Cryptosporidium spp. and Giardia spp. in Free-Ranging Introduced Monk Parakeets from Santiago, Chile

Simple Summary Monk Parakeets are medium-sized parrots that were internationally traded as pets and that as a byproduct have become invasive species in 19 countries. This is the case of Chile, where Monk Parakeets have thrived in the city of Santiago. Cryptosporidium spp. and Giardia spp. are parasites that can affect the digestive system of a wide variety of animals, including humans and birds. This study sought to determine the occurrence of these parasites within Monk Parakeets from the city of Santiago. To do this, 207 fecal samples from Monk Parakeet nestlings that were captured during the summer seasons of 2017 and 2018 were analyzed. Environmental data related to the trees in which the nestlings were captured was studied in order to the determine the existence of areas more prone to have infected parakeets and whether certain environmental variables influence the presence or absence of these parasites in Monk Parakeets. In total, 33 samples were positive to the presence of one or both parasites. Of the 33, 10 nestlings (30%) were infected with Cryptosporidium spp. and 25 (76%) with Giardia spp. Two nestlings were infected with both parasites (6%). Environmental analyses revealed that pruned trees might constitute a protective factor against infection with these parasites. These findings emphasize Monk Parakeet’s potential role as a disease disseminator, especially in urban environments. Abstract Monk Parakeets (Myiopsitta monachus) are medium-sized parrots that due to international pet trade currently exist as invasive species in 19 countries globally. Such is the case of Chile, where Monk Parakeets have thrived in the city of Santiago. Cryptosporidium spp. and Giardia spp. are worldwide distributed gastrointestinal parasites whose potential hosts include birds and humans. The present study sought to determine the presence of these pathogens in Monk Parakeets from Santiago. During the austral summers of 2017 and 2018, 207 Monk Parakeet nestlings were captured, and fecal samples were studied via microscopical analyses. Environmental data related to the trees in which the nestlings were captured were analyzed to establish the existence of infection clusters. Associations between spatial clusters, environmental variables, and the presence or absence of these pathogens were explored. In total, 33 samples were positive to the presence of one or both protozoa. Of the 33, Cryptosporidium spp. oocysts were detected in 10 nestlings (30%) while Giardia spp. cysts were detected in 25 (76%). Two nestlings presented poly-parasitism (6%). Statistical analyses established pruned trees as a potential protective factor against infection with these parasites. The present study corresponds to the second report of Cryptosporidium spp. in Monk Parakeets in Chile and the first worldwide report of Giardia spp. in these birds, emphasizing Monk Parakeet’s potential role as a reservoir and pathogen disseminator, especially in urban environments.

Considering that biological invasions can be a source of pathogens, some of them zoonotic, and can thus eventually affect the health of people [3,[32][33][34], and that Monk Parakeets are synanthropic birds, keeping a close contact with human populations by building their nests within urban public squares and parks [20,21], investigating the sanitary state of these invasive birds becomes important. Therefore, the aim of this study was to perform a thorough inquest into the occurrence of these pathogens within Monk Parakeets located in Santiago. We additionally sought to determine the existence of infection clusters within the city and to study the correlation between environmental variables associated with the trees in which Monk Parakeets nest and positive rates of infection.

Study Area
The sampling area compromised twenty-one municipalities of Santiago (33 • 27 S; 70 • 38 W), the capital city of Chile. Santiago is located in the Metropolitan Region in Central Chile, a Mediterranean bioclimatic zone defined by dry summers, wet winters, and interannual variability caused by the El Niño-Southern Oscillation phenomenon [35]. Mean annual temperature is 13.2 • C, and mean annual precipitation is 531 mm [36]. Vegetation occurs in an assortment of Acacia caven shrubland on lower hillslopes and evergreen sclerophyllous forest on watersheds and south-facing slopes [36,37]. The Metropolitan Region is currently divided into 52 municipalities and is the highest densely populated region in the country, concentrating 40% of the national population with 7,112,808 inhabitants and a density of 462 people/km 2 [38]. Due to its accelerated and unorganized expansion, Santiago is characterized by urban sprawl and deep environmental degradation [39]. Green areas within the city are highly stratified, from a total of 3825 ha, 62% (2387 ha) are distributed in only 3% of the total green areas. These areas are associated with large public parks [40].

