Global trade of feed ingredients that may be contaminated with significant concentrations of swine viruses is a concern for the potential transmission of swine diseases because viable virus particles can survive in feed ingredients and complete feed for several weeks or months. No global swine virus surveillance and monitoring system exists to determine the possible presence and concentrations of swine viruses in feed ingredients. Biosecurity protocols based on hazard analysis and risk-based preventive controls must be developed and implemented in feed ingredient supply chains to prevent virus contamination. In addition, mitigation strategies including the use of extended storage time, thermal and irradiation processing, and certain feed additives have been shown to provide partial virus inactivation in contaminated ingredients and complete feeds under specific conditions. However, analytical methods capable of accurately determining viable virus concentrations that can lead to infection are lacking and need to be developed. Effective feed mill decontamination strategies are also needed for various swine viruses. Several functional ingredients and nutrients such as spray-dried animal plasma, medium-chain fatty acids, and soy isoflavones have antiviral properties and have been shown to alleviate adverse health of pigs undergoing a viral disease challenge when included in diets.No system nor standardized analytical procedures at commercial laboratories exist to facilitate and accurately measure potential viable virus contamination in feed ingredients and complete feeds globally. As a result, there is high uncertainty of the extent of swine virus contamination in global feed supply chains. Many knowledge gaps need to be addressed to improve our ability to prevent virus contamination and transmission in swine feed. This review summarizes the current state of knowledge involving: (1) the need for biosecurity protocols to identify production, processing, storage, and transportation conditions that may cause virus contamination of feed ingredients and complete feed; (2) challenges of measuring virus inactivation; (3) virus survival in feed ingredients during transportation and storage; (4) minimum infectious doses; (5) differences between using a food safety objective versus a performance objective as potential approaches for risk assessment in swine feed; (6) swine virus inactivation from thermal and irradiation processes, and chemical mitigants in feed ingredients and complete feed; (7) efficacy of virus decontamination strategies in feed mills; (8) benefits of functional ingredients, nutrients, and commercial feed additives in pig diets during a viral health challenge; and (9) considerations for improved risk assessment models of virus contamination in feed supply chains. Concerns about the transboundary transmission of swine viruses through international trade and travel  have ledThe major swine viruses of concern for potential transmission through global feed supply chains are PEDV, African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine respiratory and reproductive syndrome virus (PRRSV), Seneca Valley A virus (SVV-A), and foot and mouth disease virus (FMDV). Although a few reports have provided evidence that contamination of some viruses such as ASFV , CSFV 1, and SVVSalmonella enterica, PEDV, and ASFV. However, accurate interpretation of results from this study is difficult because no uncertainty estimates nor detailed assumptions used to make these determinations were provided.Because of the high uncertainty, mathematical models have been developed to evaluate the likelihood of virus transmission through feed. Galvis et al.  evaluateSeveral laboratory-based inoculation studies have shown that most swine viruses of concern can survive in some feed ingredients for several weeks or months ,25,26,27Identifying conditions during production, processing, transportation, and storage that can lead to virus contamination of feed ingredients;Determining the likelihood of swine virus contamination in feed;Understanding the chemical and physical characteristics of feed ingredients that allow various types of viruses to survive;Understanding the unique characteristics of various types of viruses that enable their survival and make them vulnerable to inactivation and loss of infectivity;Developing and validating highly sensitive and specific assays that accurately quantify viable and infectious virus particles for various viruses in different types of feed ingredients;Identifying time and temperature conditions that effectively inactivate viruses without degrading the nutritional value of ingredients;Identifying chemical mitigants that effectively inactivate viruses without degrading the nutritional value or safety of ingredients;Determining effective practices for decontaminating feed mills;Determining minimum concentrations of viruses and feeding conditions that prevent disease when pigs consume contaminated feed.Because of the global trade of feed ingredients and subsequent movement of imported ingredients to feed mills and swine farms, feed biosecurity programs to minimize the risk of virus contamination and transmission have emerged as a relatively new component of feed safety and biosecurity protocols. Approaches to risk management of ASFV transmission through imported feed ingredients vary among countries ,29. In tBecause of our inability to accurately and routinely determine the presence, concentration, viability, and infectivity of virus contamination in feed ingredient supply chains, this high uncertainty of potential virus contamination requires development and implementation of biosecurity protocols that focus on specific swine viruses of concern. However, until more strict biosecurity regulations and programs are implemented, effective mitigation strategies are also needed to reduce viral load and viability in feed ingredients suspected of being contaminated. The biosecurity of swine farms is an essential component for preventing introduction and controlling pathogens that cause foreign and endemic disease, and for maintaining high health to optimize productivity. International biosecurity guidelines have been developed that involve minimizing the exposure of animals to external hazards that are potential routes of pathogen transmission . Major rFeed manufacturing facilities are a collection, storage, proportioning, mixing, and processing point for various types of feed ingredients sourced from many geographic regions before finished complete feeds are delivered to multiple farms. Therefore, the biosecurity of feed mills must be a major part of the overall biosecurity program for swine farms to prevent pathogen introduction. Cochrane et al.  describeMechanisms for evaluating the quality, safety, and biosecurity procedures used by suppliers in the production of ingredients, including auditing and verification that protocols are followed;Facility design and maintenance protocols that prevent or reduce the introduction of pathogens;Routine housekeeping procedures that adequately prevent or reduce the introduction of pathogens;Standard operating procedures (SOPs) and surveillance programs for biosecurity that include ingredient sourcing, receiving, and storage;Biosecurity and personal hygiene protocols for visitors, employees, and drivers to control access to the facility;Manufacturing practices that are effective for maintaining the biosecurity protocols of the facility;Biosecure transportation of finished feed using sealed containers and disinfection practices.Feed manufacturers are responsible for the biosecurity of the feed supply chain, which begins with sourcing, receiving, and processing feed ingredients used to manufacture complete feeds until the delivery of finished feed to swine farms . The Amehttps://www.fda.gov/food/food-safety-modernization-act-fsma/fsma-final-rule-sanitary-transportation-human-and-animal-food) (accessed 26 March 2023). Biosecurity protocols for sanitary transport of imported ingredients should include HARPC plans to reduce the risk of adulteration or cross-contamination of viruses. Key components of sanitary transport protocols include:Documentation verifying that the manufacturing and storage facilities in the country of origin have been decontaminated;One-way driveways for dirty vehicles and containers should be used to separate potentially contaminated vehicles and containers from those that are empty, clean, and disinfected using approved and effective disinfectants;Washing and disinfection facilities should be provided, and their use required for all trucks and equipment used for feed transport;After disinfection, transport vessels should be loaded and sealed at the manufacturing facility before transport to the destination;After ingredients are loaded and sealed, trucks should enter the delivery destination through a “clean” driveway;After unloading, transport time and temperatures conditions should be recorded and considered when estimating required holding times during storage at the destination;Upon arrival at the destination, only trucks that are empty, clean, and disinfected should be used to transport bulk ingredients for quarantine in a heated temporary warehouse;For bagged ingredients, new or properly cleaned and disinfected pallets should be used;Documentation of storage conditions and holding times for each lot of each feed ingredient should be provided to end users.Sanitary transport is commonly overlooked in biosecurity protocols. However, trucks, driver shoes, bags, and totes have been identified as the primary route of pathogen transmission associated with feed in several studies ,13,14,17Upon completion of a comprehensive feed mill biosecurity plan, all new procedures must be correctly implemented to reduce the likelihood of pathogen introduction. Therefore, employee training must be provided so that they can demonstrate an understanding of each risk factor being controlled and that they are capable of following procedures and protocols to minimize each risk. Unannounced internal audits are useful to ensure compliance with existing procedures and to identify aspects of protocols where more employee training is needed. Biosecurity plans should be re-evaluated at least once annually or when a new feed ingredient source is acquired at the feed manufacturing facility. External third-party audits, review, and consultation from qualified feed mill biosecurity experts are also useful practices for identifying potential hazards that may have been overlooked when initial protocols were developed.Salmonella, Escherichia coli, and Clostridium perfringens contamination in feed samples have been routinely used for many decades [50 [50) [Accurate and repeatable analytical assays for routine evaluation and monitoring of common bacterial pathogens including  decades . Virus-sades [50  and hema[50 [50)  and in v[50 [50) . Unfortu3 TCID50/g or 105 TCID50/g PEDV and collected samples using individual probes or composite sampling. These researchers reported that composite samples were more sensitive to virus RNA than probes in bulk soybean meal and suggested a minimum of 10 subsamples be collected for creating a composite sample for analysis. Elijah et al. [Accurate determination of virus concentrations in feed ingredients begins with collecting representative samples. However, viruses in contaminated feed may not be uniformly distributed and may be present in low concentrations. No sampling methods have been validated for use in collecting representative feed samples for virus analysis. Jones et al.  inoculath et al.  evaluateh et al.  and suggThere are also numerous challenges with using existing analytical assays for the accurate detection of viable virus particles in feed ingredients that can lead to infection, especially for ASFV . MeasuriTargeted virus diagnostic methods must have high specificity (accurately identify negative results) and high sensitivity (accurately identify positive results) to accurately quantify the amount of viable virus particles capable of causing infection if ingested by pigs . Results50/mL) is another common assay used to calculate virus concentrations by incubating a red blood cell suspension with an infected cell culture to measure 50% of the replicates, showing the amount of virus hemadsorption per milliliter of blood [50) per gram of sample, which measures the amount of virus capable of infecting 50% of cell culture replicates, but it does not directly infer infectivity in pigs. More recently, surrogate assays have been developed for some swine viruses in research applications because using the actual virus of interest, such as ASFV [For some viruses, PCR methodologies have been modified to create viability PCR assays that distinguish between viable and free DNA in feed samples . Howeverof blood . Howeverof blood . The mos as ASFV , CSFV, o as ASFV , is rest as ASFV  to confi as ASFV . 