Download Prenatal passive transfer of maternal immunity in Asian elephants

Veterinary Immunology and Immunopathology 153 (2013) 308–311
Contents lists available at SciVerse ScienceDirect
Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm
Short communication
Prenatal passive transfer of maternal immunity in Asian
elephants (Elephas maximus)
Sally A. Nofs a , Robert L. Atmar b , Wendy A. Keitel a,b , Cathleen Hanlon c ,
Jeffrey J. Stanton a,1 , Jie Tan a , Joseph P. Flanagan d ,
Lauren Howard d , Paul D. Ling a,∗
a
b
c
d
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
The Rabies Laboratory, Kansas State University, 2005 Research Park Circle, Manhattan, KS 66502, USA
Houston Zoo, Inc., 1513 Cambridge, Houston, TX 77030, USA
a r t i c l e
i n f o
Article history:
Received 21 January 2013
Received in revised form 5 March 2013
Accepted 19 March 2013
Keywords:
Asian elephant
Endotheliochorial
Passive immunity
Rabies
Tetanus
a b s t r a c t
Asian (Elephas maximus) and African (Loxodonta africana) elephants exhibit characteristics
of endotheliochorial placentation, which is common in carnivore species and is associated with modest maternal to fetal transplacental antibody transfer. However, it remains
unknown whether the bulk of passive immune transfer in elephants is achieved prenatally
or postnatally through ingestion of colostrum, as has been documented for horses, a species
whose medical knowledgebase is often extrapolated for elephants. To address this issue,
we took advantage of the fact that many zoo elephants are immunized with tetanus toxoid
and/or rabies vaccines as part of their routine health care, allowing a comparison of serum
antibody levels against these antigens between dams and neonates. Serum samples were
collected from 3 newborn Asian elephant calves at birth (before ingestion of colostrum);
2–4 days after birth; and 2–3 months of age. The findings indicate that the newborns had
anti-tetanus toxoid and anti-rabies titers that were equivalent to or higher than the titers
of their dams from birth to approximately 3 months of age, suggesting that the majority of maternal-to-fetal transfer is transplacental and higher than expected based on the
architecture of the Asian elephant placenta.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
The placentas of eutherian mammals are classified
according to two major characteristics: (1) placental shape
and contact sites between fetal and maternal tissue, and
(2) the number of cellular layers that separate maternal
and fetal circulation systems (Chucri et al., 2010; Moffett
∗ Corresponding author. Tel.: +1 713 798 8474; fax: +1 713 798 3170.
E-mail address: [email protected] (P.D. Ling).
1
Current address: Oregon National Primate Research Center, Division of Comparative Medicine, Mail Code L584, 505 NW 185th Avenue,
Beaverton, OR 97006, USA.
0165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetimm.2013.03.008
and Loke, 2006). Passive transfer of maternal antibodies
to the fetus is determined by the structure of the fetomaternal barrier (Chucri et al., 2010; Moffett and Loke, 2006).
Species such as horses, swine, and ruminants develop
epitheliochorial or synepitheliochorial placentas, which
generally impede passage of antibodies from maternal to
fetal circulation systems (Tizard, 2001). In these animals,
passive transfer of antibodies is dependent on the ingestion
and absorption of colostrum. In contrast, endotheliochorial placentation, which occurs in dogs and cats, has been
estimated to allow 5–10% of maternal transplacental
antibody transfer (Chucri et al., 2010; Heddle and Rowley,
1975). Hemochorial placentation, exhibited by humans
and rodents, allows significant transplacental antibody
S.A. Nofs et al. / Veterinary Immunology and Immunopathology 153 (2013) 308–311
transfer (Chucri et al., 2010; Roopenian and Akilesh, 2007).
