Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques.

Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

[Prevalence and Characteristics of Campylobacter and Salmonella in Meat Derived from Spent Hens].

Meat derived from spent hens as well as broilers is destined for human consumption. There are many reports on the prevalence and antimicrobial resistance of Campylobacter and Salmonella in broiler meat, but few in spent hen meat. Therefore, we investigated the prevalence and antimicrobial resistance of these genera in spent hen meat collected at chicken processing plants. Campylobacter and Salmonella were isolated from 47 (92.2%) and 18 (35.5%), respectively, of breast meat derived from 51 spent hen flocks. Campylobacter jejuni accounted for 87.5% of Campylobacter isolates. The highest resistant rate in C. jejuni isolates was found for ampicillin (45.3%), followed by tetracycline (14.3%) and ciprofloxacin (14.3%). There was no Campylobacter isolate resistant to erythromycin, which is recommended as a first-choice antimicrobial for humans when Campylobacter enteritis is strongly suspected. Of Salmonella isolates, the first and second most frequent serovars were Salmonella Corvallis (30.4%) and S. Braenderup (21.7%), respectively. Of Salmonella isolates, 30.4% were resistant to streptomycin. There was no Salmonella isolate resistant to ciprofloxacin, which is one of the recommended antimicrobials for humans against Salmonella enteritis. This study shows that one third of spent hen meat is contaminated with Campylobacter or Salmonella, and administration of erythromycin or cefotaxime is an effective option for patients with Campylobacter- or Salmonella- enteritis, respectively, caused by consumption of spent hen meat.

[Quantitative Survey of Campylobacter on Chicken Livers in Japan].

Chicken liver is a potential source of campylobacteriosis in humans. Therefore, we determined the number of Campylobacter in chicken liver. In total, 33 vacuum-packed liver products were obtained from retail stores, and found that 27 of the 33 products (81.8%) were contaminated with Campylobacter. Moreover, Campylobacter was isolated from 138 of 149 livers (92.6%) collected from the 27 Campylobacter-positive products. The mean Campylobacter count was 2.3 log10 CFU/g, while Campylobacter count in 22 of the 138 contaminated livers (15.9%) was >3.0 log10 CFU/g. Furthermore, gastrointestinal tract, liver, and bile samples were collected from 35 broilers at chicken processing plants. We isolated Campylobacter from the gastrointestinal tract of 27 broilers (77.1%). Of these 27 broilers, liver of 24 broilers (88.9%) was Campylobacter-positive, with a mean Campylobacter count of 2.8 log10 CFU/g. Of these 24 broilers, bile of 13 broilers (54.2%) was contaminated with Campylobacter (mean Campylobacter count, 3.5 log10 CFU/mL). Among them, bile of 2 broilers had a Campylobacter count of >8.3 log10 CFU/mL. Collectively, these results indicate that livers derived from broilers colonized with Campylobacter are contaminated with Campylobacter at the time of evisceration. Therefore, to prevent foodborne campylobacteriosis in humans, chicken livers should be thoroughly heated before consumption.

Zoonotic Infection with Oz Virus, a Novel Thogotovirus.

Oz virus is a novel thogotovirus isolated from ticks that causes lethal infection in mice. We conducted serosurveillance of Oz virus infection among humans and wild mammals in Japan using virus-neutralization tests and ELISAs. Results showed that Oz virus may be naturally infecting humans and other mammalian hosts.

Histopathological Characterization of Cases of Spontaneous Fatal Feline Severe Fever with Thrombocytopenia Syndrome, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tickborne infectious disease caused by SFTS virus (SFTSV). We report 7 cases of spontaneous fatal SFTS in felines. Necropsies revealed characteristic lesions, including necrotizing lymphadenitis in 5 cases and necrotizing splenitis and SFTSV-positive blastic lymphocytes in all cases. We detected hemorrhagic lesions in the gastrointestinal tract in 6 cases and lungs in 3 cases, suggesting a more severe clinical course of SFTS in felids than in humans. We noted necrotic or ulcerative foci in the gastrointestinal tract in 3 cases, the lung in 2 cases, and the liver in 4 cases. We clarified that blastic lymphocytes are predominant targets of SFTSV and involved in induction of necrotic foci. We also found that thymic epithelial cells were additional targets of SFTSV. These results provide insights for diagnosing feline SFTS during pathological examination and demonstrate the similarity of feline and human SFTS cases.

