# Maternal antibody transfer in yellow-legged gulls

Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier

► To cite this version:

Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier. Maternal antibody transfer in yellow-legged gulls. Emerging Infectious Diseases, 2009, 15 (7), pp.1147-1149. 10.3201/eid1507.090036. hal-03325692

HAL Id: hal-03325692

https://hal.inrae.fr/hal-03325692

Submitted on 25 Aug 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
spp.) that cohabitated the pond, but
showed no external signs of disease,
were positive for ranavirus by PCR us-
ing primers M153 and M154. Further
sequence analyses are ongoing, and
additional investigations of other am-
phibians and fishes are needed.

Live freshwater fish from sev-
eral countries have been imported
into Japan. However, large amounts
(≤1,300 tons in 2007) of live aquacul-
ture products, including eels and other
fishes, have been imported from Tai-
wan into Japan (www.customs.go.jp/
tariff/2007_4/data/03.htm). Given that
viruses that originate in Japan and Tai-
wan are similar, the ranavirus we de-
tected was likely imported into Japan
in an infected aquatic organism. How-
ever, an epidemiologic survey will be
necessary to determine the source of
the ranavirus in the pond studied. Like-
wise, this virus may be endemic to Ja-
pan, and a survey of native and foreign
free-ranging amphibians should be
conducted. Molecular analysis of rana-
viruses detected in these surveys will
be necessary to differentiate endemic
viruses from introduced viruses.

Japan is located at middle lati-
tudes and has a temperate climate. This
country has long been geologically iso-
lated from Asia. This isolation has re-
sulted in the development of many di-
verse species of amphibians in Japan;
23 species of the order Caudata and 35
species of the order Anura. Of these
species, 49 (84%) are native and 36
(62%) are listed by the Ministry of the
Environment as threatened species (8).
*R. catesbeiana* frogs were introduced
into Japan in 1918 as a food animal,
and raising them by aquaculture was
widely attempted. Although they are
no longer cultured, feral populations
have become established throughout
Japan (9). Ranavirus in *R. catesbeiana*
frogs represents a serious threat to am-
phibians throughout Japan.

This study was supported by the
Global Environment Research Fund (F-3

and F-081) of the Ministry of the Environ-
ment, Japan, 2008, and The Promotion
and Mutual Aid Corporation for Private
Schools of Japan, Grant in Aid for Match-
ing Fund Subsidy for Private Universities.

Yumi Une, Akiko Sakuma,
Hiroki Matsueda, Katsuki Nakai,
and Masaru Murakami

Author affiliations: Azabu University, Kana-
gawa, Japan (Y. Ume, A. Sakuma, H. Mat-
sueda, M. Murakami); and Nature Conser-
vation Division of Shiga Prefecture, Shiga,
Japan (K. Nakai)

DOI: 10.3201/eid1507.081636

References

1. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R. Emerging infectious diseases and amphibian population declines. Emerg Infect Dis. 1999;5:735-48.

2. Cunningham AA, Hyatt AD, Russell P, Bennett PM. Emerging epidemic diseases of frogs in Britain are dependent on the source of ranavirus agent and the route of exposure. Epidemiol Infect. 2007;135:1200-12.

3. Docherty DE, Meteyer CU, Wang J, Mao J, Case ST, Chinchar VG. Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events. J Wildl Dis. 2003;39:556-66.

4. Marsh IB, Whittington RJ, O'Rourke B, Hyatt AD, Chisholm O. Rapid differentiation of Australian, European and American ranavirus based on variation in major capsid protein gene sequence. Mol Cell Probes. 2002;16:137-51. DOI: 10.1006/mcpr.2001.0400

5. Wolf K, Bullock GL, Dunbar CE, Quimby MC. Tadpole edema virus: a viscerotropic pathogen for anuran amphibians. J Infect Dis. 1968;118:253-62.

6. Miller DL, Rajeev S, Gray MJ, Baldwin CA. Frog virus 3 infection, cultured American bullfrogs. Emerg Infect Dis. 2007;13:342-3.

