Infected Ducks Research Articles

Isolation and polymerase chain reaction-based identification of Riemerella anatipestifer from ducks in Kerala, India

Aim: The aim was to isolate and characterize Riemerella anatipestifer organisms from disease outbreaks in ducks in Kerala. Materials and Methods: Ducklings, suspected of Riemerella infection, were sacrificed and subjected to post-mortem examination. Heart blood smears and impression smears from liver and spleen were examined for the presence of pathogenic organisms. Heart blood, lung, liver, and spleen collected aseptically from the birds were subjected to isolation trials in brain heart infusion agar and 10% bovine blood agar. The isolates were characterized based on morphology, cultural characteristics and biochemical tests, and their identity were confirmed by polymerase chain reaction (PCR) and the PCR amplified DNA was sequenced. The antibiotic sensitivity testing of the isolates were carried out using six antibiotics viz ciprofloxacin, chloramphenicol, enrofloxacin, amoxycillin, cotrimoxazole, and gentamicin. Results: Colonies suggestive of Riemerella organisms could be isolated on blood agar. Biochemical characterization and PCR confirmed the identity of isolates as R. anatipestifer. The nucleotide sequence of the PCR product showed 99% homology to the R. anatipestifer sequences in the NCBI. The antibiogram revealed that the organisms were sensitive to ciprofloxacin, enrofloxacin, and gentamicin. Conclusion: The present study suggests that the PCR assay can facilitate fast and proper identification of R. anatipestifer infection in ducks. The assay can also differentiate between R. anatipestifer and Pasteurella multocida and can replace the traditional methods of differentiation which are cumbersome and time-consuming.

In vitro and in vivo anti-hepatitis B virus activities of the lignan nirtetralin B isolated from Phyllanthus niruri L.

Ethnopharmacological relevanceNirtetralin B, a new lignan first reported by our team, is isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of nirtetralin B using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models. Materials and methodsNirtetralin B was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after nirtetralin B was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks. ResultsIn the human HBV-transfected liver cell line HepG2.2.15, nirtetralin B effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC50 values for HBsAg of 17.4μM, IC50 values for HBeAg of 63.9μM. In DHBV-infected ducklings, nirtetralin B significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. And analysis of the liver pathological changes confirmed the hepatoprotective effect of nirtetralin B. ConclusionThe experimental data demonstrated that nirtetralin B exhibits anti-hepatitis B virus activity both in vitro and in vivo.

Development of an indirect ELISA with epitope on nonstructural protein of Muscovy duck parvovirus for differentiating between infected and vaccinated Muscovy ducks.

The aim of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of Muscovy duck parvovirus (MDPV). Sera (100) from negative and vaccinated Muscovy ducks were compared with infected sera (240) to establish the cut-off value of this i-ELISA. There was a significant difference between the positive and negative populations (P<0·05). The adoption of this positive-negative threshold value for this i-ELISA assay resulted in specificity of 98·0%. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus. In this study, we developed an i-ELISA based on epitope AA503-509 (RANEPKE), which is on nonstructural protein of MDPV. This i-ELISA could be used as a diagnostic tool for differentiating infected Muscovy ducks from Muscovy ducks vaccinated with inactivated virus.

Evidence of possible vertical transmission of duck circovirus

To test the hypothesis that duck circovirus (DuCV) may be vertically transmitted from infected breeder ducks to their ducklings, we investigated 120 newly hatched ducklings, 30 dead duck embryos and 80 non-embryonated duck eggs with the duplex polymerase chain reaction (PCR). DuCV DNA was present in 15 newly hatched ducklings, 4 duck embryos and 3 non-embryonated eggs. Four ducklings from two flocks were co-infected by DuCV-1 and DuCV-2, three ducklings from three flocks were DuCV-1 single infection, and eight ducklings from six flocks were DuCV-2 single infection. One duck embryo and one non-embryonated egg were positive for both DuCV-1 and DuCV-2 DNAs, one embryo for DuCV-1 DNA, and two embryos and two non-embryonated eggs for DuCV-2 DNA. The findings provide evidence of possible vertical transmission of DuCV and simultaneous transmission of DuCV-1 and DuCV-2 from breeder ducks to ducklings.

