Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite.

BACKGROUND: As a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes. RESULTS: To increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups. CONCLUSIONS: In defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Defining cat mitogenome variation and accounting for numts via multiplex amplification and Nanopore sequencing.

Hair shed by domestic cats is a potentially useful source of forensic evidence. Analysable hair DNA is predominantly mitochondrial, but the recent domestication history of cats means that mtDNA diversity is low. A 402-bp control region segment is usually sequenced, defining only a small number of distinct haplotypes in populations. Previously, we used a long-amplicon approach to sequence whole mitogenomes in a sample of blood DNAs from 119 UK cats, greatly increasing observed diversity and reducing random match probabilities. To exploit this variation for forensic analysis, we here describe a multiplex system that amplifies the cat mitogenome in 60 overlapping amplicons of mean length 360 bp, followed by Nanopore sequencing. Variants detected in multiplex sequence data from unrooted hair completely mirror those from long-amplicon data from blood from the same individuals. However, applying the multiplex to matched blood DNA reveals additional sequence variants which derive from the major feline nuclear mitochondrial insertion sequence (numt), which covers 7.9 kb of the 17-kb mitogenome and exists in multiple tandem copies. We use long-amplicon Nanopore sequencing to investigate numt variation in a set of cats, together with an analysis of published genome sequences, and show that numt arrays are variable in both structure and sequence, thus providing a potential source of uncertainty when nuclear DNA predominates in a sample. Forensic application of the multiplex was demonstrated by matching hairs from a cat with skeletal remains from its putative mother, both of which shared a globally common haplotype at the control region. The random match probability in this case with the CR 402-bp segment was 0.21 and this decreased to 0.03 when considering the whole mitogenome. The developed multiplex and sequencing approach, when applied to cat hair where nuclear DNA is scarce, can provide a reliable and highly discriminating source of forensic genetic evidence from a single hair. The confounding effect of numt co-amplification in degraded samples where mixed sequences are observed can be mitigated by variant phasing, and by comparison with numt sequence diversity data, such as those presented here.

Challenges in the diagnosis and management of skin diseases in alpacas, goats, pigs, and sheep.

The primary aim of this article is to provide an overview of several selected skin conditions in livestock species. Topics include ectoparasites in alpacas, antler velvet in reindeer, immune-mediated disease in goats, ectoparasites in pigs, Culicoides allergic dermatitis and parapox infection in sheep. When dealing with skin disease in livestock, it is important to collect a detailed history and undertake a thorough clinical examination to include the axilla, groin, limbs and feet. While the diagnosis will often be anticipated from the history and presentation, it is important to consider a differential diagnosis list and appropriate diagnostic testing before embarking on a poly-pharmacy approach to "rule out" causes of disease. This is particularly important where morbidity is high and the livestock of perceived high value to the keeper/owner, such as goats and small-breed pigs, or when the skin condition is long standing/chronic. Ideally, the management plan should sequentially clarify the role of microbial infection and then ectoparasites before considering less common allergic and autoimmune conditions. Skin cytology is an invaluable in-house diagnostic method that can support the findings of culture. Taking skin samples for histopathology and possibly culture may prove valuable once other diagnostic methods have been explored. Given the need to protect the use of parenteral antimicrobials, topical antimicrobial therapies can be deployed successfully. The repeated use of macrocyclic lactones (avermectins) must be balanced in terms of the risks of promoting anthelmintic resistance versus controlling or eradicating the ectoparasites that have, ideally, been specifically identified.

A survey of enteric disease agents in UK deer populations.

