Analyze Diet

Topic:DNA

DNA in horses refers to the genetic material that carries the hereditary information necessary for the growth, development, functioning, and reproduction of equine species. It consists of sequences of nucleotides that encode the genetic instructions used in the development and functioning of horses. DNA analysis in horses can provide insights into genetic diversity, lineage, and breed characteristics. It is also utilized in identifying genetic disorders, understanding hereditary traits, and assisting in selective breeding programs. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and applications of DNA analysis in equine genetics and breeding.
Sequencing and Assembling the Genome of Przewalski’s Horse in the Classroom.
Journal of equine veterinary science    February 15, 2025   105383 doi: 10.1016/j.jevs.2025.105383
Faulk C.Sequencing a genome by students has now become practical as we demonstrated with our recent publication of the Przewalski's horse (Equus ferus przewalskii) genome. In this review, I describe my experience teaching genome assembly in the classroom. In my course, students sequenced, assembled, and published a high-quality genome for Przewalski's horse using Oxford Nanopore long-read sequencing with only $4000 of materials. Along with the genome, we assembled the mitochondrial genome, sequence variants, predicted gene annotations, and DNA methylation levels. Our genome statistics far exceeded the...
Comparative phylogenetic and sequence identity analysis of internal transcribed spacer 2 and cytochrome C oxidase subunit I as DNA barcode markers for the most common equine Strongylidae species. Diekmann I, Krücken J, Kuzmina TA, Bredtmann CM, Louro M, Kharchenko VA, Tzelos T, Matthews JB, Madeira de Carvalho LM, von Samson-Himmelstjerna G.Morphologically, 64 strongylid species have been described in equines. Co-infections are common, with up to 29 species reported in a single horse. Morphological identification of these species is time consuming and requires expert knowledge due to their similar appearance. Therefore, non-invasive identification methods are needed. DNA barcoding offers a rapid and reliable tool for species identification and the discovery of cryptic species for these most common parasitic nematodes of equines. In total, 269 COI and 312 ITS-2 sequences from 27 equine Strongylidae species, including sequences fro...
Genome sequence of Equine Erythroparvovirus 1, identified in the United States.
Microbiology resource announcements    January 29, 2025   e0089724 doi: 10.1128/mra.00897-24
Yu YT, Olarte Castillo X, Reboul G, Zehr J, Sun Y, Anderson R, Wang M, Sun Q, Tallmadge R, Sams K, Brown J, Marra N, Stanhope B, Grenier J.... is a parvovirus that was identified in the blood of four horses in the United States. Here, we report one genome from a horse in New York State. This genome may represent a new species within the genus .
Nebulization of 2% lidocaine has no detectable impact on the healthy equine respiratory microbiota.
PloS one    January 24, 2025   Volume 20, Issue 1 e0316079 doi: 10.1371/journal.pone.0316079
Holley L, Creasey HN, Bedenice D, Reed S, Romualdo da Silva DR, Trautwein V, Mazan M, Widmer G.Glucocorticosteroids remain the most common pharmaceutical approach for the treatment of equine asthma but can be associated with significant side effects, including respiratory microbiome alterations. The goal of the study was to assess the impact of 2% lidocaine nebulization, a projected alternative treatment of equine asthma, on the healthy equine respiratory microbiota. A prospective, randomized, controlled, blinded, 2-way crossover study was performed, to assess the effect of 1 mg/kg 2% lidocaine (7 treatments over 4 days) on the equine respiratory microbiota compared to control horses (s...
Blood-Based Whole-Genome Methylation Analysis of Yili Horses Pre- and Post-Racing.
Animals : an open access journal from MDPI    January 24, 2025   Volume 15, Issue 3 doi: 10.3390/ani15030326
Wang J, Ren W, Li Z, Ma S, Li L, Wang R, Zeng Y, Meng J, Yao X.This study aims to analyze the whole-genome DNA methylation differences in Yili horses before and after racing, with the goal of identifying differentially methylated genes associated with racing performance and exploring the epigenetic mechanisms underlying exercise in horses. Blood samples were collected from the jugular veins of the top 3 Yili horses in a 5000 m race, which included 25 competitors, both prior to and within 5 min after the race. Genomic DNA was extracted, followed by sequencing using Whole-Genome Bisulfite Sequencing (WGBS) to assess DNA methylation levels, differentially me...
