Sage Journals: Discover world-class research

Abstract

Faecal samples were collected from 57 clinically healthy kittens presented for initial vaccination, in the UK. Routine bacteriological examination identified Salmonella species in one and Campylobacter species in five samples. Polymerase chain reaction (PCR) detected the presence of Campylobacter species in a further four samples. Routine parasitological examination revealed Toxocara species ova in nine (including four kittens stated to have been administered an anthelmintic) and Isospora species in four samples. No Giardia or Cryptosporidium species were detected by routine methods. A Giardia species enzyme-linked immunosorbent assay (ELISA) test kit designed for use in cats was positive in three kittens. A similar test kit designed for use in humans was negative in all samples and produced negative results even when known positive samples were tested. Potentially pathogenic enteric organisms were detected in 19 kittens by routine methods and 26 (prevalence 45%) by all methods. The high prevalence in asymptomatic kittens highlights the possibility that the detection of these organisms in kittens with gastrointestinal disease may be an incidental finding.

Campylobacter species, Salmonella species protozoa, and helminths are frequently implicated as the cause of gastrointestinal disease when they are isolated from cats exhibiting clinical signs. The true significance of these organisms in causation of clinical signs is unclear as the prevalence of these organisms in asymptomatic animals in the UK has not been investigated. Spain et al,¹ who investigated potentially zoonotic enteric organisms in cats <1 year of age in New York, concluded that infection did not correlate with the presence of gastrointestinal signs. Thus it is possible that detection of these organisms in symptomatic cats may be an incidental finding.

Certain of these enteric organisms also has the potential to cause disease in humans and their presence in asymptomatic cats may be a source of infection for their owners. Although studies have shown that cat ownership is an increased risk factor for the development of some diseases, such as campylobacteriosis,^2,3 others have reported this to be an insignificant variable when investigating the incidence of these diseases in humans.^4–6 In addition, Heyworth et al⁷ demonstrated that dog or cat ownership appeared to be protective against the incidence of gastroenteritis in children. Thus the role of companion animals in transmission of these organisms to humans is unclear. Veterinary surgeons and doctors may be asked for advice regarding the risk of owning a cat, especially if a member of the household is immunocompromised. Knowledge of the prevalence of these enteric organisms in clinically healthy pet cats would assist veterinary surgeons and doctors in counselling at-risk owners.

The aim of this study was to sample clinically healthy kittens presented to veterinary surgeons in the UK in order to identify: (1) the prevalence of Campylobacter species, Salmonella species and enteric parasites using standard laboratory methods, (2) the prevalence of Campylobacter species and Salmonella species using polymerase chain reaction (PCR) techniques, and (3) the prevalence of Giardia species using two commercially available rapid immunoassays; one designed for cats and dogs (Snap Giardia, Idexx Laboratories, Westbrook, USA) and one for humans (Giardia-Strip, Coris Bioconcept, Gembloux, Belgium). Information on signalment, source of the kitten, anthelmintic history and presence of gastrointestinal disease was also sought.

Materials and methods

Sampling

Between May 2006 and June 2007, 14 first opinion veterinary practices in mainland UK distributed faecal sampling kits consisting of three sample pots, disposable gloves and a questionnaire to owners who presented their kittens (age range 9–20 weeks) for the initial vaccination course.

The owners were instructed to collect one faecal sample each day for 3 days from their kitten and complete the questionnaire. The questionnaire (^{Fig 1}) requested details on signalment, where the owner acquired the kitten, previous anthelmintic treatment (including preparation if known) and faecal consistency. The samples and completed questionnaires were then posted to Langford Veterinary Diagnostics at the University of Bristol.

Fig 1.

Questionnaire included in faecal sampling kits.

On arrival, an aliquot of each of the three samples from each kitten was mixed and standard laboratory methods were employed on this pooled sample for bacteriological culture and parasitological examination, while the remaining samples were stored at −20°C. The questionnaires and laboratory test results were faxed to the Hospital for Small Animals, Royal (Dick) School of Veterinary Studies for analysis. The result was reported to the veterinary surgeon with primary care of the kitten and any treatment was ultimately under their direction.

