Conjunctival flora of clinically normal and diseased turtles and tortoises

The number of people who raise reptiles as pets has increased, but information about microbiological is limited. Among reptiles, pet turtles and tortoises are also becoming increasingly popular. The animals involved in this survey are almost all captive chelonians, that are routinely or occasionally subjected to medical examination.

Except the presence of Bacillus spp. (13 isolates), Staphylococcus xylosus was the most frequent isolate in tortoises (10 isolates). Sphingomonas paucimobilis and Aeromonas hydrophila/caviae were likewise the most frequent isolates in turtles (6 and 5 isolates, respectively).

From Table 1 a clear predominance of Gram positive isolates in tortoises and Gram negative isolates in turtles can be noticed (p < 0.001). A preponderance of Gram positive species was already observed by the Authors in the normal conjunctival bacterial flora of green iguana, in accordance with data reported for other terrestrial animal species [7]. The prevalence of Gram negative isolates in water turtles is also a not surprising result. Captive water turtles, as those included in the present study, often live in small water volumes of aquaria and ponds, where fecal matter and other waste material tend to be concentrated. Therefore, these animals live immersed in a medium were proliferation of enteric bacteria is favoured. To date, little is known about the microbial communities or potential pathogens associated with aquarium water. However, in a survey of Smith et al. [8] the bacterial community of ornamental fish aquarium freshwater was characterized by molecular methods. In accordance with our results, they found in their samples the presence of Aeromonas and Vibrio genera, which include species that can become pathogenic for fish as for turtles in stressful conditions. On the contrary, they did not found the presence of Salmonella spp. This is not unexpected, as it is well known that reptiles are frequently carriers of Salmonella spp. [9]. However, it was reported that also bacteria belonging to Salmonella genus, as well as Aeromonas and Vibrio genera, can be present in home aquaria and probably be responsible of illness in humans due to exposure to ornamental fish [10].

The occurrence of conjunctivitis was higher in tortoises than in turtles: three out of eighteen tortoises (n. 10, 11 and 14) and one out of sixteen turtles (n. 23), showed bilateral conjunctivitis (Table 1). However, the data is not statistically significant (p = 0.282) and to be confirmed would require a greater number of samples. As already it is clear looking at the data, the statistical analysis confirms the absence of association (p = 0.398) between the presence of conjunctivitis and the presence of infection with multiple bacterial species. Bacterial flora of tortoises 11 and 14 did not differed from that of other healthy tortoises. In these animals, as well as in tortoise 10, the only common isolate was represented by Staphylococcus xylosus. However, tortoise 10 was the only one in which it was possible to detect the presence of Chlamydia spp. by PCR. Unlike in the other clinical cases, in this animal conjunctivitis was particularly severe and the animal was not able to open the eyelids themselves. This finding may indicate a role of chlamydia in inducing severe ocular disease. As reviewed by Corsaro and Venditti [11], Chlamydia or Chlamydia-like organisms infections have occasionally been reported in reptiles, including both turtles and tortoises, and almost all the reported cases concerned captive animals. Common symptoms are lethargy, anorexia and chronic respiratory disease. However, almost all Chlamydiaceae species are able to infect the conjunctiva of their vertebrate host and, with regard to reptiles, exudative conjunctivitis attributed to Chlamydia spp. infection was reported in farmed juvenile crocodiles [11,12].

On the other hand, it is noteworthy that bacterial isolates from turtle 23, which is the only acquatic turtle showing conjunctivitis, that is Staphylococcus haemolyticus and Morganella morganii, were not found in any of the other turtles. Staphylococcus haemolyticus belong to the group of coagulase-negative staphylococci (CNS). CNS are a major component of the normal microflora of the human skin and are mostly considered to be saprophytes. However, S. haemolyticus can be also an opportunistic pathogen and is the second most frequently encountered species of CNS in human clinical infections [13]. S. haemolyticus is a frequently isolated pathogen in dairy cows and small ruminants and can be sporadically involved in subclinical mastitis [14]. Less is known about the relation of this bacterium and other animal species. Instead, it is well known the ability of S. haemolyticus to acquire multi-antibiotic resistance [15]. In our case, S. haemolyticus, as well as S. lentus, was found in acquatic animals, while, on the contrary, all the other species of staphylococci were detected in animals with terrestrial habitat. One isolate of S. aureus was found in tortoise n. 1. S. aureus may be involved in case of ocular disease induced by hypovitaminosis in tortoises [16], but in our case the animal was health. M. morganii is a Gram negative facultative anaerobe, belonging to the Enterobacteriaceae family. It is commonly found in the environment and as normal flora in the intestinal tracts of humans, mammals other than humans and reptiles and is considered an opportunistic bacterium. In human, despite its wide distribution, is an uncommon cause of nosocomial infections in adults and is mostly isolated from urinary tract or wound infections, as well as can be involved in neonatal sepsis [17]. A case of postoperative bacterial endophtalmitis caused by M. morganii was also reported [18]. In animals, M. morganii was isolated from mixed infections of the marine mammal Dugong dugon [19] and was reported as one of the most common pathogens identified in human secondary wound infections following snakebites [20]. Recently, a sporadic case of fatal infection in chickens, caused by a highly pathogenic M. morganii strain, was also reported [21]. Our isolate of Morganella morganii was resistant to a wide variety of antibiotics suitable for chelonians treatment (data not showed). The presence of multiple drug resistance is significant, since the bacterium can, although rarely, cause the occurrence of zoonotic disease, particularly in weak persons.

