Detection of chicken chapparvovirus 2 in chickens with hemorrhagic hepatitis in Japan.

Chicken chaphamaparvovirus causes diarrheal symptoms and can be detected in fecal samples. This study reports the detection of chicken chapparvovirus 2 in debilitated chickens with hemorrhagic hepatitis at a broiler farm in Japan. After euthanasia and necropsy, liver hemorrhage was observed. Nuclear inclusion bodies in the hepatocytes were identified using histological analysis. High-throughput sequencing analysis using RNA from livers of three affected chickens revealed infection by chicken chapparvovirus 2 and chicken anemia virus. Polymerase chain reaction analysis showed that all three chickens were positive for chicken chapparvovirus 2, and only one was positive for both chicken chapparvovirus 2 and chicken anemia virus. In conclusion, chicken chapparvovirus 2 causes infection in chickens in Japan and might be involved in hemorrhagic hepatitis.

Evidence of vertical transmission of fowl adenovirus 8b in ducks.

Fowl adenovirus mainly causes hydropericardium hepatitis syndrome (HHS), inclusion body hepatitis (IBH) and gizzard erosion (GE), etc. In 2015, the first outbreak of HHS was reported in broiler chickens in central China, followed by an outbreak in waterfowl. The first outbreak of HHS in broiler flocks in central China in 2015, followed by outbreaks in waterfowl, has severely restricted the healthy development of the poultry industry. During the investigation, fowl adenovirus was detected in ducklings from a total of seven hatcheries in Shandong, Inner Mongolia and Jiangsu provinces. In addition, the DNA of fowl adenovirus was detected in breeding ducks and their progeny. To test the hypothesis that FAdV can be transmitted vertically, sixty 250-day-old Cherry Valley breeder ducks were divided equally into three groups for experimental infection. FAdV-8b SDLY isolate (duck/Shandong/SDLY/2021, SDLY) preserved in our laboratory was injected intramuscularly into group A and inoculated orally into group B. FAdV-8b DNA was detected in the yolk membranes, embryos and allantoic fluid of duck embryos in the FAdV-infected group after inoculation. In addition, the FAdV-8b hexon gene isolated from yolk membranes, embryos, allantoic fluid and duck eggs was close to 100% nucleotide homology to the FAdV-8b hexon gene isolated from laying duck ovaries, indicating that fowl adenovirus can be transmitted vertically in ducks. These findings provide evidence for the possible vertical transmission of fowl adenovirus from breeder ducks to ducklings.

Necrotizing hepatitis caused by Clostridium novyi type B in a dog with no predisposing liver lesions: a case report.

BACKGROUND: Infectious necrotic hepatitis (INH) is typically a disease of ruminants caused by Clostridium novyi type B. Growth of the causative agent is supported by development of an anaerobic environment within the liver. In dogs, C. novyi is rare and has only been previously reported as a post-mortem diagnosis. In one case, infection was secondary to metastatic pancreatic adenocarcinoma and the other was presumptively diagnosed on histopathology of a hepatic lesion in a dog initially presented for acute collapse. CASE PRESENTATION: An 8-year-old spayed, female mixed breed dog was presented for acute onset of hyporexia and vomiting. Serum biochemistry revealed elevated hepatocellular injury and cholestatic liver enzymes. Ultrasound revealed peritoneal fluid accumulation and multiple hepatic masses. Cytologic examination of liver aspirates and peritoneal fluid revealed frequent 4 × 1 µm bacilli with a terminal endospore. Anaerobic bacterial growth isolated from the fluid sample could not be identified using typical laboratory identification techniques. Long-read, whole genome sequencing was performed, and the organism was identified as Clostridium novyi type B. Antimicrobial and hepatic support treatment were initiated. The patient re-presented 27 days later, and the follow up liver aspirate with cytology revealed no appreciable bacteria and anaerobic culture was negative. The patient was presented four months later and a large hepatic mass and peritoneal fluid were again identified on abdominal ultrasound. Cytologic examination of the peritoneal fluid revealed bacilli similar to those identified on initial presentation. The patient was euthanized. The most significant finding on necropsy was necrotizing hepatitis with intralesional endospore-forming bacilli compatible with recurrence of Clostridium novyi type B. There was no identifiable cause of an anaerobic insult to the liver. CONCLUSIONS: This case demonstrates the diagnostic utility of using cytology as part of the initial diagnostic work up for infectious hepatitis. The cytologic findings coupled with whole genome sequencing and anaerobic culture were crucial for the identification and classification of the organism identified on fine needle aspirate. Clostridium novyi type B should be considered when bacilli organisms containing a terminal endospore are identified on liver aspirates collected from canine patients.