The vaccine efficacy of recombinant duck enteritis virus expressing secreted E with or without PrM proteins of duck tembusu virus

A newly emerged tembusu virus that causes egg-drop has been affecting ducks in China since 2010. Currently, no vaccine is available for this disease. A live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine has been used routinely to control lethal DEV in ducks since the 1960s. Here, we constructed two recombinant DEVs by transfecting overlapping fosmid DNAs. One virus, rDEV-TE, expresses the truncated form of the envelope glycoprotein (TE) of duck tembusu virus (DTMUV), and the other virus, rDEV-PrM/TE, expresses both the TE and pre-membrane proteins (PrM). Animal study demonstrated that both recombinant viruses induced measurable anti-DTMUV neutralizing antibodies in ducks. After two doses of recombinant virus, rDEV-PrM/TE completely protected ducks from DTMUV challenge, whereas rDEV-TE only conferred partial protection. These results demonstrate that recombinant DEV expressing the TE and pre-membrane proteins is protective and can serve as a potential candidate vaccine to prevent DTMUV infection in ducks.

Experimental infection of duck origin virulent Newcastle disease virus strain in ducks.

BackgroundNewcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is an acute, highly contagious and fatal viral disease affecting most species of birds. Ducks are generally considered to be natural reservoirs or carriers of NDV while being resistant to NDV strains, even those most virulent for chickens; however, natural ND cases in ducks have been gradually increasing in recent years. In the present study, ducks of different breeds and ages were experimentally infected with duck origin virulent NDV strain duck/Jiangsu/JSD0812/2008 (JSD0812) by various routes to investigate the pathogenicity of NDV in ducks.ResultsSix breeds (mallard, Gaoyou, Shaoxing, Jinding, Shanma, and Pekin ducks) were infected intramuscularly (IM) with JSD0812 strain at the dose of 5 × 108 ELD50. Susceptibility to NDV infection among breeds varied, per morbidity and mortality. Mallard ducks were the most susceptible, and Pekin ducks the most resistant. Fifteen-, 30-, 45-, 60-, and 110-day-old Gaoyou ducks were infected with JSD0812 strain at the dose of 5 × 108 ELD50 either IM or intranasally (IN) and intraocularly (IO), and their disease development, viral shedding, and virus tissue distribution were determined. The susceptibility of ducks to NDV infection decreased with age. Most deaths occurred in 15- and 30-day-old ducklings infected IM. Ducks infected IN and IO sometimes exhibited clinical signs, but seldom died. Clinical signs were primarily neurologic. Infected ducks could excrete infectious virus from the pharynx and/or cloaca for a short period, which varied with bird age or inoculation route; the longest period was about 7 days. The rate of virus isolation in tissues from infected ducks was generally low, even in those from dead birds, and it appeared to be unrelated to bird age and infection route.ConclusionsThe results confirmed that some of the naturally occurring NDV virulent strains can cause the disease in ducks, and that ducks play an important role in the epidemiology of ND. The prevention of NDV spread in ducks should receive more attention and research in terms of preventing the occurrence and prevalence of ND.

In vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L.

Ethnopharmacological relevanceNiranthin is a lignan isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of niranthin using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models. Materials and methodsNiranthin was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after niranthin was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks. ResultsIn the human HBV-transfected liver cell line HepG2.2.15, the secretion of HBsAg and HBeAg were significantly decreased after treatment with niranthin for 144h, with IC50 values for HBsAg of 15.6µM, IC50 values for HBeAg of 25.1µM. In DHBV-infected ducklings, niranthin significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of niranthin. ConclusionThe experimental data demonstrated that niranthin exhibits anti-hepatitis B virus activity both in vitro and in vivo.

Two functional type VI secretion systems in avian pathogenic Escherichia coli are involved in different pathogenic pathways.

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenic Escherichia coli (APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucial clpV clusters of these two T6SS loci and their vgrG genes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines cultured in vitro, decreased pathogenicity in duck and mouse infection models in vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.

