Large-scale spatial and temporal genetic diversity of feline calicivirus.

Feline calicivirus (FCV) is an important pathogen of domestic cats and a frequently used model of human caliciviruses. Here we use an epidemiologically rigorous sampling framework to describe for the first time the phylodynamics of a calicivirus at regional and national scales. A large number of FCV strains cocirculated in the United Kingdom at the national and community levels, with no strain comprising more than 5% and 14% of these populations, respectively. The majority of strains exhibited a relatively restricted geographical range, with only two strains (one field virus and one vaccine virus) spreading further than 100 km. None of the field strains were identified outside the United Kingdom. Temporally, while some strains persisted locally for the majority of the study, others may have become locally extinct. Evolutionary analysis revealed a radial phylogeny with little bootstrap support for nodes above the strain level. In most cases, spatially and temporally diverse strains intermingled in the phylogeny. Together, these data suggest that current FCV evolution is not associated with selective competition among strains. Rather, the genetic and antigenic landscape in each geographical location is highly complex, with many strains cocirculating. These variants likely exist at the community level by a combination of de novo evolution and occasional gene flow from the wider national population. This complexity provides a benchmark, for the first time, against which vaccine cross-protection at both local and national levels can be judged.

Pet ownership, dog types and attachment to pets in 9-10 year old children in Liverpool, UK.

BACKGROUND: Little is known about ethnic, cultural and socioeconomic differences in childhood ownership and attitudes to pets. The objective of this study was to describe the factors associated with living with different pet types, as well as factors that may influence the intensity of relationship or 'attachment' that children have to their pet. Data were collected using a survey of 1021 9-10 year old primary school children in a deprived area of the city of Liverpool, UK. RESULTS: Dogs were the most common pet owned, most common 'favourite' pet, and species most attached to. Twenty-seven percent of dog-owning children (10% of all children surveyed) reported living with a 'Bull Breed' dog (which includes Pit Bulls and Staffordshire Bull Terriers), and the most popular dog breed owned was the Staffordshire Bull Terrier. Multivariable regression modelling identified a number of variables associated with ownership of different pets and the strength of attachment to the child's favourite pet. Girls were more likely to own most pet types, but were no more or less attached to their favourite pet than boys. Children of white ethnicity were more likely to own dogs, rodents and 'other' pets but were no more or less attached to their pets than children of non-white ethnicity. Single and youngest children were no more or less likely to own pets than those with younger brothers and sisters, but they showed greater attachment to their pets. Children that owned dogs lived in more deprived areas than those without dogs, and deprivation increased with number of dogs owned. 'Pit Bull or cross' and 'Bull Breed' dogs were more likely to be found in more deprived areas than other dog types. Non-whites were also more likely to report owning a 'Pit Bull or cross' than Whites. CONCLUSIONS: Gender, ethnicity and socioeconomic status were associated with pet ownership, and sibling status with level of attachment to the pet. These are important to consider when conducting research into the health benefits and risks of the common childhood phenomenon of growing up with pets.

A cross-sectional study of frequency and factors associated with dog walking in 9-10 year old children in Liverpool, UK.

BACKGROUND: Owning a pet dog could potentially improve child health through encouraging participation in physical activity, through dog walking. However, evidence to support this is limited and conflicting. In particular, little is known about children's participation in dog walking and factors that may be associated with this. The objective of this study was to describe the participation of children in dog walking, including their own and those belonging to somebody else, and investigate factors associated with regular walking with their own pet dog. METHODS: Primary school children (n=1021, 9-10 years) from a deprived area of Liverpool were surveyed during a 'fitness fun day' as part of the SportsLinx project. The 'Child Lifestyle and Pets' survey included questions about pet ownership, pet attachment, and dog walking. Multivariable logistic regression models were used to investigate factors associated with walking any dog, or their own dog, several times a day or more, including level of attachment to the dog, dog type, and sociodemographic factors. RESULTS: Overall, 15.4% of children reported walking with any dog (their own or belonging to a friend or family member) ≥ once daily, 14.1% several times a week, 27.6% ≤ once a week, and 42.8% never. Dog owning children (37.1% of the population) more often reported dog walking 'several times a week or more' (OR=12.30, 95% CI=8.10-18.69, P<0.001) compared to those without a dog, but were less likely to report other walking without a dog. The majority (59.3%) of dog owning children indicated that they usually walked their dog, with 34.6% reporting that they walked their dog ≥ once daily. Attachment score was highly associated with the child reporting walking their dog (lower score=higher attachment; OR=0.93, 95% CI=0.89-0.96, P<0.001). There was no evidence that gender, ethnicity, sibling status or deprivation score was associated with dog walking. Children that reported owning Pit Bulls were more likely to report friends walking with their dog than those owning non-Pit bull types (OR=10.01, 95% CI=1.52-65.76, P=0.02, respectively). CONCLUSIONS: Promotion of supervised walking of suitable pet dogs may be an opportunity for increasing physical activity in 9-10 year old children. The identification of stronger attachment to dogs regularly walked is similar to findings in adult studies.

Multilocus sequence typing of Bartonella henselae in the United Kingdom indicates that only a few, uncommon sequence types are associated with zoonotic disease.

Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections.

Detection of squirrel poxvirus by nested and real-time PCR from red (Sciurus vulgaris) and grey (Sciurus carolinensis) squirrels.

BACKGROUND: Squirrel poxvirus (SQPV) is highly pathogenic to red squirrels (Sciurus vulgaris), and is a significant contributing factor to the local extinction of the species in most parts of England and Wales, where infection is endemic in Eastern grey squirrel (Sciurus carolinensis) populations. Although a nested PCR assay has been used successfully to study the epidemiology of SQPV, samples have a long processing time and the assay is not quantifiable. RESULTS: This project describes the design and optimization of a real-time PCR for SQPV. Comparison with the nested PCR showed the real-time assay to be more sensitive by one log and able to detect approximately 144 genome copies per mg of tissue. CONCLUSIONS: The real-time PCR has been used to quantify viral genome load in tissues from diseased and apparently healthy red and grey squirrels, and suggests that the titre of virus in tissues from diseased red squirrels is considerably higher than that found even in a grey squirrel with cutaneous lesions.

Comparison of the ability of feline calicivirus (FCV) vaccines to neutralise a panel of current UK FCV isolates.

Feline calicivirus (FCV) comprises a large number of strains which are related antigenically to varying degrees. The antigenic variability creates problems for choosing antigens to include in vaccines. Historically, these have been selected for use based on their cross-reactivity with a high proportion of field strains. However, it is important to determine the current level of cross-reactivity of vaccines and whether or not this may be decreasing owing to widespread vaccine use. In this in vitro study, we have compared the ability of antisera to two vaccine viruses (FCV strain F9 and FCV strain 255) to neutralise a panel of 40 recent UK field isolates. These 40 isolates were obtained by randomised, cross-sectional sampling of veterinary practices in different geographical regions of the UK so as to ensure they were representative of viruses circulating in the veterinary-visiting population of cats in the UK. Virus neutralisation assays showed that both vaccine strains are still broadly cross-reactive, with F9 antiserum neutralising 87.5% and 255 antiserum 75% of isolates tested with antiserum dilutions of 1 in 2 or greater. However, when antibody units were used, in order to take account of differences in homologous titres between antisera, fewer isolates were neutralised, with F9 antiserum showing a slightly higher proportion of isolates neutralised than 255. Multivariable analysis of the sample population of 1206 cats from which the 40 isolates were derived found that vaccinated cats were at a decreased risk of being positive for FCV, whereas cats from households with more than one cat, and cats with mouth ulcers were at increased risk. In addition as cats became older their risk of shedding FCV decreased.

Carriage of antimicrobial resistant Escherichia coli in dogs: Prevalence, associated risk factors and molecular characteristics.

Resistance to antimicrobials, in particular that mediated by extended spectrum ß-lactamases (ESBL) and AmpC ß-lactamases are frequently reported in bacteria causing canine disease as well as in commensal bacteria, which could be a potential health risk for humans they come into contact with. This cross-sectional study aimed to estimate the prevalence and investigate the molecular characteristics of ESBL and plasmid encoded AmpC (pAmpC)-producing E. coli in the mainland UK vet-visiting canine population and, using responses from detailed questionnaires identify factors associated with their carriage. Faecal samples were cultured for antimicrobial resistant (AMR), ESBL and pAmpC-producing E. coli. A subset of ESBL and pAmpC-producing isolates were subjected to multi-locus sequence typing and DNA microarray analyses. Multivariable logistic regression analysis was used to construct models to identify risk factors associated with multidrug resistant (MDR, resistance to three or more antimicrobial classes), fluoroquinolone resistant, ESBL and AmpC-producing E. coli. AMR E.coli were isolated from 44.8% (n=260) of samples, with 1.9% and 7.1% of samples carrying ESBL and pAmpC-producing E. coli, respectively. MDR E. coli were identified in 18.3% of samples. Recent use of antimicrobials and being fed raw poultry were both identified as risk factors in the outcomes investigated. A number of virulence and resistance genes were identified, including genes associated with extra-intestinal and enteropathogenic E. coli genotypes. Considering the close contact that people have with dogs, the high levels of AMR E. coli in canine faeces may be a potential reservoir of AMR bacteria or resistance determinants.

The challenge for the next generation of feline calicivirus vaccines.

Feline calicivirus (FCV) has been shown to evolve within individual cats and in the environment of colonies. This evolution and the diversity it creates has important clinical implications, not only for the disease in cats, but also for attempts to control disease by vaccination. Generally speaking, existing vaccines appear to be very effective at controlling the majority of clinical disease. However, some concerns remain including a failure to induce sterilising immunity, occasional vaccine breakdowns, and for live vaccines, occasional vaccine-induced disease. Key areas for future vaccine development include monitoring and broadening the cross-reactivity of vaccine immunity to field viruses, especially the recently evolved highly virulent strains, and attempting to reduce/eliminate field virus shedding by vaccinated cats.

Long-term analysis of feline calicivirus prevalence and viral shedding patterns in naturally infected colonies of domestic cats.

Feline calicivirus (FCV) is a highly infectious respiratory pathogen of domestic cats. The prevalence of FCV in the general cat population is high, particularly in multi-cat households, largely because many clinically recovered cats remain persistently infected carriers. In order to assess how FCV circulates in such groups and to assess the contribution that each individual animal makes to the epidemiology of the disease, we have carried out the first detailed analysis of long-term shedding patterns of FCV in individual cats within naturally infected colonies. The prevalence of FCV in each of the groups on individual sampling occasions ranged from 0% to 91%, with averages for the individual colonies ranging from 6% to 75%. Within each of the colonies, one to three distinct strains of FCV were identified. Individual cats showed a spectrum of FCV shedding patterns over the sampling period which broadly grouped into three categories: those that shed virus relatively consistently, those that shed virus intermittently, and those that appeared never to shed virus. This is the first report identifying non-shedder cats that appear resistant to FCV infection over long periods of time, despite being continually exposed to virus. Such resistance appeared to be age related, which may have been immune-mediated, although by analogy with other caliciviruses, factors such as host genetic resistance may play a role. Given that a proportion of the population appears to be resistant to infection, clearly the cohort of cats that consistently shed virus are likely to provide an important mechanism whereby infection can be maintained in small populations.

Carriage of Staphylococcus species in the veterinary visiting dog population in mainland UK: molecular characterisation of resistance and virulence.

This study investigated the prevalence of nasal carriage of staphylococci in dogs and determined the characteristics of the isolates. A total of 724 dogs from 87 veterinary practices across the mainland UK were screened for carriage of Staphylococcus spp. All isolates were examined for meticillin resistance (MR) and the presence of the mecA gene investigated in those isolates showing resistance. All coagulase-positive staphylococci and MR coagulase-negative staphylococci (MRCoNS) were subjected to antimicrobial susceptibility testing. Spa typing and DNA microarray analysis of resistance and virulence genes was carried out on all MR S. aureus (MRSA) and a subset of meticillin susceptible S. aureus (MSSA). Staphylococci were isolated from 399 (55.1%) of the dogs; only seven (1%) carried MRSA, all of which were identified as the dominant UK healthcare-associated strain (EMRSA-15, ST22). MSSA was identified in 47 (6.5%) dogs, the sequence types of which have been suggested as precursors to successful MRSA clones. Forty (5.5%) dogs carried MRCoNS, while no dogs carried MR S. pseudintermedius, although this is increasingly reported in mainland Europe. Resistance to antimicrobials among the isolates varied between species, with multidrug resistance (MDR) in 87.5% of MRCoNS and 21.8% of coagulase positive staphylococci. Microarray analysis of MRSA and a subset of MSSA isolates identified numerous virulence genes associated with pathogenesis, which are commonly identified in isolates of human origin. However, no isolates carried Panton-Valentine leukocidin (PVL) genes. This study suggests that MRSA carriage is low in the vet visiting dog population, but there is a diverse range of virulence and resistance determinants in canine S. aureus and MRCoNS isolates.

Family pet ownership during childhood: findings from a UK birth cohort and implications for public health research.

In developed nations, approximately half of household environments contain pets. Studies of Human-Animal Interaction (HAI) have proposed that there are health benefits and risks associated with pet ownership. However, accurately demonstrating and understanding these relationships first requires a better knowledge of factors associated with ownership of different pet types. A UK birth cohort, the Avon Longitudinal Study of Parents and Children (ALSPAC), were used to collect pet ownership data from the mothers, from gestation to child age 10 years old. 14,663 children were included in the study, of which mothers of 13,557 reported pet information at gestation, and 7,800 by age 10. Pet types recorded include cat, dog, rabbit, rodent, bird, fish and tortoise/turtle. The dataset also contains a number of demographic, socioeconomic and behavioural variables relevant to human health behaviour. Logistic regression was used to build multivariable models for ownership of each pet type at age 7 years. Family pet ownership increased during childhood, in particular rabbits, rodents and fish. A number of socioeconomic and demographic factors were associated with ownership of different pet types and the effects differed depending on the pet type studied. Variables which require consideration by researchers include gender, presence of older siblings, ethnicity, maternal and paternal education, maternal and paternal social class, maternal age, number of people in the household, house type, and concurrent ownership of other pets. Whether the mother had pets during her childhood was a strong predictor of pet ownership in all models. In HAI studies, care should be taken to control for confounding factors, and to treat each pet type individually. ALSPAC and other similar birth cohorts can be considered a potential resource for research into the effects of pet ownership during childhood.

