Molecular epidemiology of rotavirus in UK cats German ACa,b#, Iturriza-Gómara Ma, Dove Wa ...

41 Pages·2014·1.22 MB·English

JCM Accepts, published online ahead of print on 19 November 2014
J. Clin. Microbiol. doi:10.1128/JCM.02266-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 Molecular epidemiology of rotavirus in UK cats
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3 German ACa,b#, Iturriza-Gómara Ma, Dove Wa, Sandrasegaram Ma, Nakagomi Tc,
4 Nakagomi Oc, Cunliffe Na, Radford ADa,b, Morgan KLb,d
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6 Institute of Infection and Global Health, University of Liverpool, Liverpool, UKa de
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7 School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston, Wirral, m

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9 University of Nagasaki, Nagasaki, Japanc m
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10 Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UKd
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12 Running head: Rotavirus in UK cats n
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14 #Address correspondence to Allison C German, [email protected] 1
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16 Key words: zoonosis, anthropozoonosis, interspecies transmission, shelter medicine, t
17 population, feline, epidemiology, G6P[9], G3P[9], RT-PCR
18
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19 ABSTRACT
20 Rotaviruses are leading causes of gastroenteritis in the young of many species.
21 Molecular epidemiological studies in children suggest that interspecies transmission
22 contributes to rotavirus strain diversity in people. However, population-based studies of
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23 rotaviruses in animals are few. We investigated the prevalence, risk factors for infection o
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24 and genetic diversity of Rotavirus A in a cross-sectional survey of cats housed within lo
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25 twenty-five rescue catteries across the UK. Morning litter tray faecal samples were e
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26 collected during winter and summer 2012 from all pens containing kittens and a random o
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27 sample of those housing adult cats. Group A rotavirus RNA was detected by real-time t
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28 reverse transcription polymerase chain reaction and positive samples were G and P /jc
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29 genotyped using nested VP4 and VP7 PCR assays. A total of 1727 faecal samples .a
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30 were collected from 1105 pens. Overall, rotavirus prevalence was 3.0% (95% CI 1.2- .o
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31 4.9). 52% (13/25; CI 31.3-72.2) centres housed at least one rotavirus-positive cat. /
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32 Prevalence was associated with season, odds ratio 14.8 (CI 1.1-200.4), p=0.04, but not J
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33 age or diarrhoea. It was higher during the summer (4.7% CI 1.2-8.3) than winter (0.8%, a
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34 CI 0.2-1.5). Asymptomatic epidemics of infection were detected in two centres. G 1
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35 genotypes were characterised for 19 (33.3%) of the 57 rotavirus positive samples and P 0
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36 genotypes for 36 (59.7%). Two rotavirus genotypes were identified: G3P[9] and G6P[9]. b
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37 This is the first population-based study of rotavirus in cats and the first report of feline u
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38 G6P[9], which questions the previous belief that G6P[9] in people was of bovine origin. t
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40 INTRODUCTION
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41 Rotavirus A (RVA), a species in the Rotavirus genus, of the Reoviridae, is an important
42 pathogen of acute diarrhea in the young of many animal species including people (1, 2).
43 With the advent of modern sequencing techniques, the extent of the contribution of
44 interspecies transmission and reassortment to rotavirus genetic diversity is increasingly
45 being realised (3-5). Despite the many reports of potential zoonotic infections in people D
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46 (6, 7), due to the scarcity of rotavirus surveillance programmes in animals, little is known n
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47 about the prevalence of potential anthroponotic and zoonotic strains in animals. d
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48 Particularly, our knowledge and understanding of rotaviruses circulating in companion fr
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49 animal populations is minimal, which is remiss when we consider the extent of contact h
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50 that occurs between pets (especially cats and dogs) and children in developed countries. :/
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51
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52 Although infections with feline rotaviruses (FRVs) rarely cause severe illness in cats (8- m
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53 11), FRVs have captured attention as perpetuating, albeit infrequent, sources of human g
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54 disease. Human RVAs with genetic homology to feline RVAs have been isolated from
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55 widespread geographical locations including Japan (12, 13), Israel (14, 15), Tunisia (16) u
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56 and America (17). Additionally, putative human/feline reassortant rotaviruses have been y
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57 identified in children in Italy (18). 2
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58 9

