Development and performance evaluation of a microbiological method for screening and LC-MS/MS for conformation of sulfonamides in animal-derived foods.

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium. After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg-1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

Development of a simple screening method for analyzing cereulide toxin in fried rice using liquid chromatography-tandem mass spectrometry.

PURPOSE: The presence of cereulide, an emetic toxin produced by Bacillus cereus, in fried rice samples is critical evidence of food poisoning even in situations where B. cereus could not be detected. This study aims to develop a screening method for analyzing cereulide in fried rice using the QuEChERS procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Cereulide was identified and quantified in fried rice samples using the QuEChERS extraction method and LC-MS/MS. The accuracies of the methods were determined by analyzing fortified blank samples at two concentrations (10 and 50 µg/kg) conducted on three samples daily for five days. RESULTS: The QuEChERS procedure removed matrix compounds from fried rice. Characteristic MS/MS spectra enabled the identification of cereulide. As the matrix effects in seven fried rice samples were within ± 6%, an external solvent calibration curve could be used for quantification. This method exhibited good accuracy ranging from 88 to 89%. The relative standard deviations for both repeatability and intra-laboratory reproducibility were < 4%. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification was 2 µg/kg. The applicability of this method was confirmed using the analysis of cereulide in fried rice samples incubated with emetic Bacillus cereus. CONCLUSIONS: The QuEChERS extraction procedure described herein showed substantial promise as a reliable screening tool for cereulide in fried rice sample.

Simultaneous determination of five carbapenems, highly polar antibiotics, in milk by LC-MS/MS.

The simultaneous determination of five carbapenems (biapenem, doripenem, ertapenem, imipenem, and meropenem) in raw and pasteurised bovine milk samples using LC-MS/MS was achieved and validated. Chromatographic separation was conducted on an InertSustain® AQ-C18 column using 0.1% formic acid in water and acetonitrile as the mobile phase. Target compounds were extracted using acetonitrile/water (20:80, v/v). After the removal of lipids with acetonitrile-saturated hexane, the dissolved protein was denatured with acetic acid. A portion of the supernatant was passed through an Oasis® PRiME HLB cartridge to remove the matrix. This novel method was validated in accordance with the Japanese validation guidelines and exhibited good trueness, ranging from 86.3% to 96.2%, using matrix-matched calibration curves. The relative standard deviation of repeatability ranged from 1.0% to 6.3%, and that of within-laboratory reproducibility ranged from 1.6% to 7.1%. The limit of quantification was 1.0 µg kg-1 for all analytes. None of the 60 milk samples commercially available in Tokyo contained any analytes. This novel method exhibited high-quality performance and can easily be implemented for the routine monitoring of carbapenems, which are highly polar antibiotics in milk.

[Single-laboratory Validation Study and Surveillance Using an Improved Multiresidue Analytical Method for Veterinary Drugs in Livestock Products by LC-MS/MS].

A method for the rapid analysis of multiclass residual veterinary drugs in poultry muscle, egg, and raw milk was validated in accordance with Japanese guidelines. Using LC-MS/MS, 20 veterinary drugs, including sulfonamides, coccidiostats, and macrolides were analyzed in one injection. Analytes were extracted from the samples with acetonitrile and then dehydrated and salted out using magnesium sulfate, trisodium citrate, and sodium chloride. This method was assessed by performing recovery tests of chicken muscle, duck muscle, egg, and raw milk spiked with 20 new target analytes at concentrations of 10 and 100 µg/kg. According to this method, 17 out of 20 target analytes satisfied the guideline criteria in chicken muscle and duck muscle, and all 20 target analytes met the criteria in egg and raw milk. The limit of quantification was less than MRLs for all analytes. Residues were detected in 4 out of 99 samples and analyzed using the validated method, finding that the levels of all residues were lower than the limits of quantification. These results suggest that continuous monitoring for a new trend of veterinary drugs is necessary.

Validation and application of an immunochromatographic test to detect four macrolides and two lincosamides in raw cow milk.