Adaptation and attenuation of duck Tembusu virus strain Du/CH/LSD/110128 following serial passage in chicken embryos.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. In the current study, a virulent strain of DTMUV, designated Du/CH/LSD/110128, was isolated from the livers of diseased ducks and attenuated by serial passage in embryonated chicken eggs. The virus was partially attenuated after 50 and 70 passages and was fully attenuated after 90 passages, based on mortality and morbidity rates and viral loads in inoculated ducklings. Fourteen amino acid substitutions were observed in the capsid, prM, envelope, NS1, NS3, NS4A, NS4B, and NS5 proteins of the fully attenuated strain of Du/CH/LSD/110128, which might be responsible for the observed changes in replication and pathogenicity. A 72-nucleotide deletion was also observed in the 3' untranslated region of the virus after 30 passages. The fully attenuated virus retained the immunogenicity of the parental strain, providing effective protection to challenge with virulent Du/CH/LSD/110128, and may represent a suitable candidate as a vaccine strain against DTMUV infection in ducks. Our results also lay the foundation for future studies on the replication and pathogenic mechanisms of DTMUV.

Immunopathological Studies on Ducks Experimentally Infected with Duck Virus Enteritis and Salmonella Enteritidis with Special References to the Effect of XPC Prebiotic

This research was performed to determine the pathogenesis of the Duck Virus Enteritis (DVE) and Salmonella Enteritidis separately or combined on experimentally infected ducks and evaluating the role of XPC prebiotic in reducing these pathological changes observed on the different organs. The experiment was carried out on 80 Muscovy ducks, equally divided into 8 groups. The group (1) was kept as control. Group (2) was administered 1 ml / L from prebiotic XPC Diamond V in drinking water from the first day of age till the end of the experiment. Group (3) at 12 days of age infected intramuscularly by (1 ml/duck) Duck Virus Enteritis (DVE) homogenates previously prepared. At the same age (12days) group (4) was infected by Salmonella Enteritidis strain orally. Group (5) was infected by both Duck Virus Enteritis homogenate suspension and Salmonella Enteritidis with the same dose and 12 days age. Groups (6,7and8) were administered 1 ml / L from prebiotic XPC in drinking water from the first day of age till the end of the experiment. In the same time groups (6,7and8) infected at 12 day of age by Duck Virus Enteritis organs homogenate suspension, Salmonella Enteritidis and both, respectively with the same dose as groups (3,4 and 5). Tissue samples were collected for viral test and pathological examination when the clinical symptom of the disease appeared. Necropsy was performed and tissue specimen were collected from liver, intestine, esophagus, heart, kidneys, spleen and bursa of Fabricius and fixed in 10% buffered neutral formalin solution for histopathological and immunohistochemistry examination. The histopathological results revealed congestion in all blood vessels of the most infected groups together with recent thrombus in portal vein of Salmonella enteritidis infected group. Degeneration and necrosis with variable degree in addition, inflammatory cells infiltration in different organs of the infected groups were observed. Intranuclear inclusion bodies were seen in the degenerating hepatocytes and in the intestinal epithelium in DVE infected groups. The lesions were alleviated in groups which administered XPC prebiotic and infected with Duck Virus Enteritis or Salmonella Enteritidis and both. It could be concluded that XPC prebiotic alleviated the immunological and pathological alteration induced from the experimentally infected ducks by Duck Virus Enteritis or Salmonella Enteritidis infection and both .

Experimentally Infected Domestic Ducks Show Efficient Transmission of Indonesian H5N1 Highly Pathogenic Avian Influenza Virus, but Lack Persistent Viral Shedding

