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Topic:Genomics

Genomics in horses involves the study and analysis of the horse genome to understand genetic variations and their implications for equine health, performance, and breeding. This field encompasses the identification and mapping of genes associated with specific traits, diseases, and conditions in horses. Techniques such as whole-genome sequencing and genome-wide association studies (GWAS) are employed to explore genetic diversity and inheritance patterns among different horse breeds. Genomics provides insights into hereditary disorders, informs selective breeding practices, and aids in the development of personalized veterinary care. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of genomic research in equine science.
Genome-wide single-nucleotide polymorphism data and mitochondrial hypervariable region 1 nucleotide sequence reveal the origin of the Akhal-Teke horse.
Animal bioscience    May 4, 2023   Volume 36, Issue 10 1499-1507 doi: 10.5713/ab.23.0044
Kang Z, Shi J, Liu T, Zhang Y, Zhang Q, Liu Z, Wang J, Cheng S.The study investigated the origin of the Akhal-Teke horse using genome-wide single-nucleotide polymorphism (SNP) data and mitochondrial hypervariable region 1 (HVR-1) nucleotide sequences. Methods: Genome-wide SNP data from 22 breeds (481 horses) and mitochondrial HVR-1 sequences from 24 breeds (544 sequences) worldwide to examine the origin of the Akhal- Teke horse. The data were analyzed using principal component analysis, linkage disequilibrium analysis, neighbor-joining dendrograms, and ancestry inference to determine the population relationships, ancestral source, genetic structure, and r...
A genome-wide landscape of mRNAs, miRNAs, lncRNAs, and circRNAs of skeletal muscles during dietary restriction in Mongolian horses.
Comparative biochemistry and physiology. Part D, Genomics & proteomics    May 2, 2023   Volume 46 101084 doi: 10.1016/j.cbd.2023.101084
Bou T, Ding W, Liu H, Gong W, Jia Z, Dugarjaviin M, Bai D.The proportion of different muscle fibers is essential for the horse breed's aptitude for athletic activities. Adaptation of locomotor muscle is correlated with altered physiologic conditions. To investigate the adaptive changes of muscle fiber phenotype and transcriptome in horse skeletal muscle during dietary restriction (DR). The muscle fiber type distribution and deep RNA-seq analysis of detecting differentially expressed mRNAs (DEGs), miRNA (DEMIRs), lncRNAs (DELs), circRNAs (DECs), and their function analysis were investigated in gluteus medius muscle of Mongolian horses during DR. A tot...
Newly identified variability of the antigen binding site coding sequences of the equine major histocompatibility complex class I and class II genes.
HLA    April 27, 2023   Volume 102, Issue 4 489-500 doi: 10.1111/tan.15078
Plasil M, Oppelt J, Klumplerova M, Bubenikova J, Vychodilova L, Janova E, Stejskalova K, Futas J, Knoll A, Leblond A, Mihalca AD, Horin P.The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in th...
Cellular and Molecular Consequences of Stallion Sperm Cryopreservation: Recent Approaches to Improve Sperm Survival.
Journal of equine veterinary science    April 25, 2023   Volume 126 104499 doi: 10.1016/j.jevs.2023.104499
Contreras MJ, Arias ME, Fuentes F, Muñoz E, Bernecic N, Fair S, Felmer R.Cryopreservation of stallion semen does not achieve the post-thaw quality or fertility results observed in other species like cattle. There are many reasons for this, but the membrane composition and intracellular changes in stallion sperm predispose them to low resistance to the cooling, freezing, and subsequent thawing process. Damage to the sperm results from different processes activated during cryopreservation, including oxidative stress, apoptosis, and structural modifications in the sperm membrane that increase the deleterious effect on sperm. In addition, significant individual variabi...
SNP-Based Heritability of Osteochondrosis Dissecans in Hanoverian Warmblood Horses.