Identification of a global gene expression signature associated with the genetic risk of catastrophic fracture in iPSC-derived osteoblasts from Thoroughbred horses.
Animal genetics    January 13, 2025   Volume 56, Issue 1 e13504 doi: 10.1111/age.13504
Palomino Lago E, Ross AKC, McClellan A, Guest DJ.Bone fractures are a significant problem in Thoroughbred racehorses. The risk of fracture is influenced by both genetic and environmental factors. To determine the biological processes that are affected in genetically susceptible horses, we utilised polygenic risk scoring to establish induced pluripotent stem cells (iPSCs) from horses at high and low genetic risk. RNA-sequencing on iPSC-derived osteoblasts revealed 112 genes that were significantly differentially expressed. Forty-three of these genes have known roles in bone, 27 are not yet annotated in the equine genome and 42 currently have ...
Molecular genotyping of Theileria spp. detected in horses from Corrientes City, Argentina.
Veterinary research communications    December 24, 2024   Volume 49, Issue 1 54 doi: 10.1007/s11259-024-10618-3
Benitez-Ibalo AP, Debárbora VN, Mangold AJ, Nava S, Sebastian PS.After the previous description and characterization of Theileria equi sensu stricto (Genotype A) in Argentina, the aim of the present study was to investigate the possible heterogeneity of 18 S rDNA genotypes circulating in this country. Therefore, blood samples from 18 horses from two different neighborhoods of Corrientes City, Corrientes Province, were analyzed for infection with Theileria species. Theileria DNA was detected in five samples (27.8%). Four nearly complete 18 S rDNA gene sequences were obtained and phylogenetic analyses were carried out. The maximum likelihood tree constructe...
A Genetic Make Up of Italian Lipizzan Horse Through Uniparental Markers to Preserve Historical Pedigrees.
Biology    December 23, 2024   Volume 13, Issue 12 1087 doi: 10.3390/biology13121087
Crisà A, Cardinali I, Giontella A, Silvestrelli M, Lancioni H, Buttazzoni L.Lipizzan is a famous horse breed dating back to 1580 when the original stud of Lipica was established by the Hasburg Archduke Charles II. Currently, the Italian State Stud of Lipizzan Horses (ASCAL) is a conservation nucleus managed through strict mating rules where mitochondrial DNA sequences are used to verify the correct assignment of mares to a historical pedigree maternal lineage. Here, we analyzed the D-loop sequences of Lipizzan horses from the ASCAL in Monterotondo (Rome, Italy) in order to confirm their pedigree assignment to known female founder families. The concurrent investigation...
Endurance Effort Affected Expression of Actinin 3 and Klotho Different Isoforms Basing on the Arabian Horses Model.
Genes    December 18, 2024   Volume 15, Issue 12 1618 doi: 10.3390/genes15121618
Myćka G, Ropka-Molik K, Cywińska A, Stefaniuk-Szmukier M.Among numerous genes that have been a focus of equine genetic research, the (Klotho) and (Alpha-actinin-3) genes stand out due to their significant roles in muscle function and overall health, as well as performance ability. Previous studies on Arabian horses and other mammalians have shown that both and occur in different isoforms that seem to have different roles in metabolism. The main purpose of this present study was to describe different isoforms (, , , , , ) expression levels affected by the endurance effort in Arabian horses. Methods: Blood samples were taken from a group of = 10 ...
Mixed strongyle parasite infections vary across host age and space in a population of feral horses.
Parasitology    December 12, 2024   1-18 doi: 10.1017/S0031182024001185
Ahn S, Redman EM, Gavriliuc S, Bellaw J, Gilleard JS, McLoughlin PD, Poissant J.Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and ...
Genetic composition of Kazakh horses of Zhabe type evaluated by SNP genotyping.