Study Species
Monk Parakeets (Myiopsitta monachus) are medium-sized, sexually monomorphic parrots originally distributed in the southeastern area of South America, specifically in Paraguay, Uruguay, Bolivia, southern Brazil, and northern and central Argentina [6,41]. As a consequence of the international pet trade, Monk Parakeets have been introduced into several countries, becoming an invasive species in 19 countries of Africa, Asia, Europe, and America, including Chile [6][7][8][9]. The invasive success this psittacine bird has exhibited can be attributed to several behavioral and ecological traits. More notably, Monk Parakeets are the only species among the order Psittaciformes that does not rely on preexisting cavities to nest, since they are able to build their own communal nests [7,12,42]. Furthermore, they feed on a flexible diet, allowing them to exploit a wide variety of fruits and seeds [13]. Additionally, their gregarious behavior leads them to engage in communal parenting, increasing their possibilities to avoid predatory attacks [6,7,12]. Ultimately, they display great tolerance to human presence and urban perturbations, often building their nests in public squares and parks [7,10,11]. All these traits provide a great capacity to adapt to different environments and lead to an accelerated population growth [11,14]. Further, their reproductive success seems to be higher in introduced versus native distributions [15].

Nestling Sampling and Processing
Sampling took place during the austral Monk Parakeet's breeding season: November and December of 2017 and 2018. Twenty-one municipalities of Santiago, Chile, were opportunistically sampled for the study (Figure 1). Monk Parakeet nestlings were considered as the sample unit and were assumed to represent the nest inhabitants' sanitary condition. Nestlings were manually counted and captured from their nest and accessed through a hydraulic aerial platform. Only one individual was captured per nest and per tree.

Nestling Sampling and Processing
Sampling took place during the austral Monk Parakeet's breeding season: November and December of 2017 and 2018. Twenty-one municipalities of Santiago, Chile, were opportunistically sampled for the study (Figure 1). Monk Parakeet nestlings were considered as the sample unit and were assumed to represent the nest inhabitants' sanitary condition. Nestlings were manually counted and captured from their nest and accessed through a hydraulic aerial platform. Only one individual was captured per nest and per tree. Since parakeet sampling depended upon the number of trees with occupied nests per municipality, accessibility to nests, technical support provided by each municipality, and daily possibilities to sample, among others, the sampling process was of convenience.
Nestlings were sacrificed via cervical dislocation following bioethical protocols. During the necropsy, individuals were aged and the distal portion of the digestive tract and its content was sectioned and preserved in 70% ethanol at 4 °C until further analyses. All procedures were conducted in compliance with national regulations established by Servicio Agrícola y Ganadero (SAG, Chilean Fish and Wildlife Service), under permit No. 716/2016, and bioethical and biosafety protocols issued by the Faculty of Animal Veterinary Sciences, University of Chile (Bioethical approval No. 19-2016 and Biosafety approval No. 82).

Protozoa Detection
Fecal content was extracted from the preserved digestive tract for macroscopic and microscopic parasitological analyses. For detection of Cryptosporidium spp. Oocysts, the modified Ziehl Neelsen method was employed using 500 µL of sediment extended onto a 1 cm × 0.5 cm slide [43]. Giardia spp. cysts were detected by means of modified Telemann method using Lugol's stain and 100 µL of sediment extended onto a 1 cm × 0.5 cm slide [44]. Since parakeet sampling depended upon the number of trees with occupied nests per municipality, accessibility to nests, technical support provided by each municipality, and daily possibilities to sample, among others, the sampling process was of convenience.
Nestlings were sacrificed via cervical dislocation following bioethical protocols. During the necropsy, individuals were aged and the distal portion of the digestive tract and its content was sectioned and preserved in 70% ethanol at 4 • C until further analyses. All procedures were conducted in compliance with national regulations established by Servicio Agrícola y Ganadero (SAG, Chilean Fish and Wildlife Service), under permit No. 716/2016, and bioethical and biosafety protocols issued by the Faculty of Animal Veterinary Sciences, University of Chile (Bioethical approval No. 19-2016 and Biosafety approval No. 82).