In contrast to other hind-gut fermenters such as equines,
both Asian and African elephants have been found to
develop endotheliochorial placentas (Cooper et al., 1964;
Perry, 1974). To the best of our knowledge, the nature and
robustness of transplacental maternal antibody transfer
in elephants has not been investigated. The importance of
addressing this issue has arisen in recent years with the
emergence of a newly recognized herpesvirus, known as
elephant endotheliotropic herpesvirus (EEHV), that has
been associated with the death of a significant number of
captive juvenile Asian elephants (Richman and Hayward,
2011). Intriguingly, peak vulnerability to this virus appears
to be between 1 and 3 years of age, which is approximately
around the time when calves are weaned, leading to some
speculation that susceptibility to lethal infections by EEHV
may correlate to loss of protective immunity from their
dams. Successful vaccines have been developed for other
herpesviruses and should theoretically be possible for
EEHV as well (Jarosinski et al., 2006; Smith and Khanna,
2010; Stegeman et al., 1994, 1995). To better understand
the role of protective maternal immunity and how to
best implement a vaccine strategy, additional information
about the nature of passive immunity is needed. To address
this issue, we took advantage of the fact that many captive
elephants are routinely vaccinated against a variety of
pathogens or toxins, including rabies and tetanus. The
purpose of this study was to determine whether significant
transplacental antibody transfer occurs in Asian elephants.
The results will have significant impact in development of
vaccine strategies for elephants, especially for future EEHV
vaccines.
2. Materials and methods
2.1. Animals and sample acquisition
All elephants in the study were Asian elephants (E.
maximus). The age, gender, and date of birth (DOB) for
the elephants are summarized in Table 1. The Rabies
(Imrab3® , Merial Inc. Athens, GA 30601) and Tetanus (Equiloid Innovator® , Ft. Dodge Animal Health, Ft. Dodge, IA,
50501) vaccines were administered intramuscularly (IM)
to elephants 1–4 on the dates summarized in Table 1.
Tetanus vaccine for elephants 5 and 6 was Tetanus Toxoid (Fort Dodge Animal Health, Ft. Dodge, IA, 50501) and
was administered IM on dates shown in Table 1. During the
study period, elephants 1–4 were located at the Houston
309
zoo and elephants 5–6 at the Oklahoma City zoo. Serum was
prepared from blood collected in Corvac serum separator
tubes (Tyco Healthcare, Mansfield, MA 02048, USA). Serum
was collected from elephants 2, 4, and 6 within 30 min after
birth and before nursing and ingestion of colostrum from
their dams (Day 0). Serum was collected opportunistically
from elephants 2, 4 and 6 on days or weeks following their
birth, as indicated in Fig. 1.
Rabies antibody titers. Anti-rabies virus titers were
determined at Kansas State University as described previously (Isaza et al., 2006; Smith et al., 1973). Briefly, elephant
sera were serially diluted and mixed with a standard
amount of live rabies virus followed by addition of baby
hamster kidney (BHK)-21 cells and then incubated on slides
for an additional 24 h. Slides were fixed in acetone and
Infectious foci detected with an anti-rabies FITC conjugate.
The calculation of endpoint titer is made from the percent virus infected cells observed on the slide. Results are
reported as RVNA (Rabies Virus Neutralizing Antibodies)
potency in International Units/ml (IU/ml). The IU is calculated from the titer by comparing it against the titer of a
standard reference serum. As described previously, RVNA
titers > 0.5 U/ml are indicative of a response to the rabies
vaccine (Isaza et al., 2006).
Tetanus antibody titers. Anti-tetanus toxoid (ATA) titers
were determined by ELISA, which was modified from a
tetanus ELISA described previously (Lindsay et al., 2010).