Development of ciclesonide analogues that block SARS-CoV-2 RNA replication.

Ciclesonide is an inhaled corticosteroid used to treat asthma and is currently undergoing clinical trials for treatment of coronavirus disease 2019 (COVID-19). An active metabolite of ciclesonide, Cic2, was recently reported to repress severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomic RNA replication. Herein, we designed and synthesized a few types of ciclesonide analogues. Cic4 (bearing an azide group) and Cic6 (bearing a chloro group) potently decreased SARS-CoV-2 viral replication and had low cytotoxicity compared with Cic2 (bearing a hydroxy group). These compounds are promising as novel therapeutic agents for COVID-19 that show significant antiviral activity.

Getah virus epizootic among wild boars in Japan around 2012.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

An unexpected case of a Japanese wild boar (Sus scrofa leucomystax) infected with the giant thorny-headed worm (Macracanthorhynchus hirudinaceus) on the mainland of Japan (Honshu).

The giant thorny-headed worm Macracanthorhynchus hirudinaceus is a zoonotic acanthocephalan species with a worldwide distribution. Its natural definitive hosts are primarily pigs and wild boars (Sus scrofa), and scarabaeid beetles serve as the intermediate host. To date, there has only been one record of this acanthocephalan in Japan: a Ryukyu wild boar (Sus scrofa riukiuanus) hunted in 1973 on Amami Island, faraway from the Japanese mainland. The present study reports the second case of this acanthocephalan in Japan: a Japanese wild boar (Sus scrofa leucomystax) hunted in May 2017 in Yamaguchi Prefecture, the westernmost part of the Japanese mainland (Honshu). More than a dozen acanthocephalans (123 to 233 mm in length and 5 to 6 mm in width) were found with their proboscides inserted deeply into the intestinal wall, forming grossly visible nodules on the external surface. Isolated worms underwent a molecular genetic characterization of the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox-1). Long rDNA nucleotide sequences (5870 to 5890 bp) spanning the beginning of the 18S rDNA through to the 28S rDNA, including the internal transcribed spacer regions, and 1384-bp cox-1 nucleotide sequences were obtained. In the future, in conjunction with the accumulation of molecular genetic data of multiple M. hirudinaceus isolates from different endemic localities abroad, our data may help to postulate the origin or present transmission status of this extremely rarely encountered acanthocephalan in Japan.

Detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses.

In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3'-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets.

Isolation of Japanese encephalitis virus and a novel insect-specific flavivirus from mosquitoes collected in a cowshed in Japan.

Cattle do not generally appear to develop severe viremia when infected with Japanese encephalitis virus (JEV), and they can be infected without showing clinical signs. However, two cattle in Japan recently died from JEV infection. In this study, we investigated the presence of different species of mosquitoes and flavivirus in a cowshed in the southwest region of Japan. In this cowshed, the two most common species of mosquitoes collected were Culex tritaeniorhynchus (including Culex pseudovishnui) and Anopheles sinensis. We performed virus isolation from the collected mosquitoes and obtained two flaviviruses: JEV and a novel insect-specific flavivirus, tentatively designated Yamadai flavivirus (YDFV). Phylogenetic analysis revealed that all three JEV isolates belonged to JEV genotype I and were closely related to a JEV strain that was isolated from the brains of cattle exhibiting neurological symptoms in Japan. Genetic characterization of YDFV revealed that the full genome RNA (10,863 nucleotides) showed homology with the Culex-associated insect-specific flaviviruses Quang Binh virus (79% identity) and Yunnan Culex flavivirus (78% identity), indicating that YDFV is a novel insect-specific flavivirus.

Geographical Variation of Antimicrobial Resistance of Salmonella in Japanese Chicken.

