Conceived and designed the experiments: RG PB. Performed the experiments: RG SS. Analyzed the data: RG PB. Contributed reagents/materials/analysis tools: RG JN PB. Wrote the paper: RG PB.
The authors have declared that no competing interests exist.
The Highly Pathogenic Avian Influenza H5N1 virus has dramatically spread throughout Southeast Asia since its first detection in 1997. Merit Release Birds, such as the Eurasian Tree Sparrow, are believed to increase one's positive karma when kissed and released during Buddhist rituals. Since these birds are often in close contact with both poultry and humans, we investigated their potential role in the spread of H5N1 virus.
Seven series of experiments were conducted in order to investigate the possible interactions between inoculated and exposed birds, including sparrow/sparrow, sparrow/chicken, duck/sparrow. Daily and post-mortem samples collected were tested for H5N1 virus by real-time RT-PCR and egg inoculation. When directly inoculated, Eurasian Tree Sparrows were highly susceptible to the H5N1 virus, with a fatality rate approaching 100% within 5 days post-inoculation. Although transmission of fatal infection between sparrows did not occur, seroconversion of the exposed birds was observed. Up to 100% chickens exposed to inoculated sparrows died of H5N1 infection, depending on the caging conditions of the birds, while a fatality rate of 50% was observed on sparrows exposed to infected ducks. Large quantities of H5N1 virus were detected in the sparrows, particularly in their feathers, from which infectious particles were recovered.
Our study indicates that under experimental conditions, Eurasian Tree Sparrows are susceptible to H5N1 infection, either by direct inoculation or by contact with infected poultry. Their ability to transmit H5N1 infection to other birds is also demonstrated, suggesting that the sparrows may play a role in the dissemination of the virus. Finally, the presence of significant quantities of H5N1 virus on sparrows' feathers, including infectious particles, would suggest that Merit Release Birds represent a risk for human contamination in countries where avian influenza virus is circulating and where this religious ritual is practiced.
The Highly Pathogenic Avian Influenza (HPAI) H5N1 virus has dramatically spread throughout Southeast Asia since its first detection in 1997
Buddhism is the major religion in several Southeast Asian countries, especially in Laos, Myanmar, Thailand and Cambodia, where 67%, 89%, 95% and 97% of the national population consider themselves as Buddhists respectively, amounting to almost 130 million people
The implications of such ritual in the transmission of zoonotic diseases have not been widely studied before
Although wild waterfowl is thought to be a natural reservoir of all influenza subtypes
In order to better understand this role, we conducted a number of experiments. We investigated the susceptibility of sparrows to HPAI H5N1 infection, their ability to transmit the virus to chickens, as well as their ability to get contaminated through contact with infected ducks. In parallel, we studied the survival of the virus on infected sparrows' feathers. This study led us to a better understanding of the potential role of the Merit Release Birds in the H5N1 virus natural contamination cycle.
All experiments using HPAI H5N1 virus and all animal experiments were carried out in the BioSafety level 3 Laboratory (BSL-3) of the Institut Pasteur in Cambodia, complying with the Animal Committee regulations of Institut Pasteur in Paris, France, in accordance with the EC 86/609/CEE directive, and approved by the Animal Ethics Committee of Institut Pasteur in Cambodia (permit number: AEC/IPC/001/2008).
Eurasian Tree Sparrows (
The HPAI H5N1 virus used in this study was the strain A/Chicken/Cambodia/LC1AL/2007 (GenBank accession numbers HQ200574-200581). The virus stock was obtained after 3 passages in SPF 9-to-11-days old embryonated hen eggs (kindly provided by the National Veterinary Research Institute of Cambodia (NaVRI), Ministry of Agriculture, Forestries and Fisheries (MAFF)) for 48 hours at 37°C. The amnio-allantoic fluid was then harvested and stored at −80°C until further use. Virus titer was determined by calculating the 50% egg infectious dose (EID50) per mL of virus stock. Titration endpoints were calculated using the method of Reed and Muench
Seven types of experiments were conducted so as to investigate 3 transmission routes: sparrows' ability to transmit H5N1 infection to each other (A), sparrows' ability to transmit H5N1 infection to chickens (B), and sparrows' ability to be contaminated through contact with infected ducks (C). See
Principles | # | Animals (or materials) inoculated on Day0 | Inoculum dose (EID50) | Animals introduced on Day1 | Containment conditions & remarks |
Sparrows' ability to transmit H5N1 virus to each other | A.1 | Sparrows (n = 8) | 106 /sparrow | Sparrows (n = 7) | All sparrows were freed in the isolator |
A.2 | Sparrows (n = 10) | 106 /sparrow | Sparrows (n = 11) | All sparrows were confined in the same cage within the isolator | |
Sparrows' ability to transmit H5N1 virus to chickens | B.1 | Sparrows (n = 12) | 106 /sparrow | Chickens (n = 20) | All birds were freed in the isolator |
B.2. | Sparrows (n = 5) | 106 /sparrow | Chickens (n = 6) | The sparrows were caged, whereas the chickens were freed in the isolator | |
B.3 | Chickens (n = 5) | 105.23 /chicken | none | All chickens were freed in the isolator | |
B.4 | Water | 105.23 in 1L of water | none | Chickens (n = 5) were introduced into the isolator containing the contaminated water on D0 | |
Sparrows' ability to be contaminated through contact with infected ducks | C | Ducks (n = 40) | 106 /duck | Sparrows (n = 6) | All birds were freed in the whole isolator |
After inoculation, oral and cloacal swabs, as well as feathers, were collected daily, and stored in VTM at −80°C until further analysis. All samples collected before and after inoculation/exposition were tested following the protocols described below.
