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Frdms.npust.edu.twVeterinary Research Communications, 31 (2007) 931–939DOI: 10.1007/s11259-007-0009-4 Salmonellae and Campylobacters in Household and Stray
Dogs in Northern Taiwan
H.-J. Tsai1,2,∗, H.-C. Huang1,2, C.-M. Lin1, Y.-Y. Lien3 and C.-H. Chou11Department of Veterinary Medicine and 2Center for Zoonoses Research, College ofBioresources and Agriculture, National Taiwan University, Taipei; 3Department ofVeterinary Medicine, National Pingtung University of Science & Technology, Pingtung,Taiwan ∗Correspondence: E-mail: [email protected] Rectal swabs were collected from 437 household and 491 stray dogs in northern Taiwan from May 2003 toJune 2005 to investigate the prevalence and antimicrobial susceptibilities of salmonellae and campylobacters.
The results revealed that 2.1% of household dogs and 6.3% of stray dogs were positive for salmonellae, withSalmonella Duesseldorf being the most dominant serotype in both. Additionally, 2.7% of the household dogsand 23.8% of the stray dogs were positive for campylobacters. Campylobacter jejuni was the most prevalentspecies (86.8%), followed by C. upsaliensis (9.3%) and C. coli (3.9%). Both salmonella and campylobacterisolation rates from the stray dogs were significantly higher than those from the household dogs ( p < 0.01).
The susceptibility of 33 C. jejuni isolates to eight antimicrobials was studied by the E-test. A high rate ofresistance was observed to azithromycin (93.9%), clindamycin (87.9%), erythromycin (81.8%), tetracycline(78.8%), chloramphenicol (69.7%), nalidixic acid (51.5%), gentamicin (33.3%), and ciprofloxacin (18.2%).
The susceptibility of 40 Salmonella isolates to 15 antimicrobials was also studied by the disc-diffusion method.
All the Salmonella isolates were susceptible to ciprofloxacin and ceftriaxone. Resistance was observed mostfrequently to tetracycline (77.5%), chloramphenicol (52.5%), and ampicillin (50%).
Keywords: antimicrobial agents, campylobacters, household dogs, salmonellae, stray dogs Abbreviations: CAT, cefoperazone amphotericin teicoplanin; MIC, minimum inhibitory concentrations; PCR,polymerase chain reaction Infectious enteric pathogens have long been recognized as a significant problem owing totheir pathogenicity potential to animals and their zoonotic risk to humans. Among them, twogastrointestinal bacterial pathogens, salmonellae and campylobacters have been consideredto be important food-borne pathogens causing human enteritis worldwide and leading toserious public health concern (Ethelberg et al., 2004). In addition to causing enteritis, theseorganisms have also been reported in association with bacteraemia, reactive arthritis, andmeningitis (Goldberg and Rubin, 1988; Peterson, 1994).
Salmonellae and campylobacters are ubiquitous and can be isolated from many kinds of farm and pet animals. The majority of human salmonellosis and campylobacteriosiscases in developed countries are most likely caused through consumption of undercookedpoultry, raw milk, or untreated surface water (Goldberg and Rubin, 1988; Kapperud et al.,1992; Altekruse et al., 1994). Furthermore, living with a household dog has previously beenidentified as a risk factor for these diseases (Kapperud et al., 1992; Robinson and Pugh, 2002). Most dogs are asymptomatic when they act as reservoirs shedding salmonellaeor campylobacters in their faeces. Pathogens in their faeces may ultimately infect otheranimals by contaminating the environment (Morse and Duncan, 1975; Fox, 1990; Haldand Madsen, 1997). Recently, among immunocompromised populations, i.e. those usingimmunosuppressive drugs having acquired immunodeficiency syndrome, and the elderlythese bacteria have become a great pathogenic risk (Robinson and Pugh, 2002).
The prevalence of salmonellae and campylobacters in dogs in Taiwan is still unknown; this study was done to determine the prevalence of these bacteria in household and straydogs and to assess bacterial antibiotic susceptibilities.
Rectal swabs were collected from household dogs at the National Taiwan University Veteri-nary Hospital, and four other private veterinary clinics located in Taipei city. For the straydogs, swabs were collected from six municipal animal shelters located in northern Taiwan.
Each shelter was visited twice in a 3-month period and all the dogs were sampled uponeach visit. The usual holding period for the stray dogs in the public shelter was 10 days, sothe dogs were sampled within 1–10 days after their arrival. The swabs were transported inCary and Blair transport medium (Oxoid, Basingstoke, Hampshire, UK) to the laboratoryon the day of collection.
Isolation and identification of salmonellae Each sample was enriched using selenite brilliant-green enrichment broth (Difco, Detroit,MI, USA) for 18 h at 37◦C. Samples were then plated onto brilliant-green phenol-redlactose sucrose agar (BPLS agar, Merck, Whitehouse Station, NJ, USA) and incubatedfor 24 h at 37◦C. Isolated pink colonies surrounded by a red zone were then subculturedon tryptic soy broth (Merck) for 18 h at 37◦C and then subjected to identification by thefollowing biochemical tests: triple sugar iron agar, sulphide-indole-motility medium, lysinedecarboxylase test, and urease test (Merck). Once salmonellae were identified, serotypingwas performed according to the Kauffmann–White scheme using a commercial antiserumkit (Difco).
Isolation and identification of campylobacters Rectal swabs were streaked on two cefoperazone amphotericin teicoplanin (CAT) agarplates (Oxoid). Under microaerophilic conditions (10% CO2 and 5% O2), one plate wasincubated at 42◦C and the other one at 37◦C in order that C. upsaliensis growth would beuninhibited (Corry et al., 1995). The plates were checked after 2–3 days and again after 5 days for growth of campylobacters. Preliminary identifications were based on phenotypiccharacteristics: colony morphology, microscopic morphology, motility and oxidase andcatalase reactions.
Polymerase chain reaction (PCR) was used to detect and differentiate three major species of campylobacters (C. jejuni, C. coli and C. upsaliensis). The genomic DNA of isolateswas extracted using a commercial kit (Genomic DNA Purification Kit, MBI FermentasGMBH, St. Leon-Rot, Germany). For the detection of C. jejuni and C. coli, the proce-dures described by Harmon and colleagues (1997) were used with a primer set of pg-3(5 -GAACTTGAACCGATTTG-3 ) and pg-50 (5 -ATGGGATTTCGTATTAAC-3 ). To fur-ther differentiate C. jejuni and C. coli, the primers 5 -TACTACAGGAGTTCAAGCTT-3and 5 -GTTGATGTAACTTGATTTTG-3 described by Nishimura and colleagues (1996)were used. The PCR described by Linton and colleagues (1996) was used to specifically de-tect C. upsaliensis. The primer set used was CHCU146F (5 -GGGACAACACTTAGAAAT-GAG-3 ) and CU1024R (5 -CACTTCCGTATCTCTACAGA-3 ).
All the 40 Salmonella isolates were tested for antimicrobial susceptibility testing by thedisc-diffusion method following the NCCLS (2002) guidelines. The following antimicro-bial agents were used at the indicated concentrations (μg/disc except where specified):amoxicillin/clavulanic acid (20/10), ampicillin (10), apramycin (15), cefoxitin (30), cef-triaxone (30), cephalothin (30), chloramphenicol (30), ciprofloxacin (5), gentamicin (10),kanamycin (30), nalidixic acid (30), nitrofurantoin (300), streptomycin (10), sulfamethox-azole/trimethoprim (23.5/1.5) and tetracycline (30).
A total of 33 C. jejuni isolates were randomly chosen and tested with the E-test system (AB BIODISC, Solna, Sweden) to determine minimum inhibitory concentrations (MICs)for eight antimicrobial agents (azithromycin, chloramphenicol, ciprofloxacin, clindamycin,erythromycin, gentamicin, nalidixic acid, and tetracycline). These eight antimicrobial agentswere those included in the National Antimicrobial Resistance Monitoring System in theUSA for the monitoring the antimicrobial resistance of Campylobacter spp by the E-testsystem (Gupta et al., 2004). The E-test was performed on Mueller–Hinton agar supple-mented with 5% sheep blood according to the manufacturer’s instructions. Inocula wereprepared by incubating the strains for 24 h at 42◦C under microaerobic conditions in tryp-ticase soy broth. After application of the E-test strips, plates were incubated at 42◦C for48 h. MIC values were read directly from the test strip according to the instructions of themanufacturer, where the elliptical zone of inhibition intersected with the MIC scale on thestrip.
Chi-squared and Fisher’s exact tests were used to evaluate the differences in prevalencewith Microsoft Excel (Microsoft, WA, USA).
In the total of 437 household dogs aged 3 months to 19 years, 9 dogs (2.1%) were positivefor salmonellae (Table I). Thirteen household dogs showed symptom of diarrhoea duringsampling. Of these dogs, only one Salmonella isolate was recovered from one dog. Therewas no statistical difference in isolation rates between male (1.8%, 4/222) and female (2.3%,5/215) groups ( p < 0.05), and no statistical difference between young (3–12 months old)(1.4%, 1/70) and adult (>1 year old) (2.2%, 8/367) groups ( p < 0.05). Six serotypes ofS. enterica were identified: Salmonella Duesseldorf (n = 3), Salmonella Derby (n = 2),Salmonella Bardo (n = 1), Salmonella Bellevue (n = 1), Salmonella Panama (n = 1)and Salmonella Stanley (n = 1). Serogroup B based on O-antigen grouping was the mostprevalent serogroup (42.5%), followed by serogroups C2 (35.0%), D1 (17.5%), and E1 (5%)(Table II).
Of the 491 stray dogs, 2/6 animal shelters (16.7%) and 31 stray dogs (6.3%) were salmonellae positive (Table I). The isolation rates between household dogs (2.1%) andstray dogs (6.3%) were significantly different ( p < 0.01). There was no statistical differ-ence in isolation rates between male (6.1%, 13/212) and female (5.7%, 16/279) stray dogs( p < 0.05). The difference between the two age groups was unavailable because their ageswere unknown. At least 17 Salmonella serotypes were recognized, among which SalmonellaDusseldorf (n = 6) and Salmonella Enteritidis (n = 5) were predominant (Table II).
Campylobacter spp. were isolated from 2.7% (12/437) of the household dogs, contrastingwith 23.8% (117/491) of the stray dogs in all the six animal shelters. Campylobacter isolation TABLE IThe numbers and percentages of salmonellae and campylobacters isolated from household dogs andstray dogs in Taiwan aPercentage values for each campylobacter species relate to the proportion relative to the total campylobacternumbers isolated.
TABLE IISalmonella serotypes and numbers isolated from dogs in Taiwan rates between household and stray dogs were significantly different ( p < 0.01). However,there was no significant difference in the isolation rates between male (2.3%, 5/222) and fe-male (3.3%, 7/215) household dogs, nor between male (22.2%, 47/212) and female (25.1%,70/279) stray dogs ( p < 0.05). There was also no significant difference in isolation ratebetween young (4.3%, 3/70) and adult household dogs (2.5%, 9/367) ( p < 0.05). C. jejuniwas the most prevalent species in both household (91.7%) and stray dogs (86.3%). C. coliwas isolated from 8.3% of the household dogs and 3.4% of the stray dogs. C. upsaliensiswas isolated from 10.3% of the stray dogs but was not found in the household dogs (Table I).
Using the disc diffusion method, 8 out of 40 strains of Salmonella (22.2%) were resistantto amoxicillin, 20 (50%) to ampicillin/clavulanic acid, 9 (22.5%) to apramycin, 1 (2.8%)to cefoxitin, none to ceftriaxone, 2 (5%) to cephalothin, 21 (52.5%) to chloramphenicol,none to ciprofloxacin, 2 (5%) to gentamicin, 2 (5%) to kanamycin, 17 (42.5%) to nalidixicacid, 4 (10%) to nitrofurantoin, 14 (38.9%) to streptomycin, 15 (37.5%) to sulfamethoxa-zole/trimethoprim and 31 (77.5%) to tetracycline.
The MICs of the eight antimicrobial agents for Campylobacter strains are shown in Table III. High rates of resistance were observed to azithromycin (93.9%), clindamycin(87.9%), erythromycin (81.8%), tetracycline (78.8%), chloramphenicol (69.7%), nalidixicacid (51.5%), gentamicin (33.3%), and ciprofloxacin (18.2%).
Multiple serotypes of salmonellae commonly exist in dog populations. In the southern USA,53 serotypes have been isolated, with Salmonella Anatum and Salmonella Typhimuriumbeing predominant (Morse and Duncan, 1975); in Trinidad, 28 serotypes were isolated,with Salmonella Javiana, Salmonella Newport, Salmonella Arechavaleta and SalmonellaHeidelberg being predominant (Seepersadsingh et al., 2004). Ten serotypes were previ-ously isolated from dogs in Taiwan in 1963–1967, with the most prevalent serotypes beingSalmonella Weltevreden, Salmonella Meleagridis, Salmonella Derby and Salmonella Tana-narive (Cheng et al., 1968). In this study, at least 20 serotypes were detected and SalmonellaDusseldorf was the most prevalent followed by Salmonella Enteritidis and SalmonellaDerby. It was concluded that the prevalent serotypes of salmonellae in dog populations arequite variable among different countries and at different times within the same country.