In this study, an immunochromatographic test (using the Charm QUAD2® Test) was used to screen for residual macrolides and lincosamides in raw cow's milk. The validation parameters (selectivity/specificity, detection capability (CCß), and ruggedness) were in agreement with the requirements of[EC] 2021. The selectivity of the immunochromatographic test was verified by the negative results of microbiological tests. The false-positive rate was 0%. The CCß values of the immunochromatographic test for various antibiotics in milk were as follows: erythromycin 0.02 mg/kg, spiramycin 0.1 mg/kg, tilmicosin 0.025 mg/kg, tylosin 0.05 mg/kg, lincomycin 0.15 mg/kg, and pirlimycin 0.15 mg/kg. The determined CCß values were lower than the respective maximum residue limits (MRLs; regulatory limits in Japan) for milk, except for lincomycin (equal to the MRL). The presence of antibiotic groups other than macrolides and lincosamides did not interfere with the specificity of the test. It showed no significant difference in lot-to-lot repeatability. The results obtained by the two researchers showed no significant differences. Finally, the test was applied to milk samples obtained from a tylosin-treated cow. The outcome was positive and in agreement with the results of the chemical analytical and microbiological methods. Therefore, this validated immunochromatographic test is expected to be suitable for routine analysis to ensure milk safety.

Proteomic identification and quantification of Clostridium perfringens enterotoxin using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

PURPOSE: Detection of Clostridium perfringens enterotoxin (CPE) in human stool is critical evidence of food poisoning. However, processing patient-derived samples is difficult and very few methods exist to confirm the presence of CPE. In this study, a technique was developed using proteomic analysis to identify and quantify CPE in artificial gut fluid as an alternative. METHODS: The standard CPE was spiked into artificial gut fluids, and effective methods were developed by employing both a stable isotope-labelled internal standard peptide and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Proteotypic peptide EILDLAAATER formed by tryptic digestion was selected for quantitation of CPE. The peptide was identified using product ion spectra. Although the nontoxic peptides originating from CPE showed very low detectability in extraction and tryptic digestion, they could be detected with sufficient sensitivity using the method we developed. Based on a spiked recovery test at two concentrations (50 and 200 µg/kg), the recovery values were 85 and 78%, respectively. The relative standard deviations of repeatability and within-laboratory reproducibility were less than 8 and 11%, respectively. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification (LOQ) was estimated to be 50 µg/kg. The combination of the product ion spectra and relative ion ratio supported CPE identification at the LOQ level. CONCLUSIONS: To the best of our knowledge, this is the first report of proteomic analysis of CPE using LC-MS/MS. The method would greatly help in assessing CPE reliably.

[Surveillance of Acaricides in Honey].

By using the LC-MS/MS method developed by us, we determined the residual amounts of acaricides in honey samples commercially available in Tokyo from April 2015 to March 2021. The results of analyzing 127 honey samples, amitraz was detected in 85 samples at the level of 1.1-34.1 µg/kg. Propargite was detected in 3 samples at 2.4-3.8 µg/kg. None of them was beyond the Japanese MRLs or uniform limits. In these survey for 6 years, amitraz was detected in high rate throughout the year. But, the present results imply that amitraz has been used properly in actual bee-keeping because of no violation of MRL and less fluctuation in the detected levels. On the other hand, propargite was detected at the levels over LOQ in domestic honey samples for the first time in 2020, which may suggest a new trend of acaricide use in apiculture in Japan.

Monitoring of residual antibacterial agents in animal and fishery products in Tokyo from 2003 to 2019: application and verification of a screening strategy based on microbiological methods.

Residual antibacterial agents in 5909 animal and fishery products in Tokyo, Japan, were investigated over 17 consecutive years (2003-2019). Monitoring of 32 antibacterial agents (lincosamides, macrolides, penicillins, quinorones and tetracyclines) per product was accomplished via two steps: screening (by microbiological methods) and confirmation (by instrumental methods). Microbiological screening methods identified presumptive groups and determined semi-quantitative values. The instrumental methods quantified 81 residues of 11 different antibacterial agents in 72 samples. The screening strategy based on microbiological methods demonstrated the following: (i) the majority of the samples (over 99%) met Japanese regulations, (ii) using multiple methods provided a reliable inspection system with accurate quantitative values and (iii) there was a constant presence of tetracyclines and unexpected residues (lincomycin and norfloxacin) in various products. Thus, this long-term monitoring and screening strategy provided evidence that the frequencies and trends of residual antibacterial agents not only enhance food safety but also help to prevent antimicrobial resistance.

[Determination of Antibacterial Agents for Animals in Swine Muscles by Microbiological Screening and LC-MS/MS].

The determination of antibacterial agents for animals in swine muscles was improved by microbiological screening and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. In the first instance, the residual drugs were extracted from the samples using the Na2EDTA-McIlvaine buffer (pH 6.0). Subsequently, the agents were purified utilizing a PLS-3 cartridge and extracted with acetonitrile. Considering the microbiological methods, the sensitivities of the investigated drugs were higher and the test plate conditions were improved using a new reference organism Geobacillus stearothermophilus. As a result, a microbiological screening approach able to detect 33 drugs at MRL was developed in Japan. Remarkable, no false positives were detected. Moreover, the same preparation method enabled rapid and reliable microbiological screening, resulting in efficient screening with no undeterminable results.