3 reduction from the initial virus concentration and should not be interpreted as 0.1% virus particles remaining in the sample. Therefore, depending on the initial virus concentration, a 99.9% or 3-log reduction in virus from thermal or chemical mitigation treatments may exceed the minimum infectious dose for a contaminated feed and potentially result in an infection. In addition, virus inactivation kinetics curves usually fit non-linear patterns with shoulders or tails and do not follow first-order kinetics (log-linear). Half-life  data for virus inactivation reported in some studies does not provide an accurate estimate of virus inactivation in a feed ingredient because it is based on the assumption of a linear relationship between the logarithmic decrease in virus concentration and time of mitigant exposure. However, using inactivation data to calculate D-values  and z-values  are useful to make comparisons of virus inactivation kinetics across studies and predict outcomes not previously determined experimentally. Unfortunately, most studies have not provided adequate data suitable for calculating D- and z-values to more accurately determine virus inactivation kinetics in feed ingredients [In addition to understanding the specific state of viruses being measured by various assays, accurate interpretation of results from virus inactivation studies is also essential. The term “complete inactivation” of viruses should be avoided because it infers zero risk of infection in pigs consuming feed that was originally contaminated, which is not possible . Althougredients . In summSeveral studies have evaluated virus survival in feed ingredients under various types of transport conditions. The initial study was conducted by Dee et al.  and inclSimulations of long-distance truck transport of virus-inoculated feed ingredients and complete feed in the U.S. have also been evaluated. Dee et al.  conducteSeveral studies have evaluated the use of extended storage time as a mitigation strategy to determine the rate and extent of inactivation of various swine viruses in different feed ingredients during various time and temperature exposures, and the results are summarized in Although extended storage times have been shown to be a simple and effective way of reducing virus concentrations in feed ingredients and complete feeds if they are contaminated, there are several unintended consequences that must also be considered. Storage facilities for feed ingredients represent a significant cost for feed handling and manufacturing facilities. As a result, frequent inventory turnover is required to minimize the cost of ingredient procurement and feed manufacturing. Furthermore, depending on the time, temperature, and relative humidity conditions of various types of feed storage and the physical and chemical characteristics  of ingredients, significant feed safety concerns and loss in the nutritional value of ingredients can occur. High temperature, humidity, and moisture conditions cause bacteria growth and mold and mycotoxin production during storage that can be detrimental to animal health and performance. These same conditions can also lead to significant losses of nutritional quality and value through reduced protein and amino acid digestibility, production of secondary lipid oxidation products, and loss of vitamin potency. Therefore, maintaining a balance between minimal storage time to reduce cost and preserve nutritional value and allowing adequate time for significant virus inactivation must be considered when using this mitigation strategy to inactivate swine viruses in contaminated feed ingredients.https://www.agriculture.gov.au/import/goods/plant-products/stockfeed-supplements) (accessed 26 March 2023) and the European Union  (accessed on 26 March 2023) also provide guidelines for assessing the risk of ASFV in imported feed ingredients but do not specifically require a standard 30-day storage period for suspect ingredients.Dee et al.  indicateThe number of studies assessing the survival of several of these important swine viruses in numerous common feed ingredients used in swine diets under different time and temperature conditions are limited. No studies have been conducted to evaluate CSFV survival during storage under controlled time and temperature conditions. Estimates for FMDV, porcine delta corona virus (PDCoV), transmissible gastroenteritis virus (TGEV), and PRRSV survival have each been determined in only one study, and the ingredients evaluated were limited to only soybean meal for PRRSV and soybean meal, DDGS, and complete feed for FMDV. Two studies have evaluated ASFV survival in four ingredients and an additional two studies have evaluated SVV-A survival in four ingredients. Although these are critically important studies to provide initial guidance on recommended storage times for virus inactivation of ingredients suspected of being contaminated, the data available from these studies is insufficient for supporting the 30-day extended storage time recommendation proposed by Dee et al. . All of To better understand the significance of the inconsistencies reported for extended storage time studies, consider the different methods, ingredients, and interpretation of results between the two studies evaluating ASFV in Feeding frequency of pigs used in bioassays and virus strain were additional factors beyond time, temperature, and assay methods that affected results for FMDV survival in soybean meal, DDGS, and complete feed during a 37-day storage period . The lasExtrapolating minimum storage times for virus inactivation is difficult if storage temperatures are below those commonly found in warm climates. For example, one extended storage study was conducted under conditions of extremely low and variable storage temperatures  to evaluate PEDV survival in 18 feed ingredients during a 30-day storage period . BecauseCoronaviridae family, of which PEDV and TGEV are classified in the Alphacorona virus genus, and PDCoV is in the Deltacorona virus genus [Extrapolating virus survival and inactivation data for a specific virus of interest from data derived from other virus types has been done, but it is not advised because of the uniqueness of each virus and the complexities of their behavior in various feed matrices. For example, PDEV, PDCoV, and TGEV are in the us genus . These cus genus . Howeverus genus  showed tOne cannot extrapolate virus inactivation results for individual feed ingredients or complete feed from studies that used non-feed matrices or cell cultures. Knight et al.  summariz5 TCID50 of SVV-A and the same concentration of PRRSV were added to feed ingredients. Results showed that only the storage temperature of 23.9 °C was effective in achieving no infectivity after 30 days. In contrast, Caserta et al. [5 TCID50 of SVV-A and showed that virus inactivation in L-lysine HCl and vitamin D occurred within 1 day at 15 °C and 30 °C compared with soybean meal and DDGS, which survived until 35 days at 15 °C and until 21 and 14 days, respectively, at 30 °C. Although Caserta et al. [Dee et al.  evaluatea et al.  inoculata et al.  did not Although soybean meal appears to be most protective for swine virus survival, it has also been the most studied of all ingredients. Other grains, grain by-products, and oilseed meals must also be evaluated for their ability to protect various swine viruses if they become contaminated because they are added to diets at much greater inclusion rates than vitamins and synthetic amino acids, and subsequently could contribute a greater virus dose for pigs to consume. However, results from several studies evaluating the survival of several viruses in soybean meal have shown that more than 30 days storage, even at relatively low temperatures, is necessary to achieve significant virus inactivation. The likelihood of a virus-contaminated ingredient causing infection is not only dependent on the initial virus concentration used to inoculate feed ingredients, and the subsequent extent of virus inactivation and loss of infectivity that may occur during storage, but also on the frequency of consumption of contaminated feed  ,61 that Structural and chemical characteristics of various feed ingredients likely play a role in the differences in survival of various viruses among different feed ingredients, but these factors have not been studied. The moisture content of feed ingredients and complete feeds is relatively low (<12%), but Trudeau et al.  reported4 for all other major swine viruses, which suggests that achieving a 3-log reduction in infectious virus concentration from an initially high theoretical contamination level of 106 in complete feed would be below MID for all viruses except for SVV-A in market-age pigs. However, all of these MID estimates are based on observations from a relatively small number of pigs used in these studies and would likely decrease when estimated using a greater number of animals in a population of pigs.Estimates for minimum infectious doses (MID) of several swine viruses have been determined through consumption of inoculated feed or direct oral inoculation in pigs . Virus sSalmonella in feed can still cause an infection when fed [7.0 TCID50 of FMDV three times, all pigs (4 of 4) were infected after two days. Likewise, after consuming 107.2 TCDI50 of FMDV only one time, all pigs (4 of 4) were infected [The minimum infectious dose cannot be interpreted as a concentration in which a product, if contaminated, is considered safe, or the safe concentration from which no infection will occur. The MID in the experiments presented in when fed . This diinfected . These rThe minimum infectious dose is different from other estimates of food and feed safety such as the food safety objective (FSO) and performance objective (PO) . These tIn addition to extended storage times, thermal processing can be an effective method of inactivating bacteria, viruses, and parasites depending on the temperature and duration . Heat isMost studies conducted with feed ingredients and complete feed have evaluated thermal processing on PEDV inactivation. A summary of the effects of various time and temperature conditions used during various types of production processes of feed ingredients and complete feed is shown in 50/mL reduction [Several studies have shown that the use of ultraviolet irradiation is an effective additional biosecurity step to further inactivate several enveloped  and non-enveloped swine viruses during the spray drying process of liquid porcine plasma . In geneeduction . Additional studies have been conducted to evaluate the effectiveness of the spray drying process used for animal plasma ,77,79,81Salmonella in poultry and swine feed, it is not approved for use in controlling swine viruses in the U.S. and numerous other