burden in this population, even when approximatively 23 samples from each individual were analyzed. Our findings also reveal a very low occurrence of the other studied protists in humans and domesticated animals from Bulindi, but do not necessarily indicate that NHP in the study area are not at risk of G. intestinalis infections or other zoonotic pathogens . The loectively ,65,66. TG. intestinalis [G. intestinalis should be taken into account. While in our study we used genotyping at the locus encoding triosephosphate isomerase (tpi), Johnston et al. [Our findings suggest that humans and livestock in Bulindi also have low levels of protist infections compared to results of surveys elsewhere in Africa  and tantalus monkeys  are transient visitors. NHP in Bulindi have experienced major habitat disturbance: between 2006 and 2014 forest fragments were reduced in size by ca. 80% and converted to farmland [Bulindi  is located in western Uganda’s Hoima District. The landscape is a mosaic of farmland, villages, and fragments of riverine forest along watercourses. A resident ‘community’ of chimpanzees, first studied in 2006–2007 , has beefarmland . Rapid hfarmland ,92 and cfarmland . Pigs, gfarmland ,54.n = 30) and colobus monkeys (n = 17) inhabiting forest fragments in Bulindi. Chimpanzees  were followed daily by the field team and fresh fecal samples were picked immediately after defecation from identified individuals. Fresh fecal samples (estimated ≤12 h old) were collected from unhabituated black and white colobus monkeys from beneath trees where the monkeys had been located. In parallel, local human participants (n = 43), livestock , poultry (n = 11), and dogs (n = 2) were sampled from 10 households in two villages located centrally within the 20 km2 home range of the chimpanzees (During October–November 2016 (in the wet season ) we non-mpanzees . We gaveGiardia Rapid Assay  and RIDA QUICK Cryptosporidium  immunochromatographic diagnostic kits were used according to the directions of the manufacturer for detection of Giardia and Cryptosporidium coproantigen in fresh and ethanol fixed feces, respectively. RIDA QUICK Cryptosporidium is primarily developed for detection of C. parvum, and therefore other Cryptosporidium spp. might not be detected. The suspension of each fecal sample in ethanol was evaporated overnight at 60 °C, before isolation of genomic DNA (gDNA). A total of 200 mg of fecal material was homogenized by bead disruption using 0.5 mm glass beads  in a FastPrep®-24 Instrument  at a speed of 5 m/s for 1 min followed by isolation/purification using the QIAamp® DNA Stool Mini Kit in accordance with the manufacturer’s instructions . Purified gDNA was stored at −20 °C prior to use in PCR. All gDNA samples obtained were analyzed by polymerase chain reaction (PCR) using sets of specific primers. A nested PCR approach was used to amplify a region of the triosephosphate isomerase gene (TPI) of G. intestinalis [SSU) of Cryptosporidium spp. [ITS) of Enterocytozoon bieneusi [Encephalitozoon spp. [Giardia microti, C. proliferans, E. hellem genotype 1A, or E. bieneusi genotype PtEbIX were used as negative and positive controls, respectively. Secondary PCR products were run on a 2% agarose gel containing 0.2 µg/mL ethidium bromide in 1 × TAE buffer at 75 volts for approximately 1 h. Bands of the predicted size were visualised using an UV light source, and then extracted using QIAquick Gel Extraction Kit (QIAgen). Gel-purified secondary products were sequenced in both directions with an ABI 3130 genetic analyzer  using the secondary PCR primers and the BigDye Terminator V3.1 cycle sequencing kit . All samples were analyzed in duplicates. In the case of positive detection, the sample was newly re-isolated and the previous finding was independently verified. Sequences have been deposited in GenBank under the accession numbers MZ048410–MZ048412  and MZ055371–MZ055373 .stinalis , small rium spp. , the intbieneusi  and Enceoon spp. . Molecul


Longissimus thoracis muscle was evaluated. At the myostatin gene, 67 homozygotes normal, 11 heterozygotes, and no double-muscled homozygote bulls were detected. Heterozygote bulls showed high values in final live weight and dressing yield; moreover, they were characterized by a low incidence of fat at steak dissection, as well as in meat chemical composition. A better muscular conformation in heterozygote bulls’ carcasses was highlighted, with a higher incidence of their carcasses in class E and evident convexity of round, back, and shoulder muscular masses compared to the carcasses of Marchigiana bulls which were normal at the myostatin gene.The aim of this work was the evaluation of slaughtering performance in a sample of 78 Marchigiana bulls with different allelic situation at the myostatin locus; in addition, the qualitative composition of meat samples collected from GDF8) is one of the most investigated loci that can be responsible for several quantitative and qualitative carcass and meat traits in double-muscled beef cattle. The objective of the study