INTRODUCTION

The green turtle of the Eastern Pacific (Chelonia Mydas) in danger of extinction, locally known as black/brown turtle, is distributed along the American Pacific coast, occupying different geographical regions during each of its life cycle stages (^{Cliffton et al., 1982}). These agencies are susceptible to specific threats of their environment and of anthropogenic origin (^{Aguirre et al., 2006}), therefore, the study of health conditions of populations in free life, including the presence of infectious agents, is receiving increasing attention for conservation.

Sea turtles in free life naturally house a wide variety of bacteria in their gastrointestinal tract. However, its opportunistic behavior and pathogenicity (^{Ahasan et al., 2018}) have also been reported. In an organism, the communities of associated microorganisms are essential for a wide variety of functions. The development of the host and its health state depend on the presence of an intact microbial community, which plays an important role in all living organisms (^{Bloodgood et al., 2020}). In different species, it has been shown that microbiota promotes the development of organs and tissues, the production of vitamins and essential amino acids that affect the use of fats (^{Koropatnick et al., 2004}), and the response of glucose and lymphocytes to the Intestinal injuries (^{Warwick et al., 2013}). The different bacteria of the gastrointestinal tract of sea turtles can provide various functions that are still unknown and suggest playing an important role in the assimilation of food and their use (^{Ahasan et al., 2017}). However, these microorganisms can also cause host damage, for example, increasing susceptibility to intestinal inflammations and infectious diseases mainly (^{Garner et al., 1995}). Internationally, the presence of various bacteria in sea turtles has been reported such as: Salmonella, Mycobacterium, Escherichia Coli, Citrobacter Freundii, Edwarsiella sp., Vibrio Alginolyticus, Vibrio Cholerae, Vibrio Fluvialis, Vibrio Furnisii, Vibrio parahaemolyticus, Aeromonas and Proteus, between others (^{Work et al., 2003}; ^{Orós et al., 2005}; ^{Santoro et al., 2006}; ^{Zavala-Norzagaray et al., 2015}). They have been identified as opportunists and potentially pathogenic for turtles. In addition, some adverse health effects have been reported in humans that consume meat and eggs of sea turtles infected with zoonotic pathogens, a common illegal practice in coastal countries around the world (^{Aguirre et al., 2006}). In Mexico, the information about these microorganisms related to diseases in sea turtles is still limited. Therefore, it is important to generate truthful information about the possible risks for turtles and human health associated with its illegal consumption. This study aimed to provide the baseline on potentially pathogenic bacterial agents for turtles and zoonotic type, through bacteriological crops and biochemical methods in black turtle samples (C. Mydas) in free life of Ojo de Liebre lagoon complex, Baja California Sur (BCS), Mexico.

MATERIAL AND METHODS

RESULTS

Many black turtles (178) were captured, which measured 72.37 ± 11.98 cm of CCL, 67.27 ± 11.26 cm CSL and weighed 44.61 ± 21.15 kg. Turtles were classified as 125 juveniles and 53 adults and their BCI was 1.38 ± 0.18. 523 isolates associated with gramnegative enterobacteria of seven different species (Table 1) were obtained.

In the mouth of turtles, the most abundant bacteria were Kleibsiella sp. (31.56%), followed by Citrobacter Freundii (29.79%), Enterobacter sp. (23.01%), Pseudomonas aeruginosa (15.34%) and Burkholderia cepacia (0.29%), while in the cloaca the most abundant was Proteus sp. (42.93%), Citrobacter Freundii (28.80%), Aeruginosa Pseudomonas (19.57%), Vibrio Fluvialis (4.89%) and finally Burkholderia cepacia (3.80%) (Figure 1).

Figure 1 Relative abundance of bacterial agents in the mouth and cloaca of black turtles (Chelonia Mydas) at the Ojo de Liebre lagoon complex, Baja California Sur, Mexico 

The Kruskal-Wallis test showed no significant differences between the presence of the different microorganisms and the CCL of turtles (Ji-square = 5.75, gl = 6, p = 0.45 value) (Figure 2).

Figure 2 Bacterial agents present in the different carapace curved length (CCL) of black turtles (Chelonia Mydas) in Ojo de Liebre lagoon complex, Baja California Sur, Mexico 

DISCUSSION

Turtles were classified as juveniles and adults according to the size reported for the area (^{Márquez, 1996}). Its BCI coincided with the previously reported for the area (^{Reséndiz et al., 2018b}) indicating that turtles had good nutritional status and presumably the capacity for favorable reproductive performance. The physical examination showed no evidence of clinical signs, serious injury or diseases that compromised the functioning of turtle organs and systems, nor that they put their normal activities at risk or attempt against their integrity (^{Norton, 2005}).