Protozoa Detection
Fecal content was extracted from the preserved digestive tract for macroscopic and microscopic parasitological analyses. For detection of Cryptosporidium spp. Oocysts, the modified Ziehl Neelsen method was employed using 500 µL of sediment extended onto a 1 cm × 0.5 cm slide [43]. Giardia spp. cysts were detected by means of modified Telemann method using Lugol's stain and 100 µL of sediment extended onto a 1 cm × 0.5 cm slide [44].

Environmental Variables
For each nestling sample, the following environmental data related to the tree in which it was captured were recorded: municipality in which it was located, tree species, sanitary state of the tree (healthy/not healthy), tree management (pruned/not pruned), tree height, tree canopy diameter, diameter at breast height (DBH), number of nests per tree, nest size, nest height, number of chambers per nest, number of nestlings per nest, and GPS coordinates. Tree canopy diameter and DBH were measured using a 30 m measuring tape, while tree height and nest height were measured using a hypsometer. Health condition of trees was determined with the collaboration of academics at the Faculty of Forestry Sciences at the University of Chile.

Statistical Analyses
Descriptive statistics were performed to summarize each recorded variable. Considering the nature of the collected data (positivity to Cryptosporidium spp. oocysts and positivity to Giardia spp. cysts), three logistic regression model analyses were performed, one for each pathogen and one for the combined two protozoan parasites (model including the results of both pathogens) [45]. Univariate logistic regression analyses were performed to assess the relationship between all recorded variables and the positivity to the pathogens (for each model). Spearman correlation, Pearson chi-square, and Fisher's exact test were performed to assess for collinearity and potential confounding factors, setting significance on p < 0.05. Variables with a p-value ≤ 0.25 were included in a multivariable logistic regression model (liberal criteria). A stepwise backward elimination procedure was conducted, using the log likelihood ratio test (LRT); the model with the lowest LRT was selected as the final model (in the three built models). Likewise, variables whose regression coefficients were not significant (p > 0.05) were removed from the multivariable logistic regression [46]. Non-significant variables, which produced a change greater than 20% in the regression coefficients of the significant variables when removed, were retained in the model to adjust for confounding factors. All the biologically feasible interaction variables were included in the model design [45]. The convergence of the models was set at a value of epsilon = e −16 in order to increase restrictions on models and to secure the representativity and power of results. Goodness-of-fit of the final model was evaluated using the Hosmer and Lemeshow test [47].
Biologically logical interactions between variables that fulfilled the liberal criteria were also analyzed. All categorical variables were analyzed using the dummy variables approach [48]. Local clustering of positive samples was assessed by means of the Bernoulli model of the spatial scan statistic, considering a purely spatial cluster analysis [49,50]. Analyses were conducted using the RStudio and the statistical software R 3.3.1 [51] plus "lme4", "ggplot2", and "gcookbook" packages, and SatScan software version 9.4.2. [52]. Odds ratios, 95% confidence intervals, and p-values were computed.
Finally, statistically significant differences between sampling years was determined by calculating the prevalence difference, equivalent to an attributable risk or excess risk, and 95% confidence intervals, based on approximation and null hypothesis testing (prevalence difference equals to 0) [53]. This analysis was performed using R version 3.6.1 [51] and "fmsb" package [54]. The presence of other parasites such as helminths and other protozoa was discarded via microscopical observation during the modified Telemann exam. in 2018 (2/109). Positivity to Giardia spp. was 10.2% in 2017 (10/98) and 13.8% in 2018 (15/109). Overall, individuals that were positive to the presence of Cryptosporidium spp. oocysts had a regular load of 5 to 10 oocysts per 500 µL of analyzed sample, while those positive to Giardia spp. presented 1 to 5 cysts detected by microscopy per 100 µL of analyzed sample. The presence of other parasites such as helminths and other protozoa was discarded via microscopical observation during the modified Telemann exam.