All birds were observed for expression of clinical signs of illness on a daily basis. The main clinical signs expected included: cloudy and/or watery eyes, head and/or face oedema, cyanosis, weakness, anorexia, depression, ruffled feathers, neurological signs (tremors, seizures, incoordination, paralysis, torticollis, etc.), respiratory difficulties, diarrhea, and death. The birds that were still alive 15 days after infection or exposure to contaminated animals or water were killed humanly.
Post-mortem examination and collection of samples were conducted on all animals. The samples collected post-mortem included: blood, oral and cloacal swabs, feathers, and organs such as trachea, muscle, heart, liver, spleen, air sacs, intestines, kidneys, lungs, and brain. All oral and cloacal swabs, and feathers, were stored in VTM at −80°C. All organs were stored in Phosphate Buffer Saline (PBS) at −80°C on one hand, in 10% neutral buffered formalin at room temperature on the other hand.
Supernatants were collected from swabs stored in VTM and directly processed for RNA extraction. Solid samples, such as feathers and organs, stored in VTM or PBS, went through a homogenization step before RNA extraction, using the MagNa Lyser Instrument (Roche Diagnostics, Mannheim, Germany). RNA was extracted using the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche) and the MagNa Pure LC Instrument (Roche). The extracted RNAs were tested with quantitative real-time Retro-Transcription and Polymerase Chain Reaction (qRT-PCR) targeting the H5 hemagglutinin gene (H5HA), using the primers H5HA-205-227v2-For (5′ – CGA TCT AGA YGG GGT GAA RCC TC – 3′) and H5HA-326-302v2-Rev (5′ – CCT TCT CCA CTA TGT ANG ACC ATT C – 3′), and the probes H5-Probe-239-RVa (5′ – (Fam) – AGC CAY CCA GCT ACR CTA CA – (MGB) – 3′) and H5-Probe-239-RVb (5′ – (Fam) – AGC
Serum samples were treated with receptor-destroying enzyme RDE (II) “SEIKEN” (Denka Seiken Co., LTD, Japan) as instructed by the manufacturer, and then heat-inactivated at 56°C for 30 minutes. Hemagglutination inhibition (HI) tests were then performed using 0.5% chicken red blood cells. HI titers were determined as the reciprocal of the highest serum dilution that inhibited the agglutination of 4 hemagglutining units (HAU) of virus. Sera with HI titers ≥16 units were considered positive for the presence of anti-influenza antibodies
Immunohistochemical staining of the tissues obtained from MRBs only was carried out for the influenza nucleoprotein using HB65 (European Veterinary Laboratories, Netherlands) as described in previously published reports
Feathers obtained from all sparrows were first homogenized using the MagNa Lyser Instrument (Roche). Each homogenized sample was then inoculated onto three 9-to-11-days old SPF hen eggs and incubated during 48 to 72 hours at 37°C. An hemagglutination (HA) assay was performed on all eggs after each passage, in order to detect infectious H5N1 particles. Positive HA results were further confirmed by qRT-PCR.
Sparrows, ducks and chickens were highly susceptible to the HPAI H5N1 strain used when directly inoculated, with overall fatality rates of 97%, 85% and 100% respectively. No symptoms were observed in chickens, which all died on D2 post-inoculation (
# |
Animals infected experimentally | Animals exposed | ||||||||||
Sp |
FR |
MDT |
MVL |
Sp |
FR |
MDT |
MVL |
|||||
Swab | Feather | Organ | Swab | Feather | Organ | |||||||
|
SPR | 100 | 4.1 | 2.74×108 | 1.13×1010 | 6.01×1010 | SPR | 0 | NA | 0 | 0 | 0 |
|
SPR | 100 | 3.7 | 2.89×106 | 1.56×106 | 3.37×109 | SPR | 0 | NA | 0 | 0 | 0 |
|
SPR | 100 | 4 | 6.42×105 | 4.34×106 | 1.88×109 | CK | 0 | NA | 0 | 0 | 0 |
|
SPR | 80 | 4 | 1.24×107 | 8.12×102 | 6.37×109 | CK | 100 | 6.5 | 1.87×107 | 1.10×108 | 1.44×1010 |
|
CK | 100 | 2 | 1.35×107 | 3.16×109 | 3.30×109 | NA | NA | NA | NA | NA | NA |
|
NA | NA | NA | NA | NA | NA | CK | 0 | NA | 0 | 0 | 0 |
|
DK | 85 | 3.7 | 8.15×106 | 2.35×1010 | 1.74×1010 | SPR | 50 | 9 | 1.93×106 | 2.59×108 | 5.44×109 |
*Sp = Species (CK = Chicken, DK = Duck, SPR = Sparrow); FR = Fatality Rate (%); MDT = Mean Death Time (days); MVL = Mean Viral Load (no. RNA copies) per mL of swab's supernatant (Swabs), per gram of feathers (Feather), per gram of organ (Organ); NA = Not Applicable.