7. Majji S, LaPatra S, Long SM, Sample R, Bryan L, Sinning A, et al. *Rana catesbeiana* virus Z (RCV-Z): a novel pathogenic ranavirus. Dis Aquat Organ. 2006;73:1-11. DOI: 10.3354/dao073001

8. Ota H. Current status of the threatened amphibians and reptiles of Japan. Population Ecology. 2000;42:5-9. DOI: 10.1007/s101440050003

9. Maeda N, Matsui M. Frogs and toads of Japan. Tokyo: Bun-ichi Sogo Shuppan Publishers; 1989. p. 100-7.

Address for correspondence: Yumi Une,
Laboratory of Veterinary Pathology, School of
Veterinary Medicine, Azabu University, 1-17-
71 Fuchinobe, Sagamihara, Kanagawa 229-
8501, Japan; email: une@azabu-u.ac.jp

Maternal Antibody
Transfer in
Yellow-legged Gulls

**To the Editor:** Avian influenza viruses (AIVs) are emerging pathogens of concern because they can cause deaths in birds and humans (1). Although wild birds likely contribute to AIV emergence because they are the natural reservoir for all known influenza virus subtypes (1), our understanding of AIV transmission and immunology in natural avian populations is incomplete (2). In this context, the transfer of maternal antibodies is a tool that should be used more often in immunologic analysis. Because antibodies in eggs and hatchlings can reflect the mother's past exposure to pathogens (3,4) and both life stages are more easily sampled than adults, quantifying antibodies found in avian young could help clarify AIV epidemiology.

We determined whether eggs of
yellow-legged gulls (*Larus micha-
hellis*) contained antibodies against
AIVs. Yellow-legged gulls can host
AIVs (C. Lebarbenchon, unpub. data),
are abundant, and nest in large, dense
colonies in coastal areas. In April
2008, we collected 466 eggs from 2
yellow-legged gull colonies located
on the Mediterranean coast: 252 eggs
from Gruissan (43.1099°N, 3.1071°E;
350 breeding pairs over 1.5 hectares),
and 212 from Villeneuve-lès-Mague-
lone (VLM; 43.4895°N, 3.8520°E;
400 pairs over 1 hectare). Villeneuve
nests formed 2 spatially clustered
subcolonies: VLM1 (129 eggs) and VLM2 (83 eggs). We also collected global positioning system coordinates for VLM1 nests.

Egg yolks were isolated and frozen at -20°C until analysis. Antibodies were obtained by using chloroform extraction (4). The yolk was diluted 1:1 in phosphate-buffered saline and homogenized. An equal volume of chloroform was added, the solution was centrifuged (6,000 × g for 15 min), and the supernatant was used in the analyses.

Extracts were tested for antibodies against the AIV nucleoprotein by using a commercial competitive ELISA (IDEXX/Institut Pourquier, Montpellier, France). The assay has been validated by using seagull serum (IDEXX, pers. comm.) and chicken egg yolk (5). A subset of samples was tested by using a second commercial competitive ELISA (IDVet, Montpellier, France). Optical density values obtained in the 2 assays were significantly correlated (r = 0.90, df = 39, p<0.001), and serostatus was consistent across assays.

Overall antibody prevalence was 14% (65/466), indicating exposure to influenza A viruses in these colonies. As expected, antibody prevalence in gulls is higher than the viral prevalence previously estimated by reverse transcription-PCR or virus isolation on fecal samples, i.e., the methods typically used by avian influenza surveillance networks. A spring and summer survey performed on feces from gulls of the Camargue region (east of our colonies) showed that only 0.9% of gulls (2 infected of 228 sampled) were excreting AIVs (C. Lebarbchenon, unpub. data).

Egg antibody prevalence did not differ significantly between colonies. The antibody prevalence of 13.5% found at Gruissan (34 of 252 eggs) was comparable with the 14.5% found at Villeneuve (31 of 214 eggs) (generalized linear model with binomial distribution, z = 0.4, p = 0.8). The sub-

colonies of Villeneuve also did not differ: 18/129 (14%) in VLM1 compared with 12/83 (14.5%) in VLM2 (z = 0.02, p = 0.9). There was no evidence of spatial autocorrelation in the distribution of antibody in eggs by using the Moran I spatial statistic. This similarity of antibody prevalence across and within colonies suggests that exposure is dictated by regional rather than local conditions, a hypothesis that should be tested by sampling across a broader range of nest densities and over time.

