[A Case of Advanced Gastric Cancer Treated with Conversion Surgery followed by Nivolumab Combination Chemotherapy].

The use of nivolumab as first-line therapy for unresectable advanced gastric cancer has now become a standard practice, and its efficacy has been established. This is the first report of a patient with advanced gastric cancer who underwent conversion surgery after first-line nivolumab combination chemotherapy. The patient was a 58-year-old woman. Her medical history included hypertension and dyslipidemia. She had advanced gastric cancer with extensive lymph node metastasis in the left supraclavicular fossa and around the abdominal aorta. After confirming the HER2-negative status and the PD-L1 CPS score to be ≥5, nivolumab was administered in combination with chemotherapy. After the treatment, she underwent a total gastrectomy with D2 dissection, combined splenectomy and pancreatic tail resection for adhesions, and para-aortic lymph node sampling as a conversion surgery. There was no obvious cancerous remnant in the resected specimen, and the pathological response was Grade 3. The patient was alive and recurrence-free at 4 months postoperatively.

Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice.

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Nationwide Monitoring of Antimicrobial-Resistant Escherichia coli and Enterococcus spp. Isolated From Diseased and Healthy Dogs and Cats in Japan.

The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was established for nationwide monitoring of antimicrobial-resistant bacteria isolated from animals. Here, antimicrobial resistance of Escherichia coli and Enterococcus spp. isolates from diseased and healthy dogs and cats was investigated. Isolates were collected from diseased dogs and cats and from healthy dogs and cats in 2018 to 2020. Minimum inhibitory concentrations were determined for 1873 E. coli and 1383 Enterococcus spp. isolates. E. coli isolates were most commonly resistant to nalidixic acid [diseased dog (DD), 62.1%; diseased cat (DC), 59.9%; healthy dog (HD), 23.5%; healthy cat (HC, 24.0%] and ampicillin (DD, 54.4%; DC, 64.1%; HD, 28.4%; HC, 25.2%), followed by ciprofloxacin (DD, 45.0%; DC, 44.0%; HD, 12.9%; HC, 10.4%). Enterococcus spp. isolates were most resistant to tetracycline (DD, 66.9%; DC, 67.8%; HD, 47.0%; HC, 52.0%), followed by erythromycin (DD, 43.2%; DC, 46.6%; HD, 27.8%; HC, 34.0%) and ciprofloxacin (DD, 27.9%; DC, 43.7%; HD, 9.7%; HC 12.9%). Only a few E. coli isolates were resistant to colistin and none were resistant to meropenem. Also, none of the Enterococcus spp. isolates we have tested were resistant to vancomycin. The significantly higher resistance rates of E. coli and Enterococcus spp. isolates from diseased, as opposed to healthy, dogs and cats against most of the tested antimicrobials indicates that the use of antimicrobials could select resistant E. coli and Enterococcus spp.

Correlation of minimum inhibitory concentrations between human and animal antimicrobials against Escherichia coli isolated from livestock.

We analyzed the correlation between minimum inhibitory concentrations (MICs) of antimicrobials used in humans and those used in animals to enable comparison of antimicrobial susceptibility between Escherichia coli isolated from humans and those from animals. We compared the following pairs of MIC data: piperacillin (PIPC) to ampicillin (ABPC), amikacin (AMK) to kanamycin (KM), minocycline (MINO) to oxytetracycline (OTC), and levofloxacin (LVFX) to enrofloxacin (ERFX) using 103 isolates of E. coli from healthy livestock (cattle, pigs, broiler chickens, and layer chickens). Kappa analysis of the agreement for resistance and susceptibility between PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX showed almost perfect (κ = 0.81), slight (κ = 0.12), fair (κ = 0.37), and moderate (κ = 0.46) agreement, respectively. Within the antimicrobial pairs, all isolates resistant to the human antimicrobial were also resistant to the veterinary antimicrobial. However, there was less agreement within the pairs for those isolates that were sensitive to the human antimicrobial. The percentage agreement for susceptibility, defined as the percentage of isolates sensitive to both antimicrobials compared with isolates sensitive to both antimicrobials, as well as those sensitive only to the human antimicrobial, was 89.9%, 87.3%, 64.0%, and 89.9% for PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX, respectively. Our results suggest that the possibility of missing the resistance for antimicrobials used in human medicine by examining MICs for the equivalent antimicrobials used in veterinary medicine is low.

A Japanese trial to monitor methicillin-resistant Staphylococcus aureus (MRSA) in imported swine during the quarantine period.