It is important to consider that the differences in the microorganism communities of sea turtles are attributed mainly to ecological and ontogenic factors (trophic level, diet, habitat, etc.) (^{Ahasan et al., 2018}), so the gastrointestinal bacterial composition differs significantly between populations in free life and rehabilitation agencies (before hospitalization and after rehabilitation) (^{Pace et al., 2019}; ^{Bloodgood et al., 2020}). In general, the high prevalence of proteibacteria has been associated with dysbiosis as well as deterioration in the health status of sea turtles (^{Ahasan et al., 2018}). Citrobacter Freundii showed the highest prevalence (86.52%). This gramnegative bacteria of the Enterobacteriaceae family has been reported earlier in sea turtles internationally (^{Santoro et al., 2006}), nationally in black turtles in Sinaloa (^{Zavala-Norzagaray et al., 2015}) and in yellow turtles (Caretta Caretta) in the Gulf of Ulloa, BCS (^{Reséndiz et al., 2019}). It is considered an opportunistic pathogen associated with infections in juvenile animals and secondary infections in immunosuppressed animals (^{Glazebrook and Campbell, 1990}), and is not zoonotic (^{Johnson-Delaney, 2014}). In sea turtles, the infectious dose and the incubation period are unknown. It has been reported that its transmission is fecal-oral by ingesting contaminated foods or by direct contact between the carrier and susceptible and immunosuppressed organisms (^{Johnson-Delaney, 2014}), this species is found in the gastrointestinal tract of animals and uses bodies of water as a reservoir (^{Work et al., 2003}). Kleibsiella sp. had a prevalence of 60.11%. This gramnegative bacteria of the Enterobacteriaceae family was previously reported in sea turtles internationally (^{Warwick et al., 2013}) and national in yellow turtles in the Gulf of Ulloa, BCS (^{Reséndiz et al., 2019}). This pathogen plays an important role as a cause of opportunistic infectious diseases, mainly in immunosuppressed organisms and frequently in juvenile animals (^{Work et al., 2003}). It has also been associated with bacteremia, injury, respiratory and urinary infections (^{Johnson-Delaney, 2014}). The infectious dose and the incubation period in sea turtles are unknown. It is not zoonotic and their vectors are unknown, although it has been reported that its most frequent transmission path is through the feces (^{Jacobson, 2007}). This genre can be isolated from the soil, bodies of water, skin, nasopharyngeal exudates, or of the gastrointestinal tract of carrier turtles (^{Glazebrook and Campbell, 1990}) and can survive in marine environments for several hours or in clinically healthy animals for long periods ( ^{Tan et al., 2009}). Pseudomonas aeruginosa presented a prevalence of 49.44%. These gram- negative bacilli belong to the Pseudomonadaceae family (^{Buller, 2004}) and have been studied in sea turtles at an international level (^{Work et al., 2003}; ^{Santoro et al., 2006}) and national (^{Reséndiz et al., 2019}). These are opportunistic pathogens suggest an increased risk of disease in immunosuppressed and fibropylomatosis juvenile turtles (^{Work et al., 2003}), most of its conditions arise from the colonization of the respiratory and urinary tract or infections of deep dissemination that can cause pneumonia, bacteremia and chronic respiratory infections (^{Buller, 2004}; ^{Jorgensen and Ferraro, 2009}). They are not zoonotic and in sea turtles, vectors are not known (^{Work et al., 2003}; ^{Johnson-Delaney, 2014}). The infectious dose in sea turtles is also unknown and its incubation period varies according to the infection (^{Orós et al., 2005}; ^{Jorgensen and Ferraro, 2009}). This species is transmitted by direct contact with contaminated water, aerosols or aspiration by contact of mucous membranes with discharges of infected conjunctivas or upper respiratory tract of infected organisms (^{Johnson-Delaney, 2014}) and can survive for several months in the marine environment with basic nutrients (^{Jacobson, 2007}). Proteus sp. had a prevalence of 44.38%. These gram-negative bacilli Morgananelaceae have been reported in other healthy and sick sea turtle species at the international level (^{Santoro et al., 2006}), in Mexico, in black turtles (^{Zavala-Norzagaray et al., 2015}) and in yellow turtles clinically healthy (^{Reséndiz et al., 2019}). They are considered part of the gastrointestinal microbiota of sea turtles; however, they can generate chronic urinary tract infections such as bacteremia, pneumonia and focal injuries in weakened and emaciated organisms (^{Work et al., 2003}). They are not zoonotic and no transmission vectors are known. In addition, in sea turtles, the infectious dose is unknown and the incubation period is not well established (^{Johnson-Delaney, 2014}), although it has been reported that it causes infections when leaving the gastrointestinal tract (^{Jorgensen and Ferraro, 2009}). This genre is not transmitted by direct contact between organisms and can be found in bodies of water (^{Jacobson, 2007}). Survive easily outside the guest, especially in areas where there is an animal protein decomposition (^{Buller, 2004}; ^{Jacobson, 2007}). Enterpose sp. showed a prevalence of 43.82%. These gramnegative enterobacteria was reported in yellow turtles in the Gulf of Ulloa, BCS (^{Reséndiz et al., 2019}) and in other marine turtle species at an international level (^{Work et al., 2003}; ^{Zavala- Norzagaray et al., 2015}). It is considered an opportunistic pathogen that has been associated with infectious outbreaks such as fibropylomatosis in juvenile turtles (^{Work et al., 2003}). It can cause numerous infections, such as pneumonia, sepsis in the intestinal tract and in the urinary tract, which can cause bacteremia (^{Work et al., 2003}; ^{Orós et al., 2005}). It is not zoonotic and do not know each other (^{Glazebrook and Campbell, 1990}; ^{Warwick et al., 2013}). In addition, its incubation and infectious dose period in sea turtles is unknown. However, it is known that it is transmitted by direct or indirect contact of mucous surfaces with the infectious agent and can be transmitted by fecal-oral (^{Johnson-Delaney, 2014}). This genus is colonizer of the lower gastrointestinal tract of humans, animals and it can often be found in plants, soil, water bodies and is able to survive with a minimum source of energy (^{Pace et al., 2019}). Vibrio Fluvialis had a prevalence of 5.06%. This gramnegative bacillus of the Vibrionaceae family has been reported in sick and healthy sea turtles at an international and national level (^{Zavala-Norzagaray et al., 2015}; ^{Reséndiz et al., 2019}). It is considered an emerging pathogen that is transmitted by eating contaminated foods (^{Ahasan et al., 2017}; ^{Franco-Monsreal et al., 2014}). All its consequences are unknown in sea turtles, but in mammals cause diarrhea similar to anger, cutaneous infections associated with exposure to aquatic environments and even sepsis in immunosuppressed individuals (^{Franco-Monsreal et al., 2014}). Its period of incubation and infectious dose in sea turtles is unknown. This microorganism is zoonotic (^{Johnson-Delaney, 2014}) and can survive for long periods in the marine environment, where it is widely distributed (^{Igbinosa and Okoh, 2010}; ^{Franco-Monsreal et al., 2014}).