Wild deer products have been linked to outbreaks of Escherichia coli 0157 in humans and bovine tuberculosis (bTB) in domestic cats, raising concerns around foodborne diseases from venison. This study investigated the prevalence of several enteric disease agents in UK deer populations, including foodborne pathogens and other diseases of import to livestock health. Intestinal samples were collected from slaughtered farmed deer (N = 211, eight farms), shot wild deer (N = 136) and ground faecal samples were collected from two farms (N = 90), six parks (N = 228) and five zoos (N = 67). DNA was extracted and multiplex qPCR assays were run to amplify targets of, C. perfringens toxins, Campylobacter spp., E. coli toxins, Mycobacterium avium subsp. Paratuberculosis (MAP), Salmonella spp. and Yersinia spp. The most commonly amplified targets were E. coli astA (61.2%) and Campylobacter spp. (43.3%), but the prevalence of C. coli and C. jejuni were ≤ 3.0% and Salmonella spp., MAP and Yersinia pseudotuberculosis also had low overall prevalences of 1.6%, 3.3% and 2.6%, respectively. However, notable targets included C. perfringens toxins α (20.4%) and ß2 (16.9%); E. coli stx1 (14.6% overall, 28.0% in abattoir samples) and stx2 (17.8% overall, 31.6% in wild samples); and Yersinia enterocolitica (10.8% overall, 25% in wild samples). The low prevalence of some foodborne pathogens is reassuring for food safety; further investigations into the commonly found targets are needed to quantify the risk to public health and the possibility of wild deer acting as reservoirs of disease.

A survey of handling and transportation of UK farmed deer.

Studies on the transport of deer (Cervidae), in the UK, were published > 15 years ago. A more recent study of deer transport is required to allow for assessments and improvements to the transport of farmed deer. Sixteen deer farmers participated in a survey describing their management practices related to transport. Their responses showed that most vehicles used to transport deer were designed for other livestock. Participating farmers estimated journey times to slaughter as 1-8 h, with an arithmetic mean of 4.8 (± 2.38) h. Specific concerns raised by the respondents, relating to the transport of deer, included a need for deer-specific vehicles, stop-off areas for long journeys, market locations and haulier experience. Furthermore, data were collected from two abattoirs between July 2019 and June 2020 comprising journey times, slaughter times, bruising, location of origin, vehicle type and the number of animals. In total, 4,922 deer were transported across 133 journeys (from farm to abattoir) from 61 farms. Median and range for journey length were 3.2 (0.4-9.8) h and 154.2 (7.1-462.2) km, whereas group size and time spent in the lairage were 24 (1-121) and 17.8 (10.2-68.9) h, respectively. Group size was found to be significantly associated with both the presence of bruising in a group and the amount of bruising per deer. This study provides a much-needed update on the transport of farmed deer in the UK and highlights key areas for future research including the welfare impact of transport in larger groups and for longer durations.

Otoscopy and aural cytological findings in a population of rescue cats and cases in a referral small animal hospital in England and Wales.

OBJECTIVES: Otitis externa is seen clinically in cats, although studies investigating this condition within the UK are lacking. The objective of this study was to investigate the prevalence of Otodectes cynotis mites and microbial infection in the ear canals of cats in various rescue centres and a referral hospital. METHODS: Otoscopy was performed in 332 cats. Otoscopic findings were noted, including the gross visualisation of Otodectes species. A sample of cerumen was collected for cytological evaluation and a cerumen smear for detection of Otodectes mites if there was a large amount of aural exudate present. RESULTS: O cynotis infestation was noted in 3/341 cats (0.9%, 95% confidence interval [CI] 0.3-2.6). A total of 129/341 (37.8%; 95% CI 32.7-43.0) cats were found to have Malassezia species within one or both ears. Bacteria were found unilaterally in 9/341 (2.6%; 95% CI 1.4-4.9) cats. Analysis of the cytological findings showed an increased likelihood for Malassezia species to be present as age increased (n = 293; Pearson r = 0.204, P <0.001). There was also an increased likelihood of finding Malassezia species in both ears if found within one ear (n = 327; r = 0.499, P <0.001). There was a positive correlation between the number of Malassezia organisms and the quantity of aural exudate (n = 338; r = 0.778, P <0.001). Cats in which Otodectes species infestation were noted (n = 3) had moderate or large quantities of cerumen. CONCLUSIONS AND RELEVANCE: This study shows that there was a low prevalence of O cynotis in this cohort of cats. In normal cats it was not unusual to find Malassezia microorganisms upon aural cytology, bacteria were noted far less frequently and in two cats this was associated with underlying anatomical pathology.

