The research was aimed at developing recipes for buns studying the nutritional value of securities. In the work, an assortment of bakery products was developed from flour, composite mixtures of leguminous crops and dry powders of sugar beets. As a result, bakery products with useful properties and improved qualities were obtained. In the recipe, sugar was completely replaced by dry powders of sugar beet. The optimal combination for making a bun from composite flour and dry sugar beet powder was 10% chickpea and 5% mung bean flour with 9.23 g of dry sugar beet powder added per 100 g flour. Physical and chemical indicators, including mineral elements, vitamin composition, and safety indicators, were determined. It was proven that the use of composite flour from leguminous crops contributes to a contraction of the technological process of the production of bakery products, reducing the time needed for dough preparation and baking. The use of technology for obtaining bakery products and recipes in production allows expanding the range of bakery products, reducing the duration of the technological process of production, improving the quality of finished products, and increasing labour productivity. It also helps to improve the socioeconomic indicators of bakery and confectionery enterprises. Recently, the entire population of Kazakhstan has taken an interest in products for a healthy lifestyle. The bakery industry today is one of the most rapidly developing sectors in Kazakhstan . At the In a market economy, the only enterprise that survives is the one that most competently and completely determines the requirements of the market, creates and organizes the production of products that are in demand, and provides high income for highly qualified workers –5. ReducBread and bakery products have always been and remain one of the main food product categories consumed by the population. Bakery products are one of the staple foods in the human diet –12.Bakery products are the most important human food. Daily bread consumption in different parts of the country ranges from 150 to 500 per capita. Bread contains many essential nutrients; these include proteins, fats, carbohydrates, vitamins, minerals, and dietary fibre –15.In addition to traditional methods of dough preparation , bakeries also use more advanced methods (reducing the duration of fermentation and using enhancers and additives).The main direction in improving the quality of bakery production and increasing the efficiency of enterprises is the introduction of innovative dough technologies, expanding the range of dietary, medicinal, and children's products. For the preparation of these products are flour mixtures with bran, unrefined and crushed grains, sunflower, sesame seeds, vitamin-mineral components, biologically active additives, etc., applied –18.An acute disease is a deficiency of vitamins, in particular, obesity C (in 60-70% of the population of Kazakhstan), folic acid (70-80%) and minerals: iron 20-40%), cholesterol (40-60%), and iodine (up to 70%). Such deficiencies reduce the functional activity of the immune system and constitute risk factors for a large number of common chronic diseases , 20.-40%, choGrain crops are grown in agriculture. Their processed products or fruits themselves are used as primary and secondary raw materials in the bakery industry. The main sources of raw materials are flour, water, salt and yeast. These are the most important components in the recipe of bakery products. Additional sources of raw materials are used to increase the nutritional value and improve the taste and aroma of bakery products. These include sugar and sugar-containing foods, fats, milk and dairy products, eggs and egg products, malt, fruit plants, various nuts, spices, and additives , 22.Wheat, buckwheat, triticale, oats, barley, and corn cereals used to make flour. Legumes (soy and peas) are used as additives that increase the nutritional value of bakery products. Essential oil crops  add a special taste and aroma to bakery products. One of the most important cereals is wheat. Wheat flour proteins turn into tissue when mixed with water. It is often used in the manufacture of bakery and pasta products and cereals. Products made from whole grains and whole grains are usually used for dietary nutrition –27.The results of literary studies show that effective technology is needed to ensure a product with high quality and useful properties. It is also necessary to develop technologies for producing sugar beet powders for use as sugar substitutes in the manufacture of bakery and flour confectionery products.In this work, a range of bakery products was developed from composite flour mixtures of legumes and dried sugar beets. Their use will help to improve the quality and beneficial properties of sugar products, shorten the production process, ensure waste-free technology, increase labour productivity, and increase the socioeconomic indicators of bakery and flour confectionery enterprises.The methodological basis of the study was a systematic analysis of the technology used in the production of bakery products enriched with useful herbal ingredients. The following main tasks were performed sequentially: (1) selection and justification of the method for introducing herbal ingredients into the recipe for bakery products and (2) improvement of the technology used in bakery products by incorporating useful plant ingredients. The theoretical basis of the research consisted of general scientific and special research methods, methods of system analysis, and experimental planning. The objects of research were wheat flour of the first grade, chickpea and mung bean flour, and sugar beet.In the work, the following indicators of the raw materials used and the resulting assortments of bakery products were investigated: organoleptic indicators by GOST 5667-85, mass fraction of moisture by GOST 21094-75, mass fraction of fat by GOST 5668-68, mass fraction the proportion of protein by GOST 10846-91, and others.The crude fiber content was also determined by the Wende method on a Fiwe-6 device . Determiα-, β-, and γ-isomers of hexachlorocyclohexane (HCH), dichlorodiphenyl trichloromethylmethane (DDT), and its metabolites, was determined by gas-liquid chromatography. Additional methods and techniques were also used.Vitamins B1, B2, and PP were determined by capillary electrophoresis on a Kapel-105M “Lumex” device. The content of pesticides, including heptachlor, We used leguminous crops such as chickpeas and mung beans. Composite flour enriched with protein, dietary fibre, B vitamins, and macro- and microelements was obtained from them.A method of drying sugar beet was developed, and a finely ground powder was obtained. Fresh sugar beet tubers were sliced thinly and dried without disturbing the structure on a Hurakan HKN-DHD10 dehydrator at a temperature of 70°C for 4.5–5 hours and subsequently crushed in an LZM-1M laboratory mill with a given stable particle size. The resulting sugar beet powder is shown in The main raw materials for baking bread are first-grade flour, chickpea flour, mung flour, water, milk, sugar, sugar beet powder, shiver, butter, and an egg. Various assortments of rolls were received. Comparing the quality of the dough and cooking and baking times, the following assortments of buns were selected for further research (for one portion of laboratory baked goods):The dough mass for one piece of bun was 50 g. In one portion (about 500 g) of laboratory baked goods prepared according to this recipe, an average of 10–12 buns were obtained.Butter buns are buns made from sweet yeast dough. Buns can be prepared for every taste: plain or with raisins, poppy seeds, striser crumbs, sugar topping, etc. In this work, ordinary buns without filling were prepared. To eliminate sugar from the bun recipe, sugar beet powder was added. Dried sugar powder contains about 70% sugar and also 7.1% moisture. Sugar contains almost no moisture and leads to a thinning of the dough. The reason for this is that increasing the osmotic pressure in the liquid phase of the dough reduces the swelling of the flour colloids. Due to the high content of free water, the dough liquefies, although the total moisture content decreases. Therefore, compared with the sugar bun recipe, there was a difference in the amount of first-grade flour in the sugar beet powder bun recipe. Also, flour from legumes, especially chickpea flour, strongly absorbs moisture. For them, first-grade flour was added depending on the consistency of the dough. The recipes for the various bun formulations are shown in Tables Saccharomyces cerevisiae and the food emulsifier E-491, was used. Since S. cerevisiae is very active and of high quality, they reduce the rising time. The yeast is placed directly in warm milk for about 10–15 min for activation before incorporation into the dough and subsequent kneading. In the work, the dough was prepared using a dry Pakmaya yeast liquid sponge with a moisture content of more than 65% for 15 min. With the sponge method of preparing the dough, the products undoubtedly have a better taste and aroma, as well as more developed and better porosity than with the unpaired one. The sponge dough is highly hydrophilic, and it contains more colloids peptized by water; its viscosity and yield point, even immediately after kneading, are less than that of a bezoparny dough. This is because when kneading the sponge dough, the starch and proteins of the part of the flour that was in the dough have already undergone certain enzymatic and colloidal changes during the fermentation of the dough and are added to the dough in a finished state.To prepare the dough, Pakmaya dry yeast, which consists of the natural yeast The buns were prepared in the following order (for one portion of baking):The milk was warmed up to room temperature, and sugar and yeast were added to it. Then, about 25 g of flour was added, and all the ingredients were mixed. After that, the container was closed with a lid and placed in a proofing cabinet with a temperature of 30–32°C for 15 min. Then, the rest of the ingredients were added, and the dough was kneaded. Flour for samples no. 4–no. 10 was added depending on the mass of chickpea and muffin flour, as well as dry sugar beet powder. Then, the dough was sent to a proofing cabinet with a temperature of 35–36°C (proofing no. 1). After the dough rose to the desired volume, it was divided; that is, small balls of 50 g were molded from it and placed on a baking sheet. Then, it was sent to a proofing cabinet with a temperature of 35–36°C (proofing no. 2). When the balls were enlarged to the desired size, they were greased (using a cooking brush) with beaten egg yolk and placed in a Unox XFT133 convection oven preheated to 150–160 degrees. It is better to place the baking sheet in the middle of the oven, so that there is approximately the same distance to the top and bottom of the oven, to ensure even baking. The buns were baked at 180°C. The baking time depends on the composition and properties of the raw materials used. Proofing and baking times are shown in Further, the obtained samples of the bun were examined in the accredited laboratory of the Scientific Research Institute of Food Safety at the Almaty Technological University.The data were analyzed using MS Excel for Windows version 10 Pro, 2010. The data collected during the study were subjected to independent tasting, and questionnaires were conducted to assess the organoleptic characteristics of control and test samples. In the process of analysis, absolute and relative statistical indicators and tabular and graphical methods for presenting the results were used.At the beginning of the work, the physicochemical and microbiological indicators of the raw materials used were investigated. The results of the study of the physicochemical and microbiological indicators of dry sugar beet powder are shown in The results of the study of the physicochemical and microbiological indicators of the first-grade flour are shown in The physical and chemical indicators of the first-grade flour presented in To determine the optimal formulations, the organoleptic properties of buns of various compositions were determined. The results are shown in 2From the data in 2Further, the physicochemical and safety indicators were investigated. The results are shown in Tables 713To exclude sugar from the bun recipe, dry sugar beet powder was added, and chickpea and muffin flour were also incorporated to enrich the dough with minerals, vitamins, and other nutrients. For comparison, buns prepared with dry sugar beet powder, chickpea, and muffin flour added separately in different ratios and quantities were studied to determine their amounts in the final product.From the data in From the data in From the data in In Vitamin E is found in large quantities in sugar beet powder and chickpea flour; therefore, its amount in sample nos. 3, 4, and 6, respectively, was higher. The content of vitamin E in sample no. 1 was 2.29 mg/100 g, and in sample no. 9, it was equal to 2.42 mg/100 g. Mung flour contains a large amount of vitamin PP; therefore, sample no. 5, made from 15% mung flour with sugar had the highest value of all the samples, about 4.874 mg/100 g. Accordingly, in test sample no. 9, the content of vitamin PP is 3.032 mg/100 g, and in the control sample, it is about 2.743 mg/100 g.Escherichia coli bacteria (colibacillus) were not detected in any bun samples tested. Yeasts were found in sample no. 4, made from 15% chickpea flour with sugar, equal to 1 CFU/g, but did not exceed the established norms. The amount of mold in control sample no. 1 was 7 CFU/g, while other samples showed less and sample nos. 4 and 9 showed none.From the data in By comparing the various bun formulations, we were able to choose the optimal one. The optimal and most effective option for baking buns is the recipe for sample no. 9, made from 5% mung flour and 10% chickpea flour with the addition of 30 g sugar beet powder (for one portion of laboratory baked goods). In this recipe, dry sugar beet powder completely replaces sugar, and this bun is enriched with useful properties, vitamins, and minerals. Therefore, it is proposed for production. A comparative photo is shown in The use of mung flour and chickpea flour for making buns shortens the baking and fermentation time and promotes the fermentation process. Dry sugar beet powder reduces the spreading of the dough pieces.Recipes and technological regimes for the reception of bakery and flour confectionery products using sugar beet powder and leguminous crops have been developed. Assortments of bakery and pastry products were obtained using sugar beet powder and composite flour of leguminous crops. In order to exclude sugar from the recipe, sugar beet powder is used in the production of buns. In dry sugar powder, the concentration is about 70% sugar and 7.1% moisture. Compared with the recipe for sugar buns, in the recipe for bakery and flour confectionery products with sugar beet powder, a difference in the amount of premium and first-grade flour is established. Pulse flours, especially chickpea flour, have also been found to be highly absorbent. For this sample, first-grade flour was added depending on the consistency of the dough.As a result, it was determined that sample no. 9 is made according to the following recipe (per 100 g): first-grade wheat flour, -73.23 g; chickpea flour, 10 g; puree flour, 5 g; milk, 46.15 ml; dry yeast, 1.85 g; eggs, 15.39 g; dry sugar beet powder, 9.23 g; and butter, 15.39 g with waiting and proofing time about 95 minutes and waiting time 10 minutes.


UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases. A variety of mechanisms regulate cellular signaling pathways. One such mechanism is the control of protein degradation. Protein degradation serves as a protein homeostasis regulatory network that removes unnecessary proteins from the cellular environment when they are no longer needed, damaged, or misfolded. In eukaryotic cells, the ubiquitin–proteasome system (UPS) and the autophagic–lysosomal pathway are the two major protein degradation systems . Of thesThe UPS utilizes ubiquitin, a 76-amino acid polypeptide, as a tag to mark substrates for degradation. This process is called protein ubiquitination and is mediated by the coordinated action of a cascade of enzymes, including ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and E3 ubiquitin ligases (E3s) ,15,16,17One unique class of E3 ubiquitin ligases (UBR1 to UBR7) is a family that contains an evolutionally conserved UBR box domain, a substrate recognition domain ,22. This2H) or Cys-sulfonic acid (CysO3H) [According to the N-end rule, the lifespan of a protein depends on the character of its N-terminal residue. N-terminal residues that destabilize a protein are termed N-degrons, classified as type 1 or type 2. Type 1 N-degrons contain positively charged amino acids such as Arg, Lys, and His, and type 2 N-degrons include hydrophobic residues such as Phe, Trp, Tyr, Lue, and Ile. These N-degrons can be generated directly by nonprocessive proteases, including methionine–aminopeptidases (MetAPs), caspases, calpains, separases, or indirectly, by enzymatic cascades that mediate the post-translational arginylation of newly exposed Asn, Gln, Asp, Glu, and Cys in mammals ,28,29,30(CysO3H) ,35,36. R(CysO3H) . N-termi(CysO3H) .Seven UBR box E3 ligases have been identified in mammals (UBR 1-7) . The UBRSaccharomyces cerevisiae [In 1990, Varshavsky and colleagues identified UBR1 (ubiquitin–protein ligase E3 component N-recognin 1) whose molecular weight is around 225 kDa as the sole N-recognin in revisiae . Later, UBR1, UBR2, and UBR3 are classified as RING-type E3s, constituting the majority of E3s. The RING domain mediates the direct transfer of ubiquitin from the E2 enzyme to the target protein and possesses conserved cysteine and histidine residues which bind two zinc ions, maintaining the overall structure ,44,45,46UBR5 belongs to the second-largest group of E3s, containing the HECT (homologous to E6-AP C-terminus) domain. The HECT domain consists of two different lobes, the N-terminal lobe (N-lobe) and C-terminal lobe (C-lobe), connected by a flexible linker. The N-lobe is responsible for binding the E2s, whereas the C-lobe bears the catalytic cysteine that accepts ubiquitin from the E2 to generate an E3–ubiquitin complex ,49. TherUBR6, also named FBXO11, is an F-box protein component of an SCF ubiquitin ligase consisting of Cullin 1 (CUL1), Skp1, RBX1, and the corresponding F-box protein ,51. CUL1UBR7 possesses the plant homeodomain (PHD) finger, a 50–80 amino acid protein domain with Cys and His residue patterns similar to that of the RING domain . This doUBR boxes of N-recognins, including UBR1, UBR2, UBR4, and UBR5, are responsible for binding type-1 basic N-degrons. For the recognition of type-2 hydrophobic N-degrons, an N-domain present in UBR1 and UBR2 is required. The N-domain is a homologue of bacterial N-recognin, ClpS. In this section, the structural bases of substrate recognition by these domains are discussed.The crystal structures of the UBR box from human UBR1, human UBR2, and yeast UBR1 have been determined, greatly enhancing the understanding of the binding mechanism between the UBR box and substrates ,59,60,61The UBR box has two pockets responsible for recognizing the first and the second residues of substrate N-degrons. The first pocket consists of D118, T120, F148, D150, and D153 of human UBR1 and forms a negatively charged surface where the first amino acid of type 1 N-degrons  can bind A. T120, There is a significant difference in the UBR box binding affinity for N-degrons with the same first amino acid but different second residues based on the interaction between the second pocket of the UBR box and the second amino acid of the N-degron. This pocket consists of five and six critical residues in the human and yeast UBR box, respectively A,B. The There are some differences between the structure and sequence of human and yeast UBR boxes. For example, R135 of yeast UBR1 corresponds to S111 of human UBR2, although they are entirely different in character and size. Furthermore, the T171 loop of yeast UBR1 is located close to the ligand-binding site, whereas the loop of human UBR1 is shorter and more distant from the binding site. These differences in the pocket surface cause differences in the substrate-binding mechanism.KD = 17.7 ± 0.325 μM) than RDAA (KD = 343.2 ± 36.4 μM). However, in the case of human UBR2, RDFS showed twice the thermal stability of RRFS, indicating that RDFS has a higher affinity than RRFS [When the RR-peptide binds to yeast UBR1, R135 moves backward to create a space, and the positive charge of the second Arg is stabilized together with a water molecule and D165 G,H. Whenhan RRFS . These rE. coli  characterized by seven-(pass)-transmembrane domains are the largest and the most diverse group of membrane receptors in eukaryotes ,68,69. GAmongst RGS family proteins, RGS4, RGS5, and RGS16 are known physiological substrates of the Arg/N-degron pathway ,36,84. TApoptosis is a type of programmed cell death utilized by multicellular organisms to selectively eliminate damaged or abnormal cells to maintain homeostasis ,89. The Apoptosis is characterized by the activation of numerous proteases such as caspases and calpains, responsible for the cleavage of over 1000 cellular proteins ,96,97. TThe evidence for the apoptosis inhibitory effect of the UBR box E3 ligases comes from multiple studies. Using colony-forming and TUNEL assays, it was observed that apoptotic cell death was significantly increased in ATE1 or UBR1/2-deficient cells under apoptotic stimuli such as UV irradiation, staurosporine, and TNF-α . AccordiTherefore, UBR box E3 ligases can inhibit apoptosis through the clearance of pro-apoptotic fragments via the UPS. However, there is a report that activated caspases can functionally inhibit ATE1 and UBR1, which both contain caspase-8 cleavage sites. In the case of ATE1, it was confirmed that the function of R-transferase was significantly reduced by caspase-8. Accordingly, when the caspase-8 activity was inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK, the R-transferase function of ATE1 was restored .Neurodegenerative diseases are caused by protein aggregation and can also be generated from a failure of mitochondrial quality control . MitochoPINK1 undergoes rapid and continuous degradation in normal mitochondria. Under normal mitochondrial conditions, the N-terminus of PINK1 is inserted into the inner mitochondrial membrane (IMM) via TOM and TIMM23 translocator complexes, where PARL, an IMM protease, cuts PINK1 between residues Ala103 and Phe104, releasing a C-terminal fragment of PINK1 (Phe104-PINK1) into the cytosol ,113,114.Inflammation is a protective response induced by the evolutionarily conserved innate immune system to fight against harmful stimuli such as pathogens, damaged cells, or irritants . The innPRRs are classified into subfamilies such as Toll-like receptor (TLR), C-type lectin receptor (CLR), and NOD-like receptor (NLR), according to their location and domain composition . It is kmNLRP1B contains an autocleavage site in the FIIND domain, which consists of the ZU5 and UPA subdomains. N-terminal and C-terminal fragments are generated when mNLRP1B is auto-cleaved between ZU5 and UPA (Phe983-Ser984) ,129,130.In contrast to the NLRP1 pathway, there is a report that inflammation can be alleviated by the Arg/N-degron pathway . PAMPs a2O2) lead to the stalling of the replication fork and the release of DNA polymerase from PCNA [Replication stress, defined as the slowing or stalling of replication fork progression and DNA synthesis, can cause DNA mutations and chromosomal aberrations ,136,137.rom PCNA ,148,149.rom PCNA ,151. Accrom PCNA ,153.UFD1-NPL4 segregase complex, resulting in the release of ubiquitinated Lys78-SDE2 from the chromatin and allowing for its degradation via the UPS, thus promoting stalled fork recovery and S phase progression [SDE2, a genome surveillance protein, is a highly conserved human protein that possesses a DNA-binding SAP  domain, which is frequently found in DNA repair proteins such as PIAS1, Ku70, and RAD18 ,154. In gression . TherefoUBR box E3 ligases including UBR1, UBR2, UBR4, and UBR5 are the primary N-recognins of the N-degron pathways. The mechanisms through which some of these N-recognins bind their substrates has been well characterized by structural analysis, as detailed in this review. These N-recognins have a wide variety of physiological substrates . The intThe homology model was generated by the SWISS-MODEL program ,66. The


Within the European Union, infectious cattle diseases are categorized in the Animal Health Law. No strict EU regulations exist for control, evidence of disease freedom, and surveillance of diseases listed other than categories A and B. Consequently, EU member states follow their own varying strategies for disease control. The aim of this study was to provide an overview of the control and eradication programs (CPs) for six cattle diseases in the Netherlands between 2009 and 2019 and to highlight characteristics specific to the Dutch situation. All of these diseases were listed as C,D or E in the New Animal Health Law. In the Netherlands, CPs are in place for six endemic cattle diseases: bovine viral diarrhea, infectious bovine rhinotracheitis, salmonellosis, paratuberculosis, leptospirosis, and neosporosis. These CPs have been tailored to the specific situation in the Netherlands: a country with a high cattle density, a high rate of animal movements, a strong dependence on export of dairy products, and a high-quality data-infrastructure. The latter specifically applies to the dairy sector, which is the leading cattle sector in the Netherlands. When a herd enters a CP, generally the within-herd prevalence of infection is estimated in an initial assessment. The outcome creates awareness of the infection status of a herd and also provides an indication of the costs and time to achieve the preferred herd status. Subsequently, the herd enrolls in the control phase of the CP to, if present, eliminate the infection from a herd and a surveillance phase to substantiate the free or low prevalence status over time. The high-quality data infrastructure that results in complete and centrally registered census data on cattle movements provides the opportunity to design CPs while minimizing administrative efforts for the farmer. In the CPs, mostly routinely collected samples are used for surveillance. Where possible, requests for proof of the herd status are sent automatically. Automated detection of risk factors for introduction of new animals originating from a herd without the preferred herd status i.e., free or unsuspected, is in place using centrally registered data. The presented overview may inspire countries that want to develop cost-effective CPs for endemic diseases that are not (yet) regulated at EU level. Leptospira spp.  such as bovine viral diarrhea (BVD), paratuberculosis, infectious bovine rhinotracheitis (IBR), and salmonellosis, no or limited EU regulation exists (referred to as non-EU regulated diseases in the rest of this manuscript) (Regulation (EU) 2016/429). These diseases are often endemic and result in substantial adverse cattle health. Additionally, presence of the diseases results in reduced cattle welfare and increased labor and costs for the farmer  is applied to programs that may lead to a free or unsuspected (“low-risk”) status from a particular infection at herd level.Because these programs bring tangible benefits to participating farmers and the dairy processing industry, development of and participation in CPs are strongly supported by farmer organizations, dairy processors and the meat industry , 10. Thewww.sound-control.eu). As part of this COST Action each of the 32 participating countries, including the Netherlands, provided a comprehensive overview of the CPs in place for non-regulated cattle diseases in their country. This information will form the basis and guide the needs for an eventually developed method to compare outputs of CPs in an objective and uniform manner.In 2018, a COST Action (European Cooperation in Science and Technology) named SOUND-control was initiated that stimulated development of methods that enable the comparison of the output of heterogeneously designed CPs between countries , Mycobacterium avium subsp. paratuberculosis (Map), Leptospira serovar Hardjo (L. Hardjo) and Neospora subsp. caninum (Neospora) between 2009 and 2019.The aim of this paper is to describe the Dutch CPs for six cattle infections i.e., bovine viral diarrhea virus (BVDV), bovine herpes virus type 1 (BoHV-1), For this study, all Dutch cattle herds that participate in the Dutch Cattle Health Surveillance System (CHSS)  were incDairy herds: herds that deliver milk to dairy plants in the Netherlands .Suckler herds: These herds do not deliver milk and have more than 20 cattle. The majority of animals (>80%) are cows and annually at least one calf is born in these herds (Identification and registration (I&R) data, Netherlands enterprise agency Nederland (RVO), Assen, the Netherlands).Young stock rearing herds: the majority of the cattle (>95%) are female and younger than 2 years of age. The cattle enter the herd at a young age (<3 months) and leave the herd before first calving to a dairy herd .Beef herds: are defined as herds with calves  or older cattle (beef) that, in general, are exclusively moved off-farm to go to slaughter. The majority (>80%) of the cattle are male and in general no calves are born in these herds, and no milk is delivered .Small scale holdings: herds that do not deliver milk to dairy plants and with <20 cattle in total .Other herds: herds that do not fit into the above mentioned criteria . This group includes herds with at least 20 head of cattle, that do not deliver milk, that have no births and that do not meet the criteria for beef or veal. This group mainly includes trading farms, herds that just started or almost stopped and other small groups of herds .Some CPs involve surveillance on bulk milk samples, which is obviously only applicable to dairy herds. Other surveillance strategies are based on individual test results and can accommodate both dairy and non-dairy herds.Each of the CPs have been tailored to the specific situation in the Netherlands, i.e., a country with high risk of introduction and transmission of infections but also with a high data quality at national level. This enables the use of standardized and targeted sampling in CPs, such as bulk milk sampling (dairy) and slaughterhouse sampling (non-dairy). Additionally, this also enables use of routinely collected data for risk-based surveillance and to support the coordination of the CPs. All six cattle diseases with no or limited regulation on EU level for which CPs are in place are endemic in the Netherlands, at varying prevalence's of infection (see Results section).In general, three phases are distinguished when conducting a CP, (i) initial assessment in which the (apparent or true) prevalence of infection in the herd is estimated as the starting point for disease control, (ii) control phase in which actions are initiated to eliminate the infection if present or to reduce the prevalence if eradication is impossible and (iii) the surveillance phase that monitors the achieved preferred disease status (free or unsuspected depending on the disease) and take action when (re-)introduction of the infection is detected. Within the CPs, herds are assigned with one of six different disease statuses that are defined as follows:Free: is achieved after whole herd screening without evidence of infection (period differs depending on the disease) or after a prolonged period of proof of an unsuspected status. More information on the definition of the free status is described in sections BVDV to Leptospirosis.Unsuspected: screening of a sample of animals in the herd , yields no indication of infection.Suspended: evidence or action is needed to prove that the herd is still free or unsuspected. The herd is within the time frame that is set to deliver the requested evidence. The herd needs to test cattle to prove that they are free of infection (after introduction of cattle originating from herds that are not classified as free or after lacking to provide evidence of freedom within the standard terms set out in the CPs), or have to prove that the herd is free of infection again after reintroduction of the disease .Unknown: the herd is a still participant in the CP but evidence or action is needed to prove that the herd is still free or unsuspected. The herd is outside the time frame that is set to deliver the requested evidence.Infected: Presence of infection has been established.Controlled: actions are taken to eliminate the infection with the aim to achieve the free or unsuspected status. These actions test and cull, vaccination, or treatment and monitoring the subsequent status. An example of this status “controlled” is the “vaccinated” status for BoHV-1.L. Hardjo the highest herd status that can be achieved is the free status, although for BVDV and BoHV-1 herds can also obtain an unsuspected status. For Salmonella spp. the highest preferred health status that can be achieved in the Netherlands is “unsuspected.” For Map, the preferred herd status is status A and status 10, which are equivalent to an unsuspected (status A) or free status (status 10). For Neospora, herds cannot achieve a free or unsuspected status and the only stages that are recognized are “participating” or “not participating” in the monitoring program. Each CP has its own aim and design which is outlined below in more detail. Additional links to detailed regulatory CP information for each included infection are provided in For BVDV, BoHV-1 and Bovine viral diarrhea virus (BVDV) is a member of the pestiviruses and the causal agent of bovine viral diarrhea (BVD). The virus can be transmitted both horizontally, leading to transiently infected cattle (TI), and vertically. In transiently infected cows, infections with BVDV may sometimes lead to severe clinical signs , 13. VerIn the Netherlands, up to 2018, a voluntary BVDV CP was in place in which dairy and non-dairy herds could participate. Since 2018, the CP was implemented on a national level while its design was slightly modified. Since then, the Dutch dairy sector  has required dairy farmers to participate in this national program (at their own expense). The aim of the national BVDV program is to eliminate BVDV from dairy herds and prevent reintroduction of the virus. For non-dairy herds, up to now, participation remains voluntary. The beef and veal producing industry have committed themselves to participate in the BVDV eradication in the coming years. It is anticipated that non-dairy herds will seek to control and eliminate BVDV and hopes are that the Netherlands will eventually become BVDV free.In the BVDV program, dairy farmers choose one of four different routes to achieve the BVDV free status . These rIn all routes the risk of purchase of cattle from herds without a BVDV free status is monitored. More information on this can be found in section Management and coordination of disease control programs.Bovine herpes virus type 1 (BoHV-1) is the causal agent of infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV) and infectious pustular balanoposthitis (IPB). Infection with BoHV-1 in cattle can occur sub clinically but can also lead to severe respiratory symptoms and abortion . SeroposSince 2018, a national BoHV-1 CP was implemented in the Netherlands after more than 17 years of having a voluntary CP . Since tPrior to enrollment of participation in the BoHV-1 CP, it is advised to start with a herd screening by conducting a BoHV-1 gE-antibody test in bulk milk (in dairy herds) or individual serological screening for antibodies in a sample of at least three cattle (the oldest ones) in non-dairy herds. In dairy herds, this initial screening can result in two outcomes: more than 10% of the cattle is gE-antibody positive, or at most 10% of the cattle is antibody positive. In non-dairy herds this initial screening only provides a rough indication whether BoHV-1 is present in the herd and whether it is best to enroll in the CP or to start vaccinating. Sampling the oldest three cattle in non-dairy herds is based on the observations that BoHV-1 has a high transmission value in a susceptible herd, and a new introduction will generally result in a major outbreak with at least 70% antibody positive cows , 23. A sWhen there is indication that more than 10% of the cattle is gE-antibody positive, removal of all antibody positive cattle will often not be feasible given the high costs involved, and the herd is advised to move to the vaccination route. In this route, the veterinarian vaccinates all cattle in the herd ≥3 months old twice a year with a gE-negative marker vaccine. In the Netherlands, only gE-negative marker vaccines are allowed to be used for BoHV-1. A declaration of vaccination is sent by the veterinarian to the CP's coordinator , and the vaccinated status is assigned. Control of the infection by vaccination will prevent major BoHV-1 outbreaks in the herd  and 25)25). SubsThe first route “bulk milk route,” involves bulk milk monitoring, which takes at least 2 years. In this bulk milk route, bulk milk is screened for antibodies against BoHV1 gE. When no antibodies are found, the herd receives the status BoHV-1 unsuspected and enters the surveillance phase in which the bulk milk is screened on a monthly interval for the presence of antibodies. After 24 months of antibody negative results, the herd can opt to be classified BoHV-1 free. To obtain this status, all cattle of 6 years and older as well as cattle that were introduced in the herd after the initial assessment of the CP have to be individually screened for BoHV-1 antibodies. If no gE-antibodies are found the herd receives a BoHV-1 free status that will again be monitored by monthly bulk milk antibody testing . Any antIn the CP, the risk of purchase of cattle from herds without a BoHV-1 free status is monitored as are cattle that show clinical signs that may be indicative of a BoHV-1 infection, such as respiratory symptoms or abortion. In the CP these cattle have to be tested for presence of BoHV-1 antibodies or virus in case of respiratory symptoms and if present subsequent actions need to be taken. Further details can be found in section Management and coordination of disease control programs.Leptospira interrogans serovar Hardjo type prajitno and Leptospira borgpetersenii serovar Hardjo type bovis , to check the L. Hardjo status of the dairy herd. When antibody positive cattle are detected during this second evaluation, there is confirmation that there is an active L. Hardjo infection in the herd. In non-dairy herds a sample of contact animals is tested 4–8 weeks after removal. When active circulation with L. Hardjo is detected, treatment of all cattle in the herd with dihydrostreptomycin (25 mg/kg I.M.) is advised. After treatment, the herd status is changed into “controlled” and the dairy processors determine how long the farm can deliver milk under this status. To survey for transmission in treated herds, every 6 months a seronegative sentinel group of animals ≥2 years old are serologically examined for L. Hardjo antibodies.Because . Hardjo . In herdL. Hardjo free status is assigned to a dairy herd this status is monitored every 4 months through antibody evaluation of bulk milk. In non-dairy The L. Hardjo free status of non-dairy herds is monitored through antibody testing of sera collected at slaughter. The frequency of testing at slaughter varies between one and six cattle per year, depending on herd size, on- and off-farm movement patterns, and slaughter frequency.After the L. Hardjo free status is controlled by serology testing of introduced cattle. Additionally, farmers are obliged to submit samples of aborting cattle to evaluate the presence of L. Hardjo. More information on the logistics involved in controlling these risk factors can be found in section Management and coordination of disease control programs.The risk of introductions of cattle from herds without a Salmonella enterica subsp. enterica infections  are of concern to the cattle industry as cause of economic and welfare losses in infected herds and as risk of zoonotic infection  was initiated in 2000 by Royal GD to enable low-risk trade of cattle between herds, to alert farmers to a Salmonella spp. infection in their herd, and to reduce human exposure to Salmonella spp. , when a design within-herd prevalence of 0.1, a diagnostic Se of 94.4% (95% CI: 72.7–99.9)  and twice a year testing of sera of the 5 youngest cattle over 90 days of age that have been present in the herd for at least 70 days (non-dairy herds). Additionally, risk based surveillance is applied by surveillance for any positive test results of samples submitted from the herd from potential clinical cases  and serological testing of any cattle introduced from herds without an unsuspected status. Positive test results in any of the routes result in suspension of the unsuspected herd status until follow up testing shows that the infection is no longer spreading. Infected herds are advised to control the infection by preventive management measures and identification and culling of Salmonella spp. carriers (A voluntary CP for lla spp. . Testing.7–99.9)  and a te.7–99.9)  were usecarriers .Salmonella spp. prevalence in the dairy processing industry , herds are classified in one of three categories. Consistently bulk milk antibody negative herds are classified as Level 1. Herds with at least two subsequent antibody positive bulk milk results are classified as Level 2. Herds in which antibodies are detected in at least four out of the five most recent bulk milk evaluations are classified as Level 3. Level 2 and 3 herds are obliged by the terms of delivery of their dairy processor to take control efforts. These efforts range from either a risk assessment or participation in the voluntary CP of Royal GD at Level 2, to an annual mandatory action plan including both preventive management measures and identification and culling of active Salmonella spp. carriers at Level 3. Herds that are assigned Level 3 for more than 3 years are obliged to seek advice of one of five specifically trained veterinarians during a herd visit before drawing their next action plan. At this stage, the dairy processors closely monitor the efforts of the farmer, to ensure that the drawn action plan is followed through.In 2009, the Dutch dairy processing industry  implemented a mandatory CP in addition to the pre-existing voluntary CP. The aim of the mandatory CP is to reduce the industry . Like thMap). The disease is widespread world-wide and causes significant economic losses (Paratuberculosis (or Johne's disease) in cattle is an infectious disease caused by Mycobacterium avium subsp. paratuberculosis . At enrolment the herd is assigned status 5 and all cattle over 3 years of age are tested by serum antibody ELISA followed confirmatory fecal culture or PCR assay. If the screening is negative, the herd progresses to status 6. Subsequent annual herd examinations consist of culture or qPCR of pooled fecal samples of all cattle over 2 years of age  is an apicomplexan protozoon, an important cause of abortion in cattle worldwide  cows, culling of heifer calves born from seropositive dams, testing of purchased cattle, use of semen from beef bulls for seropositive cows and sexed semen on seronegative cows. In case of genetically valuable seropositive cows it is advised to apply embryo transfer and use seronegative donor animals.In 2003, Royal GD developed a voluntary onducted . The aimthe herd . SpecifiIn the Netherlands, a good quality infrastructure is present for collecting bulk milk samples , individual milk samples (collected for milk production registration) and routine collection of data. These samples and data can automatically be used in support of the CP with informed consent of the farmer, which is obtained at enrolment in the CPs. Using routinely collected samples and data, the CPs carried out in the Netherlands are labor sparing and cost-efficient. All programs are managed by one organization (GD) with a large commercial veterinary laboratory. This lab is accredited by the relevant authorities, as well as all diagnostic tests used for the CPs. In some cases, diagnostic results from other labs are also allowed in the CPs. These labs are on a list and accredited by the national reference institute (WBVR). The labs can use their own in-house or commercial kits and WBVR assures with proficiency tests that diagnostic test validity is comparable between the labs.Data is routinely collected in an objective and standardized way on a national level, enabling optimization, and automatization of processes within the CPs. For coordination of all control efforts, cattle movement data from the identification and registration system  is combined within automated Certification Coordination software Programs (CCP) that evaluate whether the herd meets all criteria set by the CP. When results are needed for all or a sample of cattle, the CCP automatically consults the I&R database to determine for which cattle at what moment a test result is needed and both farmer and veterinarian are notified accordingly. Notification is done by regular mail and through email. When the subsequent samples are submitted and the laboratory test results become available, the CCP automatically processes the test results in the CP, adapts the status if needed and informs farmers and veterinarians of the result and the CP status by either mail or email.L. Hardjo, Salmonella spp. (unsuspected CP) and Map (IPP as well as MQAP) programs, any introductions of cattle into participating herds are identified real-time using data from the national I&R database, in which all cattle movements are recorded. This information is processed by the CCP software within a day and an observation status is assigned to the herd if the herd of origin had a lower herd status. Subsequently the farmer and veterinarian are notified if removal or testing of the introduced cattle is required.In the BVDV, BoHV-1, L. Hardjo, Salmonella spp. and Neospora bulk milk samples are tested. These bulk milk samples are routinely collected at the time of on-farm collection of milk by the dairy processors and tested for milk quality purposes at Qlip laboratories . If a dairy farmer enrolls in a CP in which bulk milk testing is part of the intake, control or surveillance phase, an automated request is sent to Qlip to forward a bulk milk sample to Royal GD for testing. The farmer receives the test result automatically without having to take any action.In CPs for BVDV, BoHV-1, L. Hardjo, Salmonella spp. or Neospora samples are automatically screened for presence of these infections.For CPs where risk-based testing of aborting cows is included, the CCP detects samples of aborting cattle that are submitted for mandatory brucellosis surveillance. When a herd participate in the CP for BVDV, BoHV-1, Since 2004, the Dutch cattle industry monitors the prevalence of endemic cattle diseases based on antibody or virus testing. Every 2 years, the cattle industry decides on a number of non-regulated cattle diseases to include in a biennial prevalence survey. The presented survey results represent the apparent prevalence which are referred to as “prevalence” in the remainder of this paper.Diseases to be included in the survey are selected based on relevance to the industry with regard to costs, impact on animal health and welfare, public health and monitoring progress of control efforts. The selected diseases and herd types for the prevalence surveys also depend on the participation in the CPs i.e., when the participation rate of the CP approaches 100%, a dedicated prevalence survey is not relevant as the data gathered in the CPs provide sufficient information to assess disease prevalence.L. Hardjo, Salmonella spp, and Map, herds were screened for the presence of antibodies. Two-stage sampling is applied, and the sample size was determined using WinEpiscope 2.0 . For BoHV-1 and L. Hardjo, the target population includes cattle ≥2 years old. For BVDV, the target population included calves between 8 and 12 months of age, which are tested for presence of antibodies indicative for BVDV transmission in the herd. For Salmonella spp. calves in the age of 3–6 months were included as target population. The sampling process is described in more detail in Veldhuis et al.  between 2015 and 2019. For suckler herds, the association between mortality and the herd health status for three infections  was assessed in the same period. Given the large sample of included herds , only associations with a P < 0.01 were presented as significant. Interaction terms were not evaluated.The CHSS has been in place since 2002 and consists of several surveillance components that combine enhanced passive reporting, diagnostic and post-mortem examinations, random surveys for prevalence estimation of endemic diseases, and quarterly data analysis . The aimIn 2019 there were 34,316 cattle holdings in the Netherlands, of which 45% were dairy herds, 33% are small scale holdings, 9% were suckler herds and 14% were other types of cattle herds . The her2 i.e. on average 96 cattle/km2, . The remaining herds were mostly in the temporarily observational status because of purchase from a herd without a free status (non-dairy herd or import). In most cases these herds are in fact also free of infection. In the CP's on the other four infections, herds are either classified as having the highest health status i.e., free or unsuspected, or farmers are in the process of obtaining these statuses, hence have to act to achieve the highest health status. Even though the participation rate in dairy herds was close to 100%, the infections are still endemic, and there were still herds in the process of eradicating these infections from their herds.During the last decade, the participation rates for five out of six CPs increased toward almost 100% in dairy herds, following an obligation to participate in these CPs by the Dutch dairy processing industry. For Map, ~80% of the Dutch dairy herds were assigned a preferred herd status (status A in MQAP or 6-10 in IPP). In the last decade, this proportion hardly changed. The other 20% of the herds had antibody positive cattle (status B or C) that have to be removed. From the start of the mandatory CP for Salmonella spp. until 2017, ~90% of the herds had bulk milk antibody negative results. Since 2018, additional actions have been taken to guide farmers where there is evidence of ongoing infection from bulk milk monitoring. This has been associated with an increased proportion of herds classified as unsuspected [on average 96.2% per measurement in 2019 , but the CP participation rates for Salmonella spp. and Map are below five percent. In the other three CPs, for BVDV, BoHV-1 and L. Hardjo, the highest participation rates in non-dairy herds were observed in suckler herds and exceeded 10% in this herd type in 2019. The participation rates in young stock rearing farms are associated with the participation of dairy herds given that the two sectors were linked to each other. Participation rates in CPs by other herd types were negligible.Non-dairy herds can participate voluntarily in five CP  in the voluntary control programs . At the More detailed information on the survey results is presented in Neospora, no prevalence surveys were conducted. However, there was information on the post mortem findings in aborted fetuses submitted for post-mortal examination and results of serological sampling in aborting cattle at Royal GD. More than 95% of these fetuses were submitted from dairy herds.For Neospora was diagnosed as the cause of abortion has decreased from 17.5% (95% CI: 15.1–20.0%) in 2005 to 5.0% (95% CI: 3.2–8.0%) in 2019  in 2004 to 11.9% (95% CI: 11.0–12.9%) in 2019  in 2004 to 0.8% (95% CI: 0.2–2.2%) in 2014. During the studied period, the BVDV prevalence decreased from 34.8% (95% CI: 29.3–40.3%) in 2004 to 7.5% (95% CI: 4.3–11.9%) in 2020 and also the BoHV-1 prevalence decreased. During the first prevalence survey in 2012, 23.4% (95% CI: 16.4–31.2) of the non-dairy herds tested BoHV-1 positive. In 2020, this prevalence was significantly lower .During the analyzed period, in non-dairy herds, a decrease in prevalence was observed for BVDV, BoHV-1 and . Hardjo  while th. Hardjo . The L. Map, only one survey was carried out, which indicated a herd prevalence of 15.6% (95% CI: 12.2–19.1%). For Salmonella spp., the prevalence in 2006 and 2014 was similar at ~10%.For More detailed information on the survey results is presented in Salmonella spp. or Map had significantly lower mortality rates compared to herds without a free, antibody negative or A status , mandatory participation for dairy herds , or obligation to have a free (L. Hardjo) or unsuspected status for dairy herds . Each of the CPs originally started voluntarily for both non-dairy and dairy herds. Participation rates were always highest for dairy herds, although these never exceeded 50% in the voluntary stages of the CPs before incentives to participate were implemented by the dairy processors. Initiatives were taken by the dairy processing industry to control and eliminate cattle infections from individual dairy herds through mandatory participation in CPs for BVDV, BoHV-1, L. Hardjo, Salmonella spp. and Map. For non-dairy herds, participation in all six CPs has remained voluntary. The main drivers for the dairy processing industry to control infections are not only to reduce the disease prevalence and disease associated losses but also to prevent the occurrence of zoonotic infections  and to deliver high quality products derived from healthy cows. Having high health statuses is important for the license to produce and the image of the sector and lead to increased consumer confidence in the products. Some export markets even demand products to originate from healthy cows.In the Netherlands, the six infections are at very different stages of control, ranging from only voluntary participation  or when the prevalence and incidence of the infection indicates a need for stricter regulations.L. Hardjo, the dairy processing industry is close to freedom from infection, and the prevalence in non-dairy herds is low as well. However, each year, several re-introductions occur, mainly through the import of cattle from countries with higher prevalence introduction in herds with a free or unsuspected status occur, mainly because of introduction of infected cattle from cattle herds in the Netherlands. The introduction of cattle is a very important risk factor for disease introduction , or perceived a high risk of the disease (either due to severity of signs or a high probability of introduction). These reasons are not unique for Dutch farmers as similar results were found in Great Britain . Other fThe results of the biennial prevalence surveys show a decrease in prevalence over time for the infections under control. The success rate i.e., reduction in prevalence after implementing obligatory participation in the CPs, varies between infections and is multifactorial, depending among others on the differences in disease characteristics, the specific rules in the CP, and the demands set by the industry (participation vs. eradication).The prevalence surveys indicated that when disease control measures were implemented in dairy herds the prevalence for these endemic infections also significantly decreased in non-dairy herds, even though participation rates remained low for non-dairy herds. Suckler herds may play a relevant role in the transmission of the six infections between herds in the Netherlands given that (i) these cattle are kept outside, (ii) suckler herds often trade and ii) calves are born regularly. In this herd type however, we observed reduced prevalences over time for infections that were under mandatory control in dairy herds. We therefore believe that disease control in one part of the population can also benefit the disease prevalence in the population that does not participate. Nevertheless, we cannot conclude that the reduction in prevalence can entirely be attributed to the performance of the CPs. Awareness of the disease may also have resulted in improved disease control in herds without official participation in a CP. Additionally, other changes in the cattle industry may have affected disease prevalence as well. In 2015, the milk quota was abolished. Subsequently, herd size increased untill governmental phosphate regulations were put into place that resulted in herd sizes reducing back to the level of July 2015. Generally, herd size was reduced by removing cattle that performed suboptimal. This may also have resulted in a decreased prevalence of several infections over time.Given that participation in CPs is still voluntary for non-dairy herds, complete eradication from infection will probably not be reached. The prevalence of infection can become very low, but re-introduction through introduction of cattle will always be a risk. Additionally, given that eradication at national level is almost impossible without a national obligation to eliminate diseases, it will be very complicated to set demands on the disease status, when importing cattle, due to international trade regulations. However, eradication may not be necessary to achieve the goals of stakeholders, such as safeguarding future access to international dairy markets.The implementation of CPs for specific infections improves animal health and welfare, and reduce disease-related costs and labor involved in the treatment of diseased animals. In this study, we showed that herds with a free or unsuspected status for the evaluated infections had lower mortality rates in calves and cows than herds with an unknown status. Infectious diseases are risk factors for mortality and culling as previously reported for BVD , 82, salCPs to control and eradicate infections are to be supported, differences in herd health status within and between countries pose a risk when trading cattle from areas with a higher disease prevalence to areas with a lower prevalence . For cati.santman@gdanimalhealth.com.The data analyzed in this study is subject to the following licenses/restrictions: Confidential data. Requests to access these datasets should be directed to Salmonella spp. and Map. LD provided input on BVDV. HW provided input on BoHV-1. KB provided input on L. Hardjo. TD provided input on Neospora and Map. AR is the CP manager and proofread the relevant parts of the paper. All experts assisted in the writing of the parts of the paper about the CPs concerning the infection that met their expertise. JH, SS, MM, MW, MB, AV, and GS edited draft versions of the paper and provided advice for improvement. MB and AV provided veterinary epidemiologic expertise and conducted the analysis on the prevalence estimations over time. GS acted as supervisor of the group and provided scientific and epidemiologic input. All authors participated in the writing of this manuscript, have proofread the final version of the manuscript and agree with its contents.IS-B was the primary author of the manuscript. MM added knowledge on the testing process, helped in writing the paper, and proof read the paper. MW provided input on IS-B, MM, MW, LD, HW, KB, TD, AR, AV, and GS are employed by Royal GD, which runs most of the CPs described in this paper. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Afghan women face enormous challenges storing wheat flour during the winter due to high relative humidity and insect pests. These challenges result in poor-quality baking wheat flour. We conducted an experiment in Herat province, Afghanistan, to assess whether hermetic bags would preserve wheat flour during the wintertime when relative humidity is high. Forty women from two districts participated in this experiment. Each woman stored 25 kg of wheat flour in a Purdue Improved Crop Storage (PICS) bag and a polypropylene (PP) woven bag. The results showed that the moisture content, color, and baking properties of wheat flour stored in PICS bags for six months did not change. No insects were observed in wheat flour stored in PICS bags. However, wheat flour stored in PP bags had high moisture content and insect pests. The price of wheat flour stored in PICS bags increased over time, and that stored in PP bags decreased. Hermetic bags can be used to safely store wheat flour for six months in areas with high relative humidity conditions. Storing wheat flour in hermetic bags would help improve food security for millions of wheat consumers in developing countries.p < 0.001) and insect population . Wheat flour stored in PICS bags for 6 months retained its color and produced better bread than that stored in PP bags. Storing wheat flour in PICS bags for six months showed a return on investments (ROI) of +16.9% against −33% for the PP bag. Farm households and other wheat value chain actors can safely store wheat flour in hermetic bags for up to six months under high relative humidity conditions. This would help improve food security for millions of wheat consumers in developing countries.On-farm preservation of wheat flour is a challenge due to insect pests and high relative humidity. This experiment was conducted to assess the effectiveness of hermetic bags in preserving wheat flour stored by women during the wintertime when relative humidity is high. Forty women (households) from two districts in Herat province, Afghanistan, stored their wheat flour for 6 months. Each woman stored 25 kg of wheat flour in a Purdue Improved Crop Storage (PICS) bag and a polypropylene (PP) woven bag. Moisture content, insect population, flour color, bread taste, and profitability of storage were assessed. Moisture content and insect population significantly increased in PP bags after six months of storage, while no changes were observed in PICS bags. There was a significant negative correlation between wheat flour color and moisture content (r = −70.7,  Wheat is an important staple crop that occupies 70% of the cultivated land area and is the primary source of nutrition for millions of Afghans ,2. WheatThe postharvest management of stored products is a challenge among smallholder farmers in developing countries including Afghanistan . FarmersIn Afghanistan, women play an important role in wheat postharvest activities, including harvesting, threshing, storage, and processing . SecurinCommercially available hermetic bags  are viable and cost-effective approaches that could help households preserve the quality of wheat flour. Hermetic storage works by depriving insects of the oxygen needed for metabolic activities . PICS baThis study was implemented in Herat province in Afghanistan from 29 November 2013, to 29 May 2014. The period corresponds to the wintertime, with an average rainfall of 241 mm, average relative humidity of about 40% to 80%, and temperatures varying from −1 to 12 °C in December and 13 to 29 °C in May . Forty women (representing 40 households) from 10 villages in two districts (Zinde Jan and Injil) were involved in this on-farm experiment. The women were selected based on their willingness to participate and make 50 kg of wheat flour available for the duration of the experiment. Wheat flour was stored in two types of bags of 50 kg capacity—Purdue Improved Crop Storage (PICS) and polypropylene (PP) woven (control) bags. Each woman stored 50 kg of wheat flour—25 kg in a PICS bag and the other 25 kg in a PP bag. Only flour from wheat harvested during the 2013 cropping season was used in this experiment. Grain used to grind the flour was not treated with any insecticides. Each layer of the PICS bag was tied separately, following the standard procedure . PolyproFlour quality was assessed before the experiment and after three and six months of storage. Data were collected on several parameters, including insect infestation, flour color, moisture content, sensory evaluation of the baked bread, and price of flour. Insect infestation: Six samples of wheat flour, each weighing 250 g, were removed from each bag. Two samples were removed from the top layer, after which half of the flour was removed from the bag, and an additional 2 samples were collected. Finally, three-quarters of the flour was removed, and another 2 samples were collected. Each sample was sifted using a no. 25 sieve, and the number of live and dead insects were counted and recorded for each bag. All non-sifted flour was returned to the bag and sealed. Flour color: To assess the flour color, a score was used: a scale from 1(bad) to 5(excellent). A score of 5 represented flour in pristine, white condition, whereas a score of 1 represented a sample that had become thoroughly discolored and appeared dark or gray to the observer. The 250 g sample used for insect assessment was also used to assess flour color. The flour color was assessed by a panel of the four women from each village who participated in this experiment, but the samples were blinded after collection to avoid any biases. Each participant had the opportunity to assess flour stored in PICS and PP bags. Flour moisture content: Wheat flour moisture content was assessed via water loss by drying. Samples of 100 mL were removed from each bag and weighed on a scale. Then, all samples were placed in an oven to dry for 24 h. After the drying period, the samples were re-weighed, and percent moisture content was determined by the equationSensory evaluation of baked bread: A 0.5 kg sample of wheat flour was removed from each bag and taken to a baker. Baked bread was assessed by a panel of the four women from each village who participated in this experiment, but the samples were blinded to avoid any biases. Each participant had the opportunity to taste bread made with flour stored in PICS and PP bags. The bread taste was scored on a binary scale as either “Good” or “Not Good”. Bread designated as “Good” (scored as 1) indicated products that were deemed edible and could be sold at the local market. Bread designated as “Not Good” (scored as 0) indicated products that were determined to be inedible or otherwise could not be sold at the market. An average score of 1 indicated no change in quality from the initial product during storage. An average score of 0 represented the total rejection of all samples.Price of flour: The price of wheat flour was collected from the nearest local market by blindly asking potential buyers how much they were willing to pay. These price data were collected at the beginning of the experiment, and then after three and six months of storage. Values were assigned in the local currency (Afghani) and listed as Afghanis/kg of wheat flour.ANOVA and LSD tests were used to compare moisture content and insect pest infestations between treatments at different observation periods. ANOVA was also used to assess the effect between the dependent and independent variables. Data were analyzed using SPSS version 26. Estimates of the return on investments (ROI) were computed using wheat prices at the beginning of the experiment, and then after three and six months.Significant effects were observed between storage methods (PICS and PP bags) and the number of live and dead insects, and between storage methods and moisture content . The priTribolium castaneum (Herbst),  was the only species recorded in the wheat flour. No live or dead insects were observed at the beginning of the experiment or in the PICS bags after 3 and 6 months of storage .The moisture content of wheat flour stored in PICS for 6 months did not increase . However storage . In PP bp < 0.001), live insects , and dead insects . Unlike the PP bags, the color of wheat flour stored in PICS bags for six months received “good or excellent” scores by all the participants  of wheat flour stored in PP bags for 6 months was significantly reduced, whereas it remained the same in PICS bags . A littlicipants . About tp < 0.001). However, negative correlations were observed between the price of wheat flour and moisture content , number of live insects , and number of dead insects . The return on investments (ROI) varied between storage methods and was positive for PICS bags, whereas it was negative for the PP bags  would help millions of households become food secure by storing wheat flour for home consumption when humidity is high. Value chain actors involved in processing or storing wheat flour can use hermetic bags to preserve its quality.


Brazil, as a major pig producer, is currently experiencing the widespread use of antimicrobials as a serious issue to be addressed. For measures to be taken in this direction, the extent of the problem must be known. The goal of this study was to evaluate the use of antimicrobials in 25 Brazilian swine herds. Antimicrobial use from birth to slaughter was correlated with biosecurity and productivity. After the first assessment , 13 herds implemented good practices to reduce antimicrobial use. Four years after the implementation of these measures , data about antimicrobial usage from these herds were collected. The results of the first assessment (M0) demonstrated a troublesome scenario: the mean value of antimicrobials used was 358.4 mg/kg of pig produced; the median of the pig’s lifetime exposure to antimicrobials was 73.7%, and the median number of drugs used was seven. A positive correlation between the antimicrobials consumed and the pig’s antimicrobial exposure time was detected. Nevertheless, these data did not correlate with biosecurity score or productivity. A significant difference was detected in M1, where a median 30% reduction in antimicrobials consumed was detected. There was also a 44.3% reduction of the pig’s lifetime exposure to antimicrobials. The median number of drugs used was reduced from seven to five. Antimicrobial use did not always reflect the sanitary condition or the real therapeutic needs, easily leading to overuse. Large amounts of antimicrobials have been used for disease prevention and control in intensive food animal production. In some countries, antimicrobial agents are considered a management tool. In many cases, farmers, technicians, and veterinarians might not even be aware that they are using them for this purpose. Excessive and continued antimicrobial use provides conditions for the selection, spread, and persistence of antimicrobial-resistant bacteria. These bacteria are capable of causing infections in animals and humans . AntimicBrazil is the fourth-largest producer and fourth-largest exporter of pork in the world with a swine population of 2,017,645. From 3983 million tons of pork produced in 2019, a total of 81% was consumed in the domestic market, with an average consumption of 15.3 kg of pig meat per inhabitant, while the remaining 19% was exported to different countries [The Brazilian government has been working since 2018 on the implementation of The National Action Plan on Antimicrobial Resistance in Agriculture. The plan’s goals include health education, epidemiological studies, surveillance, and monitoring of antimicrobial use. It also aims to strengthen biosecurity and implement control measures and promote rational antimicrobial use in livestock. Nevertheless, as of this writing, no official data on the volume of antimicrobials used in livestock production is publicly available. The existing data is provided by the National Union of Animal Health Products Industries (SINDAN). It includes the sale of antibiotics for both livestock and small animals, without discrimination among species .Escherichia coli carrying CTX-M-15, CMY-2, MCR-1, or MCR-3 [Salmonella Typhimurium carrying qnrE1 [Staphylococcus aureus ST398 [Enterococcus faecalis resistant to oxazolidinones carrying cfr and optrA genes [Antimicrobial use in swine in Brazil has increased in the last 20 years, according to the perception of veterinarians and agroindustry professionals. The reason for this growth is particularly complex, and includes the greater availability of antimicrobials, fear of disease outbreaks, increased market competitiveness, lack of technical guidance and especially lack of a legal control regarding antimicrobial usage in livestock. At the same time, there was as increase in antimicrobial resistance rates in different bacterial species isolated from swine at diagnostic laboratories. There were also reports of bacteria carrying resistance genes against antimicrobials important to human health. Some examples are descriptions of or MCR-3 ,7,8, Salng qnrE1 , methicius ST398 , and EntrA genes , which wConsidering the lack of information about the pattern and amount of antimicrobials used in Brazilian swine production, in this study, we evaluated antimicrobial use as a preventive or growth promoter in 25 swine herds located in the country’s main swine-producing states. The antimicrobial usage was correlated with the biosecurity score and productivity index of studied herds. The use of antimicrobials was reevaluated four years after the first assessment in 13 of the initially selected herds. The implementation of a good management practices program was assessed in these 13 production systems, and the observed results were discussed.A convenience sample of 25 pig herds was approached with an invitation to participate in the study. Herds included a minimum of 150 and a maximum of 15,000 sows and originated from nine Brazilian states. Data on production parameters, antimicrobial usage, biosecurity practices, vaccination programs, and other management practices were collected. This study was approved by the FMVZ-USP ethics committee number 5446170717.Mycoplasma hyopneumoniae was reported. Swine herds were classified as farrow-to-finish herds , piglet production unit and finishing herds , or only piglet production unit.All swine herds visited are independent producers who provide animals for slaughter in the Brazilian domestic market. But they are representative of the national swine chain management and technical levels. The main characteristics of the evaluated herds are presented in From January to December 2016, the 25 herds were submitted to at least one visit to collect all the data described above. This was called moment zero (M0). After the first assessment, 13 herds accepted the implementation of good practices and measures to reduce antimicrobial usage. The main researcher monitored these herds. From June to November 2020, they were submitted to a new data collection, called moment 1 (M1).The productivity and antimicrobials usage data were obtained through personal interviews with the owner and/or herd manager. All data were collected by the same veterinarian/researcher in all herds. The herd’s productivity was evaluated by calculating the average kg of pig produced by a sow per year.Antimicrobial usage was evaluated considering their use as a growth promoter, and preventive or metaphylactic programs involving large groups of animals, including injectable use (in suckling piglets only), feed use (in most cases), or water use .Calculations considered milligrams of antimicrobials per kilogram of pig produced in each herd and the period of use (mg/kg biomass) as described in . The folQualitative and quantitative data regarding the herd’s health status, management, structure, biosafety, and vaccines used were evaluated. The applied questionnaire  is basedBriefly, the questionnaire contains 120 questions divided into 16 areas. The questionnaire addresses herd susceptibility to external and internal risks. These risks are related to introducing exotic agents to the system, and the dissemination of agents already present on the property, respectively. Each item/question was classified as “suitable” (implemented and followed appropriately), representing 10 points; “requiring adjustments” (implemented but requiring corrections), worth 5 points; and “inadequate” (not yet implemented), with zero points. The maximum score that could be reached is 1200, and the higher this value, the better the observed biosecurity conditions.Between the two data collections (M0 and M1), in the 13 herds in the study, a series of management improvements were implemented. Their focus was on reducing the use of antimicrobials. These measures are described in p ≤ 0.05 was adopted. Quantitative variables’ normality was assessed by the Shapiro–Wilk test. The correlations between the antimicrobial usage with the biosecurity score and productivity index of studied herds were performed using Spearman’s rank correlation. The Wilcoxon test for paired samples was applied for the comparison of M0 and M1 data.Data management