Research Note: Molecular and pathologic characterization of avian adenovirus isolated from the oviducts of laying hens in eastern Japan.

Cases of poor egg production were investigated in 2 layer farms from Ibaraki Prefecture in eastern Japan. To identify any microbial agents that may have caused the problem, necropsy, bacterial isolation, histopathology, and virus detection were performed. Members of the avian adenoviruses was detected by PCR in oviduct samples from both farms; chicken anemia virus coinfection was also confirmed in one of the farms. Avian adenovirus was isolated from the oviducts of the affected chickens on each farm. Inoculation into chick embryos showed tropism for the chorio-allantoic membrane. Stunting and hemorrhaging was observed in all infected embryos, as well as death in a few. Inoculation of 1-day-old specific pathogen-free chicks, and 400-day-old commercial hens, did not result in any significant findings. The isolated viruses were analyzed by sequencing of the hexon gene and were confirmed as fowl adenovirus type-c serotype-4 (FAdV-4). The 2 virus strains were found to be 99.29% similar to each other. One of the strains, Japan/Ibaraki/Y-H6/2016, was 99.15% similar to the KR5 strain. The other, Japan/Ibaraki/M-HB2/2016, was 99.57% similar to the KR5 strain. Fiber-2 gene analysis confirmed the identity as FAdV-4 that is closely related to nonpathogenic strains. Although nonpathogenic to chicks and laying hens, this infection can possibly cause economic damage. Perhaps the bigger concern is the effect on infected breeder operations. Because the virus is fatal to 9.09% of infected embryos, this could translate to a considerable loss in chick production owing to embryonic death. This is the first report of detection and isolation of FAdV-4 from the chicken oviduct; however, further studies are needed to elucidate its impact on both layer and breeder flocks. Indeed, FAdV-4 has negative effects on the avian reproductive tract as well.

The diseases suspected of the involvement of chicken anemia virus infection in 11 to 14-weeks old replacement pullets from eastern Japan: a case report.

Three strains of chicken anemia virus (CAV) were detected in 11 to 14-weeks old chickens, showing depression, wasting, and increased mortality, from three farms in eastern Japan. Another strain was detected in 12-weeks old chickens from one farm without clinical signs. Bacterial infections were suggested in three farms with clinical signs and its involvement in the occurrence of the diseases might be suspected. Sequence analysis of the VP1, VP2, and VP3 genes of four CAV strains revealed that the three from farms with clinical signs belonged to genotype A2, whereas that from the apparently-normal farm belonged to A3. This may be a rare case report about the diseases suspected of the involvement of the CAV infection in older birds.

The Expanded Role of Roof-Rats (Rattus rattus) in Salmonella spp. Contamination of a Commercial Layer Farm in East Japan.

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.

Atypical velogenic Newcastle disease in a commercial layer flock in Japan.

In 2002, a commercial layer flock in Japan was initially diagnosed as being infected with infectious bronchitis (IB) based on clinical signs, virus isolation, and serological analysis but was later found to be atypically infected with velogenic Newcastle disease virus (NDV) following molecular diagnosis. The flock had slightly decreased egg production and an increased occurrence of soft-shelled eggs without significant mortality. IB-like viruses were isolated, which caused dwarfing and curling in 12-day-old chicken embryos. Ten years after this case, retrospective genetic analyses showed that apart from IB virus (IBV), the flock was also infected with NDV. Mean death time (MDT), intracerebral pathogenicity index (ICPI), and deduced amino acid sequence of the cleavage site of the fusion (F)-protein gene revealed that the NDV isolate was velogenic ((112)RRQKR(116)). These results indicate that poultry clinicians should look out for atypical velogenic ND, especially in vaccinated commercial chicken flocks, which may harbor hidden NDV infection.

Characterization of complete genome sequence of genotype VI and VII velogenic Newcastle disease virus from Japan.

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.

Antibody response and protective immunity of chickens vaccinated with booster dose of recombinant oil-adjuvanted Leucocytozoon caulleryi subunit vaccine.

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.

Molecular epidemiology of Newcastle disease virus isolates from vaccinated commercial poultry farms in non-epidemic areas of Japan.

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.

Epizootiologic role of feeds in the epidemiology of Salmonella Senftenberg contamination in commercial layer farms in eastern Japan.

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.

Transmission and shedding patterns of Salmonella in naturally infected captive wild roof rats (Rattus rattus) from a Salmonella-contaminated layer farm.

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 ratus) 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 x 10(8) colony-forming units (cfu), while the lowest measured quantity was 1 x 10(3) 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.

Comparison of the prevalence of Salmonella infection in layer hens from commercial layer farms with high and low rodent densities.

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.

An epidemiological analysis of Salmonella enteritidis contamination in a rat-infested chicken layer farm, an egg processing facility, and liquid egg samples by pulsed-field gel electrophoresis.

In order to determine the epidemiological link between the Salmonella Enteritidis contamination in a rat-infested chicken layer farm, an attached egg processing facility and liquid egg samples, several S. Enteritidis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. A total of 33 S. Enteritidis strains were isolated from a total of 4,081 samples. Similar pulsed-field patterns were generated by S. Enteritidis isolates from liquid eggs, rats and effluent water. Additionally, only two phage types were detected among the S. Enteritidis isolates, PT 1b and PT 6. These results suggest that S. Enteritidis isolates from rats, egg processing facility, and liquid eggs are genetically related. Furthermore, S. Enteritidis infection in rats in layer farms poses a serious public health concern and should be included in future epidemiological studies.

Pulsed-field gel electrophoresis-based subtyping of DNA degradation-sensitive Salmonella enterica subsp. enterica serovar Livingstone and serovar Cerro isolates obtained from a chicken layer farm.

Salmonella enterica serovar subsp. enterica Livingstone and serovar Cerro isolates from a commercial egg-producing farm, which had previously been untypeable by pulsed-field gel electrophoresis (PFGE) because of DNA degradation during the PFGE process, successfully gave banding patterns using electrophoresis buffer supplemented with 50 microM thiourea. By PFGE in the presence of thiourea, DNA degradation-sensitive S. enterica serovar Cerro isolates from the commercial egg-producing farm were found to be genetically unrelated to S. enterica serovar Cerro isolates that gave the patterns in the absence of thiourea. Forty-five of 50 (90%) S. enterica serovar Livingstone isolates from the farm showed arbitrarily designated XbaI-digested patterns X1 and X2 that were distinguished by one-band difference and had an identical BlnI-digested pattern. In one of the two layer houses in the farm, the numbers of isolates having the pattern X2 increased from 57% in 1997 to 89% in 1998, whereas virtually all the isolates obtained from the other house in the same period showed the profile X1. This suggests that strains having the pattern X2 might have an advantage to preferentially colonize in the former house.