Staphylococcal skin disease in livestock.

UNLABELLED: This review covers cutaneous manifestations of staphylococcal infection in livestock species. CATTLE: In cattle, staphylococcal infections may present as folliculitis or as impetigo. Both may present as mild forms of a group of conditions loosely termed udder dermatitis, which has various clinical presentations and does not always involve staphylococci. GOATS: In goats, staphylococcal infection may be secondary to chorioptic mange or contagious pustular dermatitis (parapox virus infection). While Staphylococcus aureus is usually implicated, infection with Staphylococcus chromogenes and Staphylococcus hyicus have also been reported. SHEEP: Ovine staphylococcal dermatitis typically involves the head. Trauma due to the close contact of heads over feeding troughs and abrasive plants at pasture may be predisposing factors. PIGS: In pigs, the most common cause of staphylococcal skin disease is S. hyicus, although other bacteria, including Staphylococcus sciuri, Staphylococcus chromogenes and meticillin-resistant S. aureus (MRSA), have also been isolated from some cases of greasy pig disease (exudative epidermitis). DIAGNOSIS: Routine culture methods are increasingly supplemented by molecular methods to characterize staphylococci. MANAGEMENT: As commensal bacteria, staphylococcal skin infection is presumed to develop because of predisposing factors. While topical and systemic therapies can be effective, it is important to control for predisposing factors so that recurrences can be prevented. LIVESTOCK-ASSOCIATED MRSA: In recent years, MRSA strains have emerged, particularly in pigs and cattle. While they rarely cause skin disease, they do pose a significant concern for public health authorities. Studies on livestock-associated MRSA may help to progress our understanding of staphylococci in livestock, especially how they spread between animals and humans.

Interventions for atopic dermatitis in dogs: a systematic review of randomized controlled trials.

The objective of this systematic review, which was performed following the guidelines of the Cochrane collaboration, was to assess the effects of interventions for treatment of atopic dermatitis (AD) in dogs. Citations identified from three databases (MEDLINE, Thomson's Science Citation Index Expanded and CAB Abstracts) and trials published by December 2007 were selected. Proceedings books from the major veterinary dermatology international congresses were hand searched for relevant citations. The authors selected randomized controlled trials (RCTs), published from January 1980 to December 2007, which reported the efficacy of topical or systemic interventions for treatment or prevention of canine AD. Studies had to report assessments of either pruritus or skin lesions, or both. Studies were selected and data extracted by two reviewers, with discrepancies resolved by a third arbitrator. Missing data were requested from study authors of recently published trials. Pooling of results and meta-analyses were performed for studies reporting similar interventions and outcome measures. A total of 49 RCTs were selected, which had enrolled 2126 dogs. This review found some evidence of efficacy of topical tacrolimus (3 RCTs), topical triamcinolone (1), oral glucocorticoids (5), oral ciclosporin (6), subcutaneous recombinant gamma-interferon (1) and subcutaneous allergen-specific immunotherapy (3) to decrease pruritus and/or skin lesions of AD in dogs. One high-quality RCT showed that an oral essential fatty acid supplement could reduce prednisolone consumption by approximately half. Additional RCTs of high design quality must be performed to remedy previous flaws and to test interventions for prevention of flares of this disease.

Mycoplasma bovis and otitis in dairy calves in the United Kingdom.

Signs of severe otitis media in 20% of dairy calves on one farm were associated with Mycoplasma bovis infection, based on isolation from the external ear canal and nares. Affected calves seroconverted to M. bovis and no other significant bacteria were isolated. Infection was considered likely to have originated from cows in the milking herd based on evidence of seroconversion and detection of infection in a milk sample. M. bovis infection should be considered when investigating otitis problems in calves.

Yersinia pseudotuberculosis infection in a suckler calf in the United Kingdom: a case report.

Enterocolitis caused by Yersinia pseudotuberculosis was identified by bacterial culture and histopathology in a 5-month old suckler calf. No other significant enteric pathogens were detected. Diagnosis of this condition can be missed if selective media are not used for bacteriology and if histopathology is not attempted or the alimentary tract is unsuitable for examination. This is the first case report of Y. pseudotuberculosis infection in a calf in the United Kingdom.

