Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles.

An overview of the lagomorph immune system and its genetic diversity.

Our knowledge of the lagomorph immune system remains largely based upon studies of the European rabbit (Oryctolagus cuniculus), a major model for studies of immunology. Two important and devastating viral diseases, rabbit hemorrhagic disease and myxomatosis, are affecting European rabbit populations. In this context, we discuss the genetic diversity of the European rabbit immune system and extend to available information about other lagomorphs. Regarding innate immunity, we review the most recent advances in identifying interleukins, chemokines and chemokine receptors, Toll-like receptors, antiviral proteins (RIG-I and Trim5), and the genes encoding fucosyltransferases that are utilized by rabbit hemorrhagic disease virus as a portal for invading host respiratory and gut epithelial cells. Evolutionary studies showed that several genes of innate immunity are evolving by strong natural selection. Studies of the leporid CCR5 gene revealed a very dramatic change unique in mammals at the second extracellular loop of CCR5 resulting from a gene conversion event with the paralogous CCR2. For the adaptive immune system, we review genetic diversity at the loci encoding antibody variable and constant regions, the major histocompatibility complex (RLA) and T cells. Studies of IGHV and IGKC genes expressed in leporids are two of the few examples of trans-species polymorphism observed outside of the major histocompatibility complex. In addition, we review some endogenous viruses of lagomorph genomes, the importance of the European rabbit as a model for human disease studies, and the anticipated role of next-generation sequencing in extending knowledge of lagomorph immune systems and their evolution.

Genetic characterization of interleukins (IL-1α, IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphs.

ILs, as essential innate immune modulators, are involved in an array of biological processes. In the European rabbit (Oryctolagus cuniculus) IL-1α, IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18 have been implicated in inflammatory processes and in the immune response against rabbit hemorrhagic disease virus and myxoma virus infections. In this study we characterized these ILs in six Lagomorpha species (European rabbit, pygmy rabbit, two cottontail rabbit species, European brown hare and American pika). Overall, these ILs are conserved between lagomorphs, including in their exon/intron structure. Most differences were observed between leporids and American pika. Indeed, when comparing both, some relevant differences were observed in American pika, such as the location of the stop codon in IL-1α and IL-2, the existence of a different transcript in IL8 and the number of cysteine residues in IL-1ß. Changes at N-glycosylation motifs were also detected in IL-1, IL-10, IL-12B and IL-15. IL-1α is the protein that presents the highest evolutionary distances, which is in contrast to IL-12A where the distances between lagomorphs are the lowest. For all these ILs, sequences of human and European rabbit are more closely related than between human and mouse or European rabbit and mouse.

Evolutionary Insights into IL17A in Lagomorphs.

In leporids, IL17A had been implicated in the host defense against extracellular pathogens, such as Francisella tularensis that infects hares and rabbits and causes the zoonotic disease tularemia. Here, we studied IL17A from five lagomorphs, European rabbit, pygmy rabbit, brush rabbit, European brown hare, and American pika. We observed that this protein is highly conserved between these species, with a similarity of 97-99% in leporids and ~88% between leporids and American pika. The exon/intron structure, N-glycosylation sites, and cysteine residues are conserved between lagomorphs. However, at codon 88, one of the interaction sites between IL17A and its receptor IL17RA, there is an Arg>Pro mutation that only occurs in European rabbit and European brown hare. This could induce critical alterations in the IL17A structure and conformation and consequently modify its function. The differences observed between leporids and humans or rodents might also represent important alterations in protein structure and function. In addition, as for other interleukins, IL17A sequences of human and European rabbit are more closely related than the sequences of human and mouse or European rabbit and mouse. This study gives further support to the hypothesis that European rabbit might be a more suitable animal model for studies on human IL17.

The safety of ONRAB® in select non-target wildlife.

ONRAB(®) is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB(®) has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB(®) in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(®), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB(®) DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB(®) DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB(®) DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB(®) DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB(®) in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB(®) as part of oral rabies vaccination programs in the United States.

Molecular bases of genetic diversity and evolution of the immunoglobulin heavy chain variable region (IGHV) gene locus in leporids.