OBJECTIVES: This study aimed to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in imported swine during the quarantine period in Japan. METHODS: Nasal swabs from a total of 125 swine belonging to 15 lots (unit of import) from five countries were investigated for MRSA from July 2016 to February 2017. Two isolates per positive lot were chosen for multilocus sequence typing (MLST). PCR was performed to determine the presence of the czrC gene, and antimicrobial susceptibility testing was performed by the broth dilution method. RESULTS: MRSA isolates were obtained from six lots (41 heads; 32.8%) from two countries. All 12 isolates that underwent MLST (two per positive lot) were classified as ST398, harboured the czrC gene and were resistant to ampicillin and tetracycline; some isolates showed additional resistance to erythromycin or streptomycin, but resistance to ciprofloxacin, gentamicin or chloramphenicol was not observed. CONCLUSIONS: MRSA ST398 isolates were obtained from imported swine in this first trial to monitor MRSA during the quarantine period in Japan. For the 'One Health' approach against antimicrobial resistance, monitoring imported animals and generating feedback data would be important.

Prevalence of Colistin Resistance Gene mcr-1 and Absence of mcr-2 in Escherichia coli Isolated from Healthy Food-Producing Animals in Japan.

We screened mcr-1 and mcr-2 genes in 9,306 Escherichia coli strains isolated from healthy animals in the Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) system. mcr-1 was detected in 39 strains (5, 20, and 14 strains isolated from cattle, swine, and broilers, respectively), whereas mcr-2 was not detected. mcr-2 was also not detected with the investigation sequence homology search against our curated GenEpid-J database.

Pathogenic characterization of Erysipelothrix rhusiopathiae Met-203 type SpaA strains from chronic and subacute swine erysipelas in Japan.

To characterize the Erysipelothrix rhusiopathiae Met-203 type surface protective antigen (Spa) A strains causing swine erysipelas in Japan, the nucleotide sequence of the hypervariable region of the spaA gene was determined in 80 E. rhusiopathiae (serotype 1a) isolates collected from pigs with chronic and subacute swine erysipelas in 14 prefectures in 2008-2014. In this study, 14 (17.5%) isolates were Met-203 type SpaA strains. We confirmed the pathogenicity of a Met-203 type SpaA strain in specific-pathogen-free pigs. In this experiment, the two challenged pigs displayed arthritis, urticaria and other clinical signs, but recovered within 10 days. Our results reveal the existence of the E. rhusiopathiae Met-203 type strains that have been causing chronic erysipelas in Japan.

Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Effect of maternal exposure to carbon black nanoparticle during early gestation on the splenic phenotype of neonatal mouse.

Maternal exposure to environmental factors is implicated as a major factor in the development of the immune system in newborns. Newborns are more susceptible to microbial infection because their immune system is immature. Development of lymphocytes reflects an innate program of lymphocyte proliferation. The aim of this study was to investigate the effects of maternal exposure to carbon black nanoparticle (CB-NP) during early gestation on the development of lymphoid tissues in infantile mice. Pregnant ICR mice were treated with a suspension of CB-NP (95 µg kg(-1) time(-1)) by intranasal instillation on gestational day 5 and 9. Spleen tissues were collected from offspring mice at 1, 3, 5, and 14 days postpartum. Splenocyte phenotypes were examined by investigating the pattern of surface molecules using flow cytometry. Gene expression in the spleen was examined by quantitative RT-PCR. CD3(+) (T), CD4(+) and CD8(+) cells were decreased in the spleen of 1-5-day-old offspring in the treated group. Expression level of Il15 was significantly increased in the spleen of newborn male offspring, and Ccr7 and Ccl19 were increased in the spleen of female offspring in the CB-NP group. Splenic mRNA change profiles by CBNP were similar between male and female offspring. This article concluded that exposure of pregnant mothers to CB-NP partially suppressed the development of the immune system of offspring mice. The decrease in splenic T cells in the treated group recovered at 14 days after birth. This is the first report of developmental effect of nanoparticle on the lymphatic phenotype.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish.

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Diffusion-weighted imaging of ovarian torsion: usefulness of apparent diffusion coefficient (ADC) values for the detection of hemorrhagic infarction.

PURPOSE: We undertook this study to evaluate the need for diffusion-weighted (DW) magnetic resonance (MR) imaging in detecting hemorrhagic infarction following ovarian torsion. METHODS: The study included 14 consecutive patients aged 12 to 74 years (average age, 36 years) with surgical confirmation of ovarian torsion who underwent 1.5-tesla MR imaging. Pathologically, hemorrhagic infarction was found in 7 patients. We retrospectively reviewed signal intensity on T1-, T2-, and diffusion-weighted images and apparent diffusion coefficients (ADCs) in swollen ovarian stroma. RESULTS: Fallopian tube thickening was seen in all patients. In patients with ovarian cystic lesion, maximum cyst wall thickness was significantly higher in patients with hemorrhagic infarction (mean, 13.5 ± 4.1 mm) than those without (mean, 5.0 ± 1.0 mm) (P < .05). Signal intensity did not differ significantly on T1-weighted, T2-weighted, and DW images between patients with and without hemorrhagic infarction. ADCs were significantly lower in patients with hemorrhagic infarction (1.20 ± 0.50 [× 10(-3) mm(2)/s]) than those without (2.04 ± 0.26 [× 10(-3) mm(2)/s]) (P < .01). With an ADC threshold of 1.80 [× 10(-3) mm(2)/s], sensitivity for hemorrhagic infarction was 0.88 (7 of 8), and specificity was 1.00 (6 of 6). CONCLUSION: ADC measurements were useful for detecting hemorrhagic infarction in patients with ovarian torsion.

Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan.

Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45-1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan.

Effect of high-fat diet prior to pregnancy on hepatic gene expression and histology in mouse offspring.

Maternal overnutrition and obesity are associated with fetal development and cause long-term effects in offspring. However, the effects of a high-fat diet specific to the pre-pregnancy period are not determined. The present study aimed to examine the effect of high-fat diet prior to pregnancy on the liver of mouse offspring. Female C57BL/6J mice were fed a normal chow (15.2% fat by energy) [control diet (CTR) and CTR pre-pregnancy (PP) groups] or a high-fat chow (31.2% fat by energy) [high-fat diet (HFD) and HFD-pre-pregnancy (PP) groups] for 3-4 weeks and then mated with male C57BL/6J mice fed normal chow. Some mothers continued on the same diet until pups reached 21 days of age (CTR and HFD), and others were fed the different chows from gestational day 0 (CTR-PP and HFD-PP) to determine the effects of a high-fat diet during the pre-pregnancy period in HFD-PP/CTR and HFD/CTR-PP comparisons. Liver tissues from pups were subjected to gene expression analysis by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and microarray, and histological analysis using Oil Red O staining (Sigma Chemical Co., Ltd., Balcatta, WA, USA). Lipid droplets were increased in hepatocytes of mice in HFD-PP compared to CTR and those in HFD compared to CTR-PP. Expression of stearoyl-coenzyme A desaturase 1 (Scd1), acetyl-coenzyme A carboxylase beta (Acacb), and fatty acid binding protein 5 (Fabp5) was increased by maternal high-fat diet during pre-pregnancy. The results showed that maternal high-fat diet intake prior to pregnancy uniquely affects metabolic phenotype related to health and disease in the liver of the next generation.

Development of an in vitro assay based on humoral immunity for quality control of oil-adjuvant Pseudotuberculosis vaccine in Yellowtail Seriola quinqueradiata.

Photobacterium damselae subsp. piscicida is an infectious pathogen that causes Pseudotuberculosis in Yellowtail fish. In Japan, several oil-adjuvant vaccines for Pseudotuberculosis have been approved for control of infectious diseases in aquaculture. Before distribution of an approved fish vaccine, an artificial challenge test for quality control is performed by the manufacturer and National Veterinary Assay Laboratory under Pharmaceutical Law of Japan to confirm potency. In this study, artificial challenge tests with a range of five diluted or undiluted approved vaccines was performed to determine the relationship between antigen levels and vaccine efficacy. Immunization of fish with the undiluted vaccine prevented Pseudotuberculosis. Results of artificial challenge tests demonstrated vaccine efficiency was dose dependent. Agglutination assays using immune sera were performed to determine agglutination titers, which were also dose dependent. These results suggest a link between survival rate in the artificial challenge tests and agglutination titers. Western blotting analysis identified a specific protein approximately 37 kDa in size in vaccinated fish. We confirmed antibodies were produced in vaccinated fish by immunoreactions with the approved vaccine. An agglutination assay based on humoral immunoreactions would be a useful alternative to the artificial challenge test for quality control of vaccines for aquaculture.

Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines.

In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10 in whole-blood culture at 4h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-α and IL-1ß changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-α in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test.

Contribution of enhanced efflux to reduced susceptibility of Salmonella enterica serovar Choleraesuis to fluoroquinolone and other antimicrobials.

We examined antimicrobial susceptibility and efflux systems in laboratory-derived mutants of Salmonella enterica serovar Choleraesuis selected by culture on fluoroquinolone-containing plates. The mutants exhibited decreased susceptibilities to quinolones and several other antimicrobials. Mutations in the gyrA gene were not always found in the mutants. Accumulation assays revealed that intracellular enrofloxacin concentrations were significantly lower in the mutants compared with parent isolates. Increased expression of acrB mRNA can explain the decreased susceptibilities to several antimicrobials but not in the case of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Decreased susceptibility to CCCP may result from the increased expression of emrA mRNA. These results suggest that the enhancement of multiple efflux pumps is responsible for decreased susceptibilities to several antimicrobials in the laboratory-derived mutants.

Intracellular concentrations of enrofloxacin in quinolone-resistant Salmonella enterica subspecies enterica serovar Choleraesuis.

The emergence of fluoroquinolone-resistant strains of Salmonella enterica subspecies enterica serovar Choleraesuis is an important concern in several countries, including Japan. We examined the intracellular concentration of enrofloxacin in S. Choleraesuis to determine the existence of a relationship with the emergence of quinolone resistance. The intracellular concentration of enrofloxacin was significantly lower in nalidixic acid-resistant isolates compared with nalidixic acid-susceptible isolates. In the presence of carbonyl cyanide m-chlorophenylhydrazone, the intracellular concentration of enrofloxacin increased in all isolates, with no significant difference in the intracellular concentration between nalidixic acid-susceptible and -resistant isolates. The frequency of emergence of fluoroquinolone-resistant mutants was higher in susceptible isolates with a low intracellular concentration of enrofloxacin. The results presented suggest that a decrease in the intracellular concentration of enrofloxacin is related to active efflux pumps and contributes to the emergence of fluoroquinolone resistance.

Comparison of three DNA extraction methods for detection of Erysipelothrix rhusiopathiae in chicken blood by polymerase chain reaction.

A previously reported Erysipelothrix-specific polymerase chain reaction (PCR) was used to detect Erysipelothrix bacteremia in chickens. The sensitivity of PCR using 3 DNA extraction methods (boiling method, commercial gene matrix, and DNA extractor kit) was compared by using a serial 10-fold dilution of a chicken isolate of Erysipelothrix rhusiopathiae strain in chicken blood. Of the techniques used, the DNA extractor kit, followed by PCR, provided the most sensitive method for the detection of the E. rhusiopathiae strain in chicken blood (approximately 10(0) CFU/0.1 ml of blood). Two E. rhusiopathiae infection experiments were then attempted. In a total of 10 inoculated chickens, bacteremia developed in 9 chickens, consisting of all 5 chickens used in the first trial (ranging from 5.1 x 10(1) to 2.0 x 10(3) CFU/0.1 ml of blood) and 4 of the 5 chickens used in the second trial (ranging from 1.0 x 10(0) to 3.3 x 10(2) CFU/0.1 ml of blood). In the second trial, the 3 detection techniques were applied to the chickens with bacteremia, and the organism could be detected by using the DNA extractor kit in blood specimens from the 3 chickens exhibiting bacteremia of > or =4.2 x 10(1) CFU/0.1 ml of blood. This observation suggests that most E. rhusiopathiae-infected chickens develop more critical bacteremia than the detectable level by PCR with the DNA extractor kit, and the PCR detection method can be used as a first-line screening of avian erysipelas.

Molecular typing of Japanese field isolates and live commercial vaccine strain of Mycoplasma synoviae using improved pulsed-field gel electrophoresis and vlhA gene sequencing.

In the present study, pulsed-field gel electrophoresis (PFGE) and vlhA gene sequence analysis were applied and verified for typing the Mycoplasma synoviae live vaccine MS-H strain and field isolates from diseased chickens in Japan. The previously published PFGE protocol using SmaI digestion could not allow the discrimination of two of the 11 M. synoviae field isolates from the vaccine strain and had relatively low discrimination power (D = 0.885). On the other hand, our new PFGE protocols using BlnI and BamHI digestions as well as the vlhA sequence analysis allowed the discrimination of all 11 M. synoviae field isolates from the vaccine strain. In addition, these PFGE protocols using BlnI and BamHI digestions generated unique fragment patterns in epidemiologically unrelated isolates, including those with identical SmaI-digested patterns or vlhA gene sequences (D = 0.987 and 1.000, respectively), and generated indistinguishable or closely related patterns in epidemiologically related isolates. Therefore, we believe that they would be useful tools to determine whether M. synoviae clinical isolates from diseased chickens are derived from the vaccine strain or wild-type strain and to further elucidate the epidemiology of M. synoviae infection.