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of commercially available raw meat diets for dogs
Rachel A. Strohmeyer, DVM, MS; Paul S. Morley, DVM, PhD, DACVIM; Doreene R. Hyatt, PhD;
David A. Dargatz, DVM, DACVIM; A. Valeria Scorza, VMD, MS; Michael R. Lappin, DVM, PhD, DACVIM
JAVMA, Vol 228, No. 4, February 15, 2006 Scientific Reports: Original Study 537
Salmonella enterica, and Campylobacter spp and to determine the to 5.85 X 105 CFUs/g of sample, those with a score of 2 had
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magnitude of bacterial contamination. Polymerase chain reac- 5.85 X 105 to 5.85 X 107 CFUs/g of sample, and those with a
tion assays were used to determine whether the products con- score of 3 had > 5.85 X 107 CFUs/g of sample. Plates with a
tained DNA from Neospora spp, Toxoplasma spp, or score of 0 had no visible bacterial growth.
Cryptosporidium spp.
Enriched NTSEC culturesOne milliliter of each
DietsProducts were arbitrarily selected from an adver- processed food sample was added to 9 mL of tryptic soy
tised selection of foods available from a large number of retailers broth and incubated at 37oC for 18 to 24 hours. Samples were
with the assumption that products that were most commonly transferred to MacConkey agar with a sterile swab and incu-
advertised were those most commonly used by consumers. bated for 18 to 24 hours at 37oC. A single lactose-fermenting
Twenty-one raw meat products sold as diets for dogs were select- colony, if present, was transferred to a blood agar plate and
ed for purchase, and 3 retail sources were selected to provide the incubated at 37oC for 18 to 24 hours. Lactose-fermenting
product from among those identified in an internet search. The colonies that contained indoleb were assumed to be NTSEC.
raw meat diets were composed of beef, lamb, chicken, or turkey
meat and were produced by 7 companies. To minimize the Salmonella enterica culturesOne milliliter of each
chance of obtaining multiple samples from the same production processed food sample was mixed with 9 mL of tetrathionate
lots, products were purchased and tested on 4 dates (March brothc (containing brilliant green and iodine) and incubated for
2002, May 2002, August 2002, and October 2002). All products 18 to 24 hours at 42oC. After incubation, samples were vortexed
were obtained during each of the 4 purchase periods, with the and 100 L was added to 10 mL of Rappaport-Vassiliadis R10
exception that 1 lamb meat product could only be obtained dur- mediac and incubated at 37oC for 18 to 24 hours. After incuba-
ing the first 2 sampling periods, and a turkey meat product was tion, sterile swabs were used to transfer samples to xylose-
therefore purchased as a substitute for that diet in sampling peri- lysine-tergitol 4 agard; plates were incubated at 37oC. Plates
ods 3 and 4. As a result of that substitution, 20 of the raw meat were examined at 24 and 48 hours for growth of hydrogen sul-
products were purchased during all 4 purchasing periods. In fideproducing colonies. If colonies were present, 1 colony
addition, 2 canned and 2 extruded dry dog foods were also arbi- from each plate was transferred to a blood agar plate and incu-
trarily selected to serve as controls; those products were major bated at 37oC for 18 to 24 hours. Isolates were tested with poly-
brands that were sold nationally and purchased at local retail O grouping antisera,d and isolates with positive results were
outlets. All products were ordered and purchased without assumed to be S enterica. Salmonella serogroupspecific antisera
informing the suppliers of the intended use for the products. were used to characterize each isolate, and isolates were sent to
Raw meat products were received frozen and stored at 20oC the USDA National Veterinary Services Laboratories for deter-
until evaluated. mination of serotype.