Our study presents evidence for the presence of antibodies against AIVs in wild bird eggs, and the findings have important practical implications. The difference in prevalence estimated from virus isolation (0.9%) and antibody detection (14%), although expected, highlights the complementary nature of the 2 approaches. Most surveys estimate current infection by virus isolation, which provides information about disease risk in addition to phylogeographic tracking of strains. In contrast, information on antibody prevalence, which shows past and present population exposure and risk, has largely been ignored with few exceptions (e.g., 6,7). Future work could benefit from using both approaches in tandem with modeling to develop an understanding of avian influenza ecology in nature.

Our results also show the generalizable potential of maternal antibody transfer for tracking pathogen exposure in wild birds, notably in the case of recognized emerging zoonoses. Because eggs and hatchlings are proxies of past and present adult pathogen exposure (3,4), the difficult and sometimes disruptive sampling of adults can be circumvented by the rapid and cost-efficient sampling of their young, which will facilitate monitoring efforts. Due to the high intranest correlation in egg antibodies (4), only partial sampling of clutches (e.g., 1 of 3 eggs) is necessary to track pathogen presence and prevalence through space and time. The sampling effects could further be mini-

mized by taking blood samples from young nestlings of a standardized age. Finally, such samples provide abundant material for the simultaneous surveillance of other emerging pathogens of interest, such as *Campylobacter* spp. (8) and West Nile virus (9).

## Acknowledgments

We thank P. Peret, P. Mayet, R. Gauthier, and E. Charbonnier for egg collection and V. Staszewski for technical assistance. Sampling permits were obtained from the Direction Régionale de l'Environnement de Languedoc-Roussillon.

This study was supported by grants from the French National Agency for Research, the Agency for Inter-Institutional Promotion of Research in Developing Countries, and a Chateaubriand fellowship.

## Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, and Thierry Boulinier

Author affiliations: Centre d'Ecologie Fonctionnelle et Evolutive, Montpellier, France (J.M.C. Pearce-Duvet, T. Boulinier); Centre de Recherche de la Tour du Valat, Arles, France (M. Gauthier-Clerc); and Kalmar University, Kalmar, Sweden (E. Jourdain)

DOI: 10.3201/eid1507.090036

## References

1. Olsen B, Munster VJ, Wallensten A, Waldenström J, Osterhaus AD, Fouchier RA. Global patterns of influenza A virus in wild birds. Science. 2006;312:384–8. DOI: 10.1126/science.1122438

2. Boyce WM, Sandrock C, Kreuder-Johnson C, Kelly T, Cardona C. Avian influenza in wild birds: a moving target. Comp Immunol Microbiol Infect Dis. 2009;32:275–86. DOI: 10.1016/j.cimid.2008.01.002

3. Boulinier T, Staszewski V. Maternal transfer of antibodies: raising immuno-ecology issues. Trends Ecol Evol. 2008;23:282–8. DOI: 10.1016/j.tree.2007.12.006

4. Gasparini J, McCoy KD, Haussy C, Tveraa T, Boulinier T. Induced maternal response to the Lyme disease spirochaete *Borrelia burgdorferi* sensu lato in a colonial sea-bird, the kittiwake *Rissa tridactyla*. Proc R Soc Lond B Biol Sci. 2001;268:647–50. DOI: 10.1098/rspb.2000.1411
# Prevalence of Human Bocavirus in Human Tonsils and Adenoids

**To the Editor:** Recently, Longtin et al. (1) reported a high rate (43%) of human bocavirus (HBoV) infection in a group of children chosen to serve as controls in a study of HBoV prevalence among hospitalized children and adults. In contrast, previous reports had found low HBoV prevalence rates of 0%–1% in control groups (2,3). Attempting to explain this surprising difference in rates, Lu et al. (4) suggested that selection of control patients may be related to the difference in rates. The control group used in the Longtin study was primarily (71%) made up of children undergoing elective surgery; previous studies had selected control groups from other sources, including well children on routine visits and outpatients with nonrespiratory symptoms. Because the Longtin study surgeries were mainly tonsillectomies, adenoidectomies, and myringotomies, Lu et al. examined the possibility that tonsillar tissues, which include the adenoids, are sites of persistent HBoV infection. When these researchers extracted DNA from tonsillar lymphocytes obtained from pediatric patients who had undergone tonsillectomies or adenoidectomies, they detected HBoV DNA in 32% of the samples (4). These findings strongly suggest a connection between HBoV and tonsillar tissue. Therefore, we tested a number of tonsillar samples for the presence of HBoV DNA.