countries. Various individual MCFAs and MCFA blends have also been extensively evaluated for their potential viral mitigation effects. Unlike FMPA, MCFAs such as C6:0 (caproic acid), C8:0 (caprylic acid), C10:0 (capric acid), and C:12:0 (lauric acid) are naturally found in triglycerides present in common fats and oils used in animal feeds, and their use is generally not restricted in commercial swine feeds. A few commercial products that have been evaluated as chemical mitigants in virus-contaminated swine feed contain certain short-chain or long-chain fatty acids, but their potential viricidal effects are questionable. Glycerol monolaurate has been shown to have more potent viricidal effects than MCFA for ASFV [Limited studies have been conducted to evaluate various types of feed additives for their effectiveness as chemical mitigants for inactivating ASFV ,86, FMDVfor ASFV . In addifor ASFV , PDCoV [for ASFV , PRRSV afor ASFV , and PEDfor ASFV ,88,97. Ofor ASFV  and PEDVfor ASFV  inactivaAmong all swine viruses evaluated, ASFV is the most difficult to inactivate because of its high thermal tolerance . FortunaPathogen contamination can occur on feed and non-feed contact surfaces in feed mills if contaminated feed ingredients are introduced, despite the use of well-designed and well-implemented feed mill biosecurity protocols ,98. BecaBiosecurity and mitigation strategies to reduce the risk of bacterial and viral pathogen contamination in feed mills have been evaluated and summarized based on a limited number of studies ,99. FeedOne of the simplest methods for decontaminating virus-contaminated feed and feed ingredients is to store potentially contaminated batches for an extended period of time in a heated warehouse or feed mill. Several government protocols have been developed and implemented in Canada , the EurMechanical reduction of PEDV concentration in experimentally contaminated feed manufacturing facilities has been evaluated using batch sequencing  and flusBecause all viruses have some sensitivity to heat exposure, the heat provided during the conditioning and pelleting process in feed mills can be effective in reducing or eliminating the infectivity of swine viruses. Cochrane et al.  conducteThe effectiveness of chemical cleaning and sanitizing feed mill equipment and surfaces to reduce PEDV concentration has also been evaluated. Huss et al.  applied Several plant extracts contain compounds with antiviral properties, including flavonoids, alkaloids, phenolic acids, terpenes, coumarins, lignans, and proteins . Of thesIsoflavones are flavonoid compounds that have potent antiviral properties against a wide variety of viruses, including enveloped and nonenveloped, single-stranded and double stranded, and RNA and DNA viruses . SoybeanOf all swine viruses, PRRSV is the only virus that has received research attention relative to the dietary benefits of soy isoflavones. Several studies have shown consistent growth performance and health benefits from feeding diets with high amounts of soybean meal to pigs infected with PRRSV. Results from initial studies showed that dietary daidzein provided less improvement in the growth of weaned pigs during a PRRSV challenge  than gen4 or 105 TCID50, and fed the contaminated feed for 14 consecutive days to determine if it would cause infection in naïve weaned pigs in two separate experiments. None of the pigs in either experiment became infected, indicating that either the minimum infective dose of ASFV is greater than 105.0 log TCID50/pig or that liquid porcine plasma has significant functional properties that may reduce the infectious capability of ASFV. Additional evidence of the functional benefits of feeding spray-dried animal plasma to weaned pigs was observed in a study conducted by Crenshaw et al. [Spray-dried animal plasma contains many functional compounds, including immunoglobulins, albumin, fibrogen, lipids, growth factors, biologically active peptides, transferrin, enzymes, and hormones , that plw et al. , which sFeeding spray-dried animal plasma to weaned pigs also appears to enhance immune response to vaccines. Weaned pigs fed a starter diet containing SDPP and vaccinated for porcine circovirus type 2 and Mycoplasma hyopneumoniae supported the best long-term benefits in survival to market and carcass weight . More reMonoglycerides and MCFA have become one of the most important types of antiviral feed additives for use in swine diets, and their molecular properties and biological functions have been reviewed and summarized . Medium-Hanczakowska  summarizMost of the research conducted to study the antiviral effects of flavonoids, alkaloids, phenolic acids, terpenes, and coumarins in plant extracts has involved either human coronaviruses or influenza viruses . PositivEffective treatments for pigs infected with ASFV are desperately needed because no effective and safe vaccines are available to prevent ASFV infection in pigs. Studies have shown that nucleoside analogues, interferons, specific flavonoids, a limited number of antibiotics, and small interfering RNA molecules inhibit ASFV replication by either acting directly as antiviral compounds or specifically providing certain antiviral effects in the host . SeveralFluoroquinolones are a class of antibiotics that are approved for use in treating certain types of bacterial infections but have also been shown to exhibit potent antiviral properties. These antibiotics trap DNA gyrases and topoisomerase IV on DNA and promote the formation of drug enzyme-DNA cleavage complexes that cause disruption of DNA replication, leading to mechanisms resulting in cell death . ModificLimited studies have evaluated the effects of various salts on swine virus inactivation. One study involved experimentally infecting pigs with CSFV and ASFV, euthanizing them during the acute phase of disease when viremia was greatest, and collecting small and large intestine samples for incubation with either sodium chloride or a salt mixture  at various temperatures up to 20 °C for multiple times up to 60 days . Both soMetals such as copper and zinc possess several properties including redox, photocatalytic, and structural stability, along with antibacterial and antiviral properties , that suThe goal of feed supply chain biosecurity programs is to deliver complete feeds to swine farms that are devoid of disease-causing pathogens. However, if viral pathogen contamination is suspected in complete feed delivered at the farm level, the addition of several commercially available feed additives to swine diets may improve the health and growth performance of pigs fed contaminated feed and undergoing a disease challenge. Many antiviral commercial feed additives containing various combinations of MCFA blends, glycerol monolaurate, organic acids, essential oils, and various other compounds have been developed, approved for use, and commercially available in some countries.Fifteen commercially available feed additives were evaluated when added to nursery feed contaminated with PRRSV, SVV-A, and PEDV to determine their effectiveness in improving the health and growth performance of pigs . A serie8.3 TCID50 FMDV A24 (greater than the previously determined minimum infectious dose) to feed with or without SalCurb or Guardian feed additives 24 h prior to feeding and observed no clinical signs or positive antemortem samples for pigs fed either feed additive treatment, except for one pig fed Guardian that was considered subclinically infected. Two of the feed additives evaluated in this study  were alsBeyond the results reported in these studies, it is unknown if these additives are effective in improving health and growth rate in swine diets contaminated with ASFV or other swine viruses. However, these results suggest that several commercially available feed additives may be effective as a last defense against a biosecurity breach to minimize adverse health effects from PRRSV-, SVV-A-, and PEDV-contaminated swine feed.Quantitative risk assessment of virus transmission through various feed ingredient supply chains is greatly needed because of high uncertainty due to the lack of a global monitoring and surveillance system. Unfortunately, most publications in the literature do not provide sufficient details and data to enable the development of quantitative risk assessment models. Many experiments evaluate only a single temperature, sampling time, or measure of virus inactivation rather than evaluating a comprehensive set of conditions. Furthermore, very few studies have reported D-values or z-values for virus inactivation kinetics that allow for comparison of results among studies. Accurate estimates for kinetic parameters of virus inactivation require multiple time and temperature conditions. Typically, virus inactivation curves follow non-linear patterns, which are best modelled with at least 4–5 observations distributed along the expected range in temperatures and sampling times in virus inactivation models. A limited number of replications per timepoint or temperature is another common problem with the data provided in the published literature. Many researchers fail to recognize the appropriate experimental unit and the number of experimental units associated with the error term measurement of the virus inactivation. Pilot experiments can be a useful approach for collecting preliminary data on the variability of conditions associated with virus inactivation to determine optimal subsequent experimental design. In the predictive modeling of pathogen inactivation, there are sources of uncertainty and variation in observed predictions that are introduced due to unknown effects of independent factors. Another source of variation is actual variability in the process input range. Because this source of variation is known, it should be estimated using sensitivity analyses. In summary, researchers are encouraged to consider these key deficiencies when reporting virus inactivation data from various mitigation strategies in feed ingredients in future studies to enable subsequent quantitative risk assessment determinations. Compared with other virus transmission routes, feed ingredients and complete feed appear to be less likely contributors toward disease transmission, but because there is no monitoring and surveillance system, there is high uncertainty of the extent of swine virus contamination in global feed supply chains. Biosecurity protocols need to be developed and implemented to improve our ability to prevent virus contamination and transmission through the production, processing, storage, and transportation of swine feed. Key components of feed biosecurity protocols should also include effective mitigation practices such as extended storage times, thermal and irradiation processing, and chemical mitigants to provide inactivation of viable swine viruses if they are present. Several types of functional feed ingredients, nutrients, and feed additives that have antiviral properties need to be further evaluated for their ability to inactivate swine viruses of concern in various types of feed ingredients. Unfortunately, there are numerous challenges that must be overcome to improve our understanding and ability to accurately predict whether feed contaminated with swine viruses is capable of causing an infection, including limitations of current analytical methods for measuring virus inactivation, viability, and infectivity in feed. In addition, the use of the food safety objective and performance objective need to be developed for risk assessment of virus survival in feed ingredients. Improving data quality and quantity when reporting results in scientific publications is needed to provide sufficient detail to allow for developing risk assessment models and calculating D-values and z-values for virus inactivation kinetics that allow for comparison of results among studies.


Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the  Culex mosquitoes transmit arboviruses that are endemic in several states. These notably include West Nile virus (WNV), which caused ~ 2500 human cases of disease annually in the U.S. between 1999 and 2019 in addition to many times more asymptomatic infections, and St. Louis encephalitis virus, which also causes a small number of cases annually, including periodic outbreaks3. WNV, considered the most prevalent cause of viral encephalitis worldwide, reached New York City in 1999 and spread to the rest of North America within four years, including California in 2003. Human infections can sometimes result in severe neuroinvasive disease, especially in older patients and those with chronic medical conditions3.Transmission of arboviruses to humans by mosquitoes is a persistent public health threat around the world. In the United States, 5. Viruses in this diverse group include insect-specific viruses (ISVs)6 and those that can be transmitted to non-human organisms. Little is known about many of these viruses, or their effect on transmission of arboviruses by mosquitoes. Recent studies have presented evidence that some ISVs may decrease arbovirus loads and transmission9 similarly to what is observed with infection by the Wolbachia bacterium. Wolbachia is a genus of intracellular bacteria that has been shown, when introduced into non-native host Ae. aegypti, to significantly reduce the mosquito’s ability to transmit dengue, Zika, and other RNA viruses to humans12. The potential mechanisms of Wolbachia-mediated antiviral effects are not completely clear, but data suggest some evidence of competition for resources between the virus, host, and Wolbachia14 as well as use of host microRNAs by the bacterium to contribute to virus inhibition15. If ISVs have some effects on arbovirus infection and transmission17, they could be used as biological control mechanisms or novel vaccine platforms by exploiting the limited host range of ISVs to protect against dangerous viruses infecting humans18. Furthermore, constant monitoring of viruses may allow us to detect the re-emergence of arboviruses transmissible to humans.Many viruses that have been detected in mosquitoes do not infect humans but do establish persistent infections in the mosquito, and evoke small RNA immune responses20. Replicative intermediates in the form of dsRNA are often generated during viral infection, and these intermediates can be processed into ~ 21-nt long siRNAs by Dicer-2. The siRNAs are loaded onto the RNA-induced silencing complex (RISC) and guide it to complementary, invading viral sequences, which will then be degraded22. By contrast, the PIWI-interacting RNA (piRNA) pathway has a well-established role in silencing transposons to maintain germline integrity23. However, piRNAs, which are generally ~ 24–29 nt in length, have also been implicated in antiviral activity in mosquitoes, although this activity is not yet well-understood28. Interestingly, this expanded piRNA activity does not seem to be present in Drosophila, despite mosquitoes and fruit flies being in the same order, Diptera. Some mosquitoes, particularly Culex and Aedes species, possess an expanded repertoire of Piwi-clade proteins as compared to Drosophila. Some of the proteins are expressed in somatic cells as well as follicular cells, and when purified were found to be associated with virus-derived sequences29. piRNAs can be produced through the primary Zucchini (Zuc)—mediated biogenesis pathway, which generates antisense piRNAs with a 1U bias31 or through the “ping-pong cycle”, where primary piRNAs are used to generate sense piRNAs with a 10A bias and further 1U antisense piRNAs32. Virus-derived piRNAs have been shown to have the antisense 1U and/or sense 10A nucleotide biases33, although the mechanisms of viral piRNA generation remain unknown. Finally, a separate class of small RNAs, miRNAs, are ~ 22-nt long, are transcribed from the host genome, and have been demonstrated as critical components of gene regulation by binding to cellular mRNAs to control their translation, stability, or decay34.In insects including mosquitoes, the small RNA interference (RNAi) system has been shown to play a central role in defense against viruses, most prominently the exogenous small interfering RNA (siRNA) pathway38. In our study, we aimed to use total small RNA extracted from whole mosquitoes to not only sample the virome of mosquitoes but also to analyze patterns of viral infection and immune signatures in these mosquitoes. We therefore deep sequenced 63 pools of Culex mosquitoes, 58 of them field-caught, and showed snapshots of the Culex virome over a three-year period in southern California. We also examined the patterns and correlation of viral infection based on location, year, and mosquito species. Furthermore, as the goal of this study was not only to discover viruses, but rather to analyze the abundance of and host response to viruses, we also mapped reads to the Culex genome to elucidate miRNA responses to both viruses and Wolbachia. Finally, we generated size profiles and virus genome coverage plots from the small RNAs mapping to individual viruses to analyze induction of small RNA pathways such as siRNA and piRNA in response to viral infection in field mosquitoes. Taken together, the results demonstrate the power of our approach, which could be used not only for virus discovery, surveillance, and epidemiology, but also to improve our understanding of mosquito immune response to many vector-borne diseases around the world.RNA sequencing has been used to detect viruses in many species, including mosquitoesCx. quinquefasciatus or Cx. tarsalis mosquitoes from the Inland Empire region of southern California, as well as five Cx. quinquefasciatus pools originating from laboratory strains. The most common pool size was 50 or near to this, but pool sizes varied . We detected an average of 7 distinct viruses in each sample. Despite high quality sequencing reads for five laboratory samples (average of 72.7 million reads), we did not detect any known mosquito-associated viruses in these samples Fig. c, suggesCx. quinquefasciatus vs Cx. tarsalis) are shown in Supplementary Fig. Culex narnavirus 1, which was present in every field sample of both species. Also common were viruses from the Hepe-Virga supergroup35, a group of (+)ssRNA viruses that has been loosely defined and only recently characterized, reflecting the lack of clear understanding around invertebrate viruses. Supporting this as well is the high prevalence of unclassified (+)ssRNA viruses which could not be placed into any defined families, such as Bunyavirales, Rhabdoviridae, and Flaviviridae, which are well-known to contain ISVs or arboviruses. Interestingly, we were also able to detect viruses thought to only infect plants . For these viruses, as discussed later, we have strong evidence of specific siRNA responses , suggesting that they may indeed infect mosquitoes. Many of the viruses detected have widespread geographical range, as some of them were found in other parts of the world including China35, Mexico40, and Colombia41, suggesting many of the same or very similar Culex viruses are found throughout the world.For most samples, there were several high-identity matches by nucleotide alignment (blastn), which we considered to be high-confidence virus detections as these sequences were closely related to known reference genomes. Many samples also had more distant matches that were detected only through virtual translation of the sequences in the six reading frames (blastx). The list of viruses detected by blastn with 90% nucleotide identity or higher can be found in Supplementary Table 42, a manifold learning technique for dimension reduction (see Methods for details). The resulting numbers of mapped reads represent a combination of viral abundance and intensity of the mosquito immune response, and will be referred to as small RNA quantity. Results show that the most obvious factors determining small RNA quantity in a sample were location and mosquito species . This was true even for samples collected over multiple years , both locations’ Cx. tarsalis (region b), Coachella Valley Cx. quinquefasciatus (region c), and lab (region d) samples.To identify factors affecting viral infection, we used direct mapping of reads to viral genomes —Cx. tarsalis samples were not included in this analysis due to the lack of an extensively annotated genome assembly. Approximately 19% of Culex-aligned reads mapped to rRNA, tRNA, snRNA, or snoRNA genes, while 32% mapped to pre-miRNA or protein-coding genes, either in coding regions or putative untranslated regions (UTRs)  Fig. , indicatCx. quinquefasciatus samples into two groups based on chosen sample attributes and compared the groups against each other. This was done using DESeq245, a software often used for RNA-seq differential expression analysis, using only sense-mapped reads and restricting the analysis to miRNA genes. DESeq2 normalizes for library size (number of reads) and has been successfully used in various fields for differential expression of small RNAs including miRNAs48. Our number of samples allowed for a higher number of replicates than typical RNA-seq experiments . As a negative control, all 48 Cx. quinquefasciatus field samples were randomly assigned into two groups five separate times. Each time, 0 miRNAs were differentially expressed between the two groups . We identified thirty-five pre-miRNA genes that were significantly upregulated in highly infected samples, including twenty-nine unique miRNAs  of Culex-unmapped reads aligned to the Wolbachia genome, was associated with a lower number of differentially expressed miRNA genes  than infection by viruses (Supplementary Table Wolbachia infection in mosquitoes50 (see Discussion). These results suggest that Wolbachia infection induces a more limited but significant miRNA response in the mosquito as compared to viral infection.We next examined the effect of ion Fig. d. High/lCulex narnavirus 1 accounted by itself for 38.6% of virus-mapped reads, we generated a separate analysis between samples with high and low abundance of this virus  and Culex phasma-like virus (CphasLV), in addition to the 21-nt peak, we detected a clear enrichment for read lengths of 24–29 nt. To validate that the detected read length of 24–29 reads represent a specific piRNA pathway response, we confirmed a sequence bias for an A in the 10th position of the forward reads and for a T in the 1st position of the reverse reads of this size range, indicative of piRNA generation by the ping-pong cycle  target one region directly upstream of the nucleoprotein gene . Overall, despite clear evidence of piRNA ping-pong generation against four viruses and strong evidence for piRNAs without ping-pong generation