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The complete genome sequences of three strains of Newcastle disease virus (NDV) isolated from vaccinated commercial layer flocks in Japan in the span of three decades were characterized. All strains had genome lengths of 15,192 nucleotides consisting of six genes in the order of 3′-NP–P/V/W–M–F–HN–L-5′. The general genomic characteristics of the Japanese field strains were consistent with previously characterized class II NDV, except for those belonging to early genotypes (genotype I–IV), which lack the six nucleotide insertion at nucleotide positions 1,648–1,653 of the nucleoprotein (NP) gene. Phylogenetic analysis showed that the Japanese strains could be classified into genotypes VIc and VIIe using the complete genome sequence and the complete coding sequence of the fusion (F) gene according to the unified NDV classification system. Characterization of functional domains and neutralizing epitopes of the F and hemagglutinin-neuraminidase (HN) proteins of Japanese field strains revealed a total of 31 amino acid substitutions, as compared to vaccine strains Ishii and B1, which were widely used in Japan. Although virus neutralization (VN) test showed that poor flock immunity due to vaccination failure or partial and non-uniform immunization maybe the major factors involved in the mechanism of breakthrough infection of the Japanese field strains, approximately two to threefold decrease in the VN titers of the field NDV strains possessing a point mutation (E347K or E347G) at the linear epitope of the HN protein was observed, as compared to vaccine strain B1 and field strain 2440/69, which lack the point mutation. This study may be a useful reference in characterizing future ND outbreaks in vaccinated chickens and as a genetic map for future investigations regarding vaccine designs, reverse genetics systems, and development of molecular diagnostic tools to prevent future ND outbreaks in vaccinated poultry flocks.

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8 mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples. Graphical ► Utility of a flotation method with the use of a wire loop of 8 mm in diameter was evaluated. ► E. tenella oocysts and Heterakis gallinarum eggs in tubes could be collected at a ratio of 2.00 to 3.08. ► Our results may implicate the application for the estimated OPG/ EPG of the samples as a simple and time saving method.

Rodents serve as amplifiers of Salmonella infections in poultry flocks and can serve as a source of Salmonella contamination in the environment even after thorough cleaning and disinfection. This study aims to determine the dynamics of Salmonella occurrence in rodents and its relation to Salmonella contamination in the layer farm environment, including air dusts and eggs. From 2008 to 2017, roof rats (Rattus rattus), environmental swabs, air dusts, and eggs were collected from an intensive commercial layer farm in East Japan and were tested for Salmonella spp. using standard procedures. In roof rat samples, the Salmonella isolation rate was reached at 10% (95% confidence interval [CI] 8.1–21.9) in which Salmonella Corvallis, Salmonella Infantis, Salmonella Potsdam, and Salmonella Mbandaka were the frequent isolates from the cecal portion of the intestines. On the other hand, the prevalence rate of Salmonella in environmental swabs was at 5.1% (95% CI 2.2–7.4) while air dusts were at 0.9% (95% CI 0.2–1.8). It was observed that the prevalence of predominant Salmonella serotypes shifted over time; in roof rats, it was noted that Salmonella Potsdam gradually replaced Salmonella Infantis. In environmental swabs and eggs, Salmonella Corvallis and Salmonella Potsdam increased significantly while Salmonella Infantis became less frequent. In air dusts, Salmonella Corvallis was observed to decrease and Salmonella Potsdam became more common. Based on our findings, the role of roof rats in the epidemiology of Salmonella in layer farms was expanded from being a reservoir and an amplifier host into a shifting vessel of the most predominant serotypes.