Heliyon    December 12, 2024   Volume 11, Issue 1 e41173 doi: 10.1016/j.heliyon.2024.e41173
Pozharskiy A, Beishova I, Nametov A, Shamshidin A, Ulyanova T, Kovalchuk A, Ulyanov V, Shamekova M, Bekova G, Gritsenko D.Horses are animals traditionally playing prominent role as both food source and working animals for Kazakh people. Zhabe horses are traditional type of indigenous Kazakh horses characterized by versatility and adaptation to conditions of Central Asia. The present work focuses on examination of genetic structure of Zhabe horses using SNP genotyping with addition of previously published data. Total 1038 individuals including 403 new samples of Zhabe horses and 42 sample of white horses 'Zhetysu Asyly' have been considered. DNA was extracted from hair roots using commercial DNA isolation kit and ...
Study of Variation of ACOX1 Gene Among Different Horse Breeds Maintained in Iran.
Animals : an open access journal from MDPI    December 10, 2024   Volume 14, Issue 24 3566 doi: 10.3390/ani14243566
Boozarjomehri Amnieh S, Hassanpour A, Moghaddam S, Sakhaee F, Ropka-Molik K.The gene is vital for fatty acid metabolism and is linked to environmental stress and physical exertion adaptation. The p.Asp237Ser variant (rs782885985) in is associated with increased enzyme activity and reactive oxygen species (ROS) levels. This study examined the polymorphism across six horse breeds in Iran: Arabian, Thoroughbred, KWPN, Caspian, Kurdish, and Turkmen. The goal was to identify differences in genotype distribution, potentially serving as genetic markers under selection pressure related to breed-specific traits. In a sample of 324 horses, genomic DNA was analyzed using PCR...
The use of sex-sorted semen in horses.
Journal of equine veterinary science    December 5, 2024   Volume 145 105251 doi: 10.1016/j.jevs.2024.105251
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Unraveling the maternal heritage: identifying the complex origins of indigenous Indian horse and pony breeds through mitochondrial genome analysis.
Mammalian genome : official journal of the International Mammalian Genome Society    December 4, 2024   Volume 36, Issue 1 118-128 doi: 10.1007/s00335-024-10089-6
Ahlawat S, Sharma U, Niranjan SK, Chhabra P, Arora R, Sharma R, Singh KV, Vijh RK, Mehta SC.This study explored the maternal genetic diversity of six indigenous Indian horse and pony breeds (Bhutia, Kathiawari, Manipuri, Marwari, Spiti, and Zanskari) using comprehensive mitochondrial genome (mitogenome) analysis. Blood samples from 53 horses across diverse agro-climatic zones of India were analyzed, revealing 36 distinct haplotypes, with a haplotype diversity of 0.889 and nucleotide diversity of 0.00347. These indices suggest significant maternal genetic diversity in Indian equines. A median-joining (MJ) network, based on the hypervariable region of the D-loop along with sequences of...
Molecular Investigations of Babesia caballi from Clinically Healthy Horses in Southwestern Romania.
Veterinary sciences    November 27, 2024   Volume 11, Issue 12 600 doi: 10.3390/vetsci11120600
is a tick-borne hemoparasite that causes equine piroplasmosis. It has a significant economic impact, decreasing performance and affecting animal welfare. This study aimed to identify DNA in the blood of horses from households in the southwestern and western regions of Romania. We included 310 animals, from which blood was collected via EDTA. To test the samples for the parasite genome, we used real-time PCR and conventional PCR. The prevalence of was 5.81% (18/310) in apparently healthy horses, suggesting that this parasite is enzootic in the regions studied, although veterinarians did not...
The Role of Viral Pathogens in Horse Respiratory Diseases: A Cytological and Molecular Approach Using Next-Generation Sequencing.
Animals : an open access journal from MDPI    November 21, 2024   Volume 14, Issue 23 doi: 10.3390/ani14233347
Mazzei M, Sorvillo B, Sgorbini M, Bindi F, Perelli A, Laus F.(1) Background: Respiratory problems may be associated with pathogens among which viruses may play an important role in causing or promoting clinical signs. However, traditional diagnostic methods for equine infectious diseases, such as PCR, are limited to known pathogens and often miss rare or novel viruses. Metagenomic next-generation sequencing is a transformative method that allows the sequencing of all genomes present in a sample. Recent studies have used next-generation sequences to identify viral sequences in horses with unexplained respiratory symptoms, revealing potential links betwee...
