Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions.

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4 kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.

Characteristics of a Temperature-Sensitive Mutant Strain of Salmonella Enteritidis and Its Potential as a Live Vaccine Candidate.

Salmonella Enteritidis is a common foodborne pathogen transmitted through poultry products, which are its main carriers. Poultry are vaccinated against Salmonella Enteritidis in many countries, despite the absence of clinical symptoms, using commercially available live-attenuated vaccines. We previously constructed a highly attenuated temperature-sensitive (ts) Salmonella Enteritidis mutant, 2S-G10. In the present study, we describe the construction and attenuation-associated characteristics of 2S-G10. We infected 1-day-old chicks with 2S-G10 and the parental strains to evaluate the attenuation. One week after infection, 2S-G10 was not detected in the liver, cecum, or cecal tonsil tissues of the orally inoculated chicks, contrary to the parental strain. This indicates that 2S-G10 was highly attenuated when compared to the parental stain. In vitro experiments revealed the inability of 2S-G10 to grow at the normal body temperature of chickens and invade chicken liver epithelial cells. Moreover, single nucleotide polymorphism (SNP) analysis between the complete genome sequence of 2S-G10 and its parental strain revealed SNPs in bcsE, recG, rfaF, and pepD_1 genes, which are involved in epithelial cell invasion and persistence in host systems, growth, lipopolysaccharide core biosynthesis, and cellular survival under heat stress, respectively. These potential characteristics are consistent with the findings of in vitro experiments. Conclusively, chemical treatment-induced random genetic mutations highly attenuated 2S-G10, implying its potential to be developed as a novel live-attenuated vaccine against Salmonella Enteritidis.

Evaluation of Immune Responses and Protective Efficacy of a Novel Live Attenuated Salmonella Enteritidis Vaccine Candidate in Chickens.

An ideal vaccine for controlling Salmonella infection in chicken flocks should be safe, inducing both humoral and cellular immunity. Live attenuated vaccines against Salmonella Enteritidis (S. Enteritidis) have been used as a potential control method of Salmonella infection in the poultry industry. However, live attenuated vaccines can persistently infect poultry for long periods and can become virulent revertant strains. In this study, we assessed the immune responses and protective efficacy of a temperature-sensitive attenuated S. Enteritidis mutant as a potential vaccine candidate. In addition, we evaluated the combined vaccine administration methods to maximize both humoral and cellular immune responses in chickens induced by the vaccine candidate. Immune responses and protective efficacy were compared between the Oral/IM group, vaccinated using one oral dose at four weeks old and a booster intramuscular dose at seven weeks old, and the IM/Oral group, vaccinated using one intramuscular dose at four weeks old and a booster oral dose at seven weeks old. The Oral/IM group showed stronger immune responses than those of the IM/Oral group. Spleens from the Oral/IM group showed a promising tendency of reduction of challenged Salmonella compared with those of other groups. Overall, the results indicated that the S. Enteritidis mutant strain is a promising live attenuated vaccine candidate with good efficacy.

Whole-Genome Sequencing Analysis of a stx-Negative Escherichia coli O63:H6 Isolate Associated with Hemolytic Uremic Syndrome.

Shiga toxin-encoding genes (stx) of enterohemorrhagic Escherichia coli (EHEC) can be lost during infection or in vitro cultivation, and in clinical practice, it is difficult to distinguish EHEC that have lost stx (EHEC-LST) from enteropathogenic E. coli (EPEC), as both are stx-negative and eae-positive. In this study, we performed whole-genome sequencing (WGS) of a stx-negative, eae-positive E. coli O63:H6 isolate from a child with hemolytic uremic syndrome and compared its genome with those of nine E. coli O63:H6 strains in public databases. Virulence gene profiles were analyzed and core-genome multilocus sequence typing (cgMLST) was conducted. The virulence gene profile of our isolate was consistent with EHEC, except for the absence of stx, and the isolate clustered with seven EHEC strains but was distant from two EPEC strains in cgMLST. In genome alignment, our isolate exhibited a high nucleotide identity with EHEC strain 377323_2f but displayed a gap corresponding to the stx-harboring prophage sequence. Overall, our isolate was genetically closely related to EHEC strains, consistent with this being an EHEC-LST strain. As EHEC-LST may be misdiagnosed as EPEC in routine laboratories, comparative genomic analysis using WGS can be useful to determine whether stx-negative and eae-positive isolates are EHEC-LST or EPEC.