Feline calicivirus.

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats. It belongs to the family Caliciviridae which includes other significant pathogens of man and animals. As an RNA virus, high polymerase error rates convey upon FCV a high genome plasticity, and allow the virus to respond rapidly to environmental selection pressures. This makes the virus very adaptable and has important implications for clinical disease and its control. Being genetically diverse, FCV is associated with a range of clinical syndromes from inapparent infections to relatively mild oral and upper respiratory tract disease with or without acute lameness. More recently, highly virulent forms of the virus have emerged associated with a systemic infection that is frequently fatal. A proportion of FCV infected cats that recover from acute disease, remain persistently infected. In such cats, virus evolution is believed to help the virus to evade the host immune response. Such long-term carriers may only represent a minority of the feline population but are likely to be crucial to the epidemiology of the virus. Vaccination against FCV has been available for many years and has effectively reduced the incidence of clinical disease. However, the vaccines do not prevent infection and vaccinated cats can still become persistently infected. In addition, FCV strain variability means that not all strains are protected against equally. Much progress has been made in understanding the biology and pathogenesis of this important feline virus. Challenges for the future will necessarily focus on how to control the variability of this virus particularly in relation to emerging virulent strains and vaccination.

Evolutionary mechanisms of persistence and diversification of a calicivirus within endemically infected natural host populations.

In order to understand the evolutionary mechanisms of persistence and diversification within the Caliciviridae, we have been exploiting endemic infection of feline calicivirus within five geographically distinct household groups of cats. By sequencing immunodominant and variable regions of the capsid gene, we identified the relative contribution of the different evolutionary processes employed by the virus to ensure its long-term survival in the host population. Such strategies included progressive evolution of a given variant of a strain through mutation accumulation within an individual, sequential reinfection with either a variant of the same strain or with a different strain, and mixed infection. Recombination between different strains in this study has been reported in detail elsewhere (K. P. Coyne et al., J. Gen. Virol. 87:921-926, 2006). Here, we provide evidence to suggest that true long-term persistent infection in individuals is relatively rare, with the majority of apparent viral carriers undergoing a combination of progressive evolution and cyclical reinfection. Progressive evolution at the individual level and variant reinfection at both the individual and population levels were associated with positive selection. Two measures of evolution rate were determined; for a virus progressively evolving within an individual (1.32 x 10(-2) to 2.64 x 10(-2) substitutions per nucleotide per year, i.e., no transmission) and for a strain circulating within a population (3.84 x 10(-2) to 4.56 x 10(-2) substitutions per nucleotide per year, i.e., including transmission). Reiteration of both progressive evolution and variant reinfection appeared to lead to a gradual increase in the diversity of a given strain of virus, both in the individual and in the population, until eventually new strains emerged.

Longitudinal molecular epidemiological analysis of feline calicivirus infection in an animal shelter: a model for investigating calicivirus transmission within high-density, high-turnover populations.

The control of outbreaks of calicivirus infection in high-density, high-throughput populations is a challenge to both human and veterinary medicine. In such populations, the prevalence of infection is, in part, dependent on the levels of biosecurity and how this affects virus transmission. Here we show how longitudinal analysis of feline calicivirus (FCV) infection in an animal rescue shelter can be used as a model to examine the dynamics of calicivirus transmission and evolution in such environments. FCV was isolated from 33 of 116 cats sampled over a 15-month period (overall prevalence, 28%). Sequence analysis of the immunodominant variable regions of the viral capsid gene identified 16 strains circulating in the shelter, with no single strain appearing to predominate. The majority of these strains were introduced into the shelter from the community and did not appear to be transmitted within the population. However, for three of these strains, putative transmission events within the shelter were identified. The rates of evolution within hypervariable regions of the FCV capsid gene in individual cats ranged from 0.05 to 1.4% per week, with the highest rates generally being found in animals that either acquired the virus while in the shelter or were undergoing acute infection. These data suggest that despite the high prevalence and presence of multiple strains of FCV within the shelter, the spread of such pathogens may be restricted by various control measures, including good hygiene and biosecurity.