The Role of Viral Pathogens in Horse Respiratory Diseases: A Cytological and Molecular Approach Using Next-Generation Sequencing.
Animals : an open access journal from MDPI    November 21, 2024   Volume 14, Issue 23 doi: 10.3390/ani14233347
Mazzei M, Sorvillo B, Sgorbini M, Bindi F, Perelli A, Laus F.(1) Background: Respiratory problems may be associated with pathogens among which viruses may play an important role in causing or promoting clinical signs. However, traditional diagnostic methods for equine infectious diseases, such as PCR, are limited to known pathogens and often miss rare or novel viruses. Metagenomic next-generation sequencing is a transformative method that allows the sequencing of all genomes present in a sample. Recent studies have used next-generation sequences to identify viral sequences in horses with unexplained respiratory symptoms, revealing potential links betwee...
Genomic insights into the genetic diversity, lateral gaits and high-altitude adaptation of Chakouyi (CKY) horses.
Journal of genetics and genomics = Yi chuan xue bao    November 19, 2024   Volume 52, Issue 8 1001-1010 doi: 10.1016/j.jgg.2024.11.008
Liu YK, Fu WW, Wang ZY, Pei SW, Li KH, Wu WW, Le MZ, Yue XP.Chakouyi (CKY) horses from the Qinghai‒Xizang Plateau are well known for their unique lateral gaits and high-altitude adaptation, but genetic mechanisms underlying these phenotypes remain unclear. This study presents a comparison of 60 newly resequenced genomes of gaited CKY horses with 139 public genomes from 19 horse breeds. Population structure analyses (admixture, PCA, and neighbor-joining tree) reveal a close genetic relationship between CKY and other highland breeds (Tibetan and Chaidamu horses). Compared with other Chinese breeds, CKY horses present reduced nucleotide diversity (θπ)...
Genetic diversity and phylogenetic relationships of Theileria equi inferred from heat shock protein 70 kDa gene sequences in Brazilian horses.
Veterinary research communications    November 19, 2024   Volume 49, Issue 1 16 doi: 10.1007/s11259-024-10599-3
Paulino PG, Amaral FB, de Oliveira RT, de Andrade SG, Rabello CA, Meirelles N, de Souza Santana M, Galdino KCP, Jacob JCF, Peckle M, Massard CL....Equine piroplasmosis, a tick-borne disease caused by hemoparasites of the Babesia and Theileria genera, has significant clinical and economic impacts worldwide. This study aims to characterize the heat shock protein 70 kDa (hsp70) gene sequences of Theileria equi from naturally infected horses across the five geographic regions in Brazil, and to analyze the phylogenetic relationships between T. equi and other parasites within the order Piroplasmida. Primers specific to T. equi were designed through in silico analysis of hsp70 gene sequences available in GenBank. Forty samples of equine whole ...
Genetic characterization of phenotypic traits in endangered Taishu horse breed and their breeding strategy.
Journal of equine veterinary science    November 15, 2024   Volume 144 105233 doi: 10.1016/j.jevs.2024.105233
Yoshihara T, Tozaki T, Nakaya S, Takasu M, Kawate K, Furukawa R, Kikuchi M, Isihge T, Nagata SI, Kakoi H, Hobo S.The Taishu horse is a native Japanese breed threatened with extinction. It is important to genotype the causative genes of rare phenotypes in endangered breeds because unique genetic traits might be lost unless a breeding strategy for conservation is constructed. In the present study, nine single-nucleotide variants and three indels of nine genes related to coat color, body composition, and gait were genotyped in 56 Taishu horses. Of these, only three genes were polymorphic. The observed coat color phenotypes coincided with the estimated phenotype from the genotypes, whereas black horses showe...
Clinical and epidemiological aspects in equine aural plaques.
