Ecological Archives A021-109-A1

Guillermo Blanco, Jesús A. Lemus, and Marino García-Montijano. 2011. When conservation management becomes contraindicated: impact of food supplementation on health of endangered wildlife. Ecological Applications 21:2469–2477.

Appendix A. Tables showing details on the PCR-based methods used in the determination of pathogens in nestlings Spanish Imperial Eagles and results from multivariate GLMM evaluating the effect of supplementary feeding, drug residues, health status, and number of pathogens on the humoral immune system of nestling Spanish Imperial Eagles.

TABLE A1. Details on the PCR-based methods used in the determination of pathogens in nestlings Spanish Imperial Eagles, Aquila adalberti.

Name Type Pathogen determination Reference
Aspergillus
fumigatus
Fungus Sense amplification primer
5-GGCCCTTAAATAGCCCGGT-3
Antisense amplification primer
5-TGAGCCGATAGTCCCCCTAA-3
Hybridization probe
5-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3  
Applied
Biosystems
Haemoproteus
spp.
Protozoan HaemNFI 5-CATATATTAAGAGAAITATGGAG-3
HaemNR3 5-ATAGAAAGATAAGAAATACCATTC-3
HaemF 5-ATGGTGCTTTCGATATATGCATG-3
HaemR2 5-GCATTATCTGGATGTGATAATGGT-3  
Hellgren et al.
(2004)
Leucocytozoon
spp.
Protozoan HaemNFI [59-CATATATTAAGAGAAITATGGAG-3
HaemNR3 5-ATAGAAAGATAAGAAATACCATTC-3
HaemFL  5-ATGGTGTTTTAGATACTTACATT-39)
HaemR2L (59-CATTATCTGGATGAGATAATGGIGC-3  
Hellgren et al.
(2004)
Plasmodium
spp.
Protozoan HaemNFI 5-CATATATTAAGAGAAITATGGAG-3
HaemNR3 5-ATAGAAAGATAAGAAATACCATTC-3
HaemF 5-ATGGTGCTTTCGATATATGCATG-3
HaemR2 5-GCATTATCTGGATGTGATAATGGT-3  
Hellgren et al.
(2004)
Trypanosoma
spp.
Protozoan ESAG 6/7 forward
5-ACA TTC CAG CAG GAG TTG GAG-3
ESAG 6/7 reverse
5-CAC GTG AAT CCT CAA TTT TGT-3  
Mandal et al.
(2008)
Campylobacter
sp.
Bacteria CCCJ609F 5-AAT CTA ATG GCT TAA CCA TTA-3
CCCJ1442R 5-GTA ACT AGT TTA GTA TTC CGG-3
HIP400F 5-GAA GAG GGT TTG GGT GGT G-3
HIP1134R 5-AGC TAG CTT CGC ATA ATA ACT TG-3
CC18F 5-GGT ATG ATT TCT ACA AAG CGA G-3
CC519R 5-ATA AAA GAC TAT CGT CGC GTG-3  
Linton et al.
(1997)
Chlamydophila
psittaci
Bacteria 201CHOMP 5-GGI GCW GMI TTC CAA TAY GCI CAR TC-3
CHOMP 336 5-CAA GMT TTT CTG GAY TTM AWY TTG TT-3
Schettler et al.
(2003)
Mycoplasma spp.
(gallisepticum,
synoviae,
pneumoniae
)
Bacteria 5-GAG CTA ATC TGT AAA GTT GGT C-3
5-GCT TCC TTG CGG TTA GCA AC-3  
Mekkes and
Feberwee (2005),
Turcsányi
et al. (2005)
Escherichia coli
enterotoxigenic
strain
Bacteria stx common primers
5-GAGCGAAATAATTTATATGTG-3
5-TGATGATGGCAATTCAGTAT-3