was to bring to light the effect of the myostatin polymorphism on slaughtering performance and meat quality in Marchigiana beef cattle. The experiment was carried out on 78 bulls reared according to the “cow-calf” extensive managing system. At the end of the fattening period, in vivo and carcass data were recorded. From each carcass, a steak of Longissimus thoracis was taken and used to determine the meat’s analytical composition and colorimetric properties. Finally, from each steak a sample of Longissimus thoracis was collected, then used for DNA extraction and genotyping at the myostatin locus. The heterozygous bulls showed slight superiority in the carcass data  and meat quality parameters, although not always with statistical significance. Only fat and ashes content were significantly affected by the myostatin genotype . The greater muscularity of heterozygous animals compared to normal ones could be a starting point to improving productive efficiency in Marchigiana beef cattle.The myostatin gene also called Growth Differentiation Factor 8 gene ( Increasing food demand will lead to the application of more efficient agricultural technologies. In this perspective, the meat industry would gain an advantage from strategies aimed at improving the efficiency of beef cattle, such as genetic selection, management practices, rumen functional efficiency, and structure and composition of feed . In addiIn Italy the meat market is changing, following modern consumers’ needs and new economic dynamics . KnowledMSTN) gene, also called Growth Differentiation Factor 8 gene (GDF8), has been recognized as important in the development of the skeletal muscles and, hence, in animal growth and carcass traits [MSTN has been identified in different vertebrate species [MSTN [The myostatin  affects the myostatin protein due to the introduction of a premature stop codon, thus blocking the translation of 254 bases of the third exon. Myostatin is a negative regulator of muscle mass development which suppresses both the proliferation and differentiation of myogenic cells. The dysfunctional myostatin leads to an increase in muscle mass in animals carrying the causative mutation. Therefore, animals can have normal (G/G) or hypertrophied (heterozygous G/T or homozygous T/T) genotypes [The Marchigiana is one of the most important Italian beef cattle breeds, and its fresh meat, since 1998, can be certified by the Protected Geographical Identification (PGI) “Vitellone Bianco dell’Appennino Centrale” . The selenotypes . Double-enotypes , large denotypes , making MSTN gene. Thus, the objective of the present study was to verify and quantify the effect of the MSTN polymorphism on both slaughtering performance and meat quality in Marchigiana beef cattle.To the best of our knowledge, this is the first report focused on performance and key meat quality parameters in heterozygous Marchigiana bulls at the The study was carried out on 78 Marchigiana bulls reared according to the “cow-calf” extensive managing system , which iLongissimus thoracis (LT) muscle between the 8th and 9th thoracic vertebrae using a portable pH-meter  equipped with the automatic temperature compensation function and a food probe  calibrated with pH = 7 and pH = 4 buffers prior to measurements.At the end of the fattening period (120 ± 30 days), the final live weight (FLW) of each animal was recorded, then animals were slaughtered in a commercial abattoir according to European guidelines . The ave2 + b2). The colorimetric readings were performed in triplicate for each sample.About 24 h after slaughtering, one steak was sampled from each carcass  between the 8th and 9th thoracic rib, transferred to the laboratory under refrigerated condition (4 °C) for further meat evaluations. Firstly, steaks’ colorimetric profiles were evaluated. Chroma Meter CR-200  was used to detect the colorimetric profile of Marchigiana meat samples, determining the coordinates of lightness , redness-greenness , and yellowness-blueness  according to the CIE Lab color space system with illuminant D65, 2° observer, Diffuse/O mode, 8 mm aperture of the instrument for illumination, and 8 mm for measurement . The col® II Advanced Line, Sartorius, Goettingen, Germany) and dissected to determine the percentage of bone, LT muscle, other muscles, and fat.Afterwards, each steak was weighed with electronic balance  following the specific manufacturer protocol. The genotype of all animals was determined by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) following a modified method developed by Marchitelli et al. . The det2, 50 mM of each dNTP, 1 mM of each primer, and 1 unit of Taq® DNA Polymerase , using a TGradient 96 Thermocycler . The PCR conditions were as follows: initial denaturation step of 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at the 57 °C, 1 m at 72 °C, and a final extension of 5 min at 72 °C.PCR amplification was performed in 25 μL reactions containing 50 ng of genomic DNA as template, 3 mM MgCl2, 100 mM KCl, 0.1 mg/mL Bovine Serum Albumin—BSA), and 1 U of