Processing for microbial cultureNone of the raw Campylobacter spp culturesOne milliliter of each
meat products were accompanied by instructions for thawing processed food sample was mixed with 8 mL of
or preparation. Frozen products were thawed at room tem- Campylobacter enrichment broth and incubated at 42oC for
perature (22C) in the original packaging for 8 to 10 hours 48 hours.e After incubation, samples were transferred with a
before sampling. Three 25-g samples of each diet were swab to Campylobacter agar,a incubated in a microaerophilic
obtained from different sections of the purchase lot, mixed environmentf to promote Campylobacter growth, and incu-
with 225 mL of sterile saline (0.9% NaCl) solution in a sealed bated for 72 hours at 42oC.
plastic bag, and placed in a paddle mixer for 30 seconds. Storage of isolatesAn individual colony of each
Sterile swabs were used to transfer samples of the processed NTSEC and S enterica isolate was incubated in tryptic soy
mixture for bacteriologic analyses, and 1 mL of each product broth for 12 hours at 35oC. After incubation, 750 L was
was placed in a microcentrifuge tube for PCR assay. mixed with 750 L of sterile glycerol. The solution was vor-
Processing 3 samples from each of the 4 purchased lots for texed and stored at 70oC until further testing.
each diet resulted in 240 samples processed for analysis from
the raw meat diets, 24 samples from the dry foods, and 24 Antimicrobial susceptibility testingIsolates were
samples from the canned foods. assessed for susceptibility to 16 antimicrobials. Minimum
inhibitory concentrations of isolates were determined by use
Direct microbial cultureSamples were transferred to of a semiautomated antimicrobial susceptibility systemd and
tryptic soy agar plates with 5% sheep blooda to assess aerobic interpreted according to the Clinical and Laboratory
bacterial contamination and to MacConkey agara to assess Standards Institute (CLSI; formerly NCCLS) guidelines for
gram-negative bacterial contamination. Plates were incubat- broth microdilution methods. The group of antimicrobial
ed at 37oC for 18 to 24 hours, and bacterial growth was drugs was chosen to be analogous with those used in the
assessed semiquantitatively by use of a scale (values from 0 United States for the National Antimicrobial Resistance
to 3) to evaluate bacterial growth. Briefly, this scoring system Monitoring System for enteric bacteria. Antimicrobial sus-
was developed by use of a reference strain of E coli (ATCC ceptibility testing was performed according to the manufac-
strain 25922) inoculated into tryptic soy broth and incubat- turers instructions.g The following antimicrobials were test-
ed for 18 hours at 37oC. Ten-fold dilutions of broth were ed: amikacin, amoxicillin-clavulanic acid, ampicillin,
inoculated onto blood agar plates and incubated at 37oC for apramycin, ceftiofur, ceftriaxone, cefoxitin, cephalothin,
18 hours to estimate the concentration of bacterial CFUs in chloramphenicol, ciprofloxacin, gentamicin, kanamycin,
the broth. Aliquots of the 10-fold dilutions were transferred streptomycin, sulfamethoxazole, tetracycline, and trimetho-
into a sterilized organic matrix (finely chopped straw), and prim-sulfamethoxazole. Escherichia coli ATCC 25922,
samples of the contaminated matrix were transferred with Enterococcus faecalis ATCC 29212, S aureus ATCC 29213,
sterile swabs to blood agar plates and MacConkey agar (for and Pseudomonas aeruginosa ATCC 27853 were used as con-
raw food samples) and incubated at 37oC for 18 hours. trols in antimicrobial MIC determinations.
Bacterial growth from samples of the contaminated organic
matrix was visually scored on agar plates by use of a semi- PCR assaysPolymerase chain reaction assay was per-
quantitative scale (values from 0 to 3) for scoring numbers of formed on all samples purchased in the third and fourth sam-
CFUs. The semiquantitative scores were compared with the pling periods. A 1-mL portion of each paddle-mixed sample
estimated number of CFUs used to contaminate the matrix. was placed in a microcentrifuge tube after processing and
Results suggested that plates with a score of 1 had 5.85 X 10 tested via PCR assay in a single reaction. Samples with posi-
538 Scientific Reports: Original Study JAVMA, Vol 228, No. 4, February 15, 2006
tive results were reprocessed and retested via PCR assay to utes at 72oC (extend). That cycle was repeated 40 times and
confirm the positive results and then sequenced. was followed by 5 minutes of incubation at 72oC.