Animals : an open access journal from MDPI    April 25, 2023   Volume 13, Issue 9 doi: 10.3390/ani13091462
Zimmermann E, Distl O.Before the genomics era, heritability estimates were performed using pedigree data. Data collection for pedigree analysis is time consuming and holds the risk of incorrect or incomplete data. With the availability of SNP-based arrays, heritability can now be estimated based on genotyping data. We used SNP array and 1.6 million imputed genotype data with different minor allele frequency restrictions to estimate heritabilities for osteochondrosis dissecans in the fetlock, hock and stifle joints of 446 Hanoverian warmblood horses. SNP-based heritabilities were estimated using a genomic restricted...
A multiplex qPCR assay for transgenes detection: A novel approach for gene doping control in horseracing using conventional laboratory setup.
Drug testing and analysis    April 19, 2023   Volume 15, Issue 8 879-888 doi: 10.1002/dta.3483
Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM.Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1...
Effects of concentrated fecal microbiota transplant on the equine fecal microbiota after antibiotic-induced dysbiosis. Di Pietro R, Arroyo LG, Leclere M, Costa M.Bacterial imbalances are observed in intestinal diseases and fecal microbiota transplantation (FMT) has been used to restore the intestinal microbiota of horses. However, there is evidence that the current methods proposed for FMT in horses have limited efficacy. The objective of this study was to concentrate the bacteria present in the donor stool by centrifugation, and to test the effect in horses with antibiotic-induced dysbiosis. One healthy 11-year-old horse was selected as a fecal donor and 9 horses were given trimethoprim sulfadiazine (TMS) for 5 days to induce dysbiosis. Horses receive...
Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation.
International journal of molecular sciences    April 7, 2023   Volume 24, Issue 8 6915 doi: 10.3390/ijms24086915
Angel-Velez D, Meese T, Hedia M, Fernandez-Montoro A, De Coster T, Pascottini OB, Van Nieuwerburgh F, Govaere J, Van Soom A, Pavani K, Smits K.In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), a...
Whole genome sequencing to study antimicrobial resistance and RTX virulence genes in equine Actinobacillus isolates.
Veterinary research    April 5, 2023   Volume 54, Issue 1 33 doi: 10.1186/s13567-023-01160-2
Vereecke N, Vandekerckhove A, Theuns S, Haesebrouck F, Boyen F.Actinobacillus equuli is mostly associated with disease in horses and is most widely known as the causative agent of sleepy foal disease. Even though existing phenotypic tools such as biochemical tests, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) can be used to identify members of the Actinobacillus genus, these methods struggle to differentiate between certain species and do not allow strain, virulence, and antimicrobial susceptibility typing. Hence, we performed in-depth analysis of 24 equine Actinobacillus isolate...
Immunotherapy of Equine Sarcoids-From Early Approaches to Innovative Vaccines.
Vaccines    March 30, 2023   Volume 11, Issue 4 769 doi: 10.3390/vaccines11040769
Jindra C, Hainisch EK, Brandt S.Horses and other equid species are frequently affected by bovine papillomavirus type 1 and/or 2 (BPV1, BPV2)-induced skin tumors termed sarcoids. Although sarcoids do not metastasize, they constitute a serious health problem due to their BPV1/2-mediated resistance to treatment and propensity to recrudesce in a more severe, multiple form following accidental or iatrogenic trauma. This review provides an overview on BPV1/2 infection and associated immune escape in the equid host and presents early and recent immunotherapeutic approaches in sarcoid management.
Early dispersal of domestic horses into the Great Plains and northern Rockies.
Science (New York, N.Y.)    March 30, 2023   Volume 379, Issue 6639 1316-1323 doi: 10.1126/science.adc9691
Taylor WTT, Librado P, American Horse CJ, Shield Chief Gover C, Arterberry J, Afraid of Bear-Cook AL, Left Heron H, Yellow Hair RM, Gonzalez M....The horse is central to many Indigenous cultures across the American Southwest and the Great Plains. However, when and how horses were first integrated into Indigenous lifeways remain contentious, with extant models derived largely from colonial records. We conducted an interdisciplinary study of an assemblage of historic archaeological horse remains, integrating genomic, isotopic, radiocarbon, and paleopathological evidence. Archaeological and modern North American horses show strong Iberian genetic affinities, with later influx from British sources, but no Viking proximity. Horses rapidly sp...
Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma.