An inactivated vaccine based on artificial non-pathogenic fowl adenovirus 4 protects chickens against hepatitis-hydropericardium syndrome.

Hepatitis-hydropericardium syndrome (HHS) in birds is mainly caused by virulent fowl adenovirus 4 (FAdV-4). A novel genotype, hypervirulent FAdV-4, emerged in 2015 with a high mortality rate ranging from 30 % to 100 % in chickens. Vaccination is an economically feasible method to control HHS. Although there have been various reports of inactivated vaccines from virulent wild-type FAdV-4 against HHS, biosafety threats of inactivated vaccines from potential pathogenic components have been presented to the poultry industry, and safer vaccines are urgently needed. A non-pathogenic recombinant FAdV-4 strain, designated as rHN20, was generated based on the hypervirulent strain in our previous study. Here, we developed a novel inactivated oil-adjuvanted vaccine derived from rHN20 strain and evaluated its immunogenicity in specific-pathogen-free chickens. Chickens subcutaneously or intramuscularly immunized with the inactivated vaccine produced high titers of neutralizing antibodies and were protected from a lethal dose of virulent wild-type FAdV-4 challenge. Collectively, an inactivated vaccine was developed, which was capable of providing full protection for chickens against HHS, and significantly reduced the potential biosafety threats.

Quantitative levels of norovirus and hepatitis A virus in bivalve molluscs collected along the food chain in the Netherlands, 2013-2017.

Contamination of bivalve molluscs with viruses is well recognized as a food safety risk. A microbiological criterion for norovirus (NoV) and hepatitis A virus (HAV) in shellfish, however, does not exist in the European Union currently. The aim of this study was to evaluate the contamination levels of these viruses for fluctuation over a long period (2013-2017) in oyster (n = 266) and mussel samples (n = 490) using a method based on ISO/TS 15216-1: 2013. Samples were taken at different points in the food chain, either directly post-harvest, at Dutch dispatch centers or in retail stores, from September until March of each year. Altogether, 53.1% of the mussel and 31.6% of the oyster samples tested positive for NoV RNA. Simultaneous presence of NoV GI and GII RNA was observed in 31.6% of mussel and 10.2% of oyster samples. Contamination levels in NoV positive mussel samples collected post-harvest from B-areas were significantly higher than in those collected post-harvest from A-areas, or at dispatch centers or retail stores. Levels in oysters from dispatch were significantly lower than those collected in retail stores. Ready for sale mussels and oysters contained 2.04 and 1.76 mean log10 transformed NoV genome copies/gram (gc/g), respectively. GII levels were at a constant level in ready for sale mussels throughout all sampling periods in the study. This seemed to be true for oysters as well. HAV RNA was detected in only one of the tested mussel samples (n = 392) (typed HAV 1A) and in none of the tested oyster samples (n = 228). Critical evaluation of NoV and HAV levels in shellfish can be of help for risk assessment and risk management actions.

The investigation of hepatitis A virus and hepatitis E virus co-infection in humans and animals in China.

The aim of this study was to investigate the prevalence of co-infection of hepatitis A and hepatitis E virus (HAV/HEV) in patients with acute hepatitis as well as in different animal species. A total of 46 serum samples from patients diagnosed as hepatitis A or hepatitis E and 675 fecal samples of 11 animal species were collected. The IgM class antibodies to HEV and HAV, respectively, were detected by enzyme-linked immunosorbent assay. HEV and HAV RNAs were extracted from serum and fecal samples for the nested reverse transcription polymerase chain reaction. At least 10.9% (5/46) of the patients were co-infected with both HAV and HEV. Fifteen percent (18/120) of rabbit fecal samples and 17.5% (7/40) of swine fecal samples were positive for HEV RNA, but only 1% (2/200) of ferret fecal samples were positive for HAV RNA. Our study showed that co-infection with both HAV and HEV in patients and animals is infrequent. At least in our study, we showed that ferrets may represent the potential HAV hosts. Keywords: hepatitis A virus; hepatitis E virus; co-infection; zoonosis; prevalence.