Environmental Variable Analyses
Sample records for each nestling were analyzed using the three mentioned logistic regression models. The Cryptosporidium spp. model (Table 2) shows borderline significance (p = 0.06) of pruned trees as a protective factor (odds ratio (OR) = 0.14, 95% CI lower limit (LL) = 0.01 and upper limit (UL) = 0.83), indicating a potential trend into the exploration of the other two models. Models for Giardia spp. show consistency of a significant role of pruning trees as a protective factor (OR = 0.25, 95% CI LL = 0.09 and UL = 0.71; p = 0.01). Nest height also showed a borderline significance (p = 0.07), but the OR and 95% CI show a non-causal association (Table 3) (Table 4). Spearman correlation, chi-square, and Fisher's exact test results show no collinearity and potential confounding factors among the variables included in the multivariable logistic regression models (p > 0.05).

Spatial Analyses
All 10 Cryptosporidium spp. positive samples, 25 Giardia spp. positive samples, and 33 Protozoan positive samples were distributed throughout the study area. The Bernoulli model of the spatial scan statistic detected several clusters, none of them significant. Only one of them, a cluster for Giardia spp., presented borderline significance (p = 0.06). This cluster included 9 sample points in a radius of 1.26 km, with a relative risk of 6.95, indicating a high risk of positivity at this site, located at Santiago, San Miguel, and San Joaquin municipalities.

Sampling Season Analysis
Prevalence values showed statistically significant differences (p < 0.05) for Cryptosporidium spp. in regard to sampling years, with higher prevalence in the sampling season of 2017 in comparison to 2018 (Table 5).

Discussion
Even though introduced Monk Marakeets have been inhabiting Chile for almost 50 years, not much is known about the local impact of this invasive bird [5,20]. The present study detected two gastrointestinal protozoa in Monk Parakeets from the city of Santiago and identified a possible protective factor associated with the absence of infection with these parasites. The findings of this work coincide with Cryptosporidium spp. oocysts found in adult Monk Parakeets from Lo Barnechea municipality, where 19.1% of sampled adult specimens were positive to the presence of the parasite [5]. The current findings, however, show that infection by Cryptosporidium spp. is not restricted to the municipality of Lo Barnechea or adult birds but can also occur in nestlings from different municipalities of Santiago. In addition to the detection of Cryptosporidium spp., the present study also reports the presence of Giardia spp. in intestinal samples of Monk Parakeets from the city of Santiago, corresponding to the first report of this parasite in this psittacine bird worldwide.
Monk Parakeet nestlings rely completely on their parents for feeding and do not leave the nest until they reach approximately 40 days of age [55]. Considering that Cryptosporidium spp. and Giardia spp. are orofecally transmitted parasites [23], that Monk Parakeets are gregarious animals that live in communal nests where they maintain close contact and practice collaborative parenting, and that they defecate within these nests [6,7,12], it is highly likely that nestlings acquired these pathogens by being in close contact with infected adult individuals or through their feces.
Detection of Cryptosporidium spp. and Giardia spp. in Monk Parakeet's fecal samples is of relevance, as these widely distributed gastrointestinal parasites possess great zoonotic potential, exhibiting a wide range and variety of potential hosts that include mammals, fish, amphibians, reptiles, and birds [23,[56][57][58][59][60][61][62]. Potential host species for these parasites often coincide, creating the possibility of coinfection events [22,24]. Such an event was detected in this study, as two of the sampled parakeets were infected with both Cryptosporidium spp. and Giardia spp. parasites.
When studying the association between the presence of these pathogens and environmental variables recorded at Monk Parakeet's nesting sites, it was possible to observe a negative association between parasite detection and pruned trees. This could indicate that pruned managed trees might constitute a protective factor against Cryptosporidium spp. and Giardia spp. infections. Considering that pruning is of great importance when it comes to maintenance of tree health [63], it is possible that this activity helps creating a less suitable environment for the survival of (oo)cysts in parakeet nests and surroundings, and therefore reducing the risk of infection. One way pruning trees could be contributing to lower infection rates is by allowing for higher U.V. light penetration toward nests. Solar U.V. light has been shown to have the power to inactivate Cryptosporidium parvum oocysts [64,65] and could thus be contributing to reduce the infective (oo)cysts load within nests. Another factor that could potentially be influencing this finding is pruning season. If trees are pruned during the winter season and some nests are destroyed during the process, it could be possible that some of the nests sampled during Monk Parakeet's breeding season (spring and summer) corresponded to recently built structures. Due their novelty, new nests could present lower loads of accumulated (oo)cyst, which could translate into lower risