Microbiology. When evaluating a bacterial infection, it must be considered that the presence of bacteria in a culture does not always indicate disease since these can be bacteria of the individual's normal microbiota [25, 26].
Most bacterial septicemias are caused by aerobic gram-negative bacteria, although there are some reports of septicemia caused by gram-positive bacteria [13]. This statement coincides with the findings of the present study, in which 78% of the bacteria identified were Gram negative and the remaining 22% were Gram positive.
The potentially pathogenic bacteria identified agree with those detailed in the literature on amphibians: Streptococcus [27], Klebsiella spp. [14, 27], H. alvei [27, 28], Pseudomonas spp. [10, 27], Enterobacter spp. [22], A. hydrophila [15, 29], Salmonella spp. [30], and Enterococcus spp. [31]. However, not all bacteria detected have pathogenic potential. A previous study detected cultivable bacteria in G. riobambae and G. plumbea associated with the skin of these frogs in Ecuador and found several isolates with known anti-Bd activity (P. fluorescens and A. hydrophila) and other genera and species similar to those reported in our study (e.g., Enterobacter spp., Burkholderia cepacia, Pseudomonas palleroniana and Aeromonas veronii) [32].
Although most of the bacteria in the present study have been described as typical of the amphibian microbiota, they could be part of a secondary infection caused by a primary viral or fungal infection [11, 22, 26, 27]. A. hydrophila, Pseudomonas spp. and Streptococcus spp. have been identified as the cause of red-leg syndrome [33].
Cytology. In the case of the genus Gastrotheca, cytology reference values do not exist. Additionally, the morphology of the cells varies between anuran species and is usually compared to that of other vertebrates [11, 24, 34, 35].
It is necessary to mention that in amphibians, knowledge of the response of the leukocyte series to diseases is limited, partly because leukocyte evaluation is not a routine methodology used in anurans [34] and because the cells identified in the edema fluid samples came from a blood extravasation, not from peripheral blood [36].
Studies show that the red blood cell count in females is lower than that in males and that the leukocyte count is uniform [37]. In general, normal cytology values and values with variations derived from pathologies can be influenced by sex [38]. In this case, females had a higher percentage of erythrocytes, neutrophils, basophils, and monocytes, and males had a higher percentage of lymphocytes and eosinophils; however, no relationship was found between the statistical results analyzed and the sex of the individuals.
The basophil count was low; these cells play a similar function to those mammals because they are peroxidase positive and have hydrolytic enzymes [34]. Similarly, thrombocytes were found in small quantities, and these cells have a function comparable to the function of those of reptiles and birds [24, 39]. Eosinophils also exhibited low counts; these cells are active in parasitic infections or in polluted areas [40, 41], but they have an inferior phagocytosis ability [34]. These low numbers are observed even in noninflammatory effusions, where there are low counts of mesothelial cells, macrophages, granulocytes, and lymphocytes [11].
Neutrophils and monocytes of amphibians share similar characteristics of migration and phagocytic activity and have the same enzymes that occur in the same cells of other vertebrates, and an increased count suggests an inflammatory response [34]. Only neutrophils are present in high numbers in both sexes of Gastrotheca.
B lymphocytes are capable of phagocytosis in response to bacterial infections [42]. In the present study, it was possible that there was a relationship between the results obtained in the microbiological characterization and the lymphocytosis and neutrophilia observed due to the presence of bacteria.
Likewise, a study carried out on individuals from the herpetofauna of Turkey determined that 80% of the leukocytes present in peripheral blood were lymphocytes and monocytes [43]. These results are partially compatible with the results regarding lymphocytes of our study. Their presence is related to an excitement response, stimulation of the immune system, or possibly lymphoid leukemia [34].
Despite the presence of the bacteria and types of cells in this study, all the animals did not show evidence or clinical signals congruent with infectious disease. In this case, it may be a consequence of captive management; since amphibians are demanding in terms of environmental and nutritional requirements, those factors most likely increased the number of bacteria but did not cause disease or death. Systemic bacterial infections are common in debilitated, stressed, or crowded amphibians [11].
One kind of effusion fluid type is septic exudate within phagocytes [21]. In amphibians and reptiles, the inflammatory response depends on the temperature and is classified according to the predominant cell type [39]. In addition, if phagosomes or vacuoles from phagocytosis are observed in leukocytes due to phagocytosis of bacteria, septic inflammation is considered [40]. In contrast, samples with higher total protein (>3 g/dL) and cell counts (> 7000/mL) are likely exudates, and inflammatory conditions should be considered [11]. A unique sample was observed with one phagosome, but it is necessary to add information such as specific gravity, total protein, and cell count to categorize the type of cavity fluid.
Another type of fluid is the transudate type that most likely derives from metabolic imbalance, such as hypoproteinemia [44]. The increase in solute attracts solvent, in this case, water, which leads to a weight increase. Here, it is possible to consider edema as a chronic process, which facilitates the passage of water over a large time period and, consequently, an increase in weight and abdominal fluid with low densities.
It is important to understand that edema syndrome has a strong relation to a deficit in osmoregulation, which could be caused by many pathogens and failure of a variety of body systems, causing difficulty in antemortem diagnosis [45]. In this study, the cause of edema was most likely related to a fusion of chronic osmoregulation failure and pathogens. Other causes of edema are a low solute concentration in water or acute renal disease (captivity) [46]. Thus, additional studies on the level of management and pathology are necessary to complement a microbiological and cytological characterization of this problem in captive anurans.