Development and evaluation of reverse transcription-insulated isothermal PCR assay to detect duck hepatitis A virus type A in liver samples using the POCKITTM system.

Duck hepatitis A virus (DHAV) infection is characterized by severe hepatitis. In recent years, DHAV-A has become widespread in Asia and has led to economic losses. Conventional methods of DHAV-A detection must often be performed in the laboratory with inconvenience equipment. We have developed a rapid reverse transcription insulated isothermal (RT-iiPCR) technique for the on-site detection of DHAV-A based on the POCKITTM system in a convenient minitype device. We optimized the PCR primers and probes for the amplification of the DHAV-A 3C/3D genes, and successfully amplified a specific fragment of DHAV-A, but no fragment from 18 other duck pathogens. The limit of detection for viral RNA was 49 copies per reaction, and the sensitivity and specificity were each 100% in the analysis of 60 liver samples. By comparison, the sensitivities of RT-iiPCR was comparable in sensitivity to existing rRT-PCR. Furthermore, the RT-iiPCR results were 98.3% in agreement with those of the rRT-PCR, with a kappa value of 0.938. In conclusion, this new method not only offers a higher sensitivity and specificity than existing techniques, but also time-saving and better suited to field diagnoses because device is portable.

A reverse transcription-insulated isothermal PCR assay for the detection of duck hepatitis A virus type 3 based on the POCKIT™ system.

Duck hepatitis A virus type 3 (DHAV-3) infection is characterized by severe hepatitis. In recent years, DHAV-3 has become widespread in Asia and has led to economic losses. Conventional methods for DHAV-3 detection usually depend on the use of larger equipment that is not portable and is not fit for on-site diagnoses. In this study, a rapid reverse transcription insulated isothermal (RT-iiPCR) technique was developed for the on-site detection of DHAV-3 based on the POCKIT™ system in a convenient device. The concentration of primer pairs and probes were optimized for amplification of the DHAV-3 VP3 gene of DHAV-3, with no amplification of 12 other duck pathogens. The detection limit of viral RNA was 3.85 × 101 copies/µL, and the analytical sensitivity and specificity levels were both 100% in the detection of 40 liver samples. Furthermore, 97.5% of the RT-iiPCR results were in agreement with those of rRT-PCR, with a kappa value of 0.93. This method is time-saving and better suited to field diagnoses because of its portable device.

[Markers of hepatitis A in the monkeys of the Adlers primate center.]

Hepatitis A is a widespread viral infection. The HAV strains of "human" and "monkey" origin are similar in their morphological and antigenic properties, but differ genotypically. OBJECTIVES: The aim of this research was a comparative study of serological and molecular-genetic markers of HAV infection in monkeys born at the Adler Primate Center and in those imported from different countries. MATERIAL AND METHODS: Fecal samples (n = 313) and serum (n = 266) from various species of monkey using ELISA and RT-PCR were studied. RESULTS AND DISCUSSION: The frequency of anti-HAV-IgG was high (78.9%) in imported animals (vervet monkeys from Tanzania and cynomolgus monkeys from Vietnam) and as well as in various species of monkeys (rhesus monkeys, cynomolgus monkeys, green monkeys and papio hamadryas) of the Center (88.6%). At the same time, in the imported monkeys, the markers of "fresh" HAV infection (IgM-27.2%, Ag-HAV-16.7%, RNA-22.0%) were detected significantly more often (p> 0.05) than in monkeys kept at the Colony (IgM-7.5%, HAV-Ag - 5.2%, RNA - 3.6%). In general, anti-IgG reactivity ranged from 1.064 to 2.073 OD450, anti-IgM ranged from 0.546 to 1.059 OD450. The number of HAV-Ag was 0.496 - 1.995 OD450. RNA HAV only in rhesus monkeys and cynomolgys monkeys born at the Colony, as well as in imported vervet monkeys was detected. CONCLUSIONS: The data obtained indicate a wide circulation of HAV among monkeys born in the Adler Primate Center and among the imported animals. Markers of "fresh" HAV infection varied depending on the species of monkeys and their origin.