A follow-up questionnaire was completed and submitted by the owners 2–9 months after the submission of the initial questionnaire. This questionnaire was identical to the first one with the addition of one question, enquiring whether the kitten had required veterinary attention for gastrointestinal problems since the last questionnaire.

Bacteriological cultures

Campylobacter

Pooled faeces were inoculated onto plates with Campylobacter blood-free selective agar plates (Oxoid CM0739B, Oxoid, Basingstoke, UK) with CCDA selective supplement (Oxoid SR0155E). Plates were then incubated at 37°C in a 2.5 l jar containing ‘Campygen’ (Oxoid CD0025A) for 3 days. Colonies which produced a positive oxidase test and revealed curved/‘seagull’ shaped negative rods under microscopy after Gram staining, were reported as Campylobacter species.

Salmonella

Pooled faeces were inoculated onto MacConkey agar (Oxoid CM007B), DCLS agar (Oxoid CM0393B) plates and into 20 ml universal containers with Selenite enrichment broth (Oxoid CM0395B+LP0121). Plates and broth were incubated at 37°C under normal atmospheric conditions overnight. After incubation, the Selenite broth was subcultured to a DCLS agar plate which was incubated overnight. Non-lactose fermenting colonies were picked off into urea broth and incubated at 37°C for 4 h. Urea negative cultures were subcultured to triple sugar iron (TSI, Oxoid CM0277) slopes and incubated at 37°C overnight. TSI slopes generating positive reactions for Salmonella species were subcultured to AP120E identification strips (BioMerieux 20100, BioMerieux, Marcy, France) for confirmation. Agglutination testing using polyvalent and monovalent Salmonella species anti-sera was also carried out.

PCR techniques

Preparation of DNA extracts

Faecal samples were thawed, the three samples from each kitten were mixed, and DNA was extracted using the QIAamp DNA Stool mini kit (Qiagen, Crawley, UK) following the manufacturer's instructions. PCRs were performed in a DNA Engine (MJ Research, Waltham, MA, USA). All PCR amplicons were electrophoresed on a 1.5% agarose gel containing 1 μg/ml ethidium bromide (Sigma–Aldrich, Dorest, UK), and visualised on an ultra violet transilluminator (UVP BioDoc It imaging system, Cambridge, UK).

Triplex PCR for the identification of Salmonella, Campylobacter and Escherichia coli species

A novel triplex PCR assay, developed in-house using previously described primers^8–12 was used to enable the simultaneous detection of Salmonella species and Campylobacter species. The third primer combination detected E coli (data not shown). The control strains used were C jejuni NCTC1168, S enterica subspecies Enteritidis LA5.

Multiplex PCR

A modification of the method described by Wang et al¹³ was used for Campylobacter species speciation (referred to in text as multiplex PCR). Each PCR reaction contained 1× HotstarTaq (Qiagen), 1.5 mM MgCl₂, 0.3 μM of each C jejuni primer, 0.6 μM of each of the C coli, C lari and C fetus primers, 0.13 μM of each 23S rDNA primer, 1.25 μM of each C upsaliensis primer and 1 μl of whole-cell template DNA. This multiplex was used to speciate Campylobacter detected with triplex PCR or routine bacteriological culture.

The PCR assay was modified further to improve the speciation of C jejuni and C coli by combining only C jejuni and C coli primers (referred to in the text as duplex PCR). Each PCR reaction contained 1× HotstarTaq (Qiagen), 1.5 mM MgCl₂, 0.65 μM of each C jejuni primer, 1.3 μM of each C coli primer, 0.13 μM of each 23S rDNA primer and 1 μl of whole-cell template DNA. This duplex assay was performed only on Campylobacter species culture positive samples. The control strains used were C upsaliensis NCTC 1141, C fetus NCTC 10842 and C coli number 17.

Parasitological examination

Following centrifugation of faecal samples in a zinc sulphate solution (specific gravity 1.18), a sample of the surface fluid was removed via a 10 μl loop or plastic pipette and positioned on a glass slide which was then examined using a ×40 objective for the microscopic identification of parasitic ova, oocysts and cysts. Thin, air-dried faecal smears were also prepared, stained with a modified Ziehl–Neelsen stain and examined microscopically using a ×100 oil objective for the identification of oocysts with morphological characteristics typical of Cryptosporidium species.

Rapid immunoassays

Faecal samples were thawed in batches of five and both immunoassays for the detection of Giardia antigen (Snap and Strip) were performed according to the manufacturers' instructions. A faecal sample positive for Giardia oocysts on microscopy was also included and tested with each batch and acted as an internal positive control.

Statistical analysis

Questionnaire and laboratory data were entered into a spreadsheet (Microsoft Excel, Washington, USA). A statistical package (GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego, USA) was used to calculate prevalence rates with 95% confidence intervals (CIs) and χ² test for significant associations. Significance was set at P<0.05.

Results

Questionnaires

Fifty-seven sets of faecal samples and completed questionnaires were returned from 14 veterinary practices (^{Fig 2}). The median age was 11.6 weeks (range: 9–20 weeks). Fifty-four percent of the kittens were male. Domestic short- and longhair cats made up 81% of the population. Siamese and Burmese each represented 5%. Eighteen percent of kittens had been acquired from a rescue centre or charity, 61% from an owner whose cat had kittens and 14% from a breeder (all pedigree cats). The remaining kittens had been acquired from a pet shop (three kittens) or bred from the owner's cat (one). Anthelmintic medication was stated to have been administered in 77% of kittens. Fenbendazole was the most frequently stated anthelmintic (25%), although 43% of owners could not recall the name or type of the wormer used.

Fig 2.

Location of participating veterinary practices in the UK.

Bacteriological culture

Bacterial cultures were performed on 57 samples.

Salmonella species was detected in one kitten. The follow-up questionnaire for this kitten 7 months later reported absence of any clinical signs and no need for veterinary attention.

Campylobacter species was cultured from five kittens (8.7%, 95% CI 3.4–19.3%). No gastrointestinal signs were reported on the follow-up questionnaire from these five kittens.

PCR assays

Triplex PCR to identify Campylobacter species and Salmonella species was performed on 54 samples.

All samples were negative for Salmonella species, including the culture positive sample.

Campylobacter species was detected in four samples, but not in any of the five culture positive samples. Further characterisation using the multiplex PCR identified two of the four Campylobacter species as C jejuni.

Of the five Campylobacter species culture positive samples, the multiplex PCR was negative in four and positive (C upsaliensis) in one sample. The duplex PCR was negative for all five culture positive samples. Positive control strains were used in all PCRs and always indicated that the PCR was working effectively.

Parasitological examination

Routine parasitological examination was performed on 57 samples. All samples were negative for Giardia cysts and Cryptosporidium species oocysts. Isospora felis cysts were present in four samples (7%, 95% CI 2.3–17.2%). Three of these kittens were reported to have soft faeces between once to 50% of the time. From the follow-up questionnaire 2–8 months later, none of these kittens had required veterinary attention for gastrointestinal disease and faecal consistency was reported to be normal.

Toxocara species ova were identified in nine kittens (15.7%, 95% CI 8.3–27.5%). The questionnaires revealed that an anthelmintic had been administered in five of these animals. The difference in prevalence of Toxocara species between those kittens stated to have an anthelmintic administered (9%) and those not (33%), was not statistically significant (P=0.082).

Rapid immunoassays

Snap and Strip tests for the detection of Giardia species antigen were performed in 55 samples. The Snap test was positive in three samples and consistently generated a positive result when the internal positive control sample was tested. The Strip test was negative on all 55 samples but also generated a negative result every time the internal positive control sample was analysed.

Prevalence of enteropathogenic organisms

Campylobacter species, Salmonella species and/or enteric parasites were detected in 19 kittens by standard laboratory methods, a prevalence of 33% (95% CI 22.5–46.3%). Using PCR assays, Campylobacter species was detected in four kittens, a prevalence of 7.4% (95% CI 2.4–18%). No Salmonella species was identified using PCR. The prevalence of Giardia using the Snap test was 7.2% (95% CI 2.4–17.7%). Combining the results of all techniques, organisms were detected in 26 animals, a prevalence of 45% (95% CI 33–58%). No animal had required veterinary attention for gastrointestinal disease at follow-up. No kitten had more than one organism of pathogenic potential isolated.

Discussion

The population of cats employed in the present study was selected as the most likely to cause transmission of enteric organisms to humans; kittens may not be fully house-trained and can cause faecal contamination in the house. It was thought that kittens would be most likely to use a litter tray and transmission to humans may occur during emptying of these. In addition, a number of studies have shown that young cats have a higher prevalence of enteric organisms of zoonotic potential than older cats.^14–18

The prevalence of Toxocara species in the present study (15.7%) is lower than the one reported (27.2%) by Spain et al¹ in healthy, client-owned, less than 1-year-old cats (New York area). However, the proportion of cats that received anthelmintic treatment was not reported in that study. Although in the present study there was no significant difference in the prevalence of Toxocara species between kittens that were administered an anthelmintic and those that were not, a limited sample size was used and it is possible that a larger sample size would have demonstrated a statistically significant difference. The presence of Toxocara species in animals which were stated to have had an anthelmintic is a potential concern. Patent infection can occur with infrequent worming or administration of an incorrect dose. In the current study, the questionnaire did not elicit the timing of anthelmintic administration and it is possible that administration may have occurred after or during sample collection, and the animal would have been subsequently negative for Toxocara species. The presence of Toxocara species underlines the need for continued client education and regular anthelmintic treatment in this population.

No samples were found to contain Cryptosporidium species oocysts. Tzannes et al¹⁸ detected a prevalence of only 1% in a UK study employing 1355 cats, most with gastrointestinal signs. Although Cryptosporidium species may be absent from the present population due to an association with gastrointestinal disease, even if the prevalence of this organism in this population was similar to the one reported by Tzannes et al,¹⁸ the small sample size would have generated negative results. Hill et al¹⁹ after testing 205 cats (<1 to >10 years old) from Colorado, with and without diarrhoea stated that Cryptosporidium species was the most prevalent zoonotic agent detected (5.4%). The majority of positive results were generated by employing an enzyme-linked immunosorbent assay (ELISA) test rather than the Ziehl–Neelsen stain. Using the ELISA test may have resulted in the detection of the organism in our sample population.

The 8.7% prevalence of Campylobacter species by culture in the current study is similar to the prevalence of 5%, reported by Hald and Madsen²⁰ who sampled 42 healthy kittens aged between 11 and 17 weeks in Denmark, but much lower than the 41.9% prevalence in the study of healthy Swiss cats reported by Wieland et al.²¹ In both studies, stricter sample handling was undertaken with culture being performed within 48 h of sampling. Routine culture for Campylobacter species by Bender et al,²² using samples from both ill and healthy cats from the USA which were posted to the laboratory in a similar method to the current study detected a prevalence of 24%. A study comparing sampling handling and culture techniques would be required to investigate if these apparent differences in prevalence are due to true population difference or study methodology. Hill et al¹⁹ detected a prevalence of 1.9% in 52 healthy client-owned cats in New York although only C jejuni was investigated.

The prevalence of Salmonella species (1/57) in this study is of a similar magnitude to the one reported by Van Immerseel et al²³ where out of 278 healthy Belgian house cats only one found to be excreting the organism. Similar prevalence was also reported by Hill et al¹⁹ where all faecal samples from 52 healthy client-owned cats from Colorado were negative for Salmonella by culture.

Triplex PCR detected the presence of Campylobacter species in four kittens but did not detect the organism in any of the five samples with positive cultures. This discrepancy between Campylobacter culture and PCR results could suggest that sensitivity of each may have caused underestimation of prevalence. This discrepancy may be due to multiple factors. A PCR will detect non-viable bacteria with intact DNA sequences, generating positive results in culture negative samples. A positive PCR result depends on sufficient quantity and quality of the sequence under investigation as well as lack of substances which may interfere with amplification. Freezing and storage of samples prior to PCR analysis may have caused disruption of bacterial DNA. Mixing of the three samples may have reduced the quantity of DNA to below detection threshold especially if the organism was only excreted intermittently. These factors may explain the samples with culture positive but PCR negative results. The PCR testing of each sample separately at the time of arrival at the laboratory may have increased the likelihood of obtaining a positive result. However, the PCR sensitivity may be inherently lower than cultures even in samples with intact bacterial DNA because the faecal material may contain PCR inhibitory components which can cause negative results.²⁴

Speciation by multiplex PCR failed to identify the type of Campylobacter species in 4/5 culture positive samples and in 2/4 triplex PCR positive samples. This may have been due to inherent PCR problems discussed above or the presence of a species that the primers were not designed to detect. For example, Weiland et al²¹ found a very high prevalence of C helveticus amongst their population of healthy cats in Switzerland. Primers for this species were not present in the multiplex PCR.

Giardia species was detected only using the Snap immunoassay. Mekaru et al²⁵ found that this test had an identical sensitivity to flotation when compared to direct immunofluorescence as the gold standard. Although no gold standard test was performed in the current study, Mekaru et al²⁵ found this test to be 100% specific, therefore, it is likely that these are true positives. The prevalence in the current study (5.4%) is very similar to the prevalence of 6.1% found by Vasiloupulos et al²⁶ in healthy cats in Mississippi and Alabama when direct immunofluorescent antibodies were used. Tzannes et al¹⁸ also found a similar prevalence in healthy UK cats using another commercial ELISA (4%). The human Giardia species immunoassay failed to detect Giardia species in any samples or the positive control samples. This human immunoassay kit has been reported to have a relatively low sensitivity; 58²⁷ and 44% compared with 88–80% sensitivity of the other kits tested.²⁸ It is possible that the lack of detection in these feline samples is partly a reflection of this low sensitivity. Giardia species immunoassays designed for human samples also performed poorly in the Mekaru et al²⁵ study. Mekaru et al²⁵ postulated that genetic differences between common animal/human Giardia species isolates may explain the poor performance of the human assay. There is evidence that the more common genotypic isolate from cats (ie, assemblage type F) is not involved in human infection, although the zoonotic assemblage type A has been identified in cats.²⁹ The sensitivity to different Giardia species assemblages of the two kits used in the current study is unknown. Mekaru et al²⁵ concluded that caution should be exercised when using human-based immunoassays for parasite detection in animals. This current study supports this warning.

This study is likely to have underestimated the prevalence of these organisms for a number of reasons. Excretion of many of these organisms is intermittent.^30,31 Although three samples were submitted, these were collected within a short time period and episodes of excretion may have been missed. The sampling method may not have been optimal. Acke et al¹⁴ found a higher rate of detection of Campylobacter species in cats where collection was by rectal swab rather than faecal sample. It is unknown if this difference was due to sampling method or increased prevalence in the animals swabbed. Toxocara species may have been present but not excreting eggs at the time of sampling or not detected by flotation.^32,33 The delay and transport conditions between sample collection and receipt at the laboratory may have reduced the viability of Campylobacter species for culture³⁴ and/or disrupted protozoal organisms, making identification impossible.³⁰ Even so, the protocol for sampling and transport mirrors sampling used in first opinion veterinary practices that submit samples to external laboratories and, therefore, the generated prevalence could be compared with the prevalence in symptomatic animals.

The use of PCR and ELISA techniques more than doubled the apparent detection rate of potentially enteropathogenic organisms. With any diagnostic laboratory test, there is the possibility of false-positive results. Although regular quality control assessments are performed at the laboratory and both positive and negative controls were used during PCR testing, this possibility cannot be ruled-out, especially as no ‘gold-standard’ test was available to evaluate further the disparate results between routine laboratory tests and the more advance techniques.

The high prevalence of these enteric organisms in this ostensibly healthy population raises a number of issues. These kittens were presented for initial vaccination and not for the presence of gastrointestinal disease. If these organisms were detected in animals with gastrointestinal disease they would likely be implicated as the cause. It is possible that when detected in diseased animals they may be incidental findings or if these organisms are causing gastrointestinal disease, there may be concurrent factors which allow this. Ultimately, a case–control study using identical sample handing and laboratory methods would be required to attempt to identify any difference in prevalence of these organisms between animals with gastrointestinal disease and clinically normal animals. It is still possible that perturbations in the gastrointestinal environment (eg, change in osmolarity or transit time) due to unrelated gastrointestinal disease may allow overgrowth and, therefore, increased detection rates of organisms.

It could be argued that the high prevalence of potential zoonotic organisms suggests that cats may be a significant source of infection for humans. The mere detection of these organisms, although indicating prevalence in this population, does not necessarily translate into zoonotic risk. Many of these organisms vary in pathogenicity to humans depending on their genotype.^29,31,35,36 Thus further typing of these organisms would be required to assess their true zoonotic potential. The number and viability of organisms excreted is an important factor in environmental contamination and subsequent zoonotic risk. The PCR techniques are designed to identify presence of organisms at very low levels. A method of organism quantification may be useful in assessing risk, such as quantitative real-time PCR.

The converse argument to zoonotic risk is that if these organisms are this prevalent, it would be expected that if cats are a significant source of human infection, then more human epidemiological studies investigating risk factors for these diseases would have identified contact with cats as a risk. This does not appear to be the case, with more studies showing pet owning as having a protective affect against the development of gastroenteritis and/or infection with these organisms^7,37 or cats having no association with development of disease in humans^4–6,38 than contact with cats increasing the risk of these diseases.^2,3 It was not the aim of this study to investigate any link between detection of potential zoonotic pathogens in these kittens and the presence of gastrointestinal signs in the owners.

Treatment of these animals was under the control of the primary care veterinary surgeon. The owners were made aware of the potential zoonotic risk of all these organisms and rigorous hygiene was advised for all owners. Anthelmintic treatment was advised in Toxocara species-positive kittens. In Isospora species-positive kittens, no treatment was advised unless they exhibited signs of gastrointestinal disease as increasing age and immunocompetence would be likely to eradicate the infection without the need for medication.³⁹ None required treatment. The Snap Giardia species results were not available until after the second questionnaire was returned and it is not known if an effective anti-protozoal medication had been administered in the interim period. Treatment of Campylobacter and Salmonella species-positive kittens was discussed on a case-by-case basis with the primary care veterinary surgeon. As previous studies have shown a high prevalence of Campylobacter species in asymptomatic cats,^14,21 treatment was not advised unless owners were thought to be at high risk, eg, immunocompromised or the kitten developed compatible clinical signs. None was ultimately treated. The one Salmonella species-positive kitten was not treated after discussion with the veterinary surgeon and owner. Greene³⁴ states that antibiotic treatment is not recommended in clinically affected animals with uncomplicated salmonellosis and may induce plasmid-mediated resistance or even activate clinical illness in latent carriers.

Footnotes

Acknowledgements

The author's would like to thank the Cambridge Infectious Disease Consortium Clinical Research Outreach Programme (CROP) for providing funding for this study, all participating veterinary practices and clients, and Dr Mark Holmes for his help and comments in the preparation the paper. AG would like to thank Hill's Pet Nutrition for providing funding for his residency. Idexx is gratefully acknowledged for providing their test kits at reduced cost.

References

Spain

C.V.

Scarlett

J.M.

Wade

S.E.

McDonough

Prevalence of enteric zoonotic agents in cats less than 1 year old in central New York State, J Vet Intern Med 15, 2001, 33–38.

Deming

M.S.

Tauxe

R.V.

Blake

P.A.

Campylobacter enteritis at a university: transmission from eating chicken and from cats, Am J Epidemiol 126, 1987, 526–534.

Saeed

A.M.

Harris

N.V.

DiGiacomo

R.F.

The role of exposure to animals in the etiology of Campylobacter jejuni/coli enteritis, Am J Epidemiol 137, 1993, 108–114.

Cook

A.J.

Gilbert

R.E.

Buffolano

Sources of toxoplasma infection in pregnant women: European multicentre case–control study. European Research Network on Congenital Toxoplasmosis, BMJ 321, 2000, 142–147.

Potter

R.C.

Kaneene

J.B.

Hall

W.N.

Risk factors for sporadic Campylobacter jejuni infections in rural Michigan: a prospective case–control study, Am J Public Health 93, 2003, 2118–2123.

Tenkate

T.D.

Stafford

R.J.

Risk factors for Campylobacter infection in infants and young children: a matched case–control study, Epidemiol Infect 127, 2001, 399–404.

Heyworth

J.S.

Cutt

Glonek

Does dog or cat ownership lead to increased gastroenteritis in young children in South Australia, Epidemiol Infect 134, 2006, 926–934.

Aabo

Rasmussen

O.F.

Rossen

Sorensen

P.D.

Olsen

J.E.

Salmonella identification by the polymerase chain reaction, Mol Cell Probes 7, 1993, 171–178.

Bej

A.K.

McCarty

S.C.

Atlas

R.M.

Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring, Appl Environ Microbiol 57, 1991, 2429–2432.

10.

Croci

Delibato

Volpe

De Medici

Palleschi

Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting Salmonella in meat products, Appl Environ Microbiol 70, 2004, 1393–1396.

11.

Linton

Owen

R.J.

Stanley

Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals, Res Microbiol 147, 1996, 707–718.

12.

Steinhauserova

Fojtikova

Klimes

The incidence and PCR detection of Campylobacter upsaliensis in dogs and cats, Lett Appl Microbiol 31, 2000, 209–212.

13.

Wang

Clark

C.G.

Taylor

T.M.

Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C coli, C lari, C upsaliensis, and C fetus subsp fetus, J Clin Microbiol 40, 2002, 4744–4747.

14.

Acke

Whyte

Jones

B.R.

McGill

Collins

J.D.

Fanning

Prevalence of thermophilic Campylobacter species in cats and dogs in two animal shelters in Ireland, Vet Rec 158, 2006, 51–54.

15.

De Santis-Kerr

A.C.

Raghavan

Glickman

N.W.

Prevalence and risk factors for Giardia and Coccidia species of pet cats in 2003–2004, J Feline Med Surg 8, 2006, 292–301.

16.

McGlade

T.R.

Robertson

I.D.

Elliot

A.D.

Read

Thompson

R.C.

Gastrointestinal parasites of domestic cats in Perth, Western Australia, Vet Parasitol 117, 2003, 251–262.

17.

Shukla

Giraldo

Kraliz

Finnigan

Sanchez

A.L.

Cryptosporidium spp. and other zoonotic enteric parasites in a sample of domestic dogs and cats in the Niagara region of Ontario, Can Vet J 47, 2006, 1179–1184.

18.

Tzannes

Batchelor

D.J.

Graham

P.A.

Pinchbeck

G.L.

Wastling

German

A.J.

Prevalence of Cryptosporidium, Giardia and Isospora species infections in pet cats with clinical signs of gastrointestinal disease, J Feline Med Surg 10, 2008, 1–8.

19.

Hill

S.L.

Cheney

J.M.

Taton-Allen

G.F.

Reif

J.S.

Bruns

Lappin

M.R.

Prevalence of enteric zoonotic organisms in cats, J Am Vet Med Assoc 216, 2000, 687–692.

20.

Hald

Madsen

Healthy puppies and kittens as carriers of Campylobacter spp., with special reference to Campylobacter upsaliensis, J Clin Microbiol 35, 1997, 3351–3352.

21.

Wieland

Regula

Danuser

Campylobacter spp. in dogs and cats in Switzerland: risk factor analysis and molecular characterization with AFLP, J Vet Med B Infect Dis Vet Public Health 52, 2005, 183–189.

22.

Bender

J.B.

Shulman

S.A.

Averbeck

G.A.

Pantlin

G.C.

Stromberg

B.E.

Epidemiologic features of Campylobacter infection among cats in the upper midwestern United States, J Am Vet Med Assoc 226, 2005, 544–547.

23.

Van Immerseel

Pasmans

De Buck

Cats as a risk for transmission of antimicrobial drug-resistant Salmonella, Emerg Infect Dis 10, 2004, 2169–2174.

24.

Radstrom

Knutsson

Wolffs

Lovenklev

Lofstrom

Pre-PCR processing: strategies to generate PCR-compatible samples, Mol Biotechnol 26, 2004, 133–146.