In total, 40 commercial layer farms and 32 replacement pullet farms with a combined population of 7.5 million adult layers and 6.6 million replacement pullets from six prefectures in eastern Japan were investigated for Salmonella Senftenberg contamination. We randomly collected 17,956 environmental samples, 5816 feed samples, and 218,470 egg samples from commercial layer farms; and 427 feed samples and 2896 environmental samples from replacement pullet farms. We monitored all samples for Salmonella. Samples were primarily enriched in Hajna tetrathinoate broth for 24 hr at 37 C followed by incubation in desoxycholate hydrogen sulfide lactose agar for 18 hr at 37 C. Salmonella colonies were confirmed and identified by biochemical tests and serotyped using Salmonella O and H antigens. We recorded 171 environmental samples (0.95%) and 10 feed samples (0.17%) that were positive for Salmonella spp. in which 36 environmental samples (0.20%) and six feed samples (0.10%) were identified as Salmonella Senftenberg. All Salmonella Senftenberg strains were isolated from nine replacement pullet farms. No Salmonella Senftenberg strains were isolated from adult layer farms and from eggs. Pulse field gel electrophoresis of BlnI-digested chromosomal DNA of 19 Salmonella Senftenberg isolates from feeds and environmental samples yielded a single identical DNA pattern. Traceback information showed that all positive feed samples were from a single feed source. Timeline studies showed that Salmonella Senftenberg contamination occurred first mostly in the feeds and then spread to the environment and other farms. This study demonstrated that the prevalence of Salmonella Senftenberg contamination in commercial layer facilities in eastern Japan is very low. Moreover, feed contamination played a major role in the epizootiology and spread of this pathogen in commercial poultry flocks. Given the resilient and persistent nature of this particular Salmonella serotype, routine monitoring and strict quality control measures at the feed level are recommended to prevent the colonization of poultry facilities with Salmonella Senftenberg that may lead to future outbreaks.

BACKGROUND: Newcastle Disease (ND) is a highly contagious and economically devastating disease of poultry. At present, limited molecular epidemiological data are available regarding the causes of ND outbreaks in vaccinated commercial poultry farms. Knowing the genomic characteristics of Newcastle disease virus (NDV) infecting commercial poultry operations in spite of vaccination might give important insights on the infection dynamics of these viruses. In addition, molecular analyses at the subgenotype level and studies on the relationship of Japanese NDVs with other isolates from around the world are lacking. Therefore, in the present study, a molecular epidemiological investigation was conducted to characterize nine NDVs isolated from vaccinated commercial poultry flocks in five different Prefectures in non-epidemic areas of Japan between 1969 and 2002. METHODS: Nucleotide sequencing and phylogenetic studies were performed to characterize the complete fusion (F)-protein gene, 3-prime end of the nucleoprotein (NP)-gene and 5-prime end of the RNA dependent RNA polymerase (L)-gene. Sequence data were compared with 180 NDV strains from GenBank representing different NDV genotypes and subgenotypes from different regions of the world at different time periods. Deduced amino acids were analyzed for homologies, recombination and mutation. Recombination events were estimated using Recombination Detection Program (RDP) version 3.44. Phylogenetic trees were constructed to determine evolutionary relationships among strains. RESULTS: Mean death time (MDT: 48-56 hr), Intracerebral Pathogenicity Index (ICPI: 1.7-1.9) and deduced amino acid sequences of the F0 proteolytic cleavage site (¹¹²RRQKR¹¹⁶) revealed that all nine field isolates were velogenic. Phylogenetic analysis showed that these isolates could be classified into two genetic lineages and three sublineages namely genotypes VIa (lineage 4a), VId (lineage 4d) and VIId (lineage 5d). No recombination events were observed but a point mutation in one of the neutralizing epitope of the F-protein was identified in the field isolates from Japan. CONCLUSIONS: All field isolates from vaccinated commercial poultry in non-epidemic areas of Japan were part of much bigger outbreaks in provinces and regions and, in some cases, continents. In general, four ND panzootics occurred in Japan and that these outbreaks were mostly characterized by co-circulation of genetically distinct virus lineages due to involvements of infected wild birds. The point mutation identified in the field isolates from Japan may be due to escape from vaccine pressure. The identification of such mutation may be useful for future site-directed mutagenesis to understand the dynamics of NDV infection in vaccinated chickens.