Briefly, ninety-six well polystyrene plates (Immulon 4 HBX,
Thermo Electron Corp., Milford, MA) were coated with
1 ␮g of purified tetanus toxoid (List Biological Laboratories,
USA) at a 20 ␮g/mL concentration in Carbonate Bicarbonate Buffer (pH 9.6). The plate was then incubated overnight
at 4 ◦ C, washed with PBS/0.5% Tween, and blocked with
1% teleostean gelatin-PBS (Sigma Chemical Co., USA) for
1 h at 37 ◦ C. The plates were then blocked for 2 h at RT
with blocking buffer (5% non-fat dry milk as in 2.5 g
non-fat dry milk powder in 50 ml 0.01 PBS) and washed
twice with PBS/Tween 0.05%. Elephant serum samples
were serially diluted two-fold in blocking buffer. Tetanus
toxoid-specific IgG was detected using a horse radish peroxidase (HRP)-conjugated rabbit anti-elephant-IgG. Rabbit
antiserum to elephant immunoglobulin was prepared by
immunizing rabbits with elephant IgG, which was purified as described previously (Kania et al., 1997). The IgG
fraction from the rabbit antiserum was purified using Protein A/G affinity chromatography and then conjugated with
HRP using a Pierce Conjugate purification kit (Thermo
Scientific, Rockford, IL) according to the manufacturer’s
Table 1
Elephant demographics and relevant vaccination history.
Dam/calf pair
Elephant
Gender
DOBa
Originb
Tetanusc
Rabiesd
A
A
B
B
C
C
1
2
3
4
5
6
F
M
F
F
F
F
10/11/90
05/04/10
Est 1981
10/03/10
02/02/95
04/15/11
CB
CB
WB
CB
CB
CB
2/08, 4/09, 2/10
10/10
8/08, 4/09, 2/10
4/11
11/08, 1/11
12/12
2/08, 4/09, 2/10
10/10
8/08, 4/09, 2/10
4/11
None
12/12
a
b
c
d
DOB, date of birth month/day/year for CB. Estimate for wild born WB.
CB, captive born; WB, wild born.
Dates of tetanus vaccination (month/year).
Dates of rabies vaccination (month/year).
310
S.A. Nofs et al. / Veterinary Immunology and Immunopathology 153 (2013) 308–311
Fig. 1. Anti-rabies and tetanus titers in newborn Asian elephants and their dams. (A) The graphs show rabies virus neutralizing antibody (RVNA) titers
(y-axis) for elephant pairs A–C. Titers for dams and calves are indicated by black and gray bars respectively. Elephant pairs consist of elephants 1–2 (pair A),
elephants 3–4 (pair B) and elephants 5–6 (pair C). Samples for each elephant were tested on days indicated below the x-axis. The y-axis scale for elephant
pair C is different for pairs A and B due to barely detectable RVNA titers in these elephants (0.11). (B) The graphs show anti-tetanus antibody (ATA) titers
(y-axis) for elephant pairs A–C. Titers for dams and calves are indicated by black and gray bars respectively. Elephant pairs consist of elephants 1–2 (pair
A), elephants 3–4 (pair B) and elephants 5–6 (pair C). Samples for each elephant were tested on days indicated below the x-axis.
protocol. The ELISA was developed by addition of 3,3 ,5,5 tetramethybenzidine substrate (TMB Microwell Peroxidase Substrate System, Kirkegaard & Perry Laboratories
Inc., USA) and the reaction was stopped by the addition of
H3 PO4 . The A450 of each well was determined spectrophotometrically with an ELISA reader (EE 311SX Microplate
Autoreader, Bio-Tek Instruments, USA). Values with an
OD > 0.05 were considered positive. The antibody titer was
defined as the reciprocal of the highest dilution resulting
in an OD > 0.05. Negative cutoff was established based on
the OD value of cord blood obtained from a neonate born
from a dam with no recent tetanus toxoid vaccine history.
3. Results and discussion
The rabies antibody assay indicated that both elephant
1 and her calf, elephant 2 (elephant pair A), had detectable
concentrations of RVNAs during the time period of the
study (Fig. 1A). Interestingly, the newborn calf had RVNAs
that were slightly higher at birth than his dam (Fig. 1A).