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59 Despite the reports of FRV isolations from people, there have been no recent studies or g
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60 surveillance of rotaviruses in cats. FRV infection was first identified by serology in cats s
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61 in 1978 (19). Experimental infections have given inconsistent results, with some
62 showing association between rotavirus and reduced faecal quality in kittens (8), whilst
63 others failed to link infection with disease (9, 10, 20). Currently, rotavirus is considered
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64 to play a minor role in clinical disease and is not routinely screened for in diarrhoeic
65 cases in small animal veterinary practice (21).
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67 The prevalence of rotavirus infection in cats has been investigated by serum antibody
68 titres and faecal electron microscopy; seroprevalence studies indicated exposure D
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69 ranged from 3.5% -100% (8-11, 22), whilst electron microscopy indicated 3-6% of cats n
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70 were infected (9, 10, 20). These studies involved small numbers of cats and looked at d
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71 convenience samples of sub-populations (e.g. veterinary hospital admissions, single fr
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72 premises and research colonies) rather than a representative sample of the national h
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73 feline population. :/
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74
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75 The aim of this study was to examine, by a systematic, population-based approach m
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76 irrespective of diarrhoea status, the prevalence and genotypes of RVAs circulating in g
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77 domestic cats in the UK.
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79 MATERIALS AND METHODS y
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80 STUDY POPULATION 2
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81 The study population comprised cats held in the twenty-five UK rehoming or adoption 9

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82 centres run by Cats Protection, the UK’s largest feline welfare charity. Each year it g
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83 rehomes or reunites with owners approximately 50,000 cats. These centres are widely s
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84 distributed geographically with twenty-one in England, two in Wales, one in Northern
85 Ireland and one in Scotland (Figure 1a). The centres vary in size and construction. The
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86 number of cat accommodation spaces (pens) are fixed and range from 16 in a specially
87 adapted city house, to 202 in the purpose built National Cat Adoption Centre (NCAC).
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89 The population of cats in the centres is in constant flux. The relinquishment and
90 adoption rates are such that centres operate at capacity throughout the year, although D
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91 stocking density (cats per pen) will vary. The demographic pattern in the centres n
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92 changes with the seasonal breeding pattern of cats, such that a higher proportion of the d
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93 population are kittens during the summer months (June-Aug) (Figure 2). Additionally, fr
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94 the centres will not put cats originating from different sources into the same pen, so h
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95 relinquishment events (single cats or multicat households) will also influence the total :/
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96 population size and stocking density in each centre.
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97 m
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98 STUDY DESIGN g
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99 Two cross sectional studies were undertaken to account for the seasonal changes in
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100 demography. The first was in the UK winter months (3rd Feb – 30th Mar 2012), the nu
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101 second was in the summer months (29th May – 17th Aug 2012). The centres were y
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102 stratified by size (small, medium and large) and randomly allocated to summer and 2
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103 winter collection periods (Figure 1a). 9