In Taiwan, Salmonella Typhimurium, Salmonella Weltevreden, Salmonella Derby, Salmonella Muenchen, Salmonella Agona, and Salmonella Panama were the six most fre-quent serotypes from humans in 1983–1993 (Wang et al., 1994). Except for few host-adaptedserotypes such as Salmonella Gallinarum and Salmonella Pullorum, other serotypes are con-sidered pathogenic to humans and have various kinds of hosts. Serotypes Salmonella Derby,Salmonella Panama, and Salmonella Enteritidis, the causative agents of human disease andfound in dogs, have also been found commonly in chickens and ducks in Taiwan (Chouand Tsai, 2001; Tsai and Hsiang, 2005). Thus the human and canine infections might bothbe due to the consumption of contaminated poultry. The antimicrobial resistant patterns ofthe Taiwanese canine and human Salmonella isolates are quite different. Increasing resis-tance to ceftriaxazone and ciprofloxacin in human Taiwanese Salmonella isolates has beenreported recently (Su et al., 2005); however, we demonstrated that all the canine isolateswere susceptible to ceftriaxazone and ciprofloxacin.
The species distribution of Campylobacter isolates from dogs differs considerably be- tween publications and years. C. upsaliensis (Sandberg et al., 2002; Hald et al., 2004;Wieland et al., 2005) and C. jejuni (Hald and Madsen, 1997; Lopez et al., 2002; Workmanet al., 2005) have been demonstrated to be the predominant species in dogs in differentstudies. Additionally, younger dogs have been reported to carry higher rates of campy-lobacters (Lopez et al., 2002; Engvall et al., 2003; Hald et al., 2004), and have higherodds of carrying C. upsaliensis than older dogs (Wieland et al., 2005). In this study, nosignificantly higher isolation rate was found in young household dogs, and this might havebeen the result of most of our isolates being C. jejuni, in which age is not associated withcarriage (Wieland et al., 2005).
In Taiwan, it has been shown that human Campylobacter isolates were significantly more susceptible than chicken isolates to erythromycin, clindamycin and ciprofloxacin (Liet al., 1998). From our study, it appears that the canine isolates were also significantly more resistant to clindamycin and erythromycin than were human isolates. Whereas nearlyall human and chicken Campylobacter isolates were susceptible to gentamicin (Li et al.,1998), our results showed a higher level of resistance in canine isolates (33.3%). However,the resistance rates to ciprofloxacin were much lower in canine isolates (18.2%) in this studythan human isolates (79%) (Li et al., 1998). Different antimicrobial susceptibility of humanand canine isolates may reflect the different use of antimicrobials in pet animal veterinarymedical practice and human medical practice, and may also indicate that exchange ofCampylobacter spp. between the human and canine population is rare.
The fact that household dogs generally show lower isolation rates of salmonellae and campylobacters than stray dogs (Shimi et al., 1976; Simpson et al., 1981; Fox, 1990; Work-man et al., 2005) was shown by our findings as well. These highly prevalent gastrointestinalpathogens in shelters may increase the risks of (1) nosocomial transmission between dogs,(2) zoonotic transmission to workers in shelters and people adopting dogs from shelters, and(3) environment contamination potential (Sokolow et al., 2005; Wright et al., 2005). Be-cause of financial constraints and crowded dog housing with frequent turnover, surveillancefor specific pathogens may not be feasible and therapeutic measures to reduce the contam-ination level may also not be easily achieved. The public health importance of salmonellaeand campylobacters in dogs in Taiwan has not been established, especially in the case ofstray dogs. However, from the findings reported here, the contamination in the dog sheltersappeared to be a potential risk to public health, although this assumption would still needto be verified by molecular methods. Concerning potential zoonotic risks, the implemen-tation of nonspecific prevention methods such as prophylactic disinfection in the animalholding areas, self-sanitation procedures for the employees, quarantine before dog adop-tion, and clear emphasis on warnings of the zoonotic risk of transmission to the adopters isrecommended (Sokolow et al., 2005; Wright et al., 2005).
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United States Patent 7,311,929 Wenniger December 25, 2007 Nutritional weight loss agent and method Abstract The present invention relates to an agent that suppresses appetite and provides quick energy. According to at least one aspect of the present invention, a lollipop is provided that suppresses appetite and provides quick energy. The lollipop is made from a premixed herb