Chicken is a potent source of Salmonella infection in humans. Occasionally, patients with severe Salmonella enteritis require antimicrobial therapy. Antimicrobials are used to prevent and treat bacterial infections in broiler and breeder farms. Herein, we investigated the prevalence and antimicrobial resistance of Salmonella in 337 vacuum-packed chicken breast products manufactured in Japan between June and December 2021. Salmonella was isolated from 287 samples (85.2%). Among the products from Eastern Japan, the lowest Salmonella prevalence was observed in those processed in September (65.6%), which was significantly (p < 0.05) lower than that in November or December. Among the products from Western Japan, the lowest Salmonella prevalence was observed in those processed in August (61.9%), which was significantly (p < 0.05) lower than that in June, November, and December. The most frequent serovar was Salmonella Schwarzengrund (223 isolates), followed by S. Infantis (53 isolates), S. Manhattan (9 isolates), and S. Enteritidis (1 isolate). High rates of antimicrobial resistance were observed for streptomycin (64.5%), kanamycin (50.2%), tetracycline (65.2%), nalidixic acid (11.5%), and trimethoprim (35.9%). Resistance rates against these five antimicrobials in S. Schwarzengrund isolates were markedly higher in the isolates from Western Japan than in those from Eastern Japan. All 287 Salmonella isolates were susceptible to ciprofloxacin which belongs to fluoroquinolones and cefotaxime which belongs to third-generation cephalosporins. Salmonella prevalence in chicken products in Japan was found to be extremely high; therefore, chicken meat should be thoroughly heated before consumption. In Japan, fluoroquinolones and third-generation cephalosporins are recommended as the first- and second-choice antimicrobials for patients with severe Salmonella enteritis, respectively. The results of this study show that administering fluoroquinolones or third-generation cephalosporins is an effective option for patients with Salmonella enteritis caused by consuming chicken meat, and efficient strategies for Salmonella management on broiler farms and chicken-processing plants need to be developed.

Changes in the Phenotypes of Salmonella spp. in Japanese Broiler Flocks.

Salmonella infections represent a leading cause of foodborne illnesses; resistance to third-generation cephalosporins (TGCs), which are a first-choice antimicrobial for treating human Salmonella enteritis, has become a serious public health concern worldwide. Because the consumption of undercooked chicken meat products is a major cause of foodborne salmonellosis in Japan, we conducted three surveys at different periods between 2017 and 2022, with the cooperation of four abattoirs (two in Eastern and two in Western Japan). The first survey was conducted at abattoir A, which is located in Eastern Japan. Salmonella was detected in 84.4% of broiler flocks tested (27/32); among them, all the TGC-resistant isolates obtained from one farm (farm FA) were identified as S. Infantis. Salmonella was recovered from 62.5% of breast meat samples (20/32), with one case suggesting cross-contamination. The second survey was conducted at three other abattoirs to examine the prevalence of TGC-resistant Salmonella, in both Western (abattoirs B and C) and Eastern (abattoir D) Japan. Salmonella was detected in 90.6% of broiler flocks examined (29/32). TGC-resistant S. Infantis was isolated from 2 flocks until 2018 and not thereafter. Subsequently, isolates were identified as TGC-susceptible S. Schwarzengrund in both regions. The third survey was performed at abattoir A to elucidate whether there were changes in the phenotypes. Of the 11 broiler flocks introduced from farm FA, 10 were positive for Salmonella (90.9%); all the isolates were S. Schwarzengrund susceptible to TGC. This study shows that TGC-susceptible S. Schwarzengrund has replaced the resistant phenotypes among broiler flocks in both Eastern and Western Japan. Although chicken meat products could be cross-contaminated with Salmonella during the slaughtering process, reducing the prevalence of Salmonella in broiler flocks remains important to decrease Salmonella enteritis in humans.

Antiviral Effect of Ephedrine Alkaloids-Free Ephedra Herb Extract against SARS-CoV-2 In Vitro.