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104 g
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105 The unit of sampling was the pen. These were selected from pen occupancy data s
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106 obtained the day prior to sampling. All pens containing at least one kitten were selected.
107 In addition, a random sample of those housing one or more adult cats was also
108 selected; the sample size was chosen to allow 95% confidence of detecting one positive
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109 pen if the prevalence of faecal shedding was 2%, assuming a test with 100% specificity
110 and 95% sensitivity. This also allowed a prevalence of 2% to be estimated with 95%
111 confidence and 1.3-1.9% precision.
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113 Recording sheets were used to transcribe demographic data from a number of sources D
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114 including centre admission records, pen data recording sheets, veterinary paper n
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115 records, Cats Protection internal database (“PAWS”) and from observation of pen d
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116 content and construction, unit structure, hygiene precautions and centre management. fr
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118 SAMPLE COLLECTION AND PROCESSING :/
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119 Faecal samples were collected from litter trays and where necessary the pen floor
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120 between 6.30am and 12.00pm (noon) on the first day of the study visit; where faeces m
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121 were not present, cats were monitored through the rest of the working day and a g
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122 collection made if faeces were passed. In large centres, where the number of cats
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123 necessitated a longer visit for data transcription, cats who only defecated once every u
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124 two to three days were observed and samples were collected from these individuals y
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125 when faeces were eventually passed. 2
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127 Faecal samples were collected in sterile 30 ml universal tubes using either individual g
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128 disposable gloves or wooden applicator sticks. The colour, consistency and number of s
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129 complete deposits of faeces in and outside the litter tray were recorded. Colour was
130 recorded as brown, green, yellow, black or other (described). Consistency was graded 1
131 (watery) - 6 (hard, dry) using a modified version of the Bristol Stool Scale (Meyers
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132 Scale) (23). Where the number of faecal deposits was equal to the number of cats it
133 was assumed that each was from a different cat (as an adult cat is highly unlikely to
134 defecate twice in the morning unless it has large intestinal diarrhoea or diffuse
135 gastrointestinal disease resulting in high volume faeces); the deposits were then
136 randomly assigned to each cat. Occasionally cats were observed defecating, or dual D
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137 occupancy pens contained cats who reliably produced differently graded faeces; these n
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138 deposits were specifically assigned to an individual. In maternity pens, adult stools were d
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139 easily differentiated from kitten stools by their size. In single occupancy pens, where fr
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140 there was more than one deposit, a sample was taken from each and the sample h
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141 recorded as “pooled from an individual”. In multiple occupancy pens, where the number :/
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142 of samples exceeded the number of cats, the sample was recorded as “pooled sample
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143 from more than one cat”. Faeces were also examined for the presence or absence of m
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144 worms, mucus and blood. Samples were transferred to +40C or -200C when all pens in rg
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145 a particular building had been sampled. They were transported to the laboratory at this
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146 temperature and either aliquoted into two 1.5ml cryovials the following day or kept at - u
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147 800C until aliquoted. Samples were kept at -800C until tested. y
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148 2
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149 DETECTION AND TYPING OF ROTAVIRUS 9