A comparison on the prevalence of Salmonella infection in layer hens from commercial layer farms with high and low rodent densities was investigated. Out of 280 laying hens sampled from three commercial layer farms with high rodent densities, Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) was isolated from 20 (7.14%) hens and Salmonella enterica subsp. enterica serovar Infantis (Salmonella Infantis) from three (1.07%) hens. In contrast, layer hens sampled from four commercial layer farms with low rodent densities were negative for any salmonellae. Significant differences (P < 0.05) in the isolation rates of Salmonella from various organs of infected layer hens were also noted. For Salmonella Enteritidis, liver (55.0%) and the oviduct (55.0%) had the highest isolation rates while all Salmonella Infantis isolates were from the oviduct. Pulsed field gel electrophoresis (PFGE) analysis of BlnI-digested chromosomal DNA of Salmonella Enteritidis isolated from layer hens and rodents showed similar patterns. PFGE analysis of Salmonella Infantis isolated from layer hens, rodents, eggs, and the environment yielded identical patterns. In this study, the significantly higher prevalence rate (P < 0.05) of Salmonella Enteritidis and Salmonella Infantis in layer hens from high rodent density farms could be attributed to the high rodent population density. The persistent Salmonella Enteritidis and Salmonella Infantis infection inside layer houses may have been amplified by the increasing numbers in the rodent population over the years, which increased the opportunity for environment-rodent-chicken interaction and the transmission of salmonellae to chickens. Monitoring of salmonellae from rodents inside poultry premises is recommended to be an effective additional tool in the assessment of the Salmonella status of layer flocks.

Rodents play a major role in the transmission and maintenance of Salmonella contamination cycles in poultry facilities. However, very limited field data are available regarding the transmission routes, infection cycle, and shedding patterns of Salmonella by naturally infected wild rodents from commercial layer farms. In this study, a total of 128 resident wild roof rats (Rattus rattus) were captured from a Salmonella-contaminated layer facility. All roof rats were divided into 51 laboratory cages, and weekly monitoring of Salmonella fecal shedding patterns was conducted for 53 wk. Seven roof rats from cages that were observed to frequently shed Salmonella were isolated in individual cages, and daily Salmonella monitoring was performed for 35 days. At the end of monitoring, each roof rat was euthanatized, and isolation of Salmonella from different organs was performed. Results of weekly monitoring of Salmonella showed that 21 of 51 cages (41.2%) were positive for Salmonella Infantis, while two cages (3.92%) were positive for Salmonella Enteritidis. Moreover, 11 cages were positive for Salmonella for at least two sampling weeks. Isolation of Salmonella from fecal droppings was mainly observed during the first 12 wk of captivity. The longest interval between two Salmonella-positive fecal dropping was 24 wk. In the daily Salmonella monitoring, only Salmonella Infantis was isolated from fecal droppings, in which the highest number of Salmonella Infantis organisms per fecal dropping was at 1 × 108 colony-forming units (cfu), while the lowest measured quantity was 1 × 103 cfu. It was noted that the frequency of Salmonella shedding in fecal droppings appeared to have a linear correlation (r  =  0.85) with the number of Salmonella organisms (cfu) per fecal pellet (P < 0.05). Moreover, pulsed-field gel electrophoresis analysis of Salmonella Infantis isolates revealed a single identical pulsed-field pattern. Salmonella Enteritidis isolates from fecal droppings and internal organs also generated a single identical pulsed-field pattern. Interestingly, Salmonella Infantis was not isolated from any of the organs examined, while Salmonella Enteritidis was isolated from the spleen and liver of one roof rat. These results may indicate that wild roof rats could persistently carry Salmonella and contaminate commercial poultry facilities through intermittent fecal shedding. Moreover, Salmonella Enteritidis in wild roof rats appears to be more of a systemic infection, in which isolation is most likely to occur in internal organs, whereas Salmonella Infantis is more likely an enteric type of infection, in which isolation is most likely to occur in the intestinal contents. It is very plausible that layer chickens could become infected with Salmonella through ingestion of Salmonella-positive fecal droppings or feeds contaminated