Immune and hormonal regulation of the Boa constrictor (Serpentes; Boidae) in response to feeding.

Feeding upregulates immune function and the systemic and local (gastrointestinal tract) concentrations of some immunoregulatory hormones, as corticosterone (CORT) and melatonin (MEL), in mammals and anurans. However, little is known about the immune and hormonal regulation in response to feeding in other ectothermic vertebrates, especially snakes, in which the postprandial metabolic changes are pronounced. Here, we investigated the effects feeding have on hormonal and innate immune responses in the snake, Boa constrictor. We divided juvenile males into two groups: fasting and fed with mice (30% of body mass). We measured the rates of oxygen consumption, plasma CORT levels, heterophil/lymphocyte ratio (HL ratio), plasma bacterial killing ability (BKA), and stomach and intestine MEL in fasting snakes and 48 h after meal intake. We observed increased rates of oxygen consumption, plasma CORT levels, and HL ratio, along with a tendency of decreased stomach and intestine MEL in fed snakes compared to fasting ones. BKA was not affected by feeding. Overall, we found that feeding modulates metabolic rates, CORT levels, and immune cell distribution in boas. Increased baseline CORT may be important to mobilize energy to support the metabolic increment during the postprandial period. Increased HL ratio might be an immunoregulatory effect of increased CORT, which has been shown in different physiological situations such as in response to immune challenge. Our results suggest that feeding activates the hypothalamic-pituitary-adrenal axis and modulates immune cell redistribution, possibly contributing to fighting potential injuries and infections derived from predation and from pathogens present in ingested food.

Dehydration enhances innate immunity in a semiaquatic snake from the wet-dry tropics.

Dehydration is considered a physiological challenge, and many organisms live in environments that undergo periods of reduced water availability that can lead to dehydration. Recent studies have found a positive relationship between dehydration and innate immune function in animals adapted to xeric or semixeric environments. To explore the generality of this relationship, we examined the impact of dehydration on innate immune performance in water pythons (Liasis fuscus), a semiaquatic snake from the wet-dry tropics of Australia. We collected blood samples from male and female water pythons held in the laboratory without food and water for 4 weeks. We also collected blood from free-ranging snakes throughout the Austral dry-season. We evaluated plasma osmolality and innate immune function (agglutination, lysis, and bacterial-killing ability) and found that increased osmolality, whether manipulated in the laboratory or as a result of natural water limitation, resulted in enhanced aspects of innate immune performance. Counter-intuitively, snakes in the wild became more hydrated as the dry season progressed, suggesting the dehydrated snakes move to water sources periodically to rehydrate. Comparing our data with those from previous studies, we suspect species divergence in the level of dehydration (i.e., hyperosmolality) that triggers enhanced immune capabilities.

Egg desiccation leads to dehydration and enhanced innate immunity in python embryos.

The immune system is essential for survival and its performance can vary depending on the physiological state of the organism. Much of the current research into immune function dynamics has examined newborn to adult life stages, despite previous studies documenting physiological responses in embryos to environmental stimuli. While energy balance has been the predominant focus as the driver of changes in immune function, recent research has found a positive relationship between dehydration and innate immune performance in adult reptiles. We expanded the understanding of this relationship by examining trans-generational immune effects of female dehydration as well as the effects of egg desiccation on embryonic hydration state and innate immunity using Children's pythons, Antaresia childreni. We used a 2 × 2 experiment with hydrated or dehydrated mothers and eggs either incubated under continuous optimal conditions or experiencing desiccating conditions for 24 h. Our results demonstrate that, similar to adults, embryos enhance some metrics of innate immunity when they are dehydrated.

Generation of Anti-Boa Immunoglobulin Antibodies for Serodiagnostic Applications, and Their Use to Detect Anti-Reptarenavirus Antibodies in Boa Constrictor.