Ducks are important maintenance hosts for avian influenza, including H5N1 highly pathogenic avian influenza viruses. A previous study indicated that persistence of H5N1 viruses in ducks after the development of humoral immunity may drive viral evolution following immune selection. As H5N1 HPAI is endemic in Indonesia, this mechanism may be important in understanding H5N1 evolution in that region. To determine the capability of domestic ducks to maintain prolonged shedding of Indonesian clade 2.1 H5N1 virus, two groups of Pekin ducks were inoculated through the eyes, nostrils and oropharynx and viral shedding and transmission investigated. Inoculated ducks (n = 15), which were mostly asymptomatic, shed infectious virus from the oral route from 1 to 8 days post inoculation, and from the cloacal route from 2–8 dpi. Viral ribonucleic acid was detected from 1–15 days post inoculation from the oral route and 1–24 days post inoculation from the cloacal route (cycle threshold <40). Most ducks seroconverted in a range of serological tests by 15 days post inoculation. Virus was efficiently transmitted during acute infection (5 inoculation-infected to all 5 contact ducks). However, no evidence for transmission, as determined by seroconversion and viral shedding, was found between an inoculation-infected group (n = 10) and contact ducks (n = 9) when the two groups only had contact after 10 days post inoculation. Clinical disease was more frequent and more severe in contact-infected (2 of 5) than inoculation-infected ducks (1 of 15). We conclude that Indonesian clade 2.1 H5N1 highly pathogenic avian influenza virus does not persist in individual ducks after acute infection.

Characterization of a new genotype of avian bornavirus from wild ducks

Avian bornaviruses (ABV) are a recently described group of intranuclear negative-stranded RNA viruses (Order Mononegavirales, Family Bornaviridae). At least 13 different ABV genotypes have been described. One genotype, the Canada goose genotype (ABV-CG), has been isolated from geese and swans and is widely distributed across North America. We have isolated and characterized a previously undescribed genotype of avian bornavirus from the brains of wild ducks. This new genotype, provisionally designated ABV genotype MALL, was detected in 12 of 83 mallards, and 1 of 8 wood ducks collected at a single location in central Oklahoma. The virus was cultured on primary duck embryo fibroblasts, fragments were cloned, and its genome sequence of 8904 nucleotides determined. This new genotype has 72% nucleotide identity and 83% amino acid identity with the ABV-CG genotype previously shown to be present in geese and swans. Histologic and immunohistochemical examination of the brains and eyes of four positive ducks indicated the presence of virus-infected neurons and glia in their cerebrums and retinas in the absence of inflammation. More than one genotype of ABV is circulating in North American waterfowl. While the infected ducks were not observed to be suffering from overt disease, based on the immunohistochemistry, we speculate that they may have suffered some visual impairment.

Comparison of the Serum Proteins and Immune Responses of Velogenic Newcastle Disease Virus Infected Chickens and Ducks

The effect of velogenic Newcastle disease virus (vNDV) on the immune responses and serum proteins was investigated in six-week-old ducks and chickens. Results showed that weight loss was markedly significant (p

Evaluation of hepatocyteprotective and anti-hepatitis B virus properties of Cichoric acid from Cichorium intybus leaves in cell culture.

Hepatitis B is the most common serious liver infection in the world. To date, there is still no complete cure for chronic hepatitis B. Natural caffeic acid analogues possess prominent antiviral activity, especially anti-hepatitis B virus (HBV) and anti-human immunodeficiency virus effects. Cichoric acid is a caffeic acid derivative from Cichorium intybus. In the study, the anti-hepatitis B property of cichoric acid was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte injury model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected cell line HepG2.2.15 cells, respectively. The results showed that cichoric acid attenuated significantly D-GalN-induced HL-7702 hepatocyte injury at 10-100 µg/mL and produced a maximum protection rate of 56.26%. Moreover, cichoric acid at 1-100 µg/mL inhibited markedly DHBV DNA replication in infected duck fetal hepatocytes. Also, cichoric acid at 10-100 µg/mL reduced significantly the hepatitis B surface and envelope antigen levels in HepG2.2.15 cells and produced the maximum inhibition rates of 79.94% and 76.41%, respectively. Meanwhile, test compound at 50-100 µg/mL inhibited markedly HBV DNA replication. In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid from Cichorium intybus leaves. In addition, cichoric acid could be used to design the antiviral agents.

Comparative Study of the Pathology and Pathogenesis of a Local Velogenic Newcastle Disease Virus Infection in Ducks and Chickens

Comparative Study of the Pathology and Pathogenesis of a Local Velogenic Newcastle Disease Virus Infection in Ducks and Chickens

Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 107.2 TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV.