The calves RVNAs increased approximately 2-fold at days 2
and 8 following birth, which was after he had ingested several feedings of colostrum. The calf maintained these RVNA
titers out to almost 3 months after birth. The anti-tetanus
assay also showed that elephant 2 had higher ATA titers at
birth than his dam, and remained at this level until day 86
(Fig. 1B). Similar to elephants 1 and 2, elephant 3 and her
calf, elephant 4 (elephant pair B) also had detectable concentrations of RVNAs (Fig. 1A). Like elephant 2, elephant 4
had detectable RVNAs at birth, although she did not have an
appreciable increase in titers until day 8. At 2 months following birth, her RVNA titer appeared to be dropping from
its peak at day 8. Elephant 4’s ATA titers were equal to her
dam’s at birth until 64 days (Fig. 1B). Her Dam, elephant 3,
continued to maintain post-partum ATAs of 400 for more
than 3 months. Elephants 5 and 6 (dam and calf respectively; elephant pair C) had no known history of rabies virus
vaccination and as expected, RVNA titers were 0.11, which
are considered insignificant (Fig. 1A; note y-axis scale is different for this graph). Elephant 6 did have ATA titers of 200
at days 0 and 2, which were similar to those found in her
dam, elephant 5 (Fig. 1B). No serum samples were available
for elephant 6 after day 2.
This study indicates that elephants passively transfer
significant amounts of immunoglobulin prenatally to their
young. Evidence for this conclusion is based on consistent detection of RVNAs and ATAs in serum from newborn
calves equal to or exceeding the levels of their dams before
the ingestion of colostrum (Fig. 1). The contribution of
colostrum in our study is less certain, as RVNA and ATA
titers between days 3–8 in elephants 2 and 4 did not
consistently increase. RVNA and ATA titers did not drop
appreciably at 2–3 months in these elephants, which might
be expected. The half-life of IgG in species such as mouse
S.A. Nofs et al. / Veterinary Immunology and Immunopathology 153 (2013) 308–311
(Vieira and Rajewsky, 1988), pig (Curtis and Bourne, 1973),
horse (Wilson et al., 2001), and humans (Paul, 2013) has
been estimated to range from 6 to 26 days and so we were
surprised to observe that RVNA and ATA titers did not drop
appreciably at 2–3 months in these elephants. One potential explanation for this is that elephants might express
a neonatal receptor for IgG (FcRn) in their intestines like
rodents, which would enable them to acquire IgG through
milk for a sustained period of time. There are several other
possibilities: elephant IgGs may have a longer half-life;
the sensitivity of our assays is limited; and/or the lack of
later time points prohibited our ability to observe declining
titers. While the ATA titers are IgG specific, the role of other
immunoglobulin isotypes in the rabies assays is possible,
although the ATA and RVNA data are roughly comparable. Although endotheliochroial placentation was expected
to allow some prenatal antibody transfer, one surprising
aspect of our data was the amount of prenatal immune
transfer: antibody titers in the neonates were similar to
or higher than the titers in the dams. Expression of FcRn
receptors in elephant trophoblasts has not been reported
to our knowledge. Whether FcRn-mediated transfer occurs
at a highly efficient level, or if alternative mechanisms
have evolved in elephants to transport immunoglobulins
across the placental barrier remains to be determined. In
summary, this study demonstrated that the majority of
passive transfer of maternal immunity occurs prenatally
and that maternal antibody persists at levels comparable
to the dam for up to 3 months of age. This information
increases our understanding of the immunity of elephant
neonates allowing us to better manage their care and to
guide the design of optimal immunization protocols.
Acknowledgments
Financial support for these studies was provided by the
Houston zoo. Sally Nofs was supported by National Institutes of Health training grant T32-AI-07471. We thank Erin
Latimer of the National Herpesvirus laboratory for the generous gift of rabbit anti-elephant antibodies. We thank Dr.
Margaret Conner of Baylor College of Medicine for assistance with the Tetanus Toxoid ELISA refinement. We would
also like to thank the Houston Zoo, the Oklahoma City Zoo,
and the St. Louis zoo for samples provided for this study.
We especially appreciate the critical review of the study
design and manuscript by Dr. Ian Tizard.
311
References
Chucri, T.M., Monteiro, J.M., Lima, A.R., Salvadori, M.L., Kfoury Jr., J.R.,
Miglino, M.A., 2010. A review of immune transfer by the placenta.