Veterinary research communications    November 13, 2024   Volume 49, Issue 1 1 doi: 10.1007/s11259-024-10572-0
Rodrigues GG, Alves DS, Bromberger CR, Nomelini QSS, Borges AS, de Oliveira-Filho JP, Delfiol DJZ.The association between aural plaques, Equus caballus papillomavirus (EcPV), and the different breeds of horses and risk factors is poorly described. The objective of this study was to determine the clinical prevalence of aural plaques in Mangalarga Marchador (MM) and Quarter Horse (QH) horses in Brazil, to evaluate the association of this prevalence with some risk factors, and to detect the presence of EcPV DNA (types 1-10) in aural plaques biopsies. A total of 400 MM and 425 QH horses, were clinically evaluated. For each horse, an identification form was completed, containing information on ...
Preliminary Functional Analysis of the Gut Microbiome in Colic Horses.
Animals : an open access journal from MDPI    November 10, 2024   Volume 14, Issue 22 3222 doi: 10.3390/ani14223222
Thomson P, Garrido D, Santibáñez R, Lara F.The gut microbiome plays a critical role in maintaining horse health, influencing digestion, immunity, and overall well-being. However, in certain conditions like colic, there is evidence of significant alterations in the microbial community. To analyze the composition of the fecal microbiome and the enriched predicted metabolic functions of horses with colic compared to a control group, 14 horses with colic and 14 control horses were recruited. From a stool sample, DNA extraction was carried out for subsequent 16S rRNA metagenomic analysis. The composition of the microbiome was analyzed from ...
Characterization and phylogenetic analysis of the mitochondrial genome of Cylicostephanus longibursatus.
Parasitology research    October 29, 2024   Volume 123, Issue 10 363 doi: 10.1007/s00436-024-08385-w
Ma L, Li J, He J, Jiang T, Hao Y, Bu Y.Cylicostephanus longibursatus is a common parasite in equine animals. Hosts infected by these nematodes might face disease or death. This study utilized next-generation sequencing technology to sequence the complete mitochondrial genome (mt genome) of C. longibursatus. Through bioinformatics techniques, the genomic base composition, codon usage, tRNA secondary structures, evolutionary relationships, and taxonomic status were analyzed. The results revealed that the mitochondrial genome of C. longibursatus is a double-stranded, 13,807-bp closed circular molecule with an AT content of 76.0%, indi...
Stability of Gastric Fluid and Fecal Microbial Populations in Healthy Horses under Pasture and Stable Conditions.
Animals : an open access journal from MDPI    October 16, 2024   Volume 14, Issue 20 2979 doi: 10.3390/ani14202979
Bishop RC, Kemper AM, Clark LV, Wilkins PA, McCoy AM.Equine gastrointestinal microbial communities vary across the gastrointestinal tract and in response to diet or disease. Understanding the composition and stability of gastric fluid microbiota in healthy horses is a prerequisite to understanding changes associated with the development of disease. The objective of this study was to describe microbial communities in the gastric fluid and feces of healthy horses longitudinally. Horses were maintained on pasture (6 weeks), stabled (5 weeks), then returned to pasture. A consistent forage diet was provided throughout. Native gastric fluid and feces ...
Influence of Sires on Population Substructure in Dülmen Wild Horses.
Animals : an open access journal from MDPI    October 9, 2024   Volume 14, Issue 19 2904 doi: 10.3390/ani14192904
Duderstadt S, Distl O.The objectives of the present study were to analyze the influence of the stallions employed in the Dülmen wild horses on the genetic diversity and population substructure using Bayesian cluster analysis. The Dülmen wild horse is maintained as a unique horse population exposed to the natural conditions all year round in the Merfelder Bruch near Dülmen in Westphalia, Germany. Stallions selected for breeding have to prove their abilities to survive under this harsh environment. We used multilocus genotypic information from a set of 29 autosomal microsatellites to determine the paternity of 185...
Attenuation of the neuropathogenic equine herpesvirus type 1 strain Ab4p in hamsters by a single amino acid mutation (D752N) in viral DNA polymerase ORF30.
The Journal of veterinary medical science    October 9, 2024   Volume 86, Issue 12 1273-1278 doi: 10.1292/jvms.24-0338
Nishimura F, Fukushi N, Sakai H, Fukushi H.Equine herpesvirus type 1 (EHV-1) causes abortion, respiratory infection, and neurological diseases (equine herpesvirus myeloencephalopathy) in horses. A single nucleotide polymorphism (SNP) associated with a single amino acid in the DNA polymerase gene (ORF30, in which D752 is neuropathogenic and N752 is non-neuropathogenic) of EHV-1 has been associated with neuropathogenicity in horses. We constructed an EHV-1 Ab4p ORF30 N752 mutant and a repair virus to examine the effect of a D752N mutation on the neuropathogenicity of the virus in Syrian hamsters. The N752 mutation did not affect viral gr...
A Comprehensive Analysis of the Genomic and Expressed Repertoire of the T-Cell Receptor Beta Chain in Equus caballus.
Animals : an open access journal from MDPI    September 29, 2024   Volume 14, Issue 19 2817 doi: 10.3390/ani14192817
In this paper, we report a comprehensive and consistent annotation of the locus encoding the β-chain of the equine T-cell receptor (TRB), as inferred from recent genome assembly using bioinformatics tools. The horse TRB locus spans approximately 1 Mb, making it the largest locus among the mammalian species studied to date, with a significantly higher number of genes related to extensive duplicative events. In the region, 136 TRBV (belonging to 29 subgroups), 2 TRBD, 13 TRBJ, and 2 TRBC genes, were identified. The general genomic organization resembles that of other mammals, with a V cluster o...
Fecal microbiome and functional prediction profiles of horses with and without crib-biting behavior: A comparative study.
Journal of equine veterinary science    September 20, 2024   Volume 142 105198 doi: 10.1016/j.jevs.2024.105198
Martínez-Aranzales JR, Córdoba-Agudelo M, Pérez-Jaramillo JE.Crib-biting is a stereotyped oral behavior with poorly understood etiology and pathophysiology. The relationship between the gut microbiome and brain function has been described in behavioral disorders such as schizophrenia, depression and anxiety in humans. In horses, studies of behavioral problems and the microbiome are very limited. This study aimed to characterize the fecal microbiome and the predicted functional profile of horses with and without aerophagia. Fecal samples were collected from 12 Colombian Creole Horses of both sexes, divided into two groups: group 1, composed of six horses...
The Use of Genomic Screening for the Detection of Chromosomal Abnormalities in the Domestic Horse: Five New Cases of 65,XXY Syndrome in the Pura Raza Español Breed.
Animals : an open access journal from MDPI    September 3, 2024   Volume 14, Issue 17 2560 doi: 10.3390/ani14172560
Valera M, Karlau A, Anaya G, Bugno-Poniewierska M, Molina A, Encina A, Azor PJ, Demyda-Peyrás S.Sex chromosomal abnormalities are a well-established cause of reproductive failure in domestic horses. Because of its difficult diagnosis, the Pura Raza Español breeding program established a routine screening for chromosomal abnormalities in all the horses prior to enrolling in the studbook. This genomic procedure combines an initial assessment based on the results from Short Tandem Repeat (STR) parentage testing followed by a Single-Nucleotide Polymorphism (SNP) based copy number aberration (CNA) confirmative analysis in positive cases. Using this methodology, we identified five new individ...
Theileria orientalis Ikeda infection detected in red deer but not dogs or horses in New Zealand.
New Zealand veterinary journal    September 2, 2024   1-5 doi: 10.1080/00480169.2024.2393679
Lawrence KE, Gedye K, Carvalho L, Wang B, Fermin LM, Pomroy WE.To determine whether evidence for infection with (Ikeda) could be identified in samples of commercial red deer , horses, and working farm dogs in New Zealand. Unassigned: Blood samples were collected during October and November 2019 from a convenience sample of red deer (n = 57) at slaughter. Equine blood samples (n = 50) were convenience-sampled from those submitted to a veterinary pathology laboratory for routine testing in January 2020. Blood samples, collected for a previous study from a convenience sample of Huntaway dogs (n = 115) from rural regions throughout the North and ...