Search

In this project, red-backed salamanders, Plethodon cinereus, were collected from the heart of their range (Mountain Lake, Virginia, USA), in an effort to document the typical, or baseline, leukocyte profile of this species in its natural state. From microscopic examination of thin blood films, we determined relative proportions of all leukocyte types and determined the ratio of neutrophils to lymphocytes, which is a useful index of stress levels. In addition, individuals were examined for three naturally occurring endoparasites: two gastro-intestinal (trematodes and a ciliated protozoan in the genus Cepedietta) and one in the blood (a Rickettsial bacteria). This allowed us to conduct statistical comparisons of leukocyte data across parasite infection groups. Of 36 salamanders, 11 (31%) had trematode parasites, 7 (19%) contained Cepedietta parasites, 6 (17%) were parasitized by Rickettsia, and 12 had no parasites. The most common leukocytes were lymphocytes (87.4%), followed by neutrophils (4.2%), monocytes (3.3%), eosinophils (2.6%), and basophils (2.5%). The average neutrophil/lymphocyte ratio for these salamanders was remarkably low (0.05) for an amphibian population. Statistical comparisons of leukocyte profiles among parasite infection groups indicated parasitized salamanders had twice as many circulating leukocytes as nonparasitized individuals, which could be considered an "inflammation response". There was a slight but significant elevation in relative eosinophil abundance in parasitized salamanders. There were few differences in leukocyte subsets between parasite types, nor was there evidence of any proliferation of phagocytic cells in any parasitized individual. [ABSTRACT FROM AUTHOR]

Abstract: Measuring stress in animals is important in many ecological, zoological and veterinary research settings. A common method is to measure plasma levels of glucocorticoid hormones (cortisol, corticosterone, hereafter CORT). Over the past decade, an alternative method has become widely popular: assessing leucocyte profiles; in other words, the heterophil‐to‐lymphocyte ratio (H/L) in birds and reptiles or the neutrophil‐to‐lymphocyte ratio (N/L) in mammals, amphibians and fish. Recent studies have shown that although both indices reliably change after acute stress, they are not always correlated at baseline, and thus are not interchangeable. This lack of correlation has led to some confusion about which of these two measures is “better”; in other words, more biologically relevant and a truer readout of stress. In this review, we first document the dramatic expansion of the use of leucocyte profiles to assess stress over the past decade. We then review published evidence that may explain the discrepancies between leucocyte profiles and plasma CORT. We argue that these discrepancies stem from the nature of the stress reaction and the role of corticosterone (or cortisol) versus leucocytes during the stress response. The differing roles of the two responses translate to differences in the timing of each, which is important for researchers studying either acute or chronic stress. Although plasma glucocorticoids rise within minutes of the onset of acute stress and return to baseline within 1 or 2 hr after the stressor passes, H/L or N/L ratios remain low for at least 30–60 min or sometimes more. The temporal nature of these metrics differs also in the context of long‐term chronic stress. The glucocorticoid response wanes in repeatedly stressed animals, or those exposed to chronic environmental stress, which can often (but not always) lead to low baseline levels and more typically, an attenuated acute stress response. Meanwhile, H/L or N/L ratios appear to remain high under the same conditions. Understanding the temporal differences in the responses should help researchers decide which metric is the better method in their particular study, or whether both would be useful for elucidating different elements of the stress response. [ABSTRACT FROM AUTHOR]

Bullfrogs ( Rana catesbeiana) are widely believed to be nonclinical carriers of Batrachochytrium dendrobatidis (Bd), the fungal pathogen that invades keratinized tissues of amphibians and causes the disease, chytridiomycosis. Although most research on this disease focuses on adults, larval anurans are also susceptible to infections in their keratinized mouthparts, and this allows for visual diagnosis of the disease via the degree of mouthpart depigmentation. When an unplanned outbreak of chytridiomycosis occurred in a set of captive bullfrog tadpoles in our lab, we conducted the current investigation into its effects on the nonspecific immune system (i.e., the leukocyte populations) of the tadpoles. We compared leukocyte counts from blood smears of 27 tadpoles that had contracted the disease (evidenced by severe mouthpart depigmentation and confirmed by histology) to those of 21 tadpoles that had little depigmentation (i.e., with little evidence of the disease). Tadpoles with severe depigmentation had significantly more neutrophils and less eosinophils than those with little depigmentation, while numbers of lymphocytes, basophils, and monocytes were not statistically different. That there was any effect at all on circulating leukocyte numbers is surprising since leukocytes are usually not seen migrating to sites of infection in tissue sections of amphibians infected with Bd, and since most research points to this disease having little outward effect on bullfrogs. Since monocyte numbers were unchanged, the leukocyte alterations were likely not due to a simple inflammation response. It is possible that Bd infections elicit increases in glucocorticoid hormones, which can cause increased numbers of circulating neutrophils and lower numbers of eosinophils, although this is often accompanied by a reduction in lymphocyte numbers, which we did not see. Further research is warranted to clarify if this effect is limited to this species. [ABSTRACT FROM AUTHOR]

When animals become stressed, their levels of glucocorticoid hormones increase, causing white blood cells to move from tissues to circulation or vice versa. The primary alteration is an increase in the abundance of circulating neutrophils and a decrease in lymphocytes in circulation. A lesser-known effect is a decrease in the number of circulating eosinophils. Salamanders in the genus Ambystoma have unusually high numbers of circulating eosinophils, and as such, any effect of stress hormones on circulating leukocytes (especially eosinophils) of these species should be especially pronounced. We conducted an experiment to determine the effect of corticosterone administration on leukocyte counts (from blood smears) of A. talpoideum salamanders. Salamanders were captured and sampled as reference animals (n = 11), given a sham injection (n = 8), or injected with 0.1cc of a 1μg/mL corticosterone solution (n = 28). After 24 hours, relative neutrophil counts were higher and relative lymphocyte counts lower, in the corticosterone group than the sham and control groups. Absolute counts showed that this effect was driven by a reduction in lymphocytes, since neutrophil counts were statistically similar across treatments. Importantly, relative and absolute numbers of eosinophils decreased in the sham and corticosterone groups, confirming the sensitivity of this cell to stress in amphibians. [ABSTRACT FROM AUTHOR]

Herpetologists are becoming increasingly aware of the importance of hematological parameters for evaluating the welfare of their study animals. In particular, differential counts of white blood cells (from blood smears) are now commonly performed on amphibians, although interpretation of such counts relies on some knowledge of the normal range for the species in question, and for many species this range is not known. In this study we examined blood smears from 79 cricket frogs (Acris c. crepitans) from two ponds in northeastern Georgia to establish reference ranges of white blood cell differentials for this species and to explore possible differences in relative white blood cell numbers between sexes, ponds, and body sizes. For comparison, we also compiled values of many other amphibians from published and unpublished records. We found that lymphocytes made up the majority (68.3%) of white blood cells in cricket frogs, followed by neutrophils (22.4%). There was no difference in relative cell numbers of any type between sexes, nor was there an association with body size. Relative numbers of certain cell types (lymphocytes and neutrophils) varied between ponds. In general the values for cricket frogs resembled those of other amphibians, with high numbers of lymphocytes and neutrophils and low numbers of eosinophils, basophils and monocytes. However, in assembling the records for other species we discovered that all Ambystomatid salamanders examined thus far have markedly elevated values of eosinophils compared to other amphibian species. Moreover, their eosinophil counts may be the highest of all vertebrates, and clearly warrant further study to determine their significance. [ABSTRACT FROM AUTHOR]

During a recent study of red-backed salamanders ( Plethodon cinereus), we discovered an intraerythrocytic organism typified by violet-staining, intracellular inclusions, consistent with descriptions of Cytamoeba or Aegyptianella (bacteria). Here we characterize its taxonomic status using molecular techniques and ask basic questions about its nature. Blood smears from 102 salamanders were examined from Pennsylvania, New York, and Virginia to determine prevalence, and whole blood from several infected animals was tested using a PCR which targets the 16S rRNA gene of bacteria. Phylogenetic analysis of partial 16S rRNA gene sequence (1201 bp) indicated this organism was in the order Rickettsiales and is likely a member of the family Anaplasmatacea. The organism differed from currently described taxa and was clearly differentiated from Aegyptianella pullorum of birds and “ Candidatus Hemobacterium ranarum” (formally A. ranarum) of frogs. Of all salamanders, 17 (16.7%) were infected and these were significantly larger (snout–vent length) and had higher body condition scores than uninfected ones, and males were more likely to be infected than females. Erythrocytes affected by the pathogen were 5% larger than unaffected ones, but otherwise similar in morphology. Infected animals tended to have a greater number of circulating white blood cells, based on estimates from smears, indicating a nonspecific response to the pathogen by the innate immune system. Given its phylogenetic position, this pathogen is likely transmitted by an arthropod vector, and the male-biased prevalence strongly implicates trombiculid mites, which also live in leaf litter and affect male salamanders more so than females. [ABSTRACT FROM AUTHOR]

Metamorphosis in amphibians is a fascinating phenomenon that offers physiologists a number of opportunities to study aspects of tissue growth and lysis in animals. In larval amphibians, tissues are formed early on during larval growth, then are vastly reorganized and broken down during metamorphosis, and the effects of this phenomenon on the relative distribution of white blood cells in circulation (i.e., leukocyte profiles) was first examined in the 1920s. Nearly a century later, there has been little work to continue this research avenue, although our knowledge of the roles of certain leukocyte types has improved. Moreover, recent interest in counts of leukocytes in amphibians for environmental monitoring purposes emphasizes the need to understand how white blood cells naturally vary throughout larval life. In this study, I examined blood smears from wild-caught larval and adult bullfrogs ( Rana catesbeiana) of varying developmental stages and quantified the proportions and abundance (estimated from blood smears) of all leukocyte types at each stage. Lymphocytes were the most common cell found and appeared most abundant during the early growth phases of larvae. Neutrophils were the second most common cell and were also associated most with earlier phases. Eosinophils appeared directly associated with metamorphosis, generally increasing in abundance to a peak at metamorphic climax. Monocytes were also found most frequently in individuals experiencing metamorphic climax. Results in this study concerning eosinophils are consistent with those of urodeles and point to their role in the breakdown of tissues, which may resemble their role in modulating inflammation responses. [ABSTRACT FROM AUTHOR]

Measuring stress in animals is an important component of many research studies, and it has traditionally been performed via sampling levels of corticosterone in plasma. A secondary, "hematological" approach used most commonly by researchers of birds, mammals, and other taxa involves evaluating leukocyte profiles from blood smears. Such research has shown that leukocytes have a characteristic response to stress, although in amphibians this phenomenon is not as well studied. In general, stress can induce a rise in the ratio of neutrophils to lymphocytes. We evaluated the hematological response of paedomorphic Mole Salamanders (Ambystoma talpoideum) to captivity stress, specifically focusing on this parameter, but also examining other white blood cell types. Individuals captured in the wild and held in captivity for ten days before sampling had significantly more neutrophils, fewer lymphocytes, and higher ratios of neutrophils to lymphocytes than those captured from the same locations and sampled within one hour. Captive individuals also had significantly higher numbers of eosinophils. These results are consistent with hematological research in birds and other taxa and highlight the utility of this approach for measuring stress in amphibians. [ABSTRACT FROM AUTHOR]

Leukocyte profiles (relative numbers of white blood cell types) have been used by a growing number of ecological studies to assess immune function and stress in wild birds. House Finches (Carpodacus mexicanus) in eastern North America are susceptible to an eye disease caused by the bacteriumMycoplasma gallisepticum, providing the opportunity to examine whether leukocyte profiles are associated with infection status and other host characteristics. In this study, we compared blood smears from 297 wild House Finches with and without conjunctivitis to examine whether leukocyte profiles were associated with the presence and severity of mycoplasmal conjunctivitis. We also evaluated the influence of age, sex, and molt on leukocyte profiles in both diseased and nondiseased birds. Of 243 House Finches of known age and sex sampled, 30% showed clinical signs of mycoplasmal conjunctivitis. House Finches with conjunctivitis had significantly higher heterophil to lymphocyte (H/L) ratios and harbored greater numbers and proportions of heterophils and monocytes than nondiseased birds. Leukocyte profiles of noninfected birds did not differ significantly with respect to sex, but young birds had significantly greater numbers of lymphocytes and total white blood cells than adults. Molting birds had significantly more eosinophils than nonmolting birds. Finally, House Finches with the most severe outward signs of conjunctivitis showed the most dramatic leukocyte changes relative to noninfected individuals, and increasing H/L ratios and monocytes in diseased birds were paralleled in a subset of birds that were recaptured during the study period. These results are consistent with patterns observed in domestic poultry and suggest that understanding patterns of leukocyte differentials in this host-pathogen system could improve our understanding of innate immunity and infectious disease risk in other wild passerines. [ABSTRACT FROM AUTHOR]