with these fecal droppings from infected resident roof rats. This is likely one of the major reasons why layer houses can be persistently infected by Salmonella even if the facilities are thoroughly cleaned and disinfected and if replacement stocks are obtained from Salmonella-free breeders and rearing units. It is therefore a noteworthy suggestion that rodent control programs inside poultry premises comprise an essential and effective tool in the management and control of Salmonella contamination in layer flocks. Patrones de transmisión y eliminación de Salmonella en ratas negras o de tejado (Rattus rattus) provenientes de una granja de gallinas de postura contaminada con Salmonella, infectadas de forma natural y mantenidas en cautiverio. Los roedores desempeñan un papel importante en la transmisión y el mantenimiento de los ciclos de la contaminación por Salmonella en las instalaciones avícola. Sin embargo, son muy limitados los datos de campo disponibles con relación a las vías de transmisión, el ciclo de infección, y los patrones de eliminación de Salmonella por los roedores en libertad infectados de forma natural de las granjas de ponedoras comerciales. En este estudio, un total de 128 ratas negras en libertad (Rattus rattus) fueron capturadas en una granja de gallinas de postura contaminada con Salmonella. Todas las ratas se dividieron en 51 jaulas de laboratorio, y se realizó un seguimiento semanal de los patrones de eliminación fecal de Salmonella durante 53 semanas. Siete ratas en las que se observó eliminaban con frecuencia Salmonella fueron aisladas en jaulas individuales, y el seguimiento diario de Salmonella se realizó durante 35 días. Al final del periodo de observación, cada rata fue sometida a la eutanasia, y el aislamiento de Salmonella a partir de diferentes órganos se realizó. Los resultados del monitoreo semanal de Salmonella mostró que 21 de 51 jaulas (41.2%) fueron positivas para Salmonella Infantis, mientras que dos jaulas (3.92%) fueron positivos para Salmonella Enteritidis. Además, 11 jaulas fueron positivas para Salmonella durante al menos dos semanas de muestreo. El aislamiento de Salmonella a partir de las muestras fecales se observó principalmente durante las primeras 12 semanas de cautiverio. El intervalo más largo entre dos muestras fecales positivas para Salmonella fue de 24 semanas. En el seguimiento diario de Salmonella, sólo Salmonella Infantis fue aislada de heces, en la que el mayor número de organismos de Salmonella Infantis en la muestra fecal fue de 1 × 108 unidades formadoras de colonias (UFC), mientras que la menor cantidad medida fue de 1 × 103 UFC. Se observó que la frecuencia de eliminación de Salmonella en muestras fecales parecía tener una correlación lineal (r  =  0.85) con el número de organismos de Salmonella (UFC) por muestra fecal (P <0.05). Por otra parte, el análisis de electroforesis en gel con campos de pulsaciones de los aislamientos de Salmonella Infantis reveló un patrón de campo pulsado único e idéntico. Los aislamientos de Salmonella Enteritidis de muestras fecales y de los órganos internos también se generó un patrón de campo pulsado único e idéntico. Curiosamente, la Salmonella Infantis no fue aislada de cualquiera de los órganos examinados, mientras que Salmonella Enteritidis se aisló a partir del bazo y el hígado de una rata negra. Estos resultados pueden indicar que las ratas negras en libertad, acarrean persistentemente Salmonella y contaminar las instalaciones comerciales de aves de corral a través de la excreción fecal intermitente. Además, Salmonella Enteritidis en ratas de techo silvestres parece ser más de una infección sistémica, en la que el aislamiento es más probable que ocurra en los órganos internos, mientras que Salmonella Infantis es más probable un tipo entérico de la infección, en el que el aislamiento es más probable que ocurra a través de la ingestión en los contenidos intestinales. Es muy probable que pudiera convertirse en gallinas ponedoras infectadas por Salmonella a través de la ingestión de positivas a Salmonella de heces o alimentos contaminados con muestras fecales de estas ratas infectadas residentes en el techo. Esto es probablemente una de las razones principales por las casetas pueden persistir infectadas por Salmonella, incluso si las instalaciones se limpian y desinfectan, y si las poblaciones de reemplazo se obtienen de los criadores libres de Salmonella y las unidades de cría. Por tanto, se sugiere destacar que los programas de control de roedores dentro de las instalaciones de aves de corral constituyen una herramienta esencial y eficaz en la gestión y el control de la contaminación por Salmonella en parvadas de ponedoras.