[Comparison of Validation Results Obtained by GC-MS/MS and LC-MS/MS for the Analysis of Residual Pesticides in Agricultural Products].

In the field of food analysis and regulation, different instruments are used to determine the accuracy of quantification values. This is essential, as inconsistencies in values are commonly encountered. To visualize the degree of these discrepancies in each food matrix, we compiled a validation study based on a routine method developed in our laboratory, for 121 pesticides in six agricultural products, namely the grapefruit, potato, paprika, cabbage, spinach, and brown rice. These were analyzed by GC-MS/MS and LC-MS/MS, and the results were compared mainly on the basis of trueness. According to the results of the validation study when using GC-MS/MS, of the 121 pesticides tested in each product class, the number of analytes that satisfied the criteria of the Japanese validation guidelines was 97 in grapefruit, 111 in potato, 110 in paprika, 118 in cabbage, 111 in spinach, and 63 in brown rice. In contrast, in the analysis of the same samples by using LC-MS/MS, the number of analytes that satisfied the criteria of the validation guidelines was 50 in grapefruit, 114 in potato, 103 in paprika, 112 in cabbage, 100 in spinach, and 103 in brown rice. Inconsistences in the differences of trueness were mainly attributed to matrix effects of each instrument, as well as to food matrices, of which the most diverged matrix was that of brown rice (over 20%).

Development of an alternative approach for detecting botulinum neurotoxin type A in honey: Analysis of non-toxic peptides with a reference labelled protein via liquid chromatography-tandem mass spectrometry.

In this study, we developed a reference labelled protein containing the partial amino acid sequence of botulinum neurotoxin type A (BoNTA). We also applied it as an internal standard to detect specific and non-toxic peptides originated from BoNTA in honey with the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Original proteins in the honey sample were collected through a two-step process that included solubilisation and trichloroacetic acid (TCA) precipitation. Solubilisation by adding water enabled processing of proteins in honey. TCA precipitation collected proteins without specific binding. The combination of protein alkylation and an appropriate enzyme-to-protein ratio ensured feasibility of tryptic digestion. A desalting process eliminated a large amount of salts and other tryptic peptides in the honey sample. The use of the reference labelled protein enabled compensation for tryptic digestion efficiency and electrospray ionisation efficiency based on LC-MS/MS measurement. After the peptide selection and protein BlastP analysis, five unique peptides were chosen. The non-toxic peptides originating from BoNTA were reliably detected using LC-MS/MS based on a multiple-reaction monitoring mode. Detection of several peptides ensured screening of BoNTA in honey samples. Based on the responses, the proteotypic peptide LYGIAINPNR was selected as the quantitative peptide. Due to maintaining the relative ion ratios, the selective transition completely identified the non-toxic peptides. The intensity of the transitions established a detection limit of BoNTA estimated to be 9.4 ng mL-1. Although extraction efficiency was not evaluated using the BoNTA standard, the results suggested this method may be used for quantification of BoNTA in honey. The method was applied to 19 honey samples purchased in Tokyo; none of them was found to contain the target toxin. Overall, the method is expected to accelerate BoNTA monitoring for food safety.

[Validation of a Method for Determining Multiple Residual Pesticides in Dried Red Pepper Using LC-MS/MS].

An analytical method has been developed and validated for determining 107 pesticide residues in dried red pepper using LC-MS/MS. LC method, the clean-up and sample dilution processes were examined to determine their impact on reducing the matrix effects. Clean up was performed using an ENVI-CarbIITM/PSA (300/600 mg, 6 mL) SPE cartridge. In the sample dilution process, eight-fold dilution was used. In the validation of the developed method at two concentrations (0.01 and 0.1 µg/g) for 107 pesticides, 96 pesticides showed recovery rates in the range of 70.1 to 112.6%, RSDs of repeatability of ≤11.5 and 3.4%, and RSDs of within-laboratory reproducibility of ≤24.3 and 19.9%. These values fulfill the criteria of the validation guidelines for pesticide residues in Japan. It is concluded that matrix effects and low recovery rates in the process of extraction are the main factors for values that do not conform to the criteria.

Surveillance of Chlorantraniliprole Residues in Vegetables and Fruits Using LC-MS/MS.