The rabbit has long been a model for studies of the immune system. Work using rabbits contributed both to the battle against infectious diseases such as rabies and syphilis, and to our knowledge, of antibodies' structure, function, and regulated expression. With the description of rabbit Ig allotypes, the discovery of different gene segments encoding immunoglobulins became possible. This challenged the "one gene-one protein" dogma. The observation that rabbit allotypic specificities of the variable regions were present on IgM and IgG molecules also led to the hypothesis of Ig class switching. Rabbit allotypes contributed to the documentation of phenomena such as allelic exclusion and imbalance in production of allelic gene products. During the last 30 years, the rabbit Ig allotypes revealed a number of unique features, setting them apart from mice, humans, and other mammals. Here, we review the most relevant findings concerning the rabbit IGHV. Among these are the preferential usage of one VH gene in VDJ rearrangements, the existence of trans-species polymorphism in the IGHV locus revealed by serology and confirmed by sequencing IGHV genes in Lepus, the unusually large genetic distances between allelic lineages and the fact that the antibody repertoire is diversified in this species only after birth. The whole genome sequence of a rabbit, plus re-sequencing of additional strains and related genera, will allow further evolutionary investigations of antibody variation. Continued research will help define the roles that genetic, allelic, and population diversity at antibody loci may play in host-parasite interactions.

Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product.

In the present study we describe type 1 cystatin, a cysteine protease inhibitor, as a major released antigen of the tropical liver fluke Fasciola gigantica (FgStefin-1). Immunohistochemical analysis showed that FgStefin-1 is abundant in (a) tissue of tegumental type, including oral and ventral sucker, pharynx, genital atrium, metraterm, cirrus and (b) the intestinal epithelium. Faint staining was observed in the epithelia of ovary and proximal uterus. Immunoblots showed the presence of FgStefin-1 in the parasite's excretion/secretion (ES) product and immunodepletion demonstrated that FgStefin-1 herein is partially complexed with cathepsin L. Furthermore, quantitation of FgStefin-1 in comparison to cathepsin L in ES product and crude worm extract of adults supports a major external function of FgStefin-1 with an estimated 50% being released in at least equimolar amounts to cathepsin L. Sera of an experimentally infected rabbit reacted with recombinant FgStefin-1 starting 8 weeks postinfection. Activity analyses of recombinant FgStefin-1 showed nanomolar inhibition constants for mammalian cathepsin B, L, and S cysteine proteases and released cysteine proteases of the parasite. The protein is active over a wide pH range and is heat stable. Our results suggest protective functions of FgStefin-1, regulating intracellular cysteine protease activity, and possibly protection against extracellular proteolytic damage to the parasite's intestinal and tegumental surface proteins. Considering inhibition kinetics and previously demonstrated immunomodulatory properties of cystatin in parasitic nematodes a comparable function of FgStefin-1 is suggested and is at present under investigation.

Fatal toxoplasmosis in brown hares (Lepus europaeus): possible reasons of their high susceptibility to the infection.

Brown hares (Lepus europaeus) trapped in the countryside and domestic rabbits were experimentally infected with Toxoplasma gondii (K7 strain) oocysts. Hares (n=12) were divided into groups of 4 and infected with 10, 10(3) and 10(5) oocysts. Rabbits (n=12) were infected in the same way. The experimentally infected animals were monitored for 33 days after infection (p.i.). Most of the infected hares demonstrated behavioural changes, and all of them died between 8 and 19 days p.i. Three of the rabbits demonstrated only clinical changes related to the concurrent pasteurellosis. The typical pathological finding in the hares were haemorrhagic enteritis, enlargement and hyperaemia of mesenteric lymph nodes, splenomegaly and multiple miliary necrotic lesions in the parenchyma of the liver and other organs. Pathological changes in the rabbits were less pronounced than in the hares. In rabbit brains, tissue cysts of the T. gondii were found. The incidence of T. gondii antibodies both in the hares and the rabbits was first ascertained on day 7 p.i. On day 12 p.i., antibodies were already found in all the animals infected. Antibody titres in indirect fluorescence antibody test (IFAT) using the anti-rabbit conjugate were markedly higher in rabbits than in hares. In all hares, T. gondii was isolated post mortem from the liver, brain, spleen, kidney, lung, heart and skeletal muscles. Although T. gondii was also isolated in all rabbits, it was not always isolated in all their organs. In all hares, parasitemia was demonstrated on days 7 and 12 p.i. The percentage of rabbits with detected parasitemia was lower. In hares, a decrease in the numbers of leukocytes during the infection was observed. No such decrease was observed in the rabbits. The lymphocyte activity after the stimulation with non-specific mitogens showed significant differences between the hares and the rabbits even before the infection. After the infection, the hares infected with 10(3) and 10(5) doses and in rabbits infected with a 10(5) dose showed a decrease of lymphocyte activity. Rabbits infected with a 10(3) dose showed an increase of the lymphocyte activity. While in hares toxoplasmosis was an acute and fatal disease, the infection in rabbits had subclinical manifestations only and easily passed to a latent stage. The different courses of toxoplasmosis in the hare and the rabbit may be due to the differences in the natural sensitivity of the two species to the T. gondii infection or a negative impact of stress to the immune status of hares.