International journal of biological macromolecules    March 28, 2023   Volume 239 124229 doi: 10.1016/j.ijbiomac.2023.124229
Alim S, Laitaoja M, Pawar SS, Talluri TR, Jänis J, Swamy MJ.Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical r...
Integrative genomics analysis highlights functionally relevant genes for equine behaviour.
Animal genetics    March 27, 2023   Volume 54, Issue 4 457-469 doi: 10.1111/age.13320
Holtby AR, Hall TJ, McGivney BA, Han H, Murphy KJ, MacHugh DE, Katz LM, Hill EW.Behavioural plasticity enables horses entering an exercise training programme to adapt with reduced stress. We characterised SNPs associated with behaviour in yearling Thoroughbred horses using genomics analyses for two phenotypes: (1) handler-assessed coping with early training events [coping] (n = 96); and (2) variation in salivary cortisol concentration at the first backing event [cortisol] (n = 34). Using RNA-seq derived gene expression data for amygdala and hippocampus tissues from n = 2 Thoroughbred stallions, we refined the SNPs to those with functional relevance to behaviour by c...
Erratum: Genetic diversity analysis and parentage verification of Taishu horses using 31 microsatellites.
Journal of equine science    March 24, 2023   Volume 34, Issue 1 19 doi: 10.1294/jes.34.19
[This corrects the article DOI: 10.1294/jes.33.63.].
A Cold Case of Equine Influenza Disentangled with Nanopore Sequencing.
Animals : an open access journal from MDPI    March 24, 2023   Volume 13, Issue 7 1153 doi: 10.3390/ani13071153
Pellegrini F, Buonavoglia A, Omar AH, Diakoudi G, Lucente MS, Odigie AE, Sposato A, Augelli R, Camero M, Decaro N, Elia G, Bányai K, Martella V....Massive sequencing techniques have allowed us to develop straightforward approaches for the whole genome sequencing of viruses, including influenza viruses, generating information that is useful for improving the levels and dimensions of data analysis, even for archival samples. Using the Nanopore platform, we determined the whole genome sequence of an H3N8 equine influenza virus, identified from a 2005 outbreak in Apulia, Italy, whose origin had remained epidemiologically unexplained. The virus was tightly related (>99% at the nucleotide level) in all the genome segments to viruses identif...
Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity.
Frontiers in veterinary science    March 23, 2023   Volume 10 1125893 doi: 10.3389/fvets.2023.1125893
Andrietti ALP, Durgam SS, Naumann B, Stewart M.Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix. Unassigned: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolate...
Diagnostic applications and limitations for the use of cell-free fetal DNA (cffDNA) in animal husbandry and wildlife management.
Research in veterinary science    March 17, 2023   Volume 158 106-116 doi: 10.1016/j.rvsc.2023.03.013
Aucamp J, van der Zwan H, Geldenhuys Z, Abera A, Louw R, van der Sluis R.In animal breeding, a species sex can influence the value of the animal. For example, in the horse breeding industry, mares are preferred as polo horses, while in wildlife breeding males with larger horns are more valuable. Therefore, the economic advantages of knowing the unborn fetus' sex are important to successful animal management. Ultrasonography is used to determine the sex of unborn fetuses, but this method places additional stress on the animal and require specialized equipment and expertise. Conversely, molecular-based sexing techniques require less invasive sampling and can determin...
Erratum: Single-cell gene expression analysis of cryopreserved equine bronchoalveolar cells.
Frontiers in immunology    March 13, 2023   Volume 14 1178189 doi: 10.3389/fimmu.2023.1178189
[This corrects the article DOI: 10.3389/fimmu.2022.929922.].
Impacts of Domestication and Veterinary Treatment on Mobile Genetic Elements and Resistance Genes in Equine Fecal Bacteria.
Applied and environmental microbiology    March 7, 2023   Volume 89, Issue 3 e0159022 doi: 10.1128/aem.01590-22
Mitchell SW, Moran RA, Elbourne LDH, Chapman B, Bull M, Muscatello G, Coleman NV.Antimicrobial resistance in bacteria is a threat to both human and animal health. We aimed to understand the impact of domestication and antimicrobial treatment on the types and numbers of resistant bacteria, antibiotic resistance genes (ARGs), and class 1 integrons (C1I) in the equine gut microbiome. Antibiotic-resistant fecal bacteria were isolated from wild horses, healthy farm horses, and horses undergoing veterinary treatment, and isolates (9,083 colonies) were screened by PCR for C1I; these were found at frequencies of 9.8% (vet horses), 0.31% (farm horses), and 0.05% (wild horses). A co...