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150 10% faecal suspensions were prepared, clarified by centrifugation at 12000 rpm for 10 g
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151 minutes and 250 μl used for RNA extraction (QIAamp RNA Kit, Qiagen, Manchester s
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152 UK), according to manufacturer’s instructions, and eluted into 50 μl RNase-free water. A
153 20 μl aliquot was used for reverse transcription using random primers and Superscipt II
154 Reverse Transcriptase (Invitrogen, Paisley, UK), following manufacturer’s instructions,
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155 giving a final cDNA volume of 35 μl. Rotavirus NSP3-specific qPCR (24) was performed
156 and samples with a CT value <40 were considered positive. The qPCR mix per reaction
157 was 12.5µl Invitrogen Platinum Mastermix; 8 pmol NSP3-F/NSP3-R primers and 3 fmol
158 NSP-3 probe with 2µl cDNA, made up to 25 µl with RNase free water. The assays were
159 run on a RotorGene Q 6000 thermal cycler at 50°C for 2 minutes, 95°C for 2 minutes D
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160 and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. n
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161 d
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162 LIMIT OF DETECTION fr
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163 The limit of detection of the NSP3 qPCR assay was investigated using a simian h
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164 rotavirus positive control SA11 (25). This was diluted in a two-fold series from 90 :/
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165 infectious virus particles (IVP)/reaction to 3 IVP/reaction, and repeatability assessed
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166 with five replicates of a ten-fold dilution series from 1x105 IVP/reaction to 1x101 m
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167 IVP/reaction and with two replicates of a two-fold dilution series from 1000 to 16 g
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168 IVP/reaction. The assay was validated further for the limit of detection of feline RVs
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169 FRV-1 (26) and FRV-64 (27). u
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170 1
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171 GENOTYPING 0
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172 Rotavirus-positive samples were further characterised using hemi-nested VP7 (G-type) b
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173 and VP4 (P-type) PCR assays, which included primers specific for G1, G2, G3, G4, G8, u
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174 G9, G10 and G12, and P[4], P[6], P[8], P[9], P[10] and P[11] (28, 29). To increase the t
175 G-typing sensitivity, a fragment of the VP7 gene segment from culture-adapted FRV
176 strains from this study was sequenced, using the consensus VP7 primers from the G-
177 typing first amplification round. The sequence data generated was used to characterise
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178 the G-types of the strains that could be propagated in cell culture, and also to design
179 feline G6- and G3-specific primers. These primers were subsequently used in single
180 typing assays to test all rotavirus-positive samples . A total of 20 pmol of primers G6F
181 AACGAGGATGATGGACTACA (nt 126-145) and G3R TARATAGATCCTGTTGGCC
182 (nt 347-329) were used in separate hemi-nested reactions, with VP7R or VP7F first D
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183 round consensus primers respectively, using Top Taq Master Mix (Qiagen, UK) and an n
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184 annealing temperature of 50oC. d
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185 fr
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186 STATISTICAL ANALYSIS h
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187 Prevalence was estimated at cat and centre levels for the combined and separate :/
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188 collection periods. The overall prevalence at cat level was estimated using the svy
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189 commands in STATA to adjust for stratification by season and clustering by centre and m
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190 pen (StataCorp 2009. Stata Statistical Software: Release 11. College Station TX: g
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191 StataCorp LP). The sampling weights were adjusted for the different sampling strategies
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192 (i.e. for pens with kittens and those with adult cats) and for those cats that did not u
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193 defaecate on the day of collection. Prevalence at centre level was the proportion of y
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194 centres with at least one positive cat, with exact binomial confidence intervals. Fisher’s 2
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195 exact tests were used to compare the centre prevalence in summer and winter. 9

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196 Hierarchical univariable and multivariable logistic regression was used to examine g
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197 associations between infection and age, season and diarrhoea using the melogit s
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198 commands in STATA. A three-level model was used, incorporating centre, pen and
199 individual. Age was modelled as a continuous variable and also as a binary variable.
200 Three categories were used for kittens: cats aged 5 months or less, 3 months or less, or
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201 2 months or less. Faecal score was also reduced to a binary variable with a cut off at
202 both scores 2 and 3. Cats with a score equal to or below 2 or 3 were considered to have
203 diarrhoea.
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205 Geographical distribution maps were constructed using QGIS 2.0.1 (Dufour) D
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207 ETHICAL APPROVAL d
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208 This study was approved by the University of Liverpool Veterinary Research Ethics fr
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209 Committee (VREC20) and the Cats Protection Ethical Review Committee. h
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210 :/
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211 RESULTS
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212 POPULATION STRUCTURE m
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213 A total of 1727 faecal samples were collected from 1105 pens across the twenty-five g
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214 centres. The median number of occupied pens per centre was 41 (interquartile range,
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215 IQR 30-79). The number of samples from each centre varied from 8 in the North London u
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216 Centre to 224 in Bridgend. The age of cats sampled ranged from one week to 21.5 y
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217 years of age. Overall, a third (30.9% 683/2213; CI 29.0-32.8%) of the Cats Protection 2
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218 Adoption Centre population was kittens (<6 months). The proportion of kittens was 9

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219 significantly greater during the summer collection period (Figure 2), 44.6% compared g
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220 with 13.9% in the winter period (P<0.0005). This was also reflected in the sample, s
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221 where the proportion of kittens was 33.6% overall (581/1727; CI31.4-35.9); 48.2% in
222 summer and 11.6% in winter. The median age of cats sampled in summer was 9.0
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