25.

Mekaru

S.R.

Marks

S.L.

Felley

A.J.

Chouicha

Kass

P.H.

Comparison of direct immunofluorescence, immunoassays, and fecal flotation for detection of Cryptosporidium spp. and Giardia spp. in naturally exposed cats in 4 Northern California animal shelters, J Vet Intern Med 21, 2007, 959–965.

26.

Vasilopulos

R.J.

Mackin

A.J.

Rickard

L.G.

Pharr

G.T.

Huston

C.L.

Prevalence and factors associated with fecal shedding of Giardia spp. in domestic cats, J Am Anim Hosp Assoc 42, 2006, 424–429.

27.

Oster

Gehrig-Feistel

Jung

Kammer

McLean

J.E.

Lanzer

Evaluation of the immunochromatographic CORIS Giardia-Strip test for rapid diagnosis of Giardia lamblia, Eur J Clin Microbiol Infect Dis 25, 2006, 112–115.

28.

Weitzel

Dittrich

Mohl

Adusu

Jelinek

Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples, Clin Microbiol Infect 12, 2006, 656–659.

29.

Vasilopulos

R.J.

Rickard

L.G.

Mackin

A.J.

Pharr

G.T.

Huston

C.L.

Genotypic analysis of Giardia duodenalis in domestic cats, J Vet Intern Med 21, 2007, 352–355.

30.

Barr

S.C.

Enteric protozoal infections, Infectious Diseases of the Dog and Cat, 3rd edn, 2006, Saunders Elsevier: St Louis, 736–750.

31.

Fox

J.G.

Enteric bacterial infections. Greene

C.E.

Infectious Diseases of the Dog and Cat, 3rd edn, 2006, Saunders Elsevier: St Louis, 339–369.

32.

Lillis

W.G.

Helminth survey of dogs and cats in New Jersey, J Parasitol 53, 1967, 1082–1084.

33.

Wolfe

Hogan

Maguire

Red foxes (Vulpes vulpes) in Ireland as hosts for parasites of potential zoonotic and veterinary significance, Vet Rec 149, 2001, 759–763.

34.

Greene

C.E.

Enteric bacterial infections, Salmonella, Infectious Diseases of the Dog and Cat, 3rd edn, 2006, Saunders Elsevier: St Louis, 355–360.

35.

Fayer

Dubey

J.P.

Lindsay

D.S.

Zoonotic protozoa: from land to sea, Trends Parasitol 20, 2004, 531–536.

36.

Palmer

C.S.

Traub

R.J.

Robertson

I.D.

Devlin

Rees

Thompson

R.C.

Determining the zoonotic significance of Giardia and Cryptosporidium in Australian dogs and cats, Vet Parasitol 154, 2008, 142–147.

37.

Robertson

Sinclair

M.I.

Forbes

A.B.

Case–control studies of sporadic cryptosporidiosis in Melbourne and Adelaide, Australia, Epidemiol Infect 128, 2002, 419–431.

38.

Glaser

Safrin

Reingold

Newman

T.B.

Association between Cryptosporidium infection and animal exposure in HIV-infected individuals, J Acquir Immune Defic Syndr Hum Retrovirol 17, 1998, 79–82.

39.

Dubey

J.P.

Greene

C.E.

Enteric coccidiosis, Infectious Diseases of the Dog and Cat, 3rd edn, 2006, Saunders Elsevier: St Louis, 775.

Prevalence of Potentially Pathogenic Enteric Organisms in Clinically Healthy Kittens in the UK

Abstract

Materials and methods

Sampling

Bacteriological cultures

Campylobacter

Salmonella

PCR techniques

Preparation of DNA extracts

Triplex PCR for the identification of Salmonella, Campylobacter and Escherichia coli species

Multiplex PCR

Parasitological examination

Rapid immunoassays

Statistical analysis

Results

Questionnaires

Bacteriological culture

PCR assays

Parasitological examination

Rapid immunoassays

Prevalence of enteropathogenic organisms

Discussion

Footnotes

Acknowledgements

References