Discovery and comparative genomic analysis of a novel equine anellovirus, representing the first complete Mutorquevirus genome.
Scientific reports    March 6, 2023   Volume 13, Issue 1 3703 doi: 10.1038/s41598-023-30875-7
Fisher M, Nebroski M, Davies J, Janzen E, Sullivan D, Lung O.The complete genome of a novel torque teno virus species (Torque teno equus virus 2 (TTEqV2) isolate Alberta/2018) was obtained by high-throughput sequencing (HTS) of nucleic acid extracted from the lung and liver tissue of a Quarter Horse gelding that died of nonsuppurative encephalitis in Alberta, Canada. The 2805 nucleotide circular genome is the first complete genome from the Mutorquevirus genus and has been approved as a new species by the International Committee on Taxonomy of Viruses. The genome contains several characteristic features of torque teno virus (TTV) genomes, including an OR...
Analysis of lncRNA and mRNA expression profiling in immature and mature DeZhou donkey (equine Taurus) testes.
Reproduction in domestic animals = Zuchthygiene    March 6, 2023   Volume 58, Issue 5 646-656 doi: 10.1111/rda.14330
Yu J, Wang Z, Wang F, Yang G, Cheng J, Ji C, Li M, Liu B, Wang Y, Dang R.Testicular development and spermatogenesis are tightly regulated by the number of genes and noncoding genes, and mRNAs and lncRNAs play vital roles in regulating posttranscriptional gene expression. However, mRNAs and lncRNAs have not been systematically identified in the testes of donkeys. In this study, mRNA and lncRNA expression profiles in the testes of DeZhou donkeys between 2 months and 2 years of age were comprehensively analysed by RNA sequencing. We identified 56,605 lncRNAs and 61,857 mRNAs by gene expression analysis, and 21,845 lncRNAs (p < .05) and 14,109 mRNAs (p < .0...
Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.
medRxiv : the preprint server for health sciences    March 6, 2023   2023.03.06.23286851 doi: 10.1101/2023.03.06.23286851
Hill V, Koch RT, Bialosuknia SM, Ngo K, Zink SD, Koetzner CA, Maffei JG, Dupuis AP, Backenson PB, Oliver J, Bransfield AB, Misencik MJ, Petruff TA....Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts...
Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses.
Genes    March 3, 2023   Volume 14, Issue 3 638 doi: 10.3390/genes14030638
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI.Thoroughbreds are some of the most famous racehorses worldwide and are currently animals of high economic value. To understand genomic variability in Thoroughbreds, we identified genome-wide insertions and deletions (INDELs) and obtained their allele frequencies in this study. INDELs were obtained from whole-genome sequencing data of 101 Thoroughbred racehorses by mapping sequence reads to the horse reference genome. By integrating individual data, 1,453,349 and 113,047 INDELs were identified in the autosomal (1-31) and X chromosomes, respectively, while 18 INDELs were identified on the mitoch...
Codon usage bias of Venezuelan equine encephalitis virus and its host adaption.
Virus research    March 3, 2023   Volume 328 199081 doi: 10.1016/j.virusres.2023.199081
Wang H, Liu S, Lv Y, Wei W.Venezuelan equine encephalitis virus (VEEV) is an emerging zoonotic virus in the alphavirus genus. It can be transmitted to humans due to spillover from equid-mosquito cycles. The symptoms caused by VEEV include fever, headache, myalgia, nausea, and vomiting. It can also cause encephalitis in severe cases. The evolutionary features of VEEV are largely unknown. In this study, we comprehensively analyzed the codon usage pattern of VEEV by computing a variety of indicators, such as effective number of codons (ENc), codon adaptation index (CAI), relative synonymous codon usage (RSCU), on 130 VEEV ...