Novel duck hepatitis A virus type 1 isolates from adult ducks showing egg drop syndrome.

Generally, duck hepatitis A virus type 1 (DHAV-1) only infects young ducklings. Since December 2016, severe outbreaks of duck viral infection with egg drop, feed consumption decline, and ovary-oviduct disease have occurred in some laying duck flocks in Shandong Province of China. DHAV-1 isolated from the affected ducks was confirmed as the causative pathogen of the egg drop. Compared with other DHAV-1 strains, the novel isolate has three special amino acid mutation points in the most variable regions at the C-terminus of VP1. The experimental infection in laying ducks indicated that successful immunization with DHAV-1 vaccine could protect laying duck from infection. To the best of our knowledge, this is the first reported incidence of a severe duck disease outbreak involving egg drop syndrome caused by DHAV-1.

Molecular genetic identification of isolates of the hepatitis A virus (HAV) from monkeys at Adler Primate Center.

BACKGROUND: The aim of this study was to assess prevalence and genetic variability of hepatitis A virus (HAV) isolates in monkeys born and kept at Adler Primate Center, as well as in imported animals. METHODS: The fecal samples from various species of monkeys (n = 119) were studied using reverse transcription seminested PCR, sequencing, and phylogenetic analysis. RESULTS: HAV RNA was detected in 2 Macaca mulatta and 1 Macaca fascicularis (3.8%) kept at Adler Primate Center (n = 79) and in 11 (27.5%) Chlorocebus pygerythrus (n = 40) imported from Tanzania. Phylogenetic analysis demonstrated that all HAV strains belonged to simian genotype V, but differed from the prototype genotype V strain (AGM-27) by 5.4%-5.5%. Sequences isolated in this study differed by only 0.1%, suggesting a common source of infection. CONCLUSIONS: This study demonstrated the asymptomatic circulation of HAV genotype V among the monkeys at Adler Primate Center, and it indicated the significant genetic diversity within this HAV genotype.

An outbreak of duck hepatitis A virus type 1 infection in Japan.

In June 2015, a highly fatal and acute disease broke out in a duckling farm in Hyogo Prefecture, Japan. The birds exhibited poor growth, reduced movement, lying in a dorsal recumbent position, depression, lethargy, ataxia and opisthotonus, with a high mortality rate of approximately 76%. By performing a reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for duck hepatitis A virus type 1 (DHAV-1), we obtained the PCR products of a predicted size. The nucleotide sequences of the PCR products showed a >96% identity with that of the DHAV-1, HB02 strain, which was isolated in China. To our knowledge, this is the first time that the DHAV-1 virus has been isolated since its outbreak in Japan in 1963.

SEROLOGICAL DETECTION OF HEPATITIS A VIRUS IN FREE-RANGING NEOTROPICAL PRIMATES (Sapajus spp., Alouatta caraya) FROM THE PARANÁ RIVER BASIN, BRAZIL.

Nonhuman primates are considered as the natural hosts of Hepatitis A virus (HAV), as well as other pathogens, and can serve as natural sentinels to investigate epizootics and endemic diseases that are of public health importance. During this study, blood samples were collected from 112 Neotropical primates (NTPs) (Sapajus nigritus and S. cay, n = 75; Alouatta caraya, n = 37) trap-captured at the Paraná River basin, Brazil, located between the States of Paraná and Mato Grosso do Sul. Anti-HAV IgG antibodies were detected in 4.5% (5/112) of NTPs, specifically in 6.7% (5/75) of Sapajus spp. and 0% (0/37) of A. caraya. In addition, all samples were negative for the presence of IgM anti-HAV antibodies. These results suggest that free-ranging NTPs were exposed to HAV within the geographical regions evaluated.