The presence of Mycoplasma negative cultures, also from those animals that resulted positive to Mycoplasma PCR, is not a surprising result [22] as mycoplasmas have many different and often fastidious requirements for growth. The seven days incubation time may have further reduced the probability of isolating slow growing mycoplasmas. In Mycoplasma PCR we found an occasional unspecific amplification, outside the 145–237 bp range of positivity, in the negative control. This could be due to an external contamination of the water used in place of template in the negative control. Conversely, we exclude the contamination of the reaction mix, because such unspecific amplification has never occurred to any of the samples tested. However, to doubtless confirm that the amplified sequences belong to the Mycoplasma genus, we repeated twice the Mycoplasma PCR and we performed sequencing analysis on Mycoplasma PCR products. We found a higher percentage of Mycoplasma PCR positive samples in turtles compared to tortoises (Table 1). Mycoplasmas are frequently found in reptile species, particularly in case of chelonians, and some of them are part of the commensal bacterial flora of the host. However, Mycoplasma agassizii and Mycoplasma testudineum can be involved in the URTD in tortoises [23]. In particular, the most frequently reported agent of URTD in tortoises is Mycoplasma agassizii [24,25]. We found by PCR the presence of Mycoplasma spp. in 23,5% of the animals (16.7% for tortoises and 31,3% for turtles). Our result for the testudo genus is similar to those obtained by Soares et al. [25], who reported a prevalence of 15.8% for Mycoplasma agassizii in captive tortoises (Testudo spp.) in the United Kingdom. In Italy, in particular on the island of Sardinia, a higher prevalence of Mycoplasma spp. (37%) was found by Lecis et al. [24] in tortoises belonging to the Testudo genus. However, in addition to the different geographical origin of the animals, Lecis et al. [24] used wild animals, while in our case, with only one exception (animal n. 27), the animals came from private collections. Moreover, they have analyzed oral and cloacal swabs and used a different technique of DNA extraction and amplification. Considering our study, two results emerge: the fact that the detection of Mycoplasma spp. is almost double in turtles than tortoises, but with a number of observations that does not allow to assign statistical significance to the data (p = 0.196), and that the presence of Mycoplasma spp. was not statistically associated with the presence of conjunctivitis (p = 0.424). It would be interesting to assess whether, in case of URTD with simultaneous presence of conjunctivitis, such an association could occur.

Regarding the presence of Candida spp., which we have found in 2 healthy tortoises, the occurrence of yeasts in reptiles, especially in those who predominantly consuming vegetable diets, has been reported, and different Candida species have been isolated from tortoises belongin to the Testudinidae family [26]. Usually reptiles colonized with yeasts do not reveal any symptoms, as in our case, and we consider this finding not worthy of further investigation for the present study. However, representatives of the Testudinidae are frequently kept as pets, as indeed in the case of our tested animals, and could play a role in transmission of yeasts to human beings, especially in immunocompromised hosts, children and elderly or ill persons who are predisposed to candidosis.

Stenotrophomonas maltophilia causes many opportunistic infections as sepsis, pneumonia, urinary tract infection, meningitis, endocarditis, septic arthritis, and peritonitis. It has also been noted to be a pathogen in many ocular infections, including conjunctivitis, keratitis, dacryocystitis, cellulitis, infected scleral buckles, and endophthalmitis, as reviewed by Chang et al. [27]. However, in our case the bacterium was isolated from an healthy animal.