stx1 specific primers
5-GCAGTTCGTGGCAAGAGCG-3
5-GCGTCGCCAGCGCACTTG-3
stx2 specific primers
5-AATTTATATGTGGCAGGGTTC-3
5-CTTCACTGTAAATGTGTCATC-3
eaeA specific primers
5-GTGGCGAATACTGGCGAGACT-3
5-CCCCATTCTTTTTCACCGTCG-3
hlyA specific primers
5{-GGTGCAGCAGAAAAAGTTGTAG-3
5{-TCTCGCCTGATAGTGTTTGGTA-3
To isolate both toxin genes
5-CGTATGGTGCTCAAGGAG-3
5-CGATAAGACTCAGTTGC-3
For stx1
5-GGAACACCTGTATATGAAGTG-3
For stx2
5-CTCAGTCATTATTAAACTGCAC-3  
Makino et al.
(2000)
Mycobacterium
avium
Bacteria AV6 3-ATGGCCGGGAGACGATCTATGCCGGCGTAC-5
AV7 3-TGTACGCGTCGAAACTGCGATCGAACG-5
IN38 3-GAACGCCCGTTGGCTGGCCATTCACGAAG-5
IN41 3-TCGAGGAACCGGTGTCCGACCGTGTTGCGC-5
Aranaz et al.
(1997)
Pasteurella
multocida
Bacteria REP1R-1Dt 3-CGGNCTACNGCNGCNNNN-5
REP2-1Dt 5-NCGNCT TATCNGGCCTAC-3  
Saxena et al.
(2002)
Pseudomonas
aeruginosa
Bacteria 5-GACAACGCCCTCAGCATCACCAGC-3
5-CGCTGGCCCATTCGCTCCAGCGCT-3
5-AGCCACATGTCGCCGATCTACACG-3
5-TTCCGCTCCCCGCCAGCCTC-3
5-AGTAGTGCAGCACGCCCTGG-3  
Khan and
Cerniglia (1994)
Salmonella spp. Bacteria F1 5-TGTGTTTTATCTGATGCAAGAGG-3
R1 5-CGTTCTTCTGGTACTTACGATGAC-3  
Deng et al.
(2009)
Adenovirus Virus Falcon adenovirus forward TaqMan primer:
5-CGCTACCCGTCATGCTACTTAA-3
Falcon adenovirus reverse TaqMan primer:
5-CCCAAGCGCCGACGTA-3
Falcon adenovirus TaqMan probe:
5-FAMAAGGCCGGGTAACTGGAGAGGTCG-3  
Schrenzel et al.
(2007)
Avian Influenza
(Serotypes
IH 5, 7 and 9)
Virus AI-For-37 5’- GACCTGATGAGTCTTCTAACCGAGGTC-3’
AI-Rev-181 5’- TGTCTTTAGCCAYTCCATGAG -3’  
Kiss et al. (2006),
Farkas et al.
(2007)
Circovirus Virus 5-GTTATACGCCGCCGTAATC-3
5-AACCCTACAGACGGCGAG-3
3-GTTAAGGGTGAAACACCAACG-3
5-GTCACAGTCCTCCTTGTACC-3
5-TGGTACAAGGAGGACTGTGAC-3
5-CCAGCACTTAATAAAGACTCAG-3
5-TAACATTTGCGTTGGAGGAC-3
Circo-s 5-CGG TGC CAG AAA ATG GTA TGT TAG-3
Circo-as 5-GAA GCT GAA GCC AAT GCC GTA-3  
Hsu et al. (2006)
Herpesvirus Virus 5 -CGCGACTATGTCCACGAGAA-3
5-TGCGTACCGCCAATCAGATA-3
5-FAMTCGGCGTTCCCAGATACGGCC-3  
Cardoso et al.
(2005)
Birnavirus causing
infectious bursal
disease  
Virus B5 5 TCTTGGGTATGTGAGGCTTG 3
B4 3 GGATGTGATTGGCTGGGTTA 5
Hamoud et al.
(2007)
Alphaherpesvirus
causing Marek’s
disease
Virus
meq (MDV1)5 -GAATCTTCCCTGCATTGTGTC-3
 5 -ATCTGGCCCGAATACAAGGAA-3_
sorf1 (HVT)5-AAGCGCTTGTATGTGTAGG-3_
 5-TATGGACGTCATGCAGTTGG-3
Gimeno et al.
(2008)
Avian
paramyxovirus
Virus Lentogens
FT NDV LF3 TCC GBA GGA TAC AAG AGT CYG TGA CC
FT NDV LF4 TCC GBA GGA TAC AAG AGT CYG TGA CT
FT NDV LR2 AGA GCY ACA CCG CCA ATA AT
FT NDV LR3 AGA GCY ACA CCA CCG ATA AT
FT NDV L probe 2 CAG GGR CGC CTT ATA