for at least four others, the most common feature among the viruses was the detection of the siRNA response pathway. For 43 out of 54 examined viruses (79.6%), 21-nt was the most common mapped small RNA length for sense-mapped reads, antisense-mapped reads, or both, even when accounting for the standard error range for viruses found in multiple samples  as a target of miR-989. This gene was predicted by all 4 algorithms and is most likely of particular interest as toll-like receptors are key to innate immunity including antiviral immunity. For mosquito samples that were highly infected with 63, to our knowledge ours is the first study to use a such wide array of viruses in field samples. Our results confirm that the siRNA pathway is the predominant small RNA response used by Culex mosquitoes in the field. For some viruses, the 21-nt size profile peak was much more pronounced for antisense reads, while in others, such as Hubei chryso-like virus 1, a strong signal was detected in both sense and antisense . Culex narnavirus 1 represents one example, with an antisense 1U-biased 24–29 nt peak similar to the one for Culex phasma-like virus. A recent study done in infected Aedes albopictus demonstrated that piRNAs are produced against a specific region of Chikungunya virus while siRNAs target the entire genome65. Our data show that Hubei mosquito 4 has a relatively high amount of antisense 24–29 nt reads with 1U bias and 10-nt overlaps with sense reads, in a peak directly outside of the coding region. By contrast, Wuhan insect virus 23 and Wuhan spider virus 10 display multiple regions of antisense 24–29 nt reads which have 1U bias, suggesting a more diffuse pattern of piRNA targeting. This pattern is more similar to those for Culex bunya-like virus, Turlock orthobunyavirus, and Hart park hapavirus, which generate widely targeting high-confidence piRNAs with the ping-pong signature. CxNV1, Hubei mosquito virus 4, Wuhan insect virus 23, and Wuhan spider virus 10 all have positive-sense ssRNA genomes, which seem to generate few antisense-mapped reads in general. All together, these data allow us to identify antisense piRNAs targeting some of these viruses in the absence of a clear ping-pong signature and suggest that different small RNA pathways covering different regions of the genome may be a common pattern across mosquito species against different type of viruses. Although our data suggest that piRNAs may be more common in Culex than previously thought, the overall scarcity of evidence for piRNAs in our data does agree with previous observations that piRNA responses occur to a wider array of viruses in Aedes compared to Culex mosquitoes66. Overall, the detection of intriguing patterns of viral infection and distinct small RNA immune response demonstrate the need to expand this type of study across different parts of the world, in a wide range of mosquitoes. Such data will allow us to generate an atlas of pathogens and the mosquito immune responses they generate to not only better understand host–pathogen interaction in field samples but to also design novel strategies against many vector-borne diseases.Interestingly, all four of the viruses with clear evidence of ping-pong piRNA response generation have negative-sense single-stranded RNA genomes, indicating that the (−)ssRNA genome itself may encourage extensive activation of this piRNA pathway in See 2 traps and gravid traps by the West Valley Mosquito and Vector Control District and Coachella Valley MVCD, respectively. Nucleic acid extraction was performed using the MagMAX Viral RNA Isolation Kit (AMB18365) and samples were deep frozen at − 75 °C or lower.For samples collected in both the Ontario and Coachella Valley areas, mosquitoes were amassed using COTRIzol was added to nucleic acid extracts for long-term storage, and RNA was extracted from this using chloroform and isopropanol precipitation. Samples were DNase-treated, checked for quality on an agarose gel, purified using Agencourt RNAclean XP beads (Beckman Coulter #A63987), and quantified using a Nanodrop spectrophotometer.Library preparation was performed using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB #E7300S/L), following the provided protocol. Size selection was performed by excising the region corresponding to small RNA on a 6% TBE PAGE gel.39. We used the default settings for maximum E-value for a hit (1e−5) and minimum percentage identity (25%) for blastn, although our analysis was mostly restricted to matches with at least 90% identity. For blastx hits, we used a cutoff of 50% percentage identity to reduce potentially inaccurate results.Illumina sequencing results were downloaded in FASTQ form, trimmed of adapter sequence, and, for analysis beyond viral detection, filtered to retain reads of length 18 bp or higher. Trimmed reads were run through VirusDetect, an automated pipeline designed for virus discovery using deep sequencing of small RNAsCx. quinquefasciatus genome (CpipJ2), we mapped reads to a combined file containing all virus genomes that had been detected with high confidence and filtered for uniquely mapped reads. We converted read counts to log-transformed frequencies, used UMAP to generate a lower dimensional visualization for the virus frequency matrix, and generated and inspected Pearson correlation matrices for correlations between samples and between viruses.After depleting reads that mapped to the Cx. quinquefasciatus samples were aligned to the Cx. quinquefasciatus genome (CpipJ2). DESeq245 was used to find differentially expressed miRNA genes between different groups of samples based on cutoffs of percentages of reads mapping to viruses (0.049% of sequenced reads) or Wolbachia . DESeq2 corrects for differences in number of reads between samples by generation of a size factor for each sample. Putative targets of differentially expressed miRNAs were predicted using sRNAtoolbox miRNAconsTarget67 with 4 algorithms: Simple seed analysis, TargetSpy68, Miranda69, and PITA70. GO enrichment was done using Fisher’s exact test through VectorBase (https://vectorbase.org).Reads from Culex-depleted reads were mapped to combined detected virus genomes. Small RNA size profiles and nucleotide bias plots were generated using custom Python and R scripts, and genome-wide coverage plots were made using the Integrative Genomics Viewer (IGV)71.Similarly to clustering analysis, Supplementary Information 1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.


IntroductionThe purpose of this study was to present the prenatal sonographic findings of external genital anomalies and determine diagnostic clues.MethodologyIn a single-center retrospective study, a total of 15,320 pregnant women underwent a routine ultrasound (US) screening between 18 and 40 weeks of gestation from March 2015 to January 2022. The fetuses with indeterminate sex and suspected genital anomalies were enrolled in the study. B-mod and three-dimensional (3D) imaging of the external genital organs were performed according to a local protocol in cases of genital anomalies. Prenatal and postnatal data were retrieved from the electronic health records.Resultshorseshoe sign on 3D images.A total of 88 fetuses were included in the study. The prevalence of external genital anomalies was found to be 0.6%, and the degree of correspondence (DC) between prenatal and postnatal diagnoses of external genital anomalies was 94.3%. The most common genital anomaly was hypospadias with a frequency of 59%. Severe hypospadias was detected in five of six cases with chordee where the penoscrotal angle was below 30°. Approximately 70% of clitoromegaly cases with labial hypertrophy had a Conclusionshorseshoe sign and penoscrotal angle may be useful in diagnosing and determining the severity of the external genital anomalies.The DC between prenatal and postnatal diagnoses of external genital anomalies is high in this study. The novel diagnostic clues, such as  Determination of fetal sex has become a part of routine prenatal sonographic examinations. Various sonographic markers of male and female external genitalia have been determined in early and late pregnancy. Although fetal sex determination is possible in most pregnancies, it can be difficult in some cases, and it may be possible to misidentify external genital organs. Many genital anomalies, such as hypospadias and clitoromegaly, may confuse fetal sex determination.tulip sign previously mentioned by Meizner [Hypospadias is a male genitourinary anomaly in which the embryological urethral groove fails to close . Therefo Meizner . The app Meizner . Despite Meizner .An accurate diagnosis of genital anomalies may be of high value to clinicians and parents by providing counseling and allowing early management of associated anomalies and syndromes. This study aims to present the prenatal sonographic findings of genital anomalies, determine diagnostic clues, and avoid pitfalls in fetal sex determination.Pregnant women who attended a secondary referral center specialized in maternal healthcare between March 2015 and January 2022 were enrolled in this single-center retrospective study. A total of 15,320 pregnant women underwent a routine US screening between 18 and 40 weeks of gestation, and fetuses with indeterminate sex and suspected genital anomalies were enrolled in the study. The fetuses who were not able to be followed up postnatally and those with bladder extrophies were excluded from the study. US screenings were performed by the same radiologist expert in the obstetric US using a high-resolution US device with a convex 6- to 1.9-MHz probe . Written informed consent was obtained from all parents, and the study was approved by the local ethics committee .In case of uncertainty of the fetal sex, genital anatomical data focused on the configuration of the genital tubercle, position of the genital folds, and visualization of the pelvic organs. Clitoromegaly was suggested by visualization of a thickened and enlarged clitoris between two and four swellings representing the labia Figure .Pelvic organs were evaluated in detail to make an accurate fetal sex determination. Demonstration of the uterus between the bladder and the rectum in the axial section of the pelvic region confirmed the diagnosis of the female gender. During routine sonographic examination, if any genital anomaly was suspected, it was considered abnormal and recorded as B-mod and also three-dimensional (3D) images as a local protocol for better evaluation of the anomaly Figure .On sonographic examination, a small incurved penis with a blunt-ended tip between the scrotal folds was diagnosed as hypospadias Figures -4. In thpenoscrotal angle, which is shown as the letter a in Figure An angle between the line extending from the ventral tip of the penis to the point where the scrotum meets the penis and the line passing