Sample acquisition was approved by the Nationwide Children's Hospital Institutional Review Board. Tonsils and adenoids were obtained from 91 patients who underwent elective surgery at Nationwide Children's Hospital from June through September 2004. Patients' ages ranged from 1 to 16 years (median 5.9 years; age was unknown for 4 patients).

Samples consisted of surgically removed tonsil or adenoid tissues. DNA was extracted and its concentration was determined as previously described (5). Two primer sets were used for HBoV detection by using real-time PCR with SYBR Green detection and melting-point determination. We designed primers 3097F (5'-GTC-CAA-TTA-CAT-GAT-CAC-GCC-TAC-TC) and 3420R (5'-TGC-GTC-CAC-AGT-ATC-AGG-TTG-TTG) that targeted the viral protein 1/2 (VP1/VP2) region of HBoV. The nonstructural protein 1 (NP1) region was targeted by using primers 188F and 542R from Alander et al. (6) Each 20-µL reaction contained SYBR Green JumpStart Taq ReadyMix (Sigma, St. Louis, MO, USA), 4 mmol/L MgCl₂, 250 nmol/L primers, double-distilled H₂O, and 2 µL of DNA (50–200 ng) cycled on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA) instrument at 94°C for 2 min, followed by 40 cycles of 94°C for 20 s, 60°C (NP1 primers) or 68°C (VP1/VP2 primers) for 20 s, and 72°C for 14 s. Amplification and melting curves were analyzed with 7900HT version 2.2.1 software (Applied Biosystems); positive samples were verified by sequence analysis. Sequenced VP- and NP1-generated amplicons were 99%–100% identical to HBoV strain ST1 (6). The detection sensitivities of the VP and NP1 assays, determined by using a plasmid construct containing the full HBoV genome, were 1–5 and 5–10 gene copies/reaction, respectively.

Our testing identified HBoV DNA in 5 (5.5%) of the 91 children who underwent elective tonsillectomy/adenoidectomy. Ages ranged from 1.9 to 4.6 years (median 3.4 years). The reason for the much lower HBoV prevalence in this group of children, compared with prevalences found in studies by Longtin et al. and Lu et al., is unclear. Lu et al. (4) reported a much higher HBoV rate of lymphocytes from adenoids (56%) than from tonsils (16%). Although we did not know

5. Trampel DW, Zhou E-M, Yoon K-J, Koehler KJ. Detection of antibodies in serum and egg yolk following infection of chickens with an H6N2 avian influenza virus. J Vet Diagn Invest. 2006;18:437–42.

6. Krauss S, Obert CA, Franks J, Walker D, Jones K, Seiler P, et al. Influenza in migratory birds and evidence of limited intercontinental virus exchange. PLoS Pathog. 2007;3:e167. DOI: 10.1371/journal. ppat.0030167

7. Stallknecht DE, Shane SM. Host range of avian influenza in free-living birds. Vet Res Commun. 1988;12:125–41. DOI: 10.1007/BF00362792

8. Sahin O, Zhang Q, Meitzler JC, Harr BS, Morishita TY, Mohan R. Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal antibodies. Appl Environ Microbiol. 2001;67:3951–7. DOI: 10.1128/AEM.67.9.3951-3957.2001

9. Baitchman EJ, Tlusty MF, Murphy HW. Passive transfer of maternal antibodies to West Nile virus in flamingo chicks (*Phoenicopterus chilensis* and *Phoenicopterus ruber ruber*). J Zoo Wildl Med. 2007;38:337–40. DOI: 10.1638/1042-7260(2007)038[0337:PTOMAT]2.0.CO;2

Address for correspondence: Jessica M.C. Pearce-Duvet, Centre d'Ecologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientifique—Unité de Mixte de Recherche 5175, 1919 Route de Mende, F-34293 Montpellier, France; email: jessica.pearce@cefe.cnrs.fr

## Letters

Letters commenting on recent articles as well as letters reporting cases, outbreaks, or original research are welcome. Letters commenting on articles should contain no more than 300 words and 5 references; they are more likely to be published if submitted within 4 weeks of the original article's publication. Letters reporting cases, outbreaks, or original research should contain no more than 800 words and 10 references. They may have 1 Figure or Table and should not be divided into sections. All letters should contain material not previously published and include a word count.