We measured the residual amounts of chlorantraniliprole in various vegetables and fruits. Sample solutions were prepared according to our routine procedure based on the QuEChERS method and analyzed by LC-MS/MS. Performance characteristics were evaluated for 8 kinds of food samples by means of recovery tests of 5 replicates at the concentration of 10 ng/g. Recoveries and RSDs (％) ranged from 50.2 to 93.4％ and from 2.1 to 9.7％, respectively. Application of this method to survey 207 vegetables and 163 fruits gave detection rates of 8.2 and 1.2％, respectively. In vegetables, detection rates were high in okra (4 out of 10 samples), paprika (4 out of 23 samples) and tomato (2 out of 6 samples), and leaf vegetables such as lettuce, mizuna, spinach and wrinkled greens also contained high concentrations of chlorantraniliprole. The highest residual concentration was 571 ng/g in mizuna. The samples containing chlorantraniliprole seemed to be mainly from Asian countries, including samples of domestic Japanese origin. However, none of them contained more than the MRL, which suggests that the use of chlorantraniliprole has been properly controlled.

Simultaneous determination of multi-class veterinary drugs in chicken processed foods and muscle using solid-supported liquid extraction clean-up.

We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography-mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70-105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0µg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.

Simultaneous determination of amantadine, rimantadine, and memantine in processed products, chicken tissues, and eggs by liquid chromatography with tandem mass spectrometry.

A simultaneous determination of amantadine, rimantadine, and memantine in processed products (deep-fried chicken, fried chicken, fried quail egg, and grilled chicken) with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This new method was also applicable for chicken tissue (muscle, liver, and gizzard) and eggs. The chromatographic separation was performed on a Kinetex® XB-C18 core-shell technology column using a mobile phase of acetonitrile and 0.1% formic acid in a 10mmol/L ammonium formate solution, resulting in the complete separation of isomers (rimantadine and memantine) and any other obstructive peaks from the sample matrices. Sample preparation was performed by a modified QuEChERS method using acetonitrile and a 0.1% acetic acid extraction solution and cleaned using an Oasis® MCX cartridge. The sample matrix had no effect on the identification of the compounds. For quantification, an external solvent calibration curve was used. This new method exhibited good accuracy ranging from 79.9% to 91.5%. The relative standard deviation of repeatability (RSDr) ranged from 1.2% to 3.6% and the relative standard deviation of within-laboratory reproducibility (RSDWR) ranged from 1.3% to 6.0%. These standard deviations satisfied the criteria for Japanese validation guidelines. The limit of quantification (LOQ) was 1.0µg/kg for all samples. Analyte residues were not detected in 55 samples using the validated method.

Determination and surveillance of hydrocortisone and progesterone in livestock products by liquid chromatography-tandem mass spectrometry.

A simple analytical method for the determination of hydrocortisone and progesterone in bovine, swine, and chicken muscle and eggs was developed. Hydrocortisone and progesterone were extracted with acetonitrile and subsequently cleaned-up using an Oasis HLB mini-cartridge. The method was validated in accordance with Japanese guidelines and exhibited trueness from 86.6% to 104.3% and precision (relative standard deviations (RSDs) of repeatability and within reproducibility were under 8.7% and 11.7%, respectively). The method was applied to 103 bovine muscle, 137 swine muscle, 69 chicken muscle and 52 egg samples that were commercially available in Tokyo, Japan. The hydrocortisone concentration was 0.9-41.2 µg kg(-1) in all bovine muscle samples, with an average of 7.7 µg kg(-1) and a median of 6.2 µg kg(-1). The progesterone concentration in 50 samples exceeded the limit of quantification (LOQ) and reached a maximum of 95.4 µg kg(-1). Hydrocortisone was also detected in all swine muscle samples at concentrations of 2.0-56.0 µg kg(-1). Its average and median concentrations amounted to 13.1 and 11.3 µg kg(-1), respectively. Twenty-three samples contained progesterone levels surpassing the LOQ, with a maximum concentration of 107.0 µg kg(-1). No chicken muscle samples contained any of the analytes. The progesterone concentration was 15.5-200.0 µg kg(-1) in all egg samples, with an average of 95.4 µg kg(-1) and a median of 90.5 µg kg(-1).

Comparison of Toxoplasma gondii Seroprevalence in Shelter Cats and Dogs during 1999-2001 and 2009-2011 in Tokyo, Japan.

Toxoplasma gondii is an important human health concern with respect to abortion, congenital hydrocephalus, and encephalitis in immunocompromised people. Cats and dogs both are potential sources of T. gondii because they have close contact with humans. However, no epidemiological surveys have been conducted in Tokyo over the past decade. Therefore, the present study investigated and compared the seroprevalence of T. gondii infection in shelter cats and dogs during 1999-2001 and 2009-2011 in Tokyo, Japan. Serum samples were collected from 337 shelter cats and 325 shelter dogs in urban and suburban areas of Tokyo, during 1999-2001 (233 cats and 219 dogs) and 2009-2011 (104 cats and 106 dogs). T. gondii antibodies were measured in the serum samples using a commercial latex agglutination test. Data were compared using the Fisher's exact test, and significance was indicated at P < 0.05. The overall seroprevalence of T. gondii infection in cats was 5.6% (13 of 233) in 1999-2001 and 6.7% (7 of 104) in 2009-2011, and that in dogs was 1.8% (4 of 219) and 1.9% (2 of 106), respectively. Significantly higher seroprevalence was observed in cats from suburban areas compared with cats in urban areas during both periods (P < 0.05). These results reveal that there has been little change in the feline and canine seroprevalence over the past decade, indicating that the risk of T. gondii exposure for cats and dogs in Tokyo is considerably low as the seroprevalence has reached a steady state.

[Determination of Amantadine in Poultry Tissues and Egg by LC-MS/MS].

An accurate and selective analytical method for amantadine, which is used as antiviral drug to treat influenza A virus infection, was developed using LC-MS/MS. Residual amantadine was extracted from 4 kinds of food sample (poultry muscle, liver, gizzard and egg) with acetonitrile-pH 3.0 McIlvaine buffer (7 : 3), then cleaned up with an Oasis® MCX mini-cartridge. An external standard calibration curve was used for quantification, after sample purification by the combination of a reverse-phase strong cation exchange mixed mode cartridge for cleanup and a HILIC column for HPLC. The method was validated by performing recovery tests in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Recovery ranged from 79.3% to 91.7%, RSDs of repeatability were under 3.3%, and RSDs of within-laboratory reproducibility were under 8.4%. This new method was applied to samples of poultry and egg purehased in Tokyo, but residual amantadine was not detected at all.

Determination and surveillance of nine acaricides and one metabolite in honey by liquid chromatography-tandem mass spectrometry.

A simple and accurate analytical method for the determination of acaricides in honey was developed and validated in accordance with Japanese validation guidelines. Analytes - amitraz, N-2,4-dimethylphenyl-N-methylformamidine (DMPF), etoxazole, fenpyroximate, fipronil, hexythiazox, propargite, pyridaben and spirodiclofen - were extracted with ethyl acetate under basic conditions and subsequently cleaned up using an InertSep(®) MA-1 polymer-based anion-exchange column. The method was validated by fortified recovery tests at three different concentrations (1, 5 and 10 µg kg(-1)) performed with three samples daily on five different days. The method exhibited recoveries of 77-116% and precision (relative standard deviations - RSDs) of repeatability and within-laboratory reproducibility ranged from 2% to 22% and from 3% to 23%, respectively. The sample solution was successfully cleaned up to enable quantification using external solvent calibration curves. The limits of quantification (LOQs) were estimated to be 1 µg kg(-1) for all analytes. The method was applied to honey samples commercially available in Tokyo, Japan. Analysis of 250 honey samples indicated that amitraz was present in 127 samples, and that its residual concentration was less than 20 µg kg(-1). Propargite was detected in 23 samples at concentrations less than 1 µg kg(-1).

Prevalence of Dirofilaria immitis among shelter dogs in Tokyo, Japan, after a decade: comparison of 1999-2001 and 2009-2011.

Changes in the seroprevalence of Dirofilaria immitis infection among shelter dogs between a decade ago and the present were evaluated. Serum samples were collected from 200 adult dogs in urban and suburban areas in Tokyo, Japan, during two 2-year periods (April 1999 to March 2001 and April 2009 to March 2011). Sera were tested for the presence of D. immitis antigen using a specific commercialized kit. The seroprevalence of D. immitis infection was 46% in 1999-2001 and 23% in 2009-2011. A decrease was observed in the prevalence of infection between 1999-2001 and 2009-2011; in particular, the prevalence in urban areas decreased significantly compared with that in suburban areas (P < 0.01). There was no significant difference in prevalence between the sexes in each period, but there was a significant difference between mixed-breed and purebred dogs (P < 0.01). The decrease in prevalence of canine heartworm disease in urban areas could be related to better veterinary care.