from this point through the most anterior-superior part of the scrotum were measured to evaluate the severity of the hypospadias with chordee. This angle was called the Prenatal data were collected from the electronic health records of the study center’s obstetrics and gynecology department. History of genital anomalies in the family, exposure to chemicals, hormone therapy, associated anomalies, and integrated risk of aneuploidy were noted.Postnatal data about infants with external genital anomalies were retrieved from the electronic health records of the pediatric and urology clinics. The demographic data of the patients, including hormonal study, molecular analyses, and clinical outcomes of the fetuses, were retrieved from the health records.P < 0.05 was considered statistically significant for all tests.IBM SPSS Statistics for Windows, Version 22.0  was used for statistical evaluations. The Shapiro-Wilk test was used to check the normality assumption of the variables. Descriptive statistics were used as number, median, mean, frequency, and standard deviation (SD). The degree of correspondence (DC) was used to quantify the degree of resemblance between two sets of data. The DC between prenatal and postnatal diagnoses of the genital anomalies was calculated with the following formula, and it is expressed as a percentage: DC = number of correspondence data/number of valid data x 100. Out of 15,320 fetuses, 92 (0.6%) fetuses with genital defects were detected on prenatal ultrasound. Four of them were excluded from the study due to a lack of postnatal data. The characteristics of the fetuses included in the study are given in Table N = 21 ), micropenis (N = 7), and isolated labial hypertrophy (N = 5) in utero were confirmed at postnatal examination  cases, clitoromegaly in 21 , micropenis in seven (7.9%), isolated labial hypertrophy in five (5.6%), and uncertainty of sex determination in three (3.4%) cases. Of the 52 cases diagnosed with hypospadias in utero, 48 (92.3%) were confirmed at postnatal examination. Of these cases, 35 (72.9%) were classified as proximal/scrotal and 13 (27%) were glandular/distal hypospadias. Of 52 hypospadias cases, four (7.6%) were diagnosed with micropenis after birth. All of the cases diagnosed with clitoromegaly  fetuses on prenatal ultrasound. The penoscrotal angle was found to be less than 30° in five 45.4%) chordee cases, which were classified as proximal/penoscrotal hypospadias in the postnatal examination. Of 11 cases, the angle was over 30° in five (45.4%) cases classified as glandular/distal hypospadias  had a  Figures -3d.Thirteen out of 21 cases (61.9%) with clitoromegaly were diagnosed with congenital adrenal hyperplasia , while eight out of 21 cases (38%) were diagnosed with isolated transient clitoromegaly in the newborn period (Table N = 4), megacystis (N = 1), ovarian cyst (N = 1), and undescended testis (N = 1). Extremity anomalies in our cases included syndactyly (N = 1) and clinodactyly (N = 2). In two cases, cardiac anomaly  was diagnosed. A central nervous system (CNS) anomaly  was detected in one case, and an abdominal wall defect  was detected in another case.The overall prevalence of associated anomalies in our study was found to be 17%  was between the reported rates of 7%-38% in the literature -14. In oThere are some limitations of this study. Since there was no standardized description of the external genital organs and no objective measurement of the genital tubercle in the literature, external genitalia could be evaluated subjectively. The absence of a definitive genetic and hormonal evaluation protocol in the prenatal period was another limitation of this study. However, the main purpose of this study was to provide clues for prenatal diagnosis of genital anomalies.horseshoe sign of the labia in 3D images should be used in the diagnosis of clitoromegaly with labial hypertrophy. In male fetuses with chordee, a penoscrotal angle below 30 degrees may indicate that hypospadias is serious. Further studies are needed to confirm the value of prenatal sonographic clues in diagnosing and determining the severity of external genital anomalies.Prenatal diagnosis of external genital anomalies is of great importance for both clinicians and parents in terms of accurate determination of fetal gender and early management. The DC between prenatal and postnatal diagnoses of external genital anomalies is high in this study. Diagnostic clues such as the


Bovine respiratory disease (BRD) is one of the most common diseases in intensively managed cattle, often resulting in high morbidity and mortality. Although several pathogens have been isolated and extensively studied, the complete infectome of the respiratory complex consists of a more extensive range unrecognised species. Here, we used total RNA sequencing  of nasal and nasopharyngeal swabs collected from animals with and without BRD from two cattle feedlots in Australia.Mollicutes in BRD cases and unaffected control animals. Of the non-Mollicutes species, Histophilus somni was detected, whereas there was a low abundance of Mannheimia haemolytica.A high abundance of bovine nidovirus, influenza D, bovine rhinitis A and bovine coronavirus was found in the samples. Additionally, we obtained the complete or near-complete genome of bovine rhinitis B, enterovirus E1, bovine viral diarrhea virus (sub-genotypes 1a and 1c) and bovine respiratory syncytial virus, and partial sequences of other viruses. A new species of paramyxovirus was also identified. Overall, the most abundant RNA virus, was the bovine nidovirus. Characterisation of bacterial species from the transcriptome revealed a high abundance and diversity of This study highlights the use of untargeted sequencing approaches to study the unrecognised range of microorganisms present in healthy or diseased animals and the need to study previously uncultured viral species that may have an important role in cattle respiratory disease.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01591-1. Respiratory disease is one of the most common causes of disease and death in intensively managed cattle. While the severity of bovine respiratory disease (BRD) varies, it can account for up to 70% of the mortality observed in intensive beef production systems  . Mild orWhile some viruses and bacteria have been widely studied and are known to be an important component of the respiratory complex, novel, emerging or unrecognised pathogens are not easily detected because they are not routinely considered in the differential diagnosis or laboratory diagnostic assays are not available. Metagenomics approaches have facilitated the detection of complete microbiomes and viromes, including emerging pathogens or pathogens that cannot be easily cultured and isolated. Untargeted RNA sequencing  can help detect the complete taxonomic profile of eukaryotic organisms, bacteria, or DNA viruses, as well as the genomic RNA of RNA viruses, enabling the identification of viral species in a single assay –6. A fewHistophilus somni, Mycoplasma bovis, Pasteurella multocida and Mannheimia haemolytica are often associated with disease . Th. ThNarmoPorphyromonadaceae family (n = 3) and Moraxella bovoculi (n = 1) were filtered out due to low relative abundance. A total of 18 species from 10 genera and 7 families were retained. Mycoplasmataceae form the majority across all libraries, with library L10 having the lowest relative abundance  and Mycoplasma arginini in 5 case libraries and no libraries from unaffected ‘control’ libraries. Of the non-Mycoplasmataceae, Fusobacterium necrophorum 4/10 cases and 1/5 unaffected controls, Histophilus somni 8/10 cases and 4/5 unaffected controls, Pasteurella multocida 3/10 cases, 1/5 unaffected controls, Mannheimia haemolytica only found in 1/10 cases.The relative abundance of bacteria was inferred across taxonomic ranks for all libraries. The p = 0.289); Shannon (p = 0.540); Simpson (p = 0.713); Fisher (p = 0.270)) , so it was considered a contaminant. The contig in which erm(B) gene found in seven libraries was also identified as part of a cloning vector (Cloning vector pZJ23).Six AMR genes were identified in the transcriptome, conferring resistance against tetracycline: tetQ tetracycline resistance gene, which was also present in unaffected “control” samples. A variable abundance of tetH, mefA and vatE was identified in all libraries, while the abundances of mphE and msrE were consistently low in samples from unaffected control animals  had a higher abundance of als Fig. .Fig. 9HeAspergillaceae, Ascobolaceae, Nectriaceae, Trichosporonaceae, Wallemiaceae, Rhabditidae, Strongyloididae, Ancylostomatidae, Chabertiidae, Cooperiidae, Babesiidae, Theileriidae, Sarcocystidae, Entamoebidae, Hypotrichomonadidae, Trichomonadidae and Simplicimonadidae. Families present with higher frequency in the libraries were Strongyloididae (n = 12), Entamoebidae (n = 12) and Trichomonadidae (n = 11). Entamoeba was present in 5 libraries from animals without BRD and only 2/10 libraries from affected animals; p = 0.003). Similarly, the presence of Simplicimonas similis was associated with “control” libraries (p = 0.005). None of the other species or genus found was associated with cases.We identified RNA that was taxonomically classified within the families Paramyxovirus and the wide diversity of Mollicutes.We have identified a variety of viruses infecting the upper respiratory tract in Australian feedlot cattle. While some of the viruses identified are known to cause respiratory disease in cattle, this study highlights that there is a variety of other viruses that may have an impact on the occurrence of BRD. Of most note was the high abundance of BoNV in all samples, the presence of IDV, the identification of a new There are no previous reports of BoNV in cattle in Australia. Globally, the previously published genome of BoNV was assembled from sequences obtained from lung samples of cattle during a severe bovine BRD outbreak in a US feedlot in 2013, but the significance of BoNV in disease was not conclusive . AlthougInfluenza D virus has been previously detected in North America, Europe and Asia. Most detections have been in cattle and swine populations, although it can also affect sheep, goats, horses and camels –41. It iBCoV was present in most animals sampled in this study, and RNA load was higher in nasal swabs compared to nasopharyngeal swabs. Although BCoV is most often known to cause gastroenteric disease, it has been associated with respiratory disease outbreaks in the past in Australia and Italy , 45. WhiBoHV-1 is the etiologic agent of infectious bovine rhinotracheitis and a known pathogen within the BRD complex (39). Although we identified BoHV-1 in 7/10 case libraries and 2/5 control libraries, the abundance estimation is not directly comparable to the abundance estimated