Experimental reproduction of an Enterococcus cecorum infection in Pekin ducks

Enterococcus cecorum (EC) was thus far only known as a pathogen for broilers and broiler breeders. Recently there was evidence of EC field outbreaks in Pekin duck flocks in Germany. In this study we experimentally reproduced an EC infection in Pekin ducks. At 12 days post hatch, groups of Pekin ducks were infected orally, via the thoracic air sac or intravenously with 1.5 × 109 colony-forming units (CFU) of EC per bird or via the air sac with 8.5 × 105 or 8.5 × 107 CFU per bird. Ducks of the intravenously infected group showed 100% mortality after 2 days post infection. The air sac inoculated high-dose group exhibited a mortality rate of 67%. Birds that were infected with 8.5 × 105 and 8.5 × 107 CFU showed 6.7% mortality after 7 days post infection. Dead birds displayed pneumonia, airsacculitis, pericarditis and splenitis and EC was re-isolated from these organs. Surviving birds of all groups apart from the orally infected ducks demonstrated clinical signs such as huddling, reduced mobility and diarrhoea. Furthermore, they showed gross pathological lesions including airsacculitis and splenitis and lower bodyweights than the control group at necropsy on days 7, 14 and 21 post infection. The present study clearly confirms that EC is pathogenic for Pekin ducks after experimental infection via the intravenous route or the respiratory tract. EC therefore has to be considered as an emerging avian pathogen not only in broilers but also in Pekin ducks.

Tembusu virus infection in Cherry Valley ducks: The effect of age at infection

Three groups of Cherry Valley ducks at 5 day, 2 week and 5 week of age were intranasally infected with the WFCL strain of Tembusu virus (TMUV) to investigate the effect of host age on the outcome of TMUV infection. For each age group, clinical signs, gross and microscopic lesions, viral copy numbers in tissues and serum neutralizing antibody titers were recorded. Age-related differences in the resistance to TMUV infection were observed with younger ducks being more susceptible. Some ducks infected at 5 day and 2 week of age developed severe clinical signs, including severe neurological dysfunction and death. However, subclinical signs and no mortality were observed in ducks infected at 5 week of age. A decline in the severity of gross and microscopic lesions was observed as ducks mature. Systemic infections were established in the three age groups post challenge. Higher viral copy numbers in the tissues, especially in vital organs such as the brain and the heart, were developed in the ducks infected at 5 day of age than older ducks, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, ducks infected at 5 week of age developed significantly higher serum neutralizing antibody titers than ducks infected at 5 day of age as determined by serum neutralization test. Therefore, age-related differences in the resistance to TMUV infection should be considered when studying the pathogenicity, pathogenesis, formulation of the vaccination and therapy strategies of TMUV infection in ducks.

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.

Avian Influenza Infection Alters Fecal Odor in Mallards

Changes in body odor are known to be a consequence of many diseases. Much of the published work on disease-related and body odor changes has involved parasites and certain cancers. Much less studied have been viral diseases, possibly due to an absence of good animal model systems. Here we studied possible alteration of fecal odors in animals infected with avian influenza viruses (AIV). In a behavioral study, inbred C57BL/6 mice were trained in a standard Y-maze to discriminate odors emanating from feces collected from mallard ducks (Anas platyrhynchos) infected with low-pathogenic avian influenza virus compared to fecal odors from non-infected controls. Mice could discriminate odors from non-infected compared to infected individual ducks on the basis of fecal odors when feces from post-infection periods were paired with feces from pre-infection periods. Prompted by this indication of odor change, fecal samples were subjected to dynamic headspace and solvent extraction analyses employing gas chromatography/mass spectrometry to identify chemical markers indicative of AIV infection. Chemical analyses indicated that AIV infection was associated with a marked increase of acetoin (3-hydroxy-2-butanone) in feces. These experiments demonstrate that information regarding viral infection exists via volatile metabolites present in feces. Further, they suggest that odor changes following virus infection could play a role in regulating behavior of conspecifics exposed to infected individuals.