Velogens/mesogens
FT NDV VF1 GAY TCY ATC CGY AGG ATA CAA GRG TC
FT NDV VR2 AAC CCC AAG AGC TAC ACY RCC
FT NDV VR3 GAC CCC AAG AGC TAC ACY RCC
FT NDV Vprobe 1 AAR CGT YTC TGY CTC C
FT NDV Vprobe 2 AGA RAC GCT TTR TAG GTG C  
Farkas et al.
(2007)
Polyomavirus Virus BamHI 5-TATGGATCCGTTATGGTTATGACTGCCATGGAGGAGTCA- 3
EcoRI 5-TATGAATTCGTCTGAGTCAGGCCCCACTTT- 3
Potti et al. (2007)
Poxvirus Virus Core protein gene (sense):
59-CAGCAGGTGCTAAACAACAA-39
Core protein gene (antisense):
59-CGGTAGCTTAACGCCGAATA-39
Core protein-II gene (sense):
59-CAGCAGGTGCTAAACAACAA-39
Core protein-II gene (antisense):
59-TCCGCCGTCGCAACTTCCGG-39  
Tadese and Reed
(2003)
Reovirus Virus PAF 5-ACTTCTTYaTCTACGCCTTTCG-3
PAR 5-ATYbAAWcDdCWeCGCATCTGCTG-3  
Zhang et al.
(2006)
West Nile
virus
Virus WN132  5' GAAAACATCAAGTATGAGG 3'
WN240  5' GAGGTTCTTCAAACTCCAT 3'  
Malkinson et al.
(2002)

TABLE A2. Results from multivariate Generalized linear mixed models (GLMM, normal error, identity link function) evaluating the effect of supplementary feeding, drug residues, health status and number of pathogens (see methods for variable descriptions) on different parameters of the humoral immune system of nestling Spanish Imperial Eagles. Degrees of freedom (d.f. = 1,40) were calculated by the Containment approximation. Nest identity was included as a random factor to avoid pseudoreplication derived from considering sibling nestlings. Dependant variables: complement classical route (CPV), complement alternative route (APW), interleukins (IL1β, IL6, IL8-like) and γ interferon (IFNγ).

Independent variables
Dependant
variables
Supplementary
feeding
Residues of
quinolones
Residues of
diclazuril
Health status No. of
pathogens
CPV 21.48 ± 17.71
F = 1.47, P = 0.228
267.86 ± 30.16
F = 78.88, P < 0.0001
-50.88 ± 30.30
F = 2.82, P = 0.095
-38.85 ± 22.01
F = 3.12, P = 0.08
-14.32 ± 4.29
F = 11.16, P = 0.001
APV -13.57 ± 14.24
F = 0.91, P = 0.34
285.20 ± 23.77
F = 143.98, P < 0.0001
-1.68 ± 23.52
F = 0.005, P = 0.94
-33.58 ± 17.40
F = 3.72, P = 0.056
-3.32 ±  3.31
F = 1.01, P = 0.32
IL1β 16.07 ± 21.74
F = 0.55, P = 0.46
238.19 ± 36.33
F = 42.99, P < 0.0001
-3.87 ± 35.99
F = 0.01, P = 0.91
-60.78 ± 26.59
F = 5.22, P = 0.024
-9.77 ± 5.07
F = 3.72, P = 0.056
IL8-like 13.79 ± 19.36
F = 0.51, P = 0.48
103.32 ± 31.65
F = 10.6, P = 0.001
10.90 ± 30.82
F = 0.13, P = 0.72
-40.03 ± 23.37
F = 2.93, P = 0.089
-13.26 ± 4.27
F = 9.64, P = 0.002
IFNγ 5.44 ± 1.90
F = 8.17, P = 0.005
-1.07 ± 3.01
F = 0.13, P = 0.72
-2.03 ± 2.87
F = 0.50, P = 0.48
-9.99 ± 2.28
F = 19.27, P < 0.001
-1.64 ± 0.38
F = 18.42, P < 0.0001

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