Whole-Genome Analysis of Multidrug-Resistant Salmonella Enteritidis Strains Isolated from Poultry Sources in Korea.

The Salmonella Enterica subsp. Enterica serovar Enteritidis is one of main serovars isolated from human patients with food poisoning and poultry without clinical signs. Consumption of poultry products contaminated with Salmonella Enteritidis is a common source of human salmonellosis; 82 Salmonella spp. were isolated from 291 samples of retail chicken meat, 201 one-day-old chicks, 30 internal organs of chickens, 156 chicken eggs, 100 duck eggs, 38 straw bedding samples, 18 samples of retail duck meat, and 19 swab samples from slaughterhouses in 2019 and 2020. An antibiotic susceptibility test was performed for all isolates, revealing 33 multidrug-resistant (MDR) strains. The whole genome of 33 MDR strains isolated in 2019 and 2020 and 10 strains isolated in 2011, 2012, and 2017 was sequenced using the MinION sequencing protocol. Within these 43 samples, 5 serovars were identified: S. Enteritidis, S. Agona, S. Virchow, S. Albany, and S. Bareilly. The most common serovar was S. Enteritidis (26/43), which showed the highest resistance to ampicillin (100%), followed by nalidixic acid (90%) and colistin (83%). Core genome multilocus sequence typing analysis showed that the S. Enteritidis strains isolated from different sources and in different years were clustered together. In addition, the S. Enteritidis strains isolated since 2011 consistently harbored the same antibiotic resistance patterns.

Genome Analysis of Nicotinamide Adenine Dinucleotide-Independent Mycoplasma synoviae Isolates From Korea.

Mycoplasma synoviae (MS) is an avian pathogen that causes respiratory disease, infectious synovitis, and eggshell apex abnormalities in chickens. Nicotinamide adenine dinucleotide (NAD)-independent MS was first reported in 1975. Despite the atypical traits of NAD-independent MS, its independence from NAD has not been studied. In this study, we isolated five NAD-independent strains from Korea and assembled their genomes using sequencing reads obtained from Illumina and Oxford Nanopore Technology platforms. The assembled genomes were compared with the genomes of MS-H vaccine strain and type strain WVU1853. We found that the coding sequences of nicotinate phosphoribosyltransferase and glycerol-3-phosphate acyltransferase, and a unique coding sequence were present only in the genomes of NAD-independent isolates.

Complete Genome Sequence of Mycoplasma gallisepticum Strain KUVMG001, an Isolate from South Korea.

Mycoplasma gallisepticum causes chronic respiratory diseases in poultry and economic losses to the chicken and turkey industry. We report the complete genome sequence of the field isolate strain KUVMG001 of Mycoplasma gallisepticum from South Korea.

Comparison of microbiota in the cloaca, colon, and magnum of layer chicken.

Anatomically terminal parts of the urinary, reproductive, and digestive systems of birds all connect to the cloaca. As the feces drain through the cloaca in chickens, the cloacal bacteria were previously believed to represent those of the digestive system. To investigate similarities between the cloacal microbiota and the microbiota of the digestive and reproductive systems, microbiota inhabiting the colon, cloaca, and magnum, which is a portion of the chicken oviduct of 34-week-old, specific-pathogen-free hens were analyzed using a 16S rRNA metagenomic approach using the Ion torrent sequencer and the Qiime2 bioinformatics platform. Beta diversity via unweighted and weighted unifrac analyses revealed that the cloacal microbiota was significantly different from those in the colon and the magnum. Unweighted unifrac revealed that the cloacal microbiota was distal from the microbiota in the colon than from the microbiota in the magnum, whereas weighted unifrac revealed that the cloacal microbiota was located further away from the microbiota in the magnum than from the microbiota inhabiting the colon. Pseudomonas spp. were the most abundant in the cloaca, whereas Lactobacillus spp. and Flavobacterium spp. were the most abundant species in the colon and the magnum. The present results indicate that the cloaca contains a mixed population of bacteria, derived from the reproductive, urinary, and digestive systems, particularly in egg-laying hens. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken's cloacal microbiota should be performed using chickens of different ages and types.

Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota.

The transferred microbiota from mother to baby constitutes the initial infant gastrointestinal microbiota and has an important influence on the development and health of infants in human. However, the reproductive tract microbiota of avian species and its inheritance have rarely been studied. We aimed to characterize the microbial community in the chicken reproductive tract and determine the origin of the chicken embryo gut microbiota. Microbiota in four different portions of chicken oviduct were determined using 16S rRNA metagenomic approach with the IonTorrent platform. Additionally, we analyzed the mother hen's magnum and cloaca, descendent egg, and embryo gut microbiota. The microbial composition and relative abundance of bacterial genera were stable throughout the entire chicken reproductive tract, without significant differences between the different parts of the oviduct. The chicken reproductive tract showed a relatively high abundance of Lactobacillus species. The number of bacterial species in the chicken reproductive tract significantly increased following sexual maturation. Core genera analysis detected 21 of common genera in the maternal magnum and cloaca, descendent egg shell, egg white, and embryo gut. Some elements of the maternal oviduct microbiota appear to be transferred to the embryo through the egg white and constitute most of the embryo gut bacterial population.

Comparative genome analysis of Korean field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis (ILT) virus (ILTV) vaccines have been used to prevent and control the outbreak of ILT worldwide. Recent studies using high-throughput sequencing technology have increased the number of complete genome sequences of ILTVs, enabling comparative genome analysis. Although 37 complete genome sequences of ILTV, including vaccine strains, have been reported, the complete genome sequence of any field strain of ILTV in South Korea is yet to be published. In this study, we determined and analyzed the complete genome sequences of three virulent Korean field strains of ILTV (40798/10/Ko, 0206/14/Ko, and 30678/14/Ko). Two of the Korean field strains (40798/10/Ko and 0206/14/Ko) displayed fewer non-synonymous single nucleotide polymorphisms than those of the Serva vaccine strain, indicating that these Korean field strains of ILTV most likely originated from the vaccine strain. The third ILTV strain, 307678/14/Ko, had two regions in the genome showing recombination between the Serva vaccine-like strain and the Australian A20 vaccine-like strain. Comparative genome analysis of ILTV using the Korean field strains with variable virulence can shed light on the recent trend of the emergence of virulent ILTV strains in the field. A few amino acid changes in the genome of ILTV vaccines could enhance the virulence in the vaccine strain, and natural recombination should be considered one of the major risks for the generation of revertant strains of ILTV under field conditions.

The possible origin of human adenovirus type 3: Evidence of natural genetic recombination between human and simian adenovirus.

We determined a complete genome sequence of the Korean field strain, KUMC-62, of human adenovirus type 3 (HAdV-3) and performed comparative genome analyses. Interestingly HAdV-3 has a distinct genomic sequence for the fiber CDS region on average 62.46% of nucleotide sequence identity to other types of HAdV-B1, while remaining genomic region of HAdV-3 is very similar (on average 95.71% of nucleotide sequence identity) to other types of HAdV-B1. The blast results showed that the fiber CDS region of HAdV-3 exhibited the highest nucleotide sequence identity with that of simian adenovirus type 32 (SAdV-32), except other strains of HAdV-3. In the Simplot analysis, a potential recombination event was detected between HAdV-7 and SAdV-32, which might have created HAdV-3 in the past. These findings suggest that HAdV-3 highly likely was created by a natural inter-species recombination event between human and non-human primate AdVs.