for RNA viruses as no genomic DNA was sequenced. However, this virus was detected by qPCR in almost all samples and the viral DNA concentration was high for several animals (Ct < 25). It is not possible to interpret the relevance of these data in the context the BRD complex because a live BoHV-1 vaccine virus had been administered intranasally to these cattle at induction to the feedlot .Picornaviruses such as BRAV, BRBV and EV-E have been infrequently associated with disease outbreaks in livestock. Recent metagenomics studies have revealed that BRAV and BRBV are often found in intensively managed cattle , 50. HowBovine respirovirus 3). While a previous study found that seroprevalence at feedlot induction was for 89% BRSV and 91% for BPI3, we detected BRSV in a low number of samples and PI3 was not detected in this study. The lack of detection may be due to animals exposed at an earlier age, or that seasonal waves of infection affect feedlots, but it is unlikely that this virus is completely absent from the feedlots sampled. BRSV is a known pathogenic virus and plays a role in the BRD complex in many countries. BRSV seroprevalence is high in Australian feedlot cattle [Two respiratory paramyxoviruses are known to circulate in Australian cattle, namely BRSV and bovine parainfluenza 3 . The current lack of comprehensive reference sequences for all viral species and genotypes in non-human hosts impedes the automation of taxonomic classification of viromes and requires specialised and labour-intensive assembly of divergent viral genomes. The automation of viral metagenomics data analyses tools depends on the creation of a thorough database with livestock viruses collected from different geographic regions to complete a species and genotype database.There are several challenges and limitations when interpreting microbial genetic data and its association with disease in cross-sectional studies. The microbial diversity observed may vary across different seasons or feedlot operations; therefore, it is only representative of the animals present at the feedlot at the time of sampling. Causation must be carefully assessed by the isolation of the pathogen candidate and followed up with reproducible clinical outcome with experimental studies. Even in cases where this can be achieved, different experimental studies may yield different results based on the specific genotype or variant used as the inoculum. Cross-sectional studies may therefore guide a deeper understanding of disease association, which may be followed up with observational longitudinal as well as experimental studies. The final outcome depends on complex interactions that are influenced by the timing of infection, the virulence of different virus strains, interactions with other viruses and microorganisms and a wide range of animal hosts, livestock management and environmental factors. For example, the failure to detect viruses such as BVDV and BoHV-1 at a single time point from a modest number of samples should not be misinterpreted and reduce their proven capacity as respiratory pathogens. Instead, the detection of novel agents should be used as a signal to investigate whether these additional viruses could also play a role in the BRD complex.With respect to the consensus genomes obtained in this study, the quality of the genomes is subject to assembly errors inherent in metagenome-assembled genomes. This becomes particularly relevant in libraries where we identified different genotypes and variants . For example, if more than one closely related virus was present in a sample, the assembly process could fail or could produce a chimeric consensus sequence . DespiteWe have found a variety of viral and bacterial species in the respiratory tract of feedlot cattle from two feedlots in Australia. From an Australian perspective, we described three previously unrecognised viral species in addition to providing sequence data for known ones. More extensive sample collections, experimental work with recently recognised viruses, automated sequencing and efficient bioinformatic analyses can each contribute to securing information that will provide a better understanding of the role of the infectome in BRD.Additional file 1. Samples collected and pooled for qPCR analysis and sequencing.Additional file 2:Table. Primers and probes used to determine viral abundance through qRT-PCR and qPCR.Additional file 3: Table. Contigs taxonomically classified as Bovine and Porcine Rotavirus and their amino acid identity to published reference. Additional file 4. Phylogenetic tree of BoNV depicting bootstrap values. Additional file 5. Overview of Influenza D virus’ segments phylogenies. 5.1. Maximum likelihood phylogenies of the seven segments of Influenza D virus. 5.2. Influenza D virus- segment 1. 5.3. Influenza D virus- segment 2. 5.4. Influenza D virus- segment 3. 5.5. Influenza D virus- segment 4. 5.6. Influenza D virus - segment 5. 5.7. Influenza D virus- segment 6. 5.8- Influenza D virus – segment 7.Additional file 6. Phylogenies with bootstrap values of BRAV, BRBV, EV-E capsid  and 3D polymerase. 6.1. Bovine rhinitis A virus – capsid . 6.2. Bovine rhinitis A virus– 3D polymerase. 6.3. Bovine rhinitis B virus – capsid . 6.4. Bovine rhinitis B virus– 3D polymerase. 6.5. Enterovirus E– capsid . 6.6. Enterovirus – 3D polymerase. Additional file 7. Overview of Bovine narmovirus-1 (Bulang virus) phylogenies. Fig 7.1. Maximum likelihood phylogenies of Bovine narmovirus-1 (Bulang virus). 7.2. Bovine narmovirus-1 (Bulang virus)– L protein. 7.3. Bovine narmovirus-1 (Bulang virus)– Nucleoprotein. 7.4. Bovine narmovirus-1 (Bulang virus)– Matrix. 7.5. Bovine narmovirus-1 (Bulang virus)– Phosphoprotein. 7.6. Bovine narmovirus-1 (Bulang virus)– Fusion protein. Additional file 8:Fig. Alpha and beta diversity of the metatranscriptomic sequence libraries obtained from animals with clinical bovine respiratory disease (cases) and without clinical disease (controls). Fig(a): The alpha diversity computed using different methods. Fig(b). NMDS plot using Bray-Curtis dissimilarities between samples in cases and controls.


Bulls’ subfertility has a major impact on the efficiency of production and profitability of cattle enterprises. Bulls typically undergo a bull breeding soundness evaluation (BBSE) to predict potential fertility. The present study investigated if a more comprehensive index of indicative fertility could be developed in bulls by including testicular ultrasonography and hormonal status in the BBSE. Bulls with homogeneous testicular parenchyma showed a higher percentage of motile sperm post-thawing compared with bulls with heterogeneous parenchyma. In bulls with homogenous parenchyma, the percentage of motile sperm, progressively motile sperm, and motility yield were positively correlated with hair DHEA-S concentration. The use of testicular ultrasonography and DHEA-S status in the BBSE would provide a more comprehensive assessment of potential fertility in bulls. In addition, ultrasonography can be used in the BBSE when the evaluation of semen parameters is not available.n = 16; 2.7 ± 0.4 years old; body condition score 3.2 ± 0.1) of five breeds were maintained under the same conditions at an accredited semen collection center. Bulls underwent routine semen collection twice weekly for 12 weeks and semen was processed and cryopreserved. Ultrasonography and hair sampling were undertaken at the last semen collection. Bulls with homogeneous testicular parenchyma (n = 8) had a higher (p < 0.05) percentage of motile sperm post-thawing compared with bulls with heterogeneous parenchyma (n = 8). There were no differences (p > 0.05) in the hair concentrations of cortisol, DHEA-S, and testosterone between bulls with homogeneous and heterogeneous parenchyma. In bulls with homogeneous parenchyma, hair DHEA-S concentration was positively correlated with percentage motile sperm (R2 = 0.76), progressively motile sperm (R2 = 0.70), and motility yield (R2 = 0.71). The findings indicate that the integration of testicular ultrasonography and hair DHEA-S status in the BBSE could provide a more comprehensive assessment of indicative fertility in bulls. Additionally, ultrasonography can be used in the BBSE when the evaluation of semen parameters is not available.Testicular ultrasonography and steroid concentrations , cortisol/DHEA-S ratio, testosterone) in hair were examined for their utility in the bull breeding soundness evaluation (BBSE). Beef and dairy bulls ( Bulls with low fertility have a major negative impact on the efficiency of production and profitability of cattle enterprises ,2. HenceSpermatogenesis is influenced by many factors including metabolic and endocrine status . The braAllostasis is a term used to describe mechanisms whereby the body adapts to stressors to maintain healthy homeostasis. Allostatic load is the build-up of stressors over time and the impact on the brain and somatic tissues. In cattle and other species, the amount of cortisol present in hair is reflective of the short- to medium-term activity of the brain–adrenal axis and provides an index of allostatic load ,22. HairAll experimental procedures complied with the Italian legislation on animal care . The study had approval from the Ethical Animal Care and Use Committee of the University of Naples Federico II (Protocol PG72021/0130477).n = 7), Holstein Friesian (n = 5), Limousine (n = 2), Charolaise (n = 1), and Chianina (n = 1). Animals were maintained under the same management at an accredited National Semen Collection Center. The study lasted 12 weeks and testicular ultrasound examination and hair sampling were undertaken at the same time as the last semen collection, according to the retrospective value provided by the hair matrix.The study involved sixteen bulls  of five breeds: Pezzata rossa italiana  equipped with a 13–3 MHz linear array probe was used to image the testes of each bull; the same settings were used for focus, gain, brightness, and contrast, standardized at the machine median settings. The ultrasound transducer was held vertically  on the caudal surface of the scrotum. The image was aligned until the mediastinum of the testes was clear and apparent . The imaThe hair in cattle grows at approximately 0.6–1.0 cm per month and animals show a full molt approximately every 3 months . The conBulls underwent semen collection twice weekly for 12 weeks as part of the routine commercial activity of the authorized National Semen Collection Center. Bulls were trained to serve an artificial vagina . A total of 384 ejaculates were collected during the study.6 sperm/mL) [2, frame rate 60 s−1, immotile AOC < 3.5 µms−1, lateral motile DSL < 15 µms−1, VSL < 20 µms−1 and VAP < 24.9 µms−1, progressively motile STR > 0.5 and LIN > 0.35, non-linear STR < 0.5 and LIN < 0.35, curvilinear DAP/Radius = 3 and LIN < 