Leucocytozoon caulleryiis an economically important poultry pathogen that causes subclinical to fatal disease in chickens. Because of limited preventive and treatment options against this disease, an oil-adjuvanted recombinant vaccine (O-rR7) targeting the R7 protein ofL. caulleryisecond-generation schizonts was developed. Different vaccination programs, namely, single vaccination at 45 days (0.1-ml dose), single vaccination at 130 days (0.25 ml), and initial vaccination at 45 days (0.1 ml) followed by a booster dose at 130 days (0.25 ml) were explored to compare the effects of single and booster vaccination on antibody response, duration of protective immunity, and degree of clinical signs after experimentalL. caulleryiinfection. Of the three treatments groups, initial vaccination at 45 days followed by a booster vaccination at 130 days of age resulted to rapid increase in antibody titers, which persisted for up to 182 days. Antibody titers reached peak values 35 days and 14 days after initial and booster vaccination, respectively. In comparison, single vaccination at 45 days of age resulted in production of antibodies above 1600 ELISA units for 56 days postvaccination, and single vaccination at 130 days of age produced peak antibody titers 35 days postvaccination, which remained above 1600 ELISA units for 126 days. Experimental infection ofL. caulleryiat 256 days, when antibody titers had waned, did not result to severe clinical disease in chickens that received booster vaccination, whereas mild to severe disease was observed in chickens that received a single vaccination. Evaluation of immune response at 15 and 21 days postinfection showed that chickens that received booster vaccination had a twofold increase (P< 0.01) in antibody titers as compared to those receiving a single vaccination. Administering booster shots of O-rR7 is therefore recommended, especially in farms located in areas whereLeucocytozoonis endemic. Leucocytozoon caulleryies un patógeno económicamente importante en la avicultura que causa una enfermedad que va de una presentación subclínica hasta una forma fatal en pollos. Debido a las opciones limitadas de prevención y tratamiento contra esta enfermedad, se desarrolló una vacuna recombinante con adyuvante oleoso (O-rR7) dirigidas a la proteína R7 de esquizontes de segunda generación deL. caulleryi. Se exploraron diferentes programas de vacunación, vacunación única a los 45 días (dosis de 0.1 ml), vacunación única a los 130 días (0.25 ml), y una vacunación inicial a los 45 días (0.1 ml), seguido de una dosis de refuerzo a los 130 días (0.25 ml), para comparar los efectos de la vacunación única y de refuerzo en la respuesta de anticuerpos, la duración de la inmunidad protectora y el grado de signos clínicos después de la infección experimental conL. caulleryi. De los tres grupos de tratamientos, la vacunación inicial a los 45 días, seguida de una dosis de refuerzo a los 130 días de edad resultó en un aumento ra pido en los títulos de anticuerpos, que persistió hasta por 182 días. Los títulos de anticuerpos alcanzaron valores máximos 35 días y 14 días después de la vacunación inicial y de refuerzo, respectivamente. En comparación, la vacunación única a los 45 días de edad dio lugar a la producción de anticuerpos por arriba de 1600 unidades ELISA durante 56 días después de la inmunización y la vacunación única a los 130 días de edad produjo los títulos más altos de anticuerpos 35 días después de la vacunación, que se mantuvo por encima de 1600 unidades ELISA durante 126 días. La infección experimental deL. caulleryia los 256 días, cuando los títulos de anticuerpos habían disminuido, no produjo la enfermedad clínica severa en los pollos que recibieron la vacunación de refuerzo, mientras que se observó la enfermedad de leve a severa en los pollos que recibieron una sola vacunación. La evaluación de la respuesta inmune a los 15 y 21 días después de la infección mostró que los pollos que recibieron la vacunación de refuerzo mostraron un incremento del doble (P<0.01) en los títulos de anticuerpos en comparación con las aves que recibieron una sola vacunación. Por lo tanto, se recomienda la administración de dosis de refuerzo de O-rR7, especialmente en explotaciones ubicadas en zonas dondeLeucocytozoones endémico.

