Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand

Paungpin, Weena; Wiriyarat, Witthawat; Chaichoun, Kridsada; Tiyanun, Ekasit; Sangkachai, Nareerat; Changsom, Don; Poltep, Kanaporn; Ratanakorn, Parntep; Puthavathana, Pilaipan

doi:10.1371/journal.pone.0186962

Cited by 11 publications

(16 citation statements)

References 50 publications

(37 reference statements)

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“…Purified recombinant EEHV1A gB, gH/gL or gL (diluted in PBS; 100 µL/well) were coated overnight on Nunc MaxiSorp high protein-binding capacity ELISA plates (Thermo Fisher Scientific). Subsequently, plates were washed three times with PBST (PBS + 0.05% Tween-20) and incubated with blocking buffer (PBS containing 0.1% Tween 20 and 3% BSA [ w / v ] for 2 h. Next, plates were washed (three times with PBST) and 100 µL serum diluted in blocking buffer was added to the wells for 1 h. Plates were washed (three times with PBST), and incubated with 100 µL HRP-conjugated recombinant Protein A/G (0.5 µg/mL diluted in blocking buffer; Pierce), previously reported to bind elephant IgG [ 25 , 26 ], for 1 h. After washing (three times with PBST), 100 µL/well TMB Substrate (Surmodics, Eden Prairie, MN, USA) was added, plates were incubated for 10 min in the dark, and the reaction was stopped by adding 100 µL 12.5% H 2 SO 4 . Optical density (OD) was measured at 450 nm in an EL-808 ELISA reader (BioTEK, Winooski, VT, USA).…”

Section: Methodsmentioning

confidence: 99%

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country

Hoornweg

Schaftenaar

Maurer

et al. 2021

Viruses

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Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

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Section: Methodsmentioning

confidence: 99%

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country

Hoornweg

Schaftenaar

Maurer

et al. 2021

Viruses

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“…Moreover, the result indicated that human H1 virus infection was common in tiger cubs originally from Kanchanaburi province, western Thailand in 2011 and 2013. Our previous study demonstrated that the source of human H1 viral infection in Thai elephants likely came from mahouts or tourists who engaged in activities involving with elephants [19].…”

Section: Discussionmentioning

confidence: 99%

“…The positive samples obtained from the HI assay were further determined for neutralizing (NT) antibody using microNT assay. The protocol for microNT assay was followed by that previously described [13, 19]. Briefly, 60 µl of the treated serum was serially 2-fold diluted in EMEM (Gibco) in 96-well microculture plates in duplicate at an initial dilution of 1:20 and then added the tested virus at the final concentration of 100 TCID50/well.…”

Section: Methodsmentioning

confidence: 99%

Serological evidence of influenza virus infection in captive wild felids, Thailand

Sangkachai

Thongdee

Chaiwattanarungruengpaisan

et al. 2019

The Journal of Veterinary Medical Science

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Influenza virus is known to affect wild felids. To explore the prevalence of influenza viruses in these animal species, 196 archival sera from 5 felid species including Panthera tigris (N=147), Prionailurus viverrinus (N=35), Panthera leo (N=5), Pardofelis temminckii (N=8) and Neofelis nebulosa (N=1) collected between 2011 and 2015 in 10 provinces of Thailand were determined for the presence of antibody to avian and human influenza viruses. Blocking enzyme-linked immunosorbent (ELISA) assay and hemagglutination inhibition (HI) assay were employed as the screening tests, which the serum samples with HI antibody titers ≥20 were further confirmed by cytopathic effect/hemagglutination based-microneutralization (CPE/HA-based microNT) test. Based on HI and microNT assays, the seropositive rates of low pathogenic avian influenza (LPAI) H5 virus, highly pathogenic avian influenza (HPAI) H5 virus and human H1 virus were 1.53% (3/196), 2.04% (4/196) and 6.63% (13/196), respectively. In addition, we also found antibody against both LPAI H5 virus and HPAI H5 virus in 2 out of 196 tested sera (1.02%). Evidences of influenza virus infection were found in captive P. tigris in Kanchanaburi, Nakhon Sawan and Ratchaburi provinces of Thailand. The findings of our study highlights the need of a continuous active surveillance program of influenza viruses in wild felid species.

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“…Next, plates were washed (three times with PBST) and 100 µl serum diluted in blocking buffer was added to the wells for 1 hour. Plates were washed (three times with PBST), and incubated with 100 µl HRP-conjugated recombinant Protein A/G (0.5 µg/ml diluted in blocking buffer; Pierce), previously reported to bind elephant IgG (36, 37), for 1 hour. After washing (three times with PBST), 100 µl/well TMB Substrate (Surmodics) was added, plates were incubated for 10 minutes in the dark, and the reaction was stopped by adding 100 µl 12,5% H2SO4.…”

Section: Methodsmentioning

confidence: 99%

“…Sera with high background values (ODvalues >1 in the wells lacking antigen) were excluded from analysis (N=8, all from Laotian elephant cohort). The rabbit sera specific for gB peptides 1-5 used in this study (36,37), for 1 hour. After washing (three times with PBST), 100 µl/well TMB Substrate (Surmodics) was added, plates were incubated for 10 minutes in the dark, and the reaction was stopped by adding 100 µl 12,5% H2SO4.…”

Section: Expression Of Recombinant Eehv Proteinsmentioning

confidence: 99%

Elephant endotheliotropic herpesvirus is omnipresent in elephants in European zoos and an Asian elephant range country

Hoornweg

Schaftenaar²,

Maurer

et al. 2020

Preprint

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Elephant endotheliotropic herpesviruses (EEHVs) are a group of evolutionary divergent herpesviruses that may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Although EEHV was first discovered over 20 years ago, its prevalence in different elephant populations is still largely unknown, partially due to the lack of readily available, sensitive serological assays. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight in its spread among elephants, we developed novel ELISAs focusing on EEHV1A gB and gH/gL as antigens. Performance of the ELISAs was assessed using sera taken from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, whereas reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. Direct comparison with another EEHV-specific ELISA previously used in two large serosurveys, showed that EEHV prevalence was underestimated before, likely due to aberrant folding of the antigen used. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.ImportanceOver the last 30 years, nearly 20% of all Asian elephants born in Western zoos succumbed to acute hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV-HD). Yet, the prevalence of EEHV in captive and wild elephant populations is still largely unknown, mainly due to the lack of readily available, sensitive serological assays. For this study two highly sensitive EEHV-specific ELISAs were developed. Using these assays, it was shown that nearly all elephants tested were seropositive for EEHV, with highest antibody levels detected in (sub)adult elephants. In contrast, antibody levels in EEHV-HD cases were very low or non-detectable. Lack of antibodies may thus be a risk factor for developing severe disease. As the novel ELISAs are low-tech in nature, these assays may easily be disseminated to local laboratories in zoos and elephant range countries in order to determine EEHV serostatus of individual animals or complete herds and (wild) populations.

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Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand

Cited by 11 publications

References 50 publications

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country

Serological evidence of influenza virus infection in captive wild felids, Thailand

Elephant endotheliotropic herpesvirus is omnipresent in elephants in European zoos and an Asian elephant range country

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