Some reports indicate that it has also been isolated from sewage, animal and human stool, as well as seafood, mainly in Bivalvos Molluscs (^{Alton et al., 2006}; ^{Igbinosa and Okoh, 2010}). Finally, Burkholderia Cepacia had a prevalence of 4.49%. This gramnegative bacteria of the Burkholderiaceae family has been previously reported at an international and national level in Tortugas Golinas (Lepidochelys Olivacea) (^{Santoro et al., 2006}; ^{Zavala-Norzagaray et al., 2015}), green turtles (C. Mydas) with and without fibropylomatosis (^{Work et al., 2003}) and in clinically healthy yellow turtles (^{Reséndiz et al., 2019}). This pathogenic microorganism can cause chronic pulmonary disease with mucopurulent secretion, characterized by multiple abscesses in the skin and subcutaneous tissues, or a severe sepsis with death in about 7-10 days (^{Orós et al., 2005}; ^{Jacobson, 2007}). In sea turtles, the infectious dose is unknown, its incubation period varies from 1 to 14 days (^{Johnson-Delaney, 2014}) and it is considered zoonotic by direct or indirect contact of the mucosa with discharges of injury from infected animals (^{Warwick et al., 2013}; Johnson-Delaney, 2014). It has been reported in mammals, reptiles and fish, considering humans as accidental guests (^{Buller, 2004}; ^{Jorgensen and Ferraro, 2009}) It can be found on the ground, bodies of water and areas close to agricultural activities, where it can survive for long periods at room temperature (^{Buller, 2004}; ^{Jacobson, 2007})

The presence of these bacterial agents in sea turtles was not related to their carving; The Kruskal-Wallis test showed no significant differences between the presence of the different microorganisms and the CCL of turtles, which indicates that the found bacteria presented interchangeably in juvenile animals and adults. Within the proteobacteria found in black turtles, Vibrio Fluvialis and Burkholderia cepacia are zoonotic pathogens, and although they have a low prevalence, they must be monitored regularly to prevent risks in turtles and public health. Since, despite the federal prohibition of capture, consumption and trade in sea turtles in Mexico since 1990, these organizations continue to be captured and consumed. This fact represents a great potential danger for human health (^{Aguirre et al., 2006}), being able to cause extreme dehydration, vomiting, diarrhea and even death to consumers due to the presence of these microorganisms (^{Alton et al., 2006}), in addition to viruses, parasites or pollutants in sea turtles (^{Zavala-Norzagaray et al., 2015}). The information generated warns about possible risks to the health of sea turtles consumers. In addition to being an illegal practice, it is potentially dangerous for public health and affects the populations of the different protected species. This study complements the health assessments of black turtles in the area and management and conservation plans of agencies and their ecosystems in the Biosphere Reserve "El Vizcaíno", along with local authorities.

CONCLUSIONS

Seven potentially pathogenic bacterial agents were reported for sea turtles in apparently healthy black turtle individuals, of which two are zoonotic. Strong clinical evidence is required to define whether these microorganisms cause diseases and studies that are more specific are needed to clarify the differences between the microbiota and the pathobiota of sea turtles, especially with molecular methods.