Leucocytozoon caulleryi is an economically important poultry pathogen that causes subclinical to fatal disease in chickens. Because of limited preventive and treatment options against this disease, an oil-adjuvanted recombinant vaccine (O-rR7) targeting the R7 protein of L. caulleryi second-generation schizonts was developed. Different vaccination programs, namely, single vaccination at 45 days (0.1-ml dose), single vaccination at 130 days (0.25 ml), and initial vaccination at 45 days (0.1 ml) followed by a booster dose at 130 days (0.25 ml) were explored to compare the effects of single and booster vaccination on antibody response, duration of protective immunity, and degree of clinical signs after experimental L. caulleryi infection. Of the three treatments groups, initial vaccination at 45 days followed by a booster vaccination at 130 days of age resulted to rapid increase in antibody titers, which persisted for up to 182 days. Antibody titers reached peak values 35 days and 14 days after initial and booster vaccination, respectively. In comparison, single vaccination at 45 days of age resulted in production of antibodies above 1600 ELISA units for 56 days postvaccination, and single vaccination at 130 days of age produced peak antibody titers 35 days postvaccination, which remained above 1600 ELISA units for 126 days. Experimental infection of L. caulleryi at 256 days, when antibody titers had waned, did not result to severe clinical disease in chickens that received booster vaccination, whereas mild to severe disease was observed in chickens that received a single vaccination. Evaluation of immune response at 15 and 21 days postinfection showed that chickens that received booster vaccination had a twofold increase (P < 0.01) in antibody titers as compared to those receiving a single vaccination. Administering booster shots of O-rR7 is therefore recommended, especially in farms located in areas where Leucocytozoon is endemic. Respuesta de anticuerpos e inmunidad protectora en pollos vacunados con una dosis de refuerzo de una vacuna recombinante de Leucocytozoon caulleryi con adyuvante oleoso. Leucocytozoon caulleryi es un patógeno económicamente importante en la avicultura que causa una enfermedad que va de una presentación subclínica hasta una forma fatal en pollos. Debido a las opciones limitadas de prevención y tratamiento contra esta enfermedad, se desarrolló una vacuna recombinante con adyuvante oleoso (O-rR7) dirigidas a la proteína R7 de esquizontes de segunda generación de L. caulleryi. Se exploraron diferentes programas de vacunación, vacunación única a los 45 días (dosis de 0.1 ml), vacunación única a los 130 días (0.25 ml), y una vacunación inicial a los 45 días (0.1 ml), seguido de una dosis de refuerzo a los 130 días (0.25 ml), para comparar los efectos de la vacunación única y de refuerzo en la respuesta de anticuerpos, la duración de la inmunidad protectora y el grado de signos clínicos después de la infección experimental con L. caulleryi. De los tres grupos de tratamientos, la vacunación inicial a los 45 días, seguida de una dosis de refuerzo a los 130 días de edad resultó en un aumento rápido en los títulos de anticuerpos, que persistió hasta por 182 días. Los títulos de anticuerpos alcanzaron valores máximos 35 días y 14 días después de la vacunación inicial y de refuerzo, respectivamente. En comparación, la vacunación única a los 45 días de edad dio lugar a la producción de anticuerpos por arriba de 1600 unidades ELISA durante 56 días después de la inmunización y la vacunación única a los 130 días de edad produjo los títulos más altos de anticuerpos 35 días después de la vacunación, que se mantuvo por encima de 1600 unidades ELISA durante 126 días. La infección experimental de L. caulleryi a los 256 días, cuando los títulos de anticuerpos habían disminuido, no produjo la enfermedad clínica severa en los pollos que recibieron la vacunación de refuerzo, mientras que se observó la enfermedad de leve a severa en los pollos que recibieron una sola vacunación. La evaluación de la respuesta inmune a los 15 y 21 días después de la infección mostró que los pollos que recibieron la vacunación de refuerzo mostraron un incremento del doble (P <0.01) en los títulos de anticuerpos en comparación con las aves que recibieron una sola vacunación. Por lo tanto, se recomienda la administración de dosis de refuerzo de O-rR7, especialmente en explotaciones ubicadas en zonas donde Leucocytozoon es endémico.

Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative 'Ca. L. asiaticus' Japanese isolate Ishi-1 was determined by metagenomic analysis of DNA extracted from 'Ca. L. asiaticus'-infected psyllids and leaf midribs. The 1.19-Mb genome has an average 36.32% GC content. Annotation revealed 13 operons encoding rRNA and 44 tRNA genes, but no typical bacterial pathogenesis-related genes were located within the genome, similar to the Floridian psy62 and Chinese gxpsy. In contrast to other 'Ca. L. asiaticus' strains, the genome of the Japanese Ishi-1 strain lacks a prophage-related region.