The ELISA for the examination of hare sera for anti-Brucella antibodies.

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.

Nomenclature of immunoglobulin A and other proteins of the mucosal immune system. IUIS/WHO Subcommittee on IgA Nomenclature.

The predominant immunoglobulin found in exocrine secretions of humans and most other mammals is secretory IgA, a polymeric form of IgA containing an additional glycoprotein chain designated "secretory component". In this article recommended abbreviations are proposed for the following forms of human IgA and other proteins of related interest: secretory IgA, secretory IgM, secretory component, polymeric immunoglobulin receptor, polymeric IgA, monomeric IgA, IgA subclass 1, IgA subclass, 2, A2 allotype marker 1, and A2 allotype marker 2.

Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). II. Early immune reactions.

As already reported, the mountain hare is much more susceptible than the domestic rabbit to oral inoculation with Toxoplasma gondii, as judged by pathological changes and dissemination of parasites within the body. In the present paper, further interspecies variations are reported. Concentrations of the acute phase reactant haptoglobin were raised in hares but not in rabbits one week post-infection (pi), probably reflecting the severe tissue damage present. No difference in the early humoral immune response of hares and rabbits was found, both species producing IgM and IgG antibodies to T. gondii one week pi. Lymphocyte stimulation tests performed before and one week after inoculation showed a high proliferative response to the parasite in blood cell cultures from rabbits but not hares. The fatal outcome of T. gondii infection in the hares is probably due, at least in part, to the lack of cellular response.

[The results of the serological examination of medium-sized mammals in the natural foci of arbovirus infections in central Siberia]. / Rezul'taty serologicheskogo obsledovaniia srednikh mlekopitaiushchikh v prirodnykh ochagakh arbovirusnykh infektsii Srednei Sibiri.

The serological survey of white hares (n = 8), squirrels (n = 118), and Asian chipmunks (n = 486) in the dark coniferous forests of Middle Siberia revealed tick-borne encephalitis virus antihemagglutinins only in the former two species (37.5 +/- 17.1 and 7.6 +/- 2.4%, respectively) and in the squirrel, there is a close seasonal relation between the parameters of immune interbred and those virophoricity of taiga tick nymphs.

Role of Lagomorpha in the wild cycle of Rickettsia conorii in Salamanca (Spain).

In an endemic zone for Mediterranean spotted fever levels of antibodies to R. conorii were evaluated in serum samples from wild rabbits (Orytolagus cuniculus) and hares (Lepus granatensis) using an indirect microimmunofluorescence antibody test. The results of the study show that the wild rabbit may carry out in this area an important function in the maintenance of R. conorii in nature.

[Radioimmunological analysis in studying the persistence of the plague antigen in the body of rodents and ectoparasites in natural foci in Siberia]. / Radioimmunologicheskii analiz pri izuchenii persistentsii chumnogo antigena v organizme gryzunov i u éktoparazitov prirodnykh ochagov Sibiri.