Immunoglobulins (Igs), the key effectors of the adaptive immune system, mediate the specific recognition of foreign structures, i.e. antigens. In mammals, IgM production commonly precedes the production of IgG in the response to an infection. The reptilian counterpart of IgG is IgY, but the exact kinetics of the reptilian immune response are less well known. Boid inclusion body disease (BIBD), an often fatal disease of captive boas and pythons has been linked to reptarenavirus infection, and BIBD is believed to be immunosuppressive. However, so far, the study of the serological response towards reptarenaviruses in BIBD has been hampered by the lack of reagents. Thus we set up a purification protocol for boa constrictor IgY and IgM, which should also be applicable for other snake species. We used centrifugal filter units, poly ethylene glycol precipitation and gel permeation chromatography to purify and separate the IgM and IgY fractions from boa constrictor serum, which we further used to immunise rabbits. We affinity purified IgM and IgY specific reagents from the produced antiserum, and labelled the reagents with horseradish peroxidase. Finally, using the sera of snakes with known exposure to reptarenaviruses we demonstrated that the newly generated reagents can be utilised for serodiagnostic purposes, such as immunoblotting and immunofluorescent staining. To our knowledge, this is the first report to show reptarenavirus-specific antibodies in boa constrictors.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius).

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

Evidence of IgY subclass diversification in snakes: evolutionary implications.

Mammalian IgG and IgE are thought to have evolved from IgY of nonmammalian tetrapods; however, no diversification of IgY subclasses has been reported in reptiles or birds, which are phylogenetically close to mammals. To our knowledge, we report the first evidence of the presence of multiple IgY-encoding (υ) genes in snakes. Two υ genes were identified in the snake Elaphe taeniura, and three υ genes were identified in the Burmese python (Python molurus bivittatus). Although four of the υ genes displayed a conventional four-H chain C region exon structure, one of the υ genes in the Burmese python lacked the H chain C region 2 exon, thus exhibiting a structure similar to that of the mammalian γ genes. We developed mouse mAbs specific for the IgY1 and IgY2 of E. taeniura and showed that both were expressed in serum; each had two isoforms: one full-length and one truncated at the C terminus. The truncation was not caused by alternative splicing or transcriptional termination. We also identified the µ and δ genes, but no α gene, in both snakes. This study provides valuable clues for our understanding of Ig gene evolution in tetrapods.

MHC class I variation associates with parasite resistance and longevity in tropical pythons.

Using restriction fragment length polymorphism (RFLP) we identified 26 unique major histocompatibility complex (MHC) genotypes in 104 water pythons. We observed a significant independent association between reduced blood parasite load (Hepatozoon sp.) and python body length/age, presence of a specific RFLP fragment (C-fragment) and the overall number of fragments. The parasite has a negative impact on several python life-history traits such as growth, nutritional status and longevity. Thus, the C-fragment could be considered a 'good gene' (a fitness-enhancing genetic element). However, while the number of fragments affected parasite load, the association between level of parasitaemia and fragment number was not linear, and, hence, minimum parasite infection level was achieved at an intermediate number of fragments. Intermediate MHC fragment numbers were also observed among the largest/oldest pythons, suggesting that both a specific fragment and intermediate levels of MHC polymorphism enhanced python longevity. Thus, our results suggest python MHC is subject to both frequency-dependent and balancing selection.

Use of an ELISA for detection of antibody responses in Argentine boa constrictors (Boa constrictor occidentalis).

OBJECTIVE: To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis. ANIMALS: Two 3-year-old Argentine boa constrictors. Procedure-Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 microg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity. RESULTS: A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas. CONCLUSIONS AND CLINICAL RELEVANCE: Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens.

Experimental study of induced inflammation in the Brazilian Boa (Boa constrictor constrictor).

The objective of this work was to identify the cellular types present in inflammatory processes in the Brazilian snake, Boa constrictor constrictor. Blood smears were first made from three normal snakes and stained by several methods to identify the cell types present, thus facilitating the identification of cells in inflammatory processes induced in 16 further snakes by the subcutaneous implantation of cotton suture threads and circular coverslips. Implanted threads induced migration of heterophils and monocytes after 4 h, more intense monocyte migration after 24 h, an intense granulocytic migration inside and around the thread after 48 h, heterophilic granulocytes, macrophages and giant cells after 7 days, and giant cells with a typical granuloma response and persistence of heterophilic cells after 15, 69 and 117 days. The cell population attached to the implanted coverslips after 4 h was composed of heterophils, thrombocytes, erythrocytes and macrophages; after 24 and 48 h heterophils predominated, and after 7 days heterophils, macrophages and giant cells predominated.

MHC variation in birds and reptiles.

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring-necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens.