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Salmonella Typhimurium ( S. Typhimurium) contamination poses a significant challenge to breeder egg hatchability and chick health, necessitating the exploration of alternative disinfection methods. This study investigates the potential of phage vB_SPuM_SP02 (SP02) as a novel disinfectant for breeder eggs contaminated with S. Typhimurium SM022. Phage SP02 was isolated from poultry farm effluent and characterized for morphology, biological properties, and genome properties. Experimental groups of specific pathogen-free (SPF) eggs were treated with Salmonella and phage SP02, and efficacy was assessed through hatching rates, chick survival, weight, Salmonella load, immune organ indices, and intestinal flora. Phage treatment effectively eradicated Salmonella contamination on eggshells within 12 h, resulting in increased hatching and survival rates compared to controls. Furthermore, phage treatment mitigated weight loss and tissue Salmonella load in chicks without causing immune organ damage while reducing Salmonella spp. abundance in the intestinal tract. This study demonstrates the potential of phage SP02 as an eco-friendly and efficient disinfectant for S . Typhimurium-contaminated breeder eggs, offering promising prospects for practical application in poultry production.

Salmonella, a prominent foodborne pathogen, has posed enduring challenges to the advancement of food safety and global public health. The escalating concern over antibiotic misuse, resulting in the excessive presence of drug residues in animal-derived food products, necessitates urgent exploration of alternative strategies for Salmonella control. Bacteriophages emerge as promising green biocontrol agents against pathogenic bacteria. This study delineates the identification of two novel virulent Salmonella phages, namely phage vB_SalS_ABTNLsp11241 (referred to as sp11241) and phage 8-19 (referred to as 8-19). Both phages exhibited efficient infectivity against Salmonella enterica serotype Enteritidis (SE). Furthermore, this study evaluated the effectiveness of two phages to control SE in three different foods (whole chicken eggs, raw chicken meat, and lettuce) at different MOIs (1, 100, and 10000) at 4°C. It's worth noting that sp11241 and 8-19 achieved complete elimination of SE on eggs after 3 h and 6 h at MOI = 100, and after 2 h and 5 h at MOI = 10000, respectively. After 12 h of treatment with sp11241, a maximum reduction of 3.17 log 10 CFU/mL in SE was achieved on raw chicken meat, and a maximum reduction of 3.00 log 10 CFU/mL was achieved on lettuce. Phage 8-19 has the same effect on lettuce as sp11241, but is slightly less effective than sp11241 on chicken meat (a maximum 2.69 log 10 CFU/mL reduction). In conclusion, sp11241 and 8-19 exhibit considerable potential for controlling Salmonella contamination in food at a low temperature and represent viable candidates as green antibacterial agents for food applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014-2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

The industrialization of farming has had an enormous impact. To most, this impact is viewed solely in the context of productivity, but the denser living conditions and shorter rearing periods of industrial livestock farms provide pathogens with an ideal opportunity to spread and evolve. For example, the industrialization of poultry farms drove the Marek's disease virus (MDV) to evolve from a mild paralytic syndrome to a highly contagious, globally prevalent, deadly disease. Fortunately, the economic catastrophe that would occur from MDV evolution is prevented through the widespread use of live imperfect vaccines that limit disease symptoms, but fail to prevent transmission. Unfortunately, the continued rollout of such imperfect vaccines is steering MDV evolution towards even greater virulence, and the ability to evade vaccine protection. Thus, there is a need to investigate alternative economically viable control measures for their ability to inhibit MDV spread and evolution. In what follows we examine the economic viability of standard husbandry practices for their ability to inhibit the spread of both virulent MDV and very virulent MDV throughout an industrialized egg farm. To do this, we parameterize a MDV transmission model and calculate the loss in egg production due to MDV. We find that MDV strain and the cohort duration have the greatest influence on both disease burden and egg production. Additionally, our findings show that for long cohort durations, conventional cages result in the least per capita loss in egg production due to MDV infection, while Aviary systems perform best over shorter cohort durations. Finally, we find that the least per capita loss in egg production for flocks infected with the more virulent MDV strains occurs when cohort durations are sufficiently short. These results highlight the important decisions that managers will face when implementing new hen husbandry practices., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

High pathogenicity avian influenza virus (HPAIV) infections in chickens negatively impact egg production and cause egg contamination. Previously, vaccination maintained egg production and reduced egg contamination when challenged with a North American H5N2 HPAIV. However, Asian H5N1 HPAIV infection has some characteristics of increased pathogenicity compared to other H5 HPAIV such as more rapid drop and complete cessation in egg production. Sham (vaccinated at 25 and 28 weeks of age), inactivated H5N1 Once (1X-H5-Vax; vaccinated at 28 weeks of age only) and inactivated H5N1 Twice (2X-H5-Vax; vaccinated at 25 and 28 weeks of age) vaccinated adult White Leghorn hens were challenged intranasally at 31 weeks of age with 6.1 log10 mean embryo infectious doses (EID50) of clade 2.3.2.1a H5N1 HPAIV (A/chicken/Vietnam/NCVD-675/2011) which was homologous to the inactivated vaccine. Sham-vaccinated layers experienced 100% mortality within 3 days post-challenge; laid soft and thin-shelled eggs; had recovery of virus from oral swabs and in 53% of the eggs from eggshell surface (35%), yolk (24%), and albumin (41%); and had very high titers of virus (average 7.91 log10 EID50/g) in all segments of the oviduct and ovary. By comparison, 1X- and 2X-H5-Vax challenged hens survived infection, laid similar number of eggs pre- and post-challenge, all eggs had normal egg shell quality, and had significantly fewer contaminated eggs with reduced virus quantity. The 2X-H5-Vax hens had significantly higher HI titers by the day of challenge (304 GMT) and at termination (512 GMT) than 1X-H5-Vax hens (45 GMT and 128 GMT). The current study demonstrated that AIV infections caused by clade 2.3.2.1a H5N1 variants can be effectively controlled by either double or single homologous vaccination., (Published by Elsevier Ltd.)

Chicks possess maternally derived antibody (MDA) against pathogens and vaccines previously encountered by the dams. This passive immunity is important in early life, when the immune system is immature and unable to fight off infection. On the other hand, MDA can also affect the development of the immune system and interfere with vaccination against avian diseases such as Newcastle disease (ND) and avian influenza (AI). The effect of MDA is generally investigated by studying the progeny of vaccinated dams, which is time-consuming, poorly flexible and expensive. Moreover, the antibody titres obtained are not homogeneous. In this study, a model was developed to offer a faster, more reproducible and cheaper way to study passive immunity in specific pathogen free chickens by injection of a polyclonal serum into the egg yolk at embryonic day 14, combined with an intraperitoneal injection at day 1. A satisfactory model, with consistent, homogeneous antibody titres, as well as persistence close to natural passive immunity, could be obtained for ND virus. On the other hand, the application of this optimized protocol in an H5 AI context induced only a low artificial passive immunity compared with that described in the literature for the progeny of AI vaccinated dams. This artificial model should facilitate future studies regarding the effect of passive immunity on vaccine efficacy at a young age and its effect on immune system development.

Infection with high-pathogenicity avian influenza virus (HPAIV) has been associated with a wide range of clinical manifestations in poultry, including severe depression in egg production and isolation of HPAIV from eggs laid by infected hens. To evaluate the pathobiology in the reproductive tract of chickens, adult hens were inoculated intranasally with 3 HPAIV strains. All 3 strains induced lesions in the reproductive tract 36 to 72 hours after inoculation. Positive immunostaining was observed in all segments of the reproductive tract, occurring predominantly in stromal cells and superficial germinal epithelium of the ovary, in mucosal epithelial cells and less often glandular epithelium throughout the oviduct, and in vascular endothelium. This study generates important data and explains previously reported virus isolation from yolk, due to ovarian virus replication, and virus recovery from albumin, due to virus replication in epithelial cells in several segments of the oviduct.

Even though low pathogenic avian influenza viruses (LPAIv) affect the poultry industry of several countries in the world, information about their transmission characteristics in poultry is sparse. Outbreak reports of LPAIv in layer chickens have described drops in egg production that appear to be correlated with the virus transmission dynamics. The objective of this study was to use egg production data from LPAIv infected layer flocks to quantify the within-flock transmission parameters of the virus. Egg production data from two commercial layer chicken flocks which were infected with an H7N3 LPAIv were used for this study. In addition, an isolate of the H7N3 LPAIv causing these outbreaks was used in a transmission experiment. The field and experimental estimates showed that this is a virus with high transmission characteristics. Furthermore, with the field method, the day of introduction of the virus into the flock was estimated. The method here presented uses compartmental models that assume homogeneous mixing. This method is, therefore, best suited to study transmission in commercial flocks with a litter (floor-reared) housing system. It would also perform better, when used to study transmission retrospectively, after the outbreak has finished and there is egg production data from recovered chickens. This method cannot be used when a flock was affected with a LPAIv with low transmission characteristics (R(0)<2), since the drop in egg production would be low and likely to be confounded with the expected decrease in production due to aging of the flock. Because only two flocks were used for this analysis, this study is a preliminary basis for a proof of principle that transmission parameters of LPAIv infections in layer chicken flocks could be quantified using the egg production data from affected flocks., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

The aim of this study was to develop and evaluate an early warning system (EWS) for commercial egg laying flocks to detect the subtle mortality and egg production changes that characterize low pathogenic avian influenza virus (LPAIV) infections. An EWS will create an alert when the recommended 'trigger point' is reached or exceeded. Previously used EWSs are based on fixed alert levels, while the proposed EWS customizes the alert level to each flock. While a fixed approach may be valid for highly pathogenic diseases, it results in a lower detection probability for low pathogenic diseases. The EWS was based on daily data collected from flocks affected by the 2000-2004 H6N2 LPAI epidemic in California. Three EWSs were evaluated: (1) EWS1, which is triggered when the observed mortality increase or production decrease exceeds more than "x" times the expected daily value (2.75-3.50 times the expected mortality), (2) EWS2, which is triggered when the observed mortality increase or production decrease exceeds more than "y" times during each of 2 consecutive days the expected daily values (1.75-2.15 times the expected mortality), and (3) a combination of the two. The EWSs were evaluated according to three parameters: detection delay (days) of a LPAI outbreak, false alerts (%) and outbreaks missed (%). Results showed that an egg production-based EWS added no benefit to a mortality-based system, mainly because H6N2 LPAI-related egg production decrease always occurred after increase in mortality. Combining the two EWSs resulted in a reduced detection delay and no missed outbreaks, but at the expense of a slight increase in the number of false alerts triggered. The system presented in this study also outperformed fixed EWSs in all three evaluated parameters. The proposed EWS, if used as part of a poultry cooperative program and combined with a rapid laboratory diagnosis, could be a useful tool in the detection and control of LPAI outbreaks and other poultry diseases. Built in a spreadsheet, the system could be inexpensively, easily and quickly incorporated into a commercial egg production farm decision support system. In addition, the proposed system could be quickly adjusted to changing epidemic situations, and easily customized to individual flocks.

H9N2 influenza viruses are endemic in many Asian countries including China and Korea, and cause a considerable economic loss to chicken industry by reduction in egg production and about 30% mortality. Here we developed live cold-adapted attenuated H9N2 influenza vaccine by adaptation of viruses in hen's eggs at 25 degrees C. Genetic analysis shows that the cold-adapted H9N2 (A/Chicken/Korea/S1/03) viruses contain a total of 44 amino acid substitutions, of which 7 amino acids are identical to the loci identified in the cold-adapted H2N2 (A/Ann Arbor/6/60) vaccine strain compared to genes in wild-type H9N2 (A/Chicken/Korea/S1/03) influenza viruses. When cold-adapted H9N2 (A/Chicken/Korea/S1/03) influenza viruses were inoculated in layers viruses were detectable in the tracheas, not in the lungs, no reduction of egg production and mortality was observed in contrast to the infection of wild-type H9N2 influenza viruses, and CD8+ T lymphocytes expressing IFN-gamma were induced. When layers vaccinated with cold-adapted attenuated H9N2 (A/Chicken/Korea/S1/03) influenza viruses were challenged with wild-type H9N2 (A/Chicken/Korea/521/04) influenza viruses, they were protected from the loss of egg production and mortality. Our results suggest that cold-adapted attenuated H9N2 vaccine can be used for controlling the infection of H9N2 influenza viruses in chickens.

Endogenous avian leukosis virus (ALVE) and the ALVE receptor (TVB*S1) status of six commercial chicken lines supplying specific-pathogen-free eggs were analyzed. All commercial chicken lines are certified free of the avian leukosis virus (ALV) by screening for expression of the p27 protein using the standard enzyme-linked immunosorbent assay. The commercial chicken lines A, E, and F contained replication competent ALVE inserts. Line A was fixed for ALVE21, and lines E and F were segregating for ALVE10. In addition, ALVE1 was detected in all the chicken lines. Chicken lines B, D, and F were essentially fixed for the TVB*S1 allele that confers susceptibility to ALVE, whereas lines A, C, B, and E were resistant, containing either the TVB*S3 or TVB*R alleles. The results show that lines selected to be ALV p27 negative give rise to two different genotypes. One genotype lacks the TVB*S1 receptor for ALVE. Chicken lines with the TVB*S1 negative genotype can retain replication competent endogenous virus inserts such as ALVE2, 10, or 21 and still display the p27 negative phenotype. These replication competent ALVE viruses are phenotypically p27 negative in the absence of the TVB*S1 receptor because their chromosomal integration sites restrict transcription and subsequent production of the p27 protein and virus particles to levels below the detection limit. If the TVB*S1 receptor is present, the limited production of ALVE virus particles reinfects and integrates into more productive chromosomal locations in the cell. Increased production of infective virus particles and detectable levels of p27 follow this reinfection and integration into more active regions of the cells genome. The other genotype observed in the commercial lines retains the ALVE receptor (TVB*S1) but either lacks replication competent inserts or expresses the envelope encoded protein from defective inserts such as ALVE3 or ALVE6. In this phenotype, the env-coded glycoprotein encoded by the defective inserts binds to the TVB*S1 receptor and blocks the reinfection of the replication competent ALVE virus. This receptor interference stops reinfection and subsequent production of detectable virus particles and the p27 protein. Mixtures of different p27 negative phenotypes can result in the p27 positive phenotype and ALVE virus production. For example, mixtures of ALVE receptor positive (TVB*S1) but ALVE negative (p27 negative and envelope negative) chick embryo fibroblasts (CEFs) with fibroblasts that are receptor negative but ALVE positive could generate cells expressing high levels of p27 and ALVE virus. In this situation, the undetectable levels of ALVE virus from the receptor negative CEFs would infect and integrate into the receptor positive CEFs and produce detectable levels of ALVE virus. The implications of these findings for vaccine manufacturers and regulatory agencies are discussed.

The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. Instructions for AI virus primary isolation and propagation, preparing antigen for an AGID test, setting up an AGID test, and interpreting results are given.

Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.

Avian influenza (AI) and Newcastle disease (ND) viruses are heat labile viruses, but exact parameters for heat inactivation at egg pasteurization temperatures have not been established. In this study we artificially infected four egg products with two AI (one low [LP] and one high pathogenicity [HP]) and three ND (two low and one highly virulent) viruses, and determined inactivation curves at 55, 57, 59, 61 and 63 degrees C. Based on D(t) values, the time to inactivation of the viruses was dependent on virus strain and egg product, and was directly related to virus titre, but inversely related to temperature. For all temperatures, the five viruses had the most rapid and complete inactivation in 10% salt yolk, while the most resistant to inactivation was HPAI virus in dried egg white. This study demonstrated that the LPAI and all ND viruses were inactivated in all egg products when treated using industry standard pasteurization protocols. By contrast, the HPAI virus was inactivated in liquid egg products but not in dried egg whites when using the low-temperature industry pasteurization protocol.

Sporadic outbreaks of Newcastle disease (ND) occurred in Taiwan during 1998-2000. In some cases, the disease occurred in broilers less than 2 wk old that originated in a broiler breeder farm, so spread of the ND virus (NDV) from the infected breeder farm to broiler ranches was suspected. The purpose of the present study was to examine the possibility of the transmission of NDV through eggs. Both clinical and experimental evidence were used to prove that this is possible. From epidemiological investigation, the possibility of transmission through eggs was suggested in two separate ND cases from a breeder farm and its progeny because two identical NDVs were isolated from both cases. In order to clarify the possibility of the transmission through eggs, one mean egg lethal dose (ELD50) of NDV was inoculated into the allantoic cavity of 155 9-to-11-day-old specific-pathogen-free (SPF) chicken embryos. Seventy-one hatching chicks from the inoculated embryos were raised for 14 days. The cloacal swabs from those chicks at the ages of 1, 4, and 7 days and the tissues after necropsy at the ages of 14 days were taken for virus isolation. The same NDV was reisolated from three hatching chicks. This experiment confirms that a few chicken embryos infected in ovo with a low titer of NDV can hatch and contain NDV after hatching, which results in NDV spreading through eggs.

The level of Salmonella enteritidis contamination in Zambian table eggs and ready market chicken carcasses was studied. Nine of the 240 (3.8%) pooled samples of table eggs and 18 of 382 (4.7%) chicken carcasses were contaminated with S. enteritidis. All the isolates from the table eggs belonged to the enteropathogenic invasive phage type 4, whereas seven isolates from the chicken carcasses belonged to phage type 4, three were phage type 7, and eight were untypable (rough type). All the isolates were sensitive to gentamycin, ampicillin, tetracycline, cotrimoxazole, amoxycillin, furazolidone, and chloramphenicol.

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.

Poultry products contaminated with pathogenic strains of Newcastle disease virus are a source of virus transmission to susceptible poultry flocks. The probability of contamination varies according to the type of product. Research conducted by various laboratories in Europe has shown that pathogenic virus can be isolated from the carcasses of chickens, whether vaccinated or not, during a brief period after experimental infection. Eggs laid by hens infected with Newcastle disease virus present a very low risk. Furthermore, feathers, bones, blood and offal present potential risks if they are incorporated in poultry feed. Finally, poultry droppings used as a fertiliser can present a major risk of infection in certain circumstances.