Tru9I. The digestion conditions were set at 65 °C for 1 h. Finally, the genotypes for all samples were assessed through a 2.5% agarose gel electrophoresis and then recorded.The PCR product underwent the digestion with restriction enzyme Tru9I . A total volume of 20 µL contained 300 ng of amplicon, 1X Buffer R (10 mM Tris-HCl (pH 8.5), 10 mM MgClData were analyzed by analysis of variance and covariance (ANOVA and ANCOVA), and multiple comparisons among means . Before performing the statistical models, normal distribution of residuals and homogeneity of variances were tested according to the Shapiro–Wilk and Barlett tests, respectively. Seven animals were excluded from the carcass data analysis because they were outliers for the length of the fattening period.ij was the dependent variable for the j-th animal, µ was the overall mean, Gi was the fixed effect of genotype , and b was the regression coefficient of the covariate: age at slaughtering (X) for FLW, ADG, HCW, CDG, and DY; hot carcass weight (X) for pH-45′ and dissection variables . The term εij was the residual error.For the analysis of final live weight (FLW), average daily gain (ADG), hot carcass weight (HCW), carcass daily gain (CDG), dressing yield (DY), carcass pH 45 min post mortem (pH-45′), and dissection variables, the following fixed model ANCOVA was applied:ij was the dependent variable for the j-th animal, µ was the overall mean, Gi was the fixed effect of genotype , and εij was the residual error.For the analysis of drip loss percentage, the following fixed model ANOVA was applied:ij was the dependent variable for the j-th animal, µ was the overall mean, Gi was the fixed effect of genotype , εij was the experimental error, and δijk was the sampling error.For the parameters of meat composition and meat colorimetric indexes, double and triplicate measurements were respectively performed for each steak. Therefore, the following fixed ANOVA model, including the sampling error, was applied to the complete data set:t-Test was performed to compare the least square means of the two genotypes at the MSTN locus. The χ2 test was applied to test the Hardy–Weinberg equilibrium for the MSTN locus and to compare the frequencies at the SEUROP grid and fat cover categorical classes of the two genotypes at the MSTN gene.Student’s MSTN gene were detected  bulls (G/G), 11 heterozygote bulls (G/T), and no double-muscled homozygotes (T/T) at the detected .p < 0.90)  , as repoMSTN locus of the present results and the ones reported by Lasagna et al. [MSTN gene is relatively low due to the breeding strategies applied to Marchigiana breed in the last decades, which do not allow MSTN homozygote bulls to be included in mating plans [Moreover, the chi-square test did not show significant differences between the genotype distribution at the a et al. , as showng plans . TherefoThe average live weight of the Marchigiana bulls was 677.02 kg (range: 454–827 kg) at an average slaughter age of 632.45 days (range: 476–731 days). Bulls were slaughtered within the age of 24 months, according to the specification of the PGI “Vitellone Bianco dell’Appennino Centrale” mark.MSTN animals are reported according to the analysis of covariance (Model 1-ANCOVA).In MSTN genotype and the heterozygous one. Despite the unbalanced dataset, heterozygous bulls showed an average final weight 17 kg higher than normal bulls. These findings may be ascribed to the ad libitum feeding regimen adopted during the trial and mainly based on concentrate, which allow hypertrophic beef cattle a better expression of their lower and late growth potential [For the final live weight reached at slaughtering, no significant differences were observed between homozygous wild-type otential . The ligotential  on Charootential  on Angusotential , in a smMSTN gene, resulting in quite a similarity in heterozygous and normal bulls. The daily gain weights observed herein were slightly lower than the daily gains observed by Casas et al. [MSTN gene, and the ones reported by Vinet et al. [In addition, the ADG was not affected by genotype at the s et al.  in Charot et al.  in a croRegarding the results for the carcasses’ post mortem parameters (Model 1-ANCOVA), none of the parameters were affected by genotype. However, heterozygotes Marchigiana bulls reached 426.09 kg in carcass weight. Carcass traits of heterozygous bulls of French beef breeds , of CharAlthough there are no significant differences between heterozygous and normal Marchigiana bulls, the carcass daily gain means suggest the effect of the mutation on this parameter, which should be supported by further research.MSTN gene, although no significant difference between means was observed (p = 0.108). This trait contributes to quantifying the production efficiency and the economic benefits either for breeders or for the beef cattle industry. Therefore, Marchigiana heterozygotes showed better performance in dressing yield as compared to heterozygotes of Blond d’Aquitaine crosses, Charolaise, Piemontese, Asturiana de los Valles, and Limousine beef cattle