Prevalence of house dust mites and dermatophagoides group 1 antigens collected from bedding, skin and hair coat of dogs in south-west England.

The house dust mites Dermatophagoides farinae (Df) and D. pteronyssinus (Dpt) are commonly implicated as allergens causing canine atopic dermatitis in the UK. However, there are few studies that characterize the exposure of UK pet dogs to these mites. The objectives of this study were to determine the prevalence of the mite species on the skin, hair coat and bedding of a population of pet dogs. Dust samples (n = 68) were collected from both dogs and their beds using a standardized vacuuming technique and stored at -20 degrees C. Mites were identified using accepted morphological criteria. House dust mite allergen concentrations were assayed using standardized ELISA for Dpt and Df group 1 allergens (Der p 1 and Der f 1). Mites were identified in 15/68 samples (22%) and Dpt was the most common. Df mites were not present. Der p 1 allergens were detected in 60% of samples, and Der f 1 in 6% of samples. There were no significant differences between the number of Der p 1 positive samples from dogs and the number of those from their bedding, or between the average Der p 1 concentrations from dogs and the number of those from their bedding. Contrary to studies elsewhere in Europe and the USA, these findings support studies of human asthma patients in the UK, where exposure to Df is rare, but to Dpt is common. As the prevalence of positive intradermal and serological reactions to Df in atopic dogs is high, further investigations are warranted to clarify true Df hypersensitivity or potential immunological cross-reactivity between mite allergens.

Immunomodulation and immunodeficiency.

This article briefly reviews the concepts of immunodeficiency and immunomodulation as they relate to selected skin diseases in the dog and cat. Immunodeficiency states are uncommon and may be associated with a subnormal or down-regulated immune system, including humoral deficiencies, such as IgA, and abnormal lymphocyte or neutrophil function. Establishing a causal relationship between a skin disease and presumed immunodeficient state has been difficult due to the rarity of such conditions, and the limited nature of the techniques used to characterise the immune system response. Severe combined immunodeficiency in dogs is a well characterised primary immunodeficiency state involving lymphocytes; retrovirus infection in cats may lead to an acquired immunodeficient state with some association with certain dermatological conditions although it remains unclear that infection is causally linked with disease. Immunomodulation usually implies stimulating the immune system along a beneficial pathway. Such a therapeutic approach may involve a wide variety of agents, for example intravenous immunoglobulin. There are few randomised controlled trials with veterinary patients that unequivocally demonstrate beneficial responses to immunomodulatory agents. Interferons are cytokines of major interest in human and veterinary medicine for their antiviral, anti-tumour and immunomodulatory effects. The advent of veterinary-licensed products containing recombinant interferon may enable demonstration of the efficacy of interferons for conditions such as canine papillomatosis and feline eosinophilic granuloma complex. Canine pyoderma has been treated with a number of presumed immunomodulatory agents with limited success. With more detailed knowledge of the pathogenesis of pyoderma it may be possible to develop efficacious immunomodulators.

Mitochondrial profiling of dog hairs.

In order to make greater use of dog hairs as forensic evidence, we have developed a robust method for duplex amplification of adjacent 306 and 332bp amplicons within the 5' hypervariable region (5' HVR) of the canine mitochondrial control region. In support of this, a 595bp region covering 35 polymorphic sites has been sequenced from the blood of 105 UK dogs. In total, 30 different haplotypes were observed, 13 only once whilst the commonest was seen 14 times; the overall exclusion capacity is 0.929. One animal was heteroplasmic in blood for a single base deletion and showed phenotypes ranging from near complete deletion to a predominance of the base among a sample of 12 hairs. In contrast, no evidence of heteroplasmy was seen in single hairs from 20 dogs which were not visibly heteroplasmic in blood. Phylogenetic analysis and comparisons with other published databases highlighted instances of possible recurrent mutation which may be relevant when interpreting single base differences between samples.