J. Reprod. Immunol. 87, 14–20.
Cooper, R.A., Connell, R.S., Wellings, S.R., 1964. Placenta of the Indian
elephant, Elephas Indicus. Science 146, 410–412.
Curtis, J., Bourne, F.J., 1973. Half-lives of immunoglobulins IgG, IgA and
IgM in the serum of new-born pigs. Immunology 24, 147–155.
Heddle, R.J., Rowley, D., 1975. Dog immunoglobulins, I. Immunochemical characterization of dog serum, parotid saliva, colostrum, milk and
small bowel fluid. Immunology 29, 185–195.
Isaza, R., Davis, R.D., Moore, S.M., Briggs, D.J., 2006. Results of vaccination
of Asian elephants (Elephas maximus) with monovalent inactivated
rabies vaccine. Am. J. Vet. Res. 67, 1934–1936.
Jarosinski, K.W., Tischer, B.K., Trapp, S., Osterrieder, N., 2006. Marek’s
disease virus: lytic replication, oncogenesis and control. Expert Rev.
Vaccines 5, 761–772.
Kania, S.A., Richman, L.K., Kennedy, M., Montali, R.J., Potgieter, L.N.D., 1997.
The isolation, detection, and cross-reactivity of Asian elephant IgG
for the development of serological diagnostic tests. Vet. Allergy Clin.
Immunol. 5, 125–128.
Lindsay, W.A., Wiedner, E., Isaza, R., Townsend, H.G., Boleslawski, M., Lunn,
D.P., 2010. Immune responses of Asian elephants (Elephas maximus)
to commercial tetanus toxoid vaccine. Vet. Immunol. Immunopathol.
133, 287–289.
Moffett, A., Loke, C., 2006. Immunology of placentation in eutherian mammals. Nat. Rev. Immunol. 6, 584–594.
Paul, W.E., 2013. Fundamental Immunology, 7th ed. Wolters Kluwer
Health/Lippincott Williams & Wilkins, Philadelphia.
Perry, J.S., 1974. Implantation, foetal membranes and early placentation
of the African elephant, Loxodonta africana. Philos. Trans. R. Soc. Lond.
B: Biol. Sci. 269, 109–135.
Richman, L.K., Hayward, G.S., 2011. Elephant herpesviruses. In: Fowler,
R.E.M.a.M (Ed.), Fowler’s Zoo and Wild Animal Medicine: Current
Therapy. Saunders, St. Louis, pp. 496–502.
Roopenian, D.C., Akilesh, S., 2007. FcRn: the neonatal Fc receptor comes
of age. Nat. Rev. Immunol. 7, 715–725.
Smith, C., Khanna, R., 2010. Herpesvirus vaccines: challenges and future
prospects. Hum. Vaccin. 6, 1062–1067.
Smith, J.S., Yager, P.A., Baer, G.M., 1973. A rapid reproducible test for determining rabies neutralizing antibody. Bull. World Health Organ. 48,
535–541.
Stegeman, A., Van Nes, A., de Jong, M.C., Bolder, F.W., 1995. Assessment
of the effectiveness of vaccination against pseudorabies in finishing
pigs. Am. J. Vet. Res. 56, 573–578.
Stegeman, A., Van Oirschot, J.T., Kimman, T.G., Tielen, M.J., Hunneman,
W.A., Berndsen, F.W., 1994. Reduction of the prevalence of pseudorabies virus-infected breeding pigs by use of intensive regional
vaccination. Am. J. Vet. Res. 55, 1381–1385.
Tizard, I., 2001. The protective properties of milk and colostrum in nonhuman species. Adv. Nutr. Res. 10, 139–166.
Vieira, P., Rajewsky, K., 1988. The half-lives of serum immunoglobulins
in adult mice. Eur. J. Immunol. 18, 313–316.
Wilson, W.D., Mihalyi, J.E., Hussey, S., Lunn, D.P., 2001. Passive transfer of maternal immunoglobulin isotype antibodies against tetanus
and influenza and their effect on the response of foals to vaccination.
Equine Vet. J. 33, 644–650.