3)

3). to immunization. Domestic cats (Felis catus) are the closest phylogenetic relatives of hyenas that have been studied in detail immunologically, and we hypothesized that anti-cat isotype-specific antibodies would cross react with hyena immunoglobulin epitopes. We used ELISA and Western blots to test isotype-specific anti-feline antibodies for specific cross-reaction to hyena Ig epitopes. Molecular weights of heavy (IgA, IgG, IgM) and light chains of hyena immunoglobulins were determined by protein electrophoresis, and as expected, they were found to be similar to feline immunoglobulins. In order to further validate the cross-reactivity of the anti-feline antibodies and document the hyena humoral response, eight spotted hyenas were immunized with dinitrophenol conjugated keyhole limpet hemocyanin (DNP-KLH) and serum anti-DNP responses were monitored by enzyme-linked immunosorbent assay (ELISA) for one year. The full array of isotype-specific antibodies identified here will allow veterinarians and other researchers to thoroughly investigate the hyena antibody response, and can be used in future studies to test hypotheses about pathogen exposure and immune function in this species. Keywords:Hyena, Crocuta, Antibody, Isotype, Humoral, Immune == 1. Introduction == Wildlife disease outbreaks can have major impacts on conservation efforts and lasting effects on cIAP1 Ligand-Linker Conjugates 3 ecosystem processes (Claude, 1996). For example, rabies and canine distemper computer virus (CDV) epizootics were associated with the extirpation of wild dogs (Lycaon pictus) in the Maasai Mara National Reserve (MMNR) in Kenya (Alexander and Appel, 1994,Kat et al., 1995,Kat et al., 1996). Additionally, a CDV outbreak in East Africa killed more cIAP1 Ligand-Linker Conjugates 3 than 1000 lions (Panthera leo) (Munson et al., 2008,Roelke-Parker et al., 1996). Animals that hunt and scavenge are likely exposed to a broad array of pathogens (Schulenburg et al., 2009). Although most carnivores, including lions and wild dogs, scavenge to some extent (Houston, 1979), theory predicts that this immune systems of carnivores exhibiting morphological specializations for carrion-feeding should have been molded by selective pressures associated with surviving microbial assaults from their food (Blount et al., 2003,Mendes et al., 2006,Schulenburg et al., 2009). Spotted hyenas (Crocuta crocuta) are capable hunters that have descended within the last million years from carrion feeding ancestors (Lewis and Werdelin, 2000,Werdelin, 1989). Despite documented exposure to anthrax, rabies, CDV and several other pathogens, spotted hyenas in East Africa have exhibited extremely low mortality rates due to infectious diseases, even when epizootics decimated sympatric carnivore populations (Alexander et al., 1995,East et al., cIAP1 Ligand-Linker Conjugates 3 2001,East et al., 2004,Harrison et al., 2004,Lembo et al., 2011,Watts and Holekamp, 2009). Spotted hyenas are the most abundant large carnivores in Africa, and may play a critical role in the ecology of disease in African wildlife and domestic animals throughout the continent (Hofer, 1998). In light of the extreme disease resistance manifested by hyenas and their potential importance for overall disease dynamics in African ecosystems, we set out to identify tools available for studying immune function in the spotted hyena. The two specific aims of this study were to identify antibodies that cross-react with hyena immunoglobulins and to assess the dynamics of the hyena humoral immune response to immunization with a nonpathogenic antigen. Domestic cats (Felis catus) were the closest phylogenetic relatives of hyenas that had been studied in detail immunologically (Bininda-Emonds IgM Isotype Control antibody (PE-Cy5) et al., 1999,OBrien and Johnson, 2005), and we hypothesized that anti-cat isotype-specific antibodies would cross react with hyena immunoglobulin (Ig) epitopes. We used ELISAs to test isotype-specific anti-feline antibodies for cross-reaction to hyena Ig epitopes and to assess temporal dynamics of hyena immunoglobulins in response to immune challenge. We used Western blots to confirm cross-reactivity and to estimate the molecular weight of hyena immunoglobulins. Reverse transcriptase polymerase chain reaction (RT-PCR), serum neutralization assessments, western blots, and agglutination assessments have been used previously to document pathogen exposure in spotted hyenas (East et al., 2001,East et al., 2004,Honer et al., 2006,Speck et al., 2008), but only a few studies have gone beyond documenting exposure and examined the immune response itself (East et al.,.