breeds [Heterozygous Marchigiana bulls reached 62.65% in dressing yield, while 60.96% was observed for normal bulls at the e breeds ,36,39,40e breeds ,32, reace breeds .MSTN genotype did not affect the pH detected on carcasses at 45′ after slaughtering. Slaughter date as random term was significant (p < 0.001), but the differences between pH means of the two genotypes was not significant. The pH-45′ results suggest that bulls had not been subjected to stressful conditions before slaughter; usually greater pH differences are referenced 12–24 h after killing, mainly due to higher carcass temperatures in double-muscled heterozygous or homozygous animals [The  animals .MSTN gene on steak dissection  are listed in Results of the genotype effect at the MSTN mutated animals reported by several authors [p < 0.05) and a significantly lower proportion of subcutaneous and intermuscular fat  than normal bulls . These results support the growth of muscular mass due to lack of myostatin protein function also in Marchigiana beef cattle, resulting in an increased number of muscle fibers (hyperplasia), fibers enlargement (hypertrophy), and a reduction in subcutaneous and intermuscular fat deposition [Heterozygotes bulls showed a low, but not significant, incidence of bone portion at steak dissection, reflecting statements about the reduction of skeleton in  authors ,30,32,35position ,41.MSTN gene on meat composition and its colorimetric profile of 78 Marchigiana bulls according to Model 3-ANOVA  are listed in Results of the genotypic effect at the MSTN locus. The lowest level in fat observed in meat from heterozygote bulls (2.01%) confirms results obtained in previous research carried out in several beef cattle breeds [Concerning the chemical composition of the LT in Marchigiana bulls, only the contents in fat and ashes were significantly affected by the genotype at the e breeds ,39,42. Te breeds  in double breeds ,32,44,45MSTN locus fall within the ranges of the quality parameters required by the PGI specification “Vitellone Bianco dell’Appennino Centrale”, according to the Commission Regulation (EC) No 134/98 [It is noteworthy that the contents in protein, fat, and ashes observed herein in the heterozygous Marchigiana bulls at the o 134/98 .MSTN. Slightly higher water losses were observed in meat from G/T Marchigiana bulls than G/G ones, thus reflecting the more glycolytic muscle metabolism of heterozygous bulls [The meat’s water-holding capacity, measured through the evaluation of drip losses, did not highlight significant differences between meat from normal and heterozygous bulls, suggesting that the presence of one copy of mutated allele did not clearly interfere with the binding systems between muscle fibers. This is in agreement with a previous study from Reardon et al.  that repus bulls .MSTN gene on colorimetric reflectance coordinates of meat from normal and heterozygous Marchigiana bulls did not detect any significant differences between means. Despite this, meat from heterozygous Marchigiana bulls showed high value in lightness (L*), thus confirming results from scientific literature reporting that meat of hypertrophic animals has a lighter color often associated to a lesser haem-pigment concentration than normal bulls [The Model 3-ANOVA performed to test the effect of al bulls ,39. L*, al bulls , at the MSTN gene are reported .In The total chi-square test highlighted an overall significant difference among the assignment of carcasses to the three SEUROP classes between the two genotypes. Orthogonal contrasts showed a significantly higher frequency in the heterozygous group of class E than class U + R carcasses, whereas no significant difference between U and R classes was found a. These Concerning the amount of fat on the outside of the carcass and in the thoracic cavity, heterozygous Marchigiana bulls showed a higher, although not significant, frequency in class 2, with a slight fat cover, than normal ones b. These Overall, a future investigation with a larger sample size would be beneficial to validate the results observed in heterozygote Marchigiana bulls for in vivo and post mortem performances and meat quality parameters.Our results confirm in the Marchigiana breed what has already been observed in other hypertrophied breeds. In particular, the mutation seems to influence important productive traits such as the final live weight, the dressing percentage, and the fat content of the meat.The muscularity of heterozygous animals could be an interesting starting point to improve producing efficiency in Marchigiana beef cattle, to meet modern consumer needs and to face the increasing demand on products of animal origin.MSTN gene in Marchigiana breed could also lead to developing new breeding schemes which will maintain heterozygotes in the breed, while limiting the diffusion of the mutate homozygous genotype to avoid the undesirable negative effects notoriously related to double-muscled phenotype.Future applications linked to programmed matings and in-depth knowledge of the variants of