MVL reported here include data recorded from lethally infected birds only.
All values appearing in the # column correspond to the 7 different experimental settings described in
Moderate nephromegaly, splenomegaly, liver discolouration, pancreatomegaly, cholecystomegaly, hyperdilatation of brain blood vessels, hemorrhagic lungs, and pericarditis were among the occasional abnormalities observed during the post-mortem examinations of all bird species used in this study.
Immunohistochemical staining of the tissues confirmed the presence of H5N1 antigen in several Passerine's tissues, namely skeletal and cardiac muscles, trachea, liver and lungs (
High viral loads were detected in all samples collected post-mortem from birds lethally infected through direct inoculation (
*NA = not available. Data presented include only birds that died following H5N1 infection.
All surviving birds seroconverted (
# |
Animals inoculated | Animals exposed | ||
Sp |
SR |
Sp |
SR |
|
|
SPR | NA | SPR | 14 |
|
SPR | NA | SPR | 36 |
|
SPR | NA | CK | 0 |
|
SPR | 100 | CK | NA |
|
CK | NA | NA | NA |
|
NA | NA | CK | 20 |
|
DK | 100 | SPR | 50 |
*Sp = Species (CK = Chicken, DK = Duck, SPR = Sparrow); SR = Seroconversion Rate in surviving birds (%); NA = Not Applicable.
All values appearing in the # column correspond to the 7 different experiments described in
Regardless of the conditions of containment of the birds, exposure of naïve sparrows to inoculated ones did not lead to any clinical manifestation nor death, despite a 100% mortality rate observed in inoculated MRBs in the A.1 and A.2 experiments (
FR = Fatality Rate; SR = Seroconversion Rate; VDR = Virus Detection Rate; NA = not available. Arrows and their respective black-filled rectangles correspond to the different experimental settings described in
When left to freely fly within the whole isolator, the sparrows did not transmit H5N1 infection to the chickens (
H5N1 viral RNA was detected in 67% of the sparrows exposed to inoculated ducks, with a case fatality rate of 50%. Deadly contaminated sparrows showed high viral loads in all organs (
Feathers of 94 sparrows were tested. A total of 50 birds tested positive for the presence of viral RNA on their feathers, and 24 were found to carry infectious particles on their feathers.
Here, Eurasian Tree Sparrows were shown to be highly susceptible to the HPAI H5N1 strain used. However, the transmission of the virus between sparrows themselves was quite low, be it released in a wide space (14% of seroconversion) or contained in a narrow cage (36% of seroconversion), and led only to asymptomatic infections with no detectable virus infection or shedding (
Similarly, when exposed to infected sparrows freed in the isolator, no chickens were infected despite their high susceptibility to the strain used (
Nonetheless, on the other hand, when caged and unable to escape from the chickens, transmission occurred. The caging, instead of protecting and separating the sparrows from the chickens, made them more vulnerable. They were placed in a stressful situation, in which chickens perpetually harassed them through the bars, and were able to easily peck them. This behavior led to the chickens' infection.
In order to better understand the absence of H5N1 virus transmission from sparrows to chickens when close contacts are not forced, we tried to evaluate the level of exposure of the chickens to the avian influenza virus during the experiment. We estimated that 24 infected sparrows shed through feces the amount of 105.23 EID50 virus per hour. We considered these conditions as extreme since the probability of having 24 infected sparrows spending one full hour dropping their feces either directly into a chicken's external orifices, or into the same liter of water, was highly unlikely. Our findings demonstrated that even at this dose, the seeded drinking water was not able to cause clinical disease in chickens, whereas the same dose was proved to be lethal when directly inoculated to chickens through nasal, ocular, oral and cloacal routes (
Efficient viral transmission also occurred from ducks to sparrows. Ducks are often incriminated in the silent spread of influenza A viruses, because of their frequent ability to undergo asymptomatic infections
Several infected sparrows were carrying infectious particles on their feathers. The presence of H5N1 virus antigens in feather follicles, associated with histological lesions, was first assessed and described by Perkins and Swayne in 2001
(PPT)
We thank the staff of the Ministry of Agriculture who raised and provided the birds, and the staff of the Virology Unit of Institut Pasteur in Cambodia for their precious technical assistance.