Experimental evidence of hepatitis A virus infection in pigs.

Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis worldwide, with HAV infection being restricted to humans and nonhuman primates. In this study, HAV infection status was serologically determined in domestic pigs and experimental infections of HAV were attempted to verify HAV infectivity in pigs. Antibodies specific to HAV or HAV-like agents were detected in 3.5% of serum samples collected from pigs in swine farms. When the pigs were infected intravenously with 2 × 10(5) 50% tissue culture infectious dose (TCID50 ) of HAV, shedding of the virus in feces, viremia, and seroconversion were detected. In pigs orally infected with the same quantity of HAV, viral shedding was detected only in feces. HAV genomic RNA was detected in the liver and bile of intravenously infected pigs, but only in the bile of orally infected pigs. In further experiments, pigs were intravenously infected with 6 × 10(5) TCID50 of HAV. Shedding of HAV in feces, along with viremia and seroconversion, were confirmed in infected pigs but not in sentinel pigs. HAV genomic RNA was detected in the liver, bile, spleen, lymph node, and kidney of the infected pigs. HAV antigenomic RNA was detected in the spleen of one HAV-infected pig, suggesting HAV replication in splenic cells. Infiltration of inflammatory cells was observed in the livers of infected pigs but not in controls. This is the first experimental evidence to demonstrate that human HAV strains can infect pigs.

SEROLOGICAL DETECTION OF HEPATITIS A VIRUS IN FREE-RANGING NEOTROPICAL PRIMATES (Sapajus spp., Alouatta caraya) FROM THE PARANÁ RIVER BASIN, BRAZIL

Nonhuman primates are considered as the natural hosts of Hepatitis A virus (HAV), as well as other pathogens, and can serve as natural sentinels to investigate epizootics and endemic diseases that are of public health importance. During this study, blood samples were collected from 112 Neotropical primates (NTPs) (Sapajus nigritus and S. cay, n = 75; Alouatta caraya, n = 37) trap-captured at the Paraná River basin, Brazil, located between the States of Paraná and Mato Grosso do Sul. Anti-HAV IgG antibodies were detected in 4.5% (5/112) of NTPs, specifically in 6.7% (5/75) of Sapajus spp. and 0% (0/37) of A. caraya. In addition, all samples were negative for the presence of IgM anti-HAV antibodies. These results suggest that free-ranging NTPs were exposed to HAV within the geographical regions evaluated.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

Genetic characterization of Duck Hepatitis A Viruses isolated in China.

In recent years, the spread of Duck Hepatitis A Viruses (DHAVs) has represented a serious threat and significant economic impact in duck industry of China. The sixteen reported DHAV isolates (15 DHAV-1 strains and one DHAV-3) were identified from infected ducks with clinical symptoms in China between 2009 and 2012. In the present study, the virulence of these viruses and complete sequences of the virion protein 1 (VP1) genes of the 16 DHAVs were characterized. The median embryonic lethal doses (ELD50) of the second generation duck embryo allantoic fluid of the 16 DHAV isolates were calculated on duck and chicken embryos. The results demonstrated that the various DHAV-1 strains have shown different pathogenic ability in embryos, and duck eggs were more susceptible to DHAV than chicken eggs. The histopathological examination revealed significant signs of virus infection, severe vacuolation, and hepatocyte necrosis. Phylogenetic analyses indicated that the 15 DHAV-1 viruses display significant correlation in their geographic distribution. The DHAV-1 strains isolated from Shandong Province were more evolutionarily divergent than the JX strains. There were two hypervariable regions in the VP1 protein, which may determine the virulence of DHAV-1 isolates in chicken eggs but not virulence in duck eggs. These results demonstrate the genetic and biological diversity of DHAVs in China and aid in understanding the epidemiology and evolution of DHAVs.

Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China.

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD(50)s) and the median lethal doses (LD(50)s), respectively. The results showed that the ELD(50)s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10(6)/mL to 1.44 × 10(7)/mL, while the LD(50)s were 2.39 × 10(5)/mL to 6.15 × 10(6)/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158-160, 180-193 and 205-219) and other variable points in VP1 protein, but which didn't cause virulence of DHAV-1 change.