We report for the first time that ephedrine alkaloids-free Ephedra Herb extract (EFE) directly inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and that the addition of EFE to the culture medium before viral infection reduces virus titers in the culture supernatant of SARS-CoV-2, including those of variant strains, by more than 99%, 24 h after infection. The addition of Ephedra Herb macromolecule condensed-tannin, which is the main active ingredient responsible for the anticancer, pain suppression, and anti-influenza effects of EFE, similarly suppressed virus production in the culture supernatant by 99% before infection and by more than 90% after infection. Since EFE does not have the side effects caused by ephedrine alkaloids, such as hypertension, palpitations, and insomnia, our results showed the potential of EFE as a safe therapeutic agent against coronavirus disease 2019.

Antimicrobial resistance profiles of Campylobacter jejuni and Salmonella spp. isolated from enteritis patients in Japan.

Understanding the antimicrobial resistance of Campylobacter jejuni and Salmonella spp. isolated from patients with enteritis will aid in therapeutic decision-making. This study aimed to characterize C. jejuni and Salmonella spp. isolates from patients with enteritis. For C. jejuni, the resistance rates against ampicillin, tetracycline, and ciprofloxacin were 17.2%, 23.8%, and 46.4%, respectively. All the C. jejuni isolates were susceptible to erythromycin, which is recommended as a first-choice antimicrobial if Campylobacter enteritis is strongly suspected. C. jejuni was classified into 64 sequence types (STs), and the five major STs were ST22, ST354, ST21, ST918, and ST50. The ciprofloxacin-resistance rate of ST22 was 85.7%. For Salmonella, the resistance rates against ampicillin, cefotaxime, streptomycin, kanamycin, tetracycline, and nalidixic acid were 14.7%, 2.0%, 57.8%, 10.8%, 16.7%, and 11.8%, respectively. All the Salmonella spp. isolates were susceptible to ciprofloxacin. Therefore, fluoroquinolones are the recommended antimicrobials against Salmonella enteritis. S. Thompson, S. Enteritidis, and S. Schwarzengrund were the three most prevalent serotypes. The two cefotaxime-resistant isolates were serotyped as S. Typhimurium and were found to harbor blaCMY-2. The results of this study would help select antimicrobials for treating patients with Campylobacter and Salmonella enteritis.

HTLV-1 Proliferation after CD8+ Cell Depletion by Monoclonal Anti-CD8 Antibody Administration in Latently HTLV-1-Infected Cynomolgus Macaques.

Human T-cell leukemia virus type 1 (HTLV-1) induces chronic asymptomatic latent infection with a substantial proviral load but without significant viral replication in vivo. Cumulative studies have indicated involvement of CD8-positive (CD8+) cells, including virus-specific CD8+ T cells in the control of HTLV-1 replication. However, whether HTLV-1 expression from latently infected cells in vivo occurs in the absence of CD8+ cells remains unclear. Here, we examined the impact of CD8+ cell depletion by monoclonal anti-CD8 antibody administration on proviral load in HTLV-1-infected cynomolgus macaques. Five cynomolgus macaques were infected with HTLV-1 by inoculation with HTLV-1-producing cells. Administration of monoclonal anti-CD8 antibody in the chronic phase resulted in complete depletion of peripheral CD8+ T cells for approximately 2 months. All five macaques showed an increase in proviral load following CD8+ cell depletion, which peaked just before the reappearance of peripheral CD8+ T cells. Tax-specific CD8+ T-cell responses were detected in these recovered CD8+ T cells. Importantly, anti-HTLV-1 antibodies also increased after CD8+ cell depletion, indicating HTLV-1 antigen expression. These results provide evidence indicating that HTLV-1 can proliferate from the latent phase in the absence of CD8+ cells and suggest that CD8+ cells are responsible for the control of HTLV-1 replication. IMPORTANCE HTLV-1 can cause serious diseases such as adult T-cell leukemia (ATL) in humans after chronic asymptomatic latent infection with substantial proviral load. Proviruses are detectable in peripheral lymphocytes in HTLV-1 carriers, and the association of a higher proviral load with a higher risk of disease progression has been observed. However, neither substantial viral structural protein expression nor viral replication was detectable in vivo. Cumulative studies have indicated involvement of CD8+ cells, including virus-specific CD8+ T cells in the control of HTLV-1 replication. In the present study, we showed that CD8+ cell depletion by monoclonal anti-CD8 antibody administration results in HTLV-1 expression and an increase in proviral load in HTLV-1-infected cynomolgus macaques. Our results indicate that HTLV-1 can proliferate in the absence of CD8+ cells, suggesting that CD8+ cells are responsible for the control of HTLV-1 replication. This study provides insights into the mechanism of virus-host immune interaction in latent HTLV-1 infection.

Detection of hepatitis E virus RNA from pig bile collected at a slaughterhouse in Japan.

Hepatitis E virus (HEV) is a zoonotic pathogen that circulates mainly between pigs and humans. In Japan, the number of confirmed HEV cases has increased over the past decade, with the majority reported as domestic HEV infections. HEV-infected pork products may be associated with this increase, but there is limited information on HEV in pork in Japanese markets. From February to March 2020, gallbladders were collected from 200 slaughtered pigs shipped from 14 farms and were surveyed to detect HEV RNA in bile using reverse transcription quantitative polymerase chain reaction. The samples were then sequenced and genotyped. Twenty pigs were positive for HEV ribonucleic acid, and seven samples had Ct values of less than 30. Among these 20 pigs, virus strains from 14 pigs were determined as genotype 3. This report indicated that HEV-contaminated pork liver was shipped to consumer markets and demonstrated the importance of detection of HEV in meat ready for shipment.

Prevalence and antimicrobial resistance of Campylobacter and Salmonella in layer flocks in Honshu, Japan.

Campylobacter and non-typhoidal Salmonella are the major causes of bacterial gastrointestinal infections in humans. Although antimicrobial therapy is typically not recommended in many cases of these infections, it may be life-saving in patients with severe symptoms. Since chicken eggs and meat derived from layers are destined for human consumption, we investigated the prevalence and antimicrobial resistance of these two bacterial genera in 82 layer flocks at chicken processing plants in Honshu, Japan. Campylobacter was isolated from 77 flocks (93.9%). Resistance to ampicillin, tetracycline, and ciprofloxacin was documented in 42.3 (30/71), 16.9 (12/71), and 14.1% (10/71) of Campylobacter jejuni, respectively. Multilocus-sequence typing identified ST4389 and ST5262 as the most frequent C. jejuni sequence types. In C. coli, resistance to ampicillin, tetracycline, and ciprofloxacin was found in 20.0 (7/35), 20.0 (7/35), and 25.7% (9/35), respectively. The most frequent sequence type in C. coli was ST8292. Erythromycin resistance was not observed among Campylobacter species. Salmonella was isolated from 14 flocks (17.1%). The two most frequent serovars were Salmonella Corvallis and S. Braenderup. Neither S. Enteritidis nor S. Infantis were isolated. Streptomycin resistance was observed in six isolates (26.1%), and all isolates were susceptible to cefotaxime and ciprofloxacin. Thus, chicken eggs and meat derived from layers are possible sources of these bacterial infections in humans. The antimicrobial susceptibility of these isolates was maintained, reflecting restrictions on the use of antimicrobial agents on layers.

Characterization of Campylobacter jejuni in large-billed crows (Corvus macrorhynchos) in Tochigi prefecture, Japan.

As free-living crows are a potential source of Campylobacter infections in broilers and cattle, we characterized Campylobacter spp. isolated from crows using multilocus sequence typing and antimicrobial susceptibility testing. We obtained 82 samples from 27 birds captured at seven different times using a trap set in Tochigi prefecture, Japan. Campylobacter jejuni was isolated from 55 (67.1%) of the 82 samples and classified into 29 sequence types, of which 21 were novel. Tetracycline and streptomycin resistance rates were 18.2% and 3.6%, respectively. These results show that most types of C. jejuni infecting crows differ from those isolated from humans, broilers, and cattle. Thus, the importance of free-living crows as reservoirs of Campylobacter infections in broilers and cattle may be limited.