To study the persistence of Y. pestis capsular antigen, or fraction 1 (F1), in the body of less important plague carriers in the Mountain Altai and Transbaikal natural foci, as well as in experimentally infected ticks, the liquid-phase competitive radioimmunoassay (RIA) was used for the first time. In this study RIA showed, due to its sensitivity, doubtless advantages over traditional methods, such as the passive hemagglutination (PHA) test and the antibody neutralization (AN) test, and made it possible to detect F1 in picogram amounts. RIA revealed that F1 persisted in Siberian long-tailed gophers for up 14 months after the infection of the animals in diffusion chambers and for 7 months after their infection by subcutaneous injection. Experiments on Daurian pikas confirmed that, in comparison with the PHA and AN tests, RIA ensured fourfold effectiveness in the detection of antigen F1. The study of infected mites revealed that antigen F1 could be retained in them for more than a year and detected by RIA techniques in 10% of cases. The data obtained in this investigation indicate that the persistence of microorganisms should be studied mainly with the use of new-generation tests, and RIA, being one of the most sensitive techniques, deserves wide approval and introduction into the practical work of institutions intended for plague control.

[Occurrence of Borrelia antibodies in field hares (Lepus europaeus)]. / Výskyt borreliových protilátek u zajíce polního (Lepus europaeus).

Using the indirect haemagglutination reaction with an antigenic extract from Borrelia recurrentis, the author assessed in the hare the incidence of Borrelia antibodies, incl. antibodies against Borrelia burgdorferi, the causal agent of Lyme borreliosis, a multisystemic disease with natural foci, a disease transmitted mostly by ticks of the genus Ixodes. Sera of 113 hares from 11 localities in the CSSR examined by this method reacted in 41.6% by titres within the range from 1 : 16 to 1 : 512, only 26.5% sera reacted by titres of 1 : 64 and more. Positive values were recorded throughout the year with a maximum in the summer months, there was no age dependence, the incidence was more frequent in adults than juveniles (28.04% and 22.2% resp.). The mean positivity of the three most frequent localities was 28%, with a variation of +/- 5.3%. Despite the hitherto not proved incidence of Borrelia in positive subjects we may assume, based on crossed reactivity of Borrelia recurrentis and Borrelia burgdorferi and analogously with North American representatives of the hare family, the presence of this infectious agent also in the field hare.

[Anti-leptospira and anti-brucella agglutinins in hares (Lepus europaeus) from Rio Cuarto Department, Province of Cordoba]. / Aglutininas anti-leptospira y anti-brucella en liebres (Lepus europaeus) del departamento de Río Cuarto, provincia de Córdoba.

Seventy-seven anti-brucella and 42 anti-leptospira agglutinin serum levels of hares in Rio Cuarto district of Córdoba Province were studied. For brucella, only one sample showed incomplete reaction at a 1:25 dilution. The isolation of brucella from liver, spleen and gastro-hepatic lymph node in this animal was negative. In the 42 serum samples examined for L. pomona, L. wolffi, L. ballum, L. hardjo, L. pyrogenes and L. grippotiphosa, 6 reacted at a dilution of 1:100 with L. ballum serovar and 1 reacted up to 1:200 dilution with L. wolffi serovar. These results indicate that the hare does not play any important role as reservoir of pathogenic brucella in this area. Meanwhile, it could act as potential reservoir for pathogenic leptospira.

A serological survey of antibodies against Francisella tularensis in some Swedish mammals.

Tularemia occurs in Sweden as an epizootic among the mountain hare. Little is known about the occurrence of the disease in other animals in this country. For this reason serum samples from 28 cattle, 83 moose, 110 beavers and 97 mountain hares were investigated for the presence of antibodies against Francisella tularensis. The antibody levels against F. tularensis found in moose and cattle were generally low and also in a low incidence. This indicates that these species are not susceptible to tularemia and not involved in the epizootiology of this disease. Beaver titres were found to vary from 1:20 to 1:1 000. Twenty-one % of the investigated sera showed titres higher than 1:100. This indicates that infections are common in the species and that the Scandinavian beaver plays an important role in the epizootiology of tularemia. It could well act as a reservoir for the disease. Ninety-six of the 97 tested sera from the mountain hare were negative. In one serum was a titer of 1:20 seen. This low titer was regarded as non-specific. The absence of titers against F. tularensis in this species could be explained by the high susceptibility to this disease. This indicates that the mountain hare is not a reservoir for tularemia in Sweden. It is one of the most susceptible and the dominating species involved in tularemia epizootics, but it is not the normal reservoir for F. tularensis.