Nanoformulation of a Trypanocidal Drug Isometamidium Chloride Ameliorates the Apurinic-Apyrimidinic DNA Sites/Genotoxic Effects in Horse Blood Cells.
Journal of xenobiotics    March 2, 2023   Volume 13, Issue 1 148-158 doi: 10.3390/jox13010012
Singh S, Kumar B, Dilbaghi N, Devi N, Prasad M, Manuja A.Isometamidium chloride (ISM) is a trypanocide for the prophylactic and therapeutic use against vector-borne animal trypanosomosis (mainly Surra caused by ) and African animal trypanosomosis caused by //). ISM was found to be an efficient trypanocide for therapeutic/prophylactic use against trypanosomosis; however, it produces some local and systemic detrimental effects in animals. We synthesized isometamidium chloride-loaded alginate gum acacia nanoformulation (ISM SANPS) to lessen the detrimental side effects of isometamidium chloride (ISM) while treating trypanosomal diseases. We intended to...
Functional annotation of the animal genomes: An integrated annotation resource for the horse.
PLoS genetics    March 2, 2023   Volume 19, Issue 3 e1010468 doi: 10.1371/journal.pgen.1010468
Peng S, Dahlgren AR, Donnelly CG, Hales EN, Petersen JL, Bellone RR, Kalbfleisch T, Finno CJ.The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and...
Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.
Viruses    February 28, 2023   Volume 15, Issue 3 650 doi: 10.3390/v15030650
Turco S, Gabbianelli F, Mavian CN, Pietrucci D, De Paolis L, Gialletti R, Mechelli L, De Ciucis CG, Cappelli K, Dell'Anno F, Mecocci S, Donato GG....Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as PV 10 (EcPV10). All EcPV genes are cons...
Behavioral and Physiological Reactions to a Sudden Novel Object in the Weanling Horse: Quantitative Phenotypes for Future GWAS.
Genes    February 26, 2023   Volume 14, Issue 3 593 doi: 10.3390/genes14030593
Powell BB, Horvath KC, Gilliam TL, Sibille KT, Keil A, Miller-Cushon EK, Wickens CL, Brooks SA.The startle response can be defined as a reflexive reaction to the sudden appearance of a novel stimulus that influences the survival and resilience of animals. In domesticated species, the behavioral component of the startle response can, in some cases, cause serious injury to the animal or human handlers if inappropriately expressed. Here, we describe a longitudinal study in a population of stock-type horses that quantified behavioral startle responses elicited by the presentation of a sudden novel object (rapidly opening umbrella). The study was performed in weanling foals across four conse...
Inbreeding depression and durability in the North American Thoroughbred horse.
Animal genetics    February 26, 2023   Volume 54, Issue 3 408-411 doi: 10.1111/age.13309
Hill EW, McGivney BA, MacHugh DE.The proportion of the genome containing runs of homozygosity (ROH) affects production traits in livestock populations. In European and Australasian Thoroughbreds inbreeding, quantified using ROH (F ), is associated with the probability of ever racing. Here, we measured F using 333 K SNP genotypes from 768 Thoroughbred horses born in North America to evaluate the effect of inbreeding on racing traits in that region. Among North American horses, F was not associated (p = 0.518) with the probability of ever racing but was significantly associated with the number of race starts (p = 0.002). ...
The first genomic insight into Chlamydia psittaci sequence type (ST)24 from a healthy captive psittacine host in Australia demonstrates evolutionary proximity with strains from psittacine, human, and equine hosts.
Veterinary microbiology    February 23, 2023   Volume 280 109704 doi: 10.1016/j.vetmic.2023.109704
White RT, Jelocnik M, Klukowski N, Haque MH, Sarker S.Chlamydia psittaci is a zoonotic pathogen that infects birds, humans, and other mammals. Notably, recent studies suggested the human-to-human transmission of C. psittaci, and this pathogen also causes equine reproductive loss in Australia. Molecular studies in Australia to date have focused on and described clonal sequence type (ST)24 strains infecting horses, wild psittacine, and humans. In contrast, the genetic identity of C. psittaci strains from captive psittacine hosts is scarce. In 2022, C. psittaci was detected in the faeces of a healthy captive blue-fronted parrot (Amazona aestiva). Ge...