was performed initially in Microsoft Excel 2010. The statistical analyses were performed with SPSS 16.0  and the level of significance of Considering antimicrobial use, we observed an average of 358.4 mg/kg of pig produced. This value varied from 5.4 to 585.6 mg/kg among the 25 herds (M0). The pig’s lifetime antimicrobial exposure was a median of 73.7%, with a variation of 2.9% to 90.4% of the time (from birth to slaughter). During this period, the animals had contact with two to 11 different antimicrobials (average 7 drugs) . A totalp = 0.693). However, in p = 0.783). Even though herds with higher biosecurity scores tended to present a better productivity index (p = 0.088). The positivity for Mycoplasma hyopneumoniae also did not correlate with the amount of antimicrobials used in the studied herds .When the antimicrobial use was correlated with the productivity index, no statistical correlation was detected  presented preventive antimicrobial use in suckling piglets with one or more drugs. Fifty percent of these herds used more than one drug at this phase. The drugs more often used were ceftiofur (40%), followed by amoxicillin (24%), gentamicin (16%), lincomycin/spectinomycin (8%), tulathromycin (8%), and bacitracin methylene disalicylate (4%). This data is presented in The frequency of antimicrobials used in the weaning and growing-finishing phases is demonstrated in The period of treatment presented a wide variation among the 25 herds in suckling piglets and the weaning/growing-finishing phases. Details about this data are shown in p = 0.034, 0.025, 0.015, and 0.019, respectively). We observed that 10 herds presented a reduction in the number of antimicrobials used. One was stable with a small use, and two presented increased usage. The reduction varied from 16% to 94.2%, with a median 30% reduction in antimicrobials consumed per kg of pig produced. Considering the lifetime exposure of pigs to antimicrobials, nine herds presented a reduction since the first assessment. An increase was observed in only two herds. The variation of reduction was from 42.9% to 78.4%. The number of drugs used reduced in nine herds, increased in two, and maintained the same in two herds. The median number of drugs used was reduced to five. The median productivity in these 13 herds increased from 2891 to 3296 kg pig/sow/year, an increase of 14%.Paired data about antimicrobial usage in the 13 study herds were collected  four yeaCurrently, in intensive pig production systems in Brazil, antimicrobials are often administered regularly by farmers. This is sometimes under the direction of a veterinarian, but without requiring a veterinary prescription . TherefoThe trust relationship between the producers and the main researcher and free access to the farm and data was important in choosing these 25 herds. The decision to work with a small group of farms was also important, but with reliable data. The herds evaluated are located in the three largest pig-producing regions in Brazil , with a total of 61,390 sows. This is very representative of our reality, although it is not a statistically significant sample considering the size of the Brazilian swine population.M. hyopneumoniae did not present a statistical correlation with the antimicrobial amount used in the 25 studied herds. Despite this result, Laanan et al. [Biosecurity level, productivity, and positivity to n et al.  describeComparing the median amount of antimicrobials used from birth to slaughter in the 25 herds in M0 (358.4 mg/kg) with the antimicrobial amounts used in food animals described by Hillerton et al.  in 30 coThe pigs’ lifetime (from birth to slaughter) exposure to antimicrobial is not easy to compare with similar studies. Nevertheless, the observed values seem to be worrisome considering that, in 12 herds, this percentage was greater than 80% of their lifetime. The high number of various drugs used during the pig’s lifetime is also a subject scarcely mentioned in recent literature.The use of preventive parenteral antimicrobials in suckling piglets in 72% of the studied herds is very expressive. The most frequently used drug in this phase was ceftiofur (40%), followed by amoxicillin (24%). Considering these herds, 50% used more than one parenteral drug as a preventive in suckling piglets. Farmers usually claim that high antimicrobial use in young piglets is to avoid infections during the lactation period  and prevent later disease problems. However, a deteriorating effect may be related to antimicrobial use at a young age on the bacterial composition of the gut or respiratory tract. This could result in higher disease susceptibility and an increase of treatment requirements at the later stages of pigs’ lives .M. hyopneumoniae infection prevention in positive herds. It can also be associated with the prevention of intestinal infections related to Lawsonia intracellularis or Brachyspyra spp. The tetracycline class was very frequent in our herds, considering the three active antimicrobials in this class . This is also common in other countries [The antimicrobials used in the weaning and growing phases presented a large variation. But the antimicrobial most frequently used was amoxicillin, which appeared in 100% of the studied herds. This usage pattern corroborates the findings of , who fouountries .The large variation of dose and period of use observed for some drugs suggest that off-label usage is a common practice in these herds. A Belgian survey that quantified antimicrobial use in pigs found several differences among oral and injectable antimicrobials used in off-label group treatments in different herds. For example, overall, 50–75% of the oral formulations were underdosed. From the four most frequently used antimicrobials, doxycycline overdosed in 50–75% of the cases. On the other hand, trimethoprim-sulfonamide was underdosed in 50–75% of the cases. Amoxicillin and colistin were underdosed in 50% and 90% of the cases, respectively .We observed the use of some critically important antimicrobials used in humans, like gentamicin, colistin, fosfomycin, third-generation cephalosporin, and fluoroquinolones, in the studied herds for preventive animal treatment. The usage of these drugs in swine was also reported in different European countries by . HoweverThe individualized assessment of the external risks present in the applied biosecurity questionnaire revealed several opportunities for improvement, considering that 56.0% of the herds did not have quarantines or adequately manage them. At least 60.0% of the farmers did not promote adequate pest control, 76.0% did not promote adequate animal water treatment, and 80.0% neglected health risks when transporting animals. The same individualized analysis of internal risks showed negligence in cleaning and disinfection programs in at least 40.0% of the evaluated systems, inadequate use of the facilities, with the “all-in/all-out” system not being adopted by 64.0% of the properties, as well as the downtime of facilities not being respected in 72.0% of the cases. Negligence in adopting measures to minimize the external and internal production systems risks tends to increase the presence of illnesses in the medium and long term. This promotes a greater environmental pressure of infection, ultimately increasing the preventive use of antimicrobials .M. hyopneumoniae, kept a low antimicrobial use and increased herd productivity during this period. Two herds showed an increase in antimicrobial usage (D and P). In these herds, the implementation of measures could not be completed due to some intrinsic characteristics of these production systems. Herd D, for example, presents a large number of animals  making management changes exceedingly difficult.Among the 13 herds agreeing to improve their management practices and biosecurity  controls, the implementation of the proposed measures was not equal. The observed results were not homogeneous. The conditions of facilities, staff capacity, and other factors affected the success of the proposed measures. Considering this fact, we saw some optimal results with a large reduction in the use of antimicrobials and improved productivity, like herd H or W, for example. Some herds, like herd L, which was negative to During the study period, no outbreaks of unexpected diseases occurred in these herds, but the decision to use antimicrobials was not controlled by the research team. Health decisions were always made by the person responsible for the farm. However, we saw that the reduction of antimicrobial use occurred in most of the herds at a significant level. We also found a reduction of exposure time and the number of different drugs used, with an increase of productivity in 10 of the 13 herds.The implementation of guidelines for the prudent use of antimicrobials in veterinary medicine in Germany presented a significant reduction of 73.0% in their antimicrobial consumption after two years. There was also a reduction from 31.6 to 13.6 days in the treatment period . SimilarThe main reason for the wide use of antimicrobials is the excessive preventive use. Although there was no well-founded justification for the repeated use of preventive group treatments, and despite the associated costs, farmers at large production systems often considered preventive antimicrobial use necessary. Seeking out to observe lower disease rates, lower mortality, and better production results, they also believed to be easier and less labor-intensive to implement preventive medication than treatment of clinically diseased animals after losses have occurred.In Brazilian systems, it was also possible to verify the existence of a cultural search for maximum productive efficiency that promotes the excessive prophylactic use of antimicrobials and other additives, often disregarding the realization of laboratory diagnosis as an auxiliary measure for making decisions about sanitary programs . Other mAfter the first assessment of the 25 herds and evaluation of results, all herds were contacted. Part of this data was presented to the herd staff, government agents, veterinarians, opinion leaders, and in local symposia and events about antimicrobial usage in swine. The information generated great interest from professionals in the area and actions to reduce antimicrobial use is emerging in different states. New studies are also being developed around the country. This is an opportune time to start a program to reduce the use of antimicrobials with the participation of all members of society and the production chain.The main conclusion of this study is that preventive use of antimicrobial did not always reflect the sanitary condition or the real therapeutic needs, easily leading to overuse in most part of the evaluated swine herds. This condition can only be improved with awareness of producers, establishment of good management practices, and implementation of a national program to reduce antimicrobial usage with the participation of all members of the production chain.