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Polymerase chain reaction assays were performed for
detection of DNA from Cryptosporidium spp, Neospora spp, Toxoplasma spp PCR assayAmplification was per-
and Toxoplasma spp, according to published protocols.11,13-16 formed according to a published protocol.11 Briefly, 5 L of
For all assays, DNA was extracted by use of a commercial- each sample was added to a thin-walled tube containing the
ly available kit.h After PCR analysis, 10 L of each sample PCR mixture (5 L of 10X piperazine-EDTA buffer, 5 L of
was analyzed on 2% agarose gels in tris-borateEDTA 25mM MgCl2, 1 L of 10mM dNTPs, 0.25 L of each primer
buffer, stained with ethidium bromide (concentration, 40 [100M], 0.5 L of goldTaq [1 U/reaction], and 33 L of dis-
g/L), and viewed by use of a UV transilluminator. tilled water) for a final volume of 50 L. The primers used
Amplifications were conducted in a thermocycler.i A posi- were 5' CGCTGCAGGGAGGAAGACGAAAGTTG-3' and 5'
tive and a negative control were included for each run of CGCTGCAGACACAGTGCATCTGGATT-3', which detect a
the PCR assays. product of 529 bp. Initial denaturation of DNA proceeded at
95oC for 7 minutes and was followed by a cycle of 60 seconds
Cryptosporidium spp PCR assayThe assay for detection at 94oC (denature), 60 seconds at 55oC (anneal), and 60 sec-
of Cryptosporidium spp included primers that recognized onds at 72oC (extend). That cycle was repeated 35 times and
genomic material from Cryptosporidium parvum, certain strains was followed by 10 minutes of incubation at 72oC.
of Cryptosporidium canis, and certain strains of Cryptosporidium
felis.j Amplification was performed via a published protocol.13 Results
Briefly, 2 L of each sample was added to a thin-walled tube con- Fifty-three percent (153/288) of samples examined
taining the PCR mixture (5 L of 10X piperazine-EDTA buffer,
3.5 L of 25mM MgCl2, 1 L of 10mM dNTPs, 1 L of each via culture in enrichment broth (including raw, dry, and
primer [100M], 0.4 L of goldTaq [1 U/reaction], and 34.1 L canned products) were contaminated with NTSEC
of distilled water) for a final volume of 50 L. The primers used (Table 1). By use of enriched culture methods, NTSEC
were awaF 995=5' TAGAGATTGGAGGTTGTTCCT-3' and were recovered at least once from raw meat products
awaR 1206=5' CTTCCACCAACTAAGAACGGCC-3', which derived from all species of source animals tested and
detect a product of 256 bp. Initial denaturation of DNA pro- 90.5% (19/21) of the specific raw meat products evaluat-
ceeded at 96oC for 10 minutes and was followed by a cycle of 30 ed contained NTSEC during at least 2 of the sampling
seconds at 96oC (denature), 30 seconds at 58oC (anneal), and 30 periods. Ten of the 21 (47.6%) raw meat products con-
seconds at 72oC (extend). That cycle was repeated 41 times and tained NTSEC at each of the 4 sampling periods. Among
was followed by 5 minutes of incubation at 72oC.
all samples of raw meat products, NTSEC were recovered
Neospora spp PCR assayAmplification was per- from 59.6% (143/240); NTSEC were isolated from raw
formed according to a published protocol.12,15 Briefly, 5 L of meat products from all vendors and all manufacturers.
each sample was added to a thin-walled tube containing the Those bacteria were also recovered from all 4 of the canned
PCR mixture (5 L of 10X piperazine-EDTA buffer, 2 L of and dry diets during the first sampling period and 1 of the
25mM MgCl2, 1 L of 10mM dNTPs, 1 L of each primer dry-food products during the second sampling period.
[100M], 0.5 L of goldTaq [1 U/reaction], and 35.5 L of
distilled water) for a final volume of 50 L. The primers15
Salmonella enterica was isolated from 17 samples, all
used were Np21=5'-GTGCGTCCAATCCTGTAAC-3' and of which were obtained from raw-meat products (7.1%
Np6=5'-CAGTCAACCTACGTCTTCT-3', which detect a [17/240] of raw meat samples; 5.9% [17/288] of all sam-
product of 328 bp. Initial denaturation of DNA proceeded at ples; Table 1). Those 17 samples were derived from 10
94oC for 3 minutes and was followed by a cycle of 60 seconds raw meat products. Overall, S enterica was isolated at
at 94oC (denature), 60 seconds at 50oC (anneal), and 2 min- least once from 47.6% (10/21) of raw meat products.