0.5, round pat area average < 40 µm2, BCF = 0, and AOC > 8 [6 spermatozoa/mL with egg yolk-based freezing extenders  before packaging into 0.25 mL straws. Straws containing extended semen were then incubated at 5 °C overnight. Freezing was initiated by transferring the straws into a fixed temperature freezing chamber  at −140 °C for 15 min, and subsequently plunging them into liquid nitrogen. Assessments on frozen-thawed semen were performed within 5 min after thawing and included the percentage of motile sperm (% motile), percentage of progressively motile sperm (% sperm PM), percentage of viable sperm (% live sperms), and motility yield (proportion of sperm migrating into the medium).Semen was assessed and processed immediately after collection. The volume of semen was estimated by weighing the ejaculates within 5 min of collection and incubation at 35 °C. Initial assessments on fresh semen included the following: ejaculate volume (mL), motility (% motile spermatozoa), and total concentration (10perm/mL) ,29. Seme AOC > 8 . Semen sHair samples were prepared for the steroid assay as previously described . BrieflyCortisol ,26, DHEAp < 0.05) were included in the regression model. Pearson correlation was also used to exclude possible intercorrelations between the independent variables. Unless otherwise stated, the results are presented as mean ± standard error and significance was set at p < 0.05.Statistical analyses were carried out using SPSS (28.0) for Windows 10 . The initial dataset was edited, discriminating both for missing information and outliers . The number of samples excluded was the same between the homogenous and heterogenous bulls, which were characterized by similar coefficients of dispersion. The final dataset consisted of 236 ejaculates (14.7 ± 1.4/bull). The normal distribution of all data was confirmed using the Shapiro–Wilk test. Bulls were used as the experimental units. Multivariate analysis of variance  was used to compare hair steroids of bulls (dependent variables); testicular parenchyma and breed were the fixed factors, and their interaction was also considered. Data on semen characteristics were analyzed by ANOVA for repeated measures with testicular parenchyma (homogenous/heterogenous) as the main factor, and breed and cortisol as random. The day of collection was the repeated measure. Multiple linear regression was performed (forward stepwise procedures) with steroid concentrations as independent variables, and fertility parameters  as dependents. Potential independent and dependent variables were first tested for potential correlations using Pearson correlations and only significant correlations  (fibrosis score 0–2) . For theFertility parameters of fresh semen did not differ between homogenous and heterogenous bulls .p < 0.05) percentage of motile sperm post-thawing , progressively motile sperm (R2 = 0.70), and motility yield (R2 = 0.71).The relationships between hair DHEA-S concentration and motile sperm (%), progressively motile sperm (%), and motility yield for bulls with homogeneous testicular parenchyma are shown in The present study examined whether the incorporation of testicular ultrasonography and hair steroid concentrations in the bull breeding soundness evaluation (BBSE) would provide a broader and more comprehensive index of indicative fertility. Another objective was to determine whether testicular ultrasonography could be used in the BBSE when semen evaluation is not available. Bulls with homogeneous testicular parenchyma had a higher percentage of motile sperm post-thawing compared with bulls with heterogeneous parenchyma. This finding could be interpreted to suggest that the sperm of bulls with homogenous parenchyma has a higher tolerance to cryopreservation and thawing compared with the sperm of bulls with heterogeneous parenchyma. This was an important observation as sperm motility is related to fertility in bulls ,29. UltrThere were no differences in hair concentrations of testosterone, cortisol, and DHEA-S between bulls with homogeneous or heterogeneous testicular parenchyma. Previous studies in cattle and other species have reported an inverse relationship between cortisol and DHEA-S, and it was suggested that DHEA-S may partly counterbalance the negative impact of elevated cortisol on physiological and endocrine functions ,22,31,32Percentage motile sperm, progressively motile sperm, and motility yield were positively correlated with hair DHEA-S concentration in bulls with homogeneous parenchyma. This relationship may have been partly due to the prohormonal role of DHEA-S and its conversion to androgens and/or estrogens in peripheral target tissues . As noteAs noted, there are conflicting reports on relationships between testicular parenchyma and testis hormonal and spermatogenic function in bulls. The present study has provided strong evidence that the condition of the parenchyma is reflective of spermatogenesis. Given the practical implementation of ultrasonography and hair sampling, the case can be made for inclusion in the BBSE, or ultrasonography can be used when semen evaluation is not available.The present study has shown that bulls with homogeneous testicular parenchyma have sperm with a greater resilience to cryopreservation than the sperm of bulls with heterogeneous testicular parenchyma. This is an important finding as bulls of high commercial value are used extensively in artificial insemination. The study also highlighted a positive relationship between hair DHEA-S and important sperm fertility parameters. A limitation of the present study was the relatively small number of bulls tested and the absence of the BBSE. Notwithstanding, it could be concluded that the inclusion of testicular ultrasonography and hair DHEA-S in the standard BBSE is practical and would provide a more integrated and comprehensive assessment of fertility in bulls. Finally, ultrasonography can be used when the evaluation of semen parameters is not available.


Campylobacter fetus subsp. venerealis (Cfv), the causative agent of bovine genital campylobacteriosis (BGC). Thus, we designed a cross-sectional study to assess the prevalence and Cfv-associated factors in cattle farms in RS, Brazil. In total, 99 farms were randomly selected to participate in the survey. Preputial mucus samples from selected bulls were collected twice  and subjected to Cfv molecular detection. A farm was considered positive when at least one sample was positive for Cfv. Our findings indicate that the farm-level Cfv prevalence in RS is 67.67%. On average, the chance of a farm using natural service to be Cfv-positive increased approximately twice compared to farms that do not use natural service. We also determined that Cfv routine tests reduce the chance of a farm being positive by 92%. Therefore, both Cfv detection tests and the reduction of natural services decrease the chance of a farm being positive for Cfv. Finally, we conclude that Cfv is widely spread in Southern Brazil cattle farms and it is urgent the implementation of control measures to reduce Cfv prevalence in the target population.Cattle farming is a major livestock activity with economic relevance in Rio Grande do Sul (RS), Brazil. However, this activity is still considered of intermediate to low technological level, and in this region, there are few epidemiologic reports of The online version contains supplementary material available at 10.1007/s42770-023-01119-7. Cattle farming is a major livestock activity in Rio Grande do Sul (RS), Brazil. In 2021, RS exported USD 308.5 million in beef to 90 countries [Campylobacter fetus subsp. venerealis (Cfv), which shows an important parasitic relationship, and a high adaptation to the bovine genital tract [Bovine genital campylobacteriosis (BGC) is the main cause of reproductive issues in extensive cattle and low-tech livestock, placing important economic losses and restrictions on animals or animal products trade . BGC is al tract . Bulls aal tract . In femaRS is the southernmost state of Brazil, bordering Uruguay in the south, Argentina in the west and northwest, and the Brazilian state of Santa Catarina in the north. According to the official data from the Secretary of Agriculture, Livestock, Sustainable Production, and Irrigation (SEAPI-RS), the RS hosts ∼178,138 cattle farms, of which approximately 80% had up to 50 animals, 17% up to 500 animals, and 1.9% up to 10,490 animals [A stratified two-level sample design was used. The primary unit was the herd, stratified along the seven RS regions and randomly selected Fig.  from a lSample collection and molecular analysis were performed between July 2020 and December 2021. The procedures were approved by the Ethics Committee for Animal Experimentation from UFRGS n. 42052. In total, 99 farmers were actively contacted by phone being invited to participate in the project. With the farmer’s agreement and the confirmation of the squeeze chute to bulls’ restraint, the visits were scheduled, and the bulls were collected. Figure Genomic DNA extraction from the collected samples was performed, as previously described , 12, usife1 (ISC1-F: 5′-GGTGGAGAGCGTAGATATAAATTAG-3′ and ISC1-R: 5′ CCATAAAGCCTAGCTGAAAAAACTG-3′) [Cfv molecular identification was performed by polymerase chain reactions (PCR) targeting ISCACTG-3′) . Each PCP-value until only significant variables remained in the model . The variable number of bulls was assumed to represent the size of the herd and used as a confounder in the analysis. The generalized linear model was implemented in routine glm from the base library; fixed effects were tested with the classic Wald test, employing an approximation by the chi-square distribution. Multicollinearity was assessed by calculating the variance inflation factor (VIF) using two as the cut-off for multicollinearity. The Wald tests and VIF were assessed from the car library. All analyses were done in R [A descriptive statistical analysis was used to explore the characteristics of the study population. To assess the factors associated with the presence or absence of Cfv in each farm, a generalized linear model  was applone in R .Among all 99 farms visited, 485 bulls were sampled, and the number of tested bulls per farm ranged from one to 20. According to the analyses, 29.3% of the sampled animals were Cfv-positive in at least one sample. Figure The overall farm-level prevalence of Cfv in RS is 67.67%, (58.1–76.35%), with 95% confidence interval (CI), showing that this agent is extensively present in the target population. Similarly, De Carli et al.  also repAfter five steps, the final regression model had Akaike criteria information of 121.96, retaining two variables -1) on average. Although the number of bulls is not statistically significant, it was pushed in the model as a confounder Below is the link to the electronic supplementary material.