Table 1Results of enriched bacterial culture for NTSEC and Salmonella enterica in 21 commercially available raw meat diets, 2 com-
mercially available dry diets, and 2 commercially available canned-food diets for dogs. Each product was purchased and sampled on 4
occasions, approximately 2 months apart.
No. of samples Positive results Positive results Positive results Positive results Total Total
Product in each period in period 1 in period 2 in period 3 in period 4 positive tested
Raw beef
NTSEC 27 12 20 22 17 71 108
Salmonella 27 2 3 2 1 8 108
Raw chicken
NTSEC 15 6 12 8 5 31 60
Salmonella 15 0 0 4 0 4 60
Raw turkey
NTSEC 12 or 9* 5 9 6 3 23 42
Salmonella 12 or 9* 0 0 3 0 3 42
Raw lamb
NTSEC 6 or 9 5 4 2 7 18 30
Salmonella 6 or 9 0 0 2 0 2 30
Canned
NTSEC 6 2 0 0 0 2 24
Salmonella 6 0 0 0 0 0 24
Dry
NTSEC 6 5 3 0 0 8 24
Salmonella 6 0 0 0 0 0 24
Overall
NTSEC 72 35 48 38 32 153 288
Salmonella 72 2 3 11 1 17 288
*Only 9 samples of turkey products were tested in periods 3 and 4. Only 6 samples of lamb products were tested in periods 1 and 2.
JAVMA, Vol 228, No. 4, February 15, 2006 Scientific Reports: Original Study 539
Salmonella enterica was recovered from multiple samples
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540 Scientific Reports: Original Study JAVMA, Vol 228, No. 4, February 15, 2006
among Salmonella spp isolates were similar to those in tially pathogenic enteric microorganisms. There was
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NTSEC. Resistance to sulfamethoxazole was most com- great variation in the susceptibility phenotypes among
mon among S enterica isolates (71.4%), and no resistance NTSEC isolates; considering that only 1 colony was
was detected to amikacin, apramycin, ciprofloxacin, ceftri- chosen for analysis per agar plate, it is likely that more
axone, or trimethoprim-sulfamethoxazole (Figure 3). phenotypes were included but not detected.
Salmonella enterica was recovered from 5.9% of all
Discussion samples. Excluding the dry and canned products
It is well recognized that raw meat may be contam- (which all had negative results for growth of
inated with a variety of microbes. Contamination is Salmonella spp), 7.1% of raw-meat diets were contam-
generally associated with the methods used for har- inated with S enterica. Previous investigators have
vesting and processing. Although many interventions reported that 20% to 35% of poultry carcasses intend-
are required by law to minimize microbial contamina- ed for human consumption have positive results of
tion of meat products sold for human consumption, tests for Salmonella spp,18 an estimate that is in contrast
those laws do not apply to meat products sold for con- to our findings, in which Salmonella spp were isolated
sumption by pets. Results of this study indicate that from only 2.1% of poultry samples.
raw meat products sold as dog food are commonly con- It is commonly presumed that half of the raw
taminated by various microbial agents. Even with chicken sold for human consumption in the United
direct (unenriched) culture methods, 99% of raw meat States is contaminated with Campylobacter spp.18 In 1
samples had some type of bacterial contamination, and study,19 Campylobacter jejuni was isolated from 98% of
more than 1 type of bacteria was recovered from most samples collected from chicken carcasses intended for
of those samples. Without enrichment, nearly 80% of human consumption. We expected that some samples
raw meat samples were contaminated with gram-nega- from poultry products in our study would contain
tive bacteria and there is a strong likelihood that at Campylobacter spp, but Campylobacter spp was not
least a small proportion of the gram-negative bacteria detected. The most likely explanation for this is that
were enteric pathogens that could cause infections in Campylobacter spp do not tolerate drying, the organism
humans or animals. Results from bacterial culture of can be killed by exposure to oxygen, and freezing is
commercial dry and canned diets suggest that those known to reduce the number of Campylobacter bacte-
products had less bacterial contamination, compared ria. Use of PCR assay to detect Campylobacter spp DNA
with raw meat diets. However, a limited number of may enhance detection of the bacterium from samples
samples of the dry and canned diets were included as in future studies.
controls, and this did not allow for statistical analyses Because of reported18 contamination levels in raw
or comparisons. Further investigation is warranted to meat samples, we expected that Toxoplasma spp,
make quantitative comparisons of the degree of bacterial Cryptosporidium spp, and Neospora spp would be detected
contamination among those types of commercial diets. in numerous samples in the present study. However, only
Many of the products contained other raw ingredients 3 samples had positive results for Cryptosporidium spp
(eg, eggs and vegetables) besides meat. Addition of those DNA, and DNA from neither Toxoplasma spp nor Neospora
ingredients, especially raw eggs, could also contribute to spp was detected. The single canned product that was con-
the risk of contamination with important bacterial taminated with Cryptosporidium spp contained fishmeal,
pathogens.16 with or without liver or intestinal tissues that could have
The USDAs Food Safety Inspection Service is served as a source of contamination. The low detection
presently responsible for ensuring that the domestic rate for those organisms may have been a consequence of
meat supply is safe and that contamination of meat the primers chosen for the PCR assay, but sensitivity and
products with bacterial pathogens is minimal, whereas specificity of those primers have been described as high in
the FDAs Centers for Food Safety and Applied earlier reports.20 Samples in the present study may have
Nutrition are responsible for overseeing the safety of contained inhibitors of the reaction, or may have been
eggs and milk. Unlike food intended for human con- contaminated with the organisms in numbers below the
sumption, no regulatory agency is responsible for detection threshold for the PCR assay. Detection of DNA
monitoring bacterial contamination in dog foods made via PCR assay does not necessarily correlate with detection
from raw meat, milk, or eggs. The FDAs Center for of live organisms or risk of infection.
Veterinary Medicine has published a guidance docu- The perceived frequency with which BARF diets
ment17 for such products, but no regulatory authority is are fed to dogs suggests that further investigation of
responsible for assuring that those products meet such diets is warranted. Determination of the rate of
guidelines for bacterial contamination. isolation of pathogens from raw meat diets in combi-
More than 60% of all samples had growth of nation with follow-up in dogs fed those diets as well as
NTSEC after enrichment. Because the heat and pres- recovery of pathogens from dogs home environments
sure applied in the manufacturing process for dry and would be useful in assessing more precisely the risks
canned pet foods are adequate to destroy most bacteria, associated with these diets. Our study was not
contamination of the products we evaluated likely designed to detect infection in dogs fed raw meat diets.
occurred after processing. Further testing to detect However, given the frequency with which microorgan-
specific pathogenic strains of E coli, such as 0157:H7, isms of fecal origin were detected and the rate of isola-
was not performed, but isolation of NTSEC is com- tion of animal and human pathogens, there may be
monly used by the Food Safety Inspection Service and potential for animal and human infections to occur as
other agencies as a marker for contamination by poten- a result of feeding raw meat diets to pets.
JAVMA, Vol 228, No. 4, February 15, 2006 Scientific Reports: Original Study 541
a. BBL, Sparks, Md. 9. Chengappa MM, Staats J, Oberst RD, et al. Prevalence of
b. Indole reagent, Anaerobe Systems, Morgan Hill, Calif. Salmonella in raw meat used in diets of racing greyhounds. J Vet Diagn
Invest 1993;5:372377.
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Pseudorabies Virus gB In vitro diagnostic test for the detec- NA USDA licensed
Antibody Test Kit (IDEXX tion of antibody to pseudorabies 11/3/05
Laboratories Inc, virus (PRV) in swine serum, plas-
Westbrook, ME, ma, and meat exudates
US Vet Lic No. 313)
542 Scientific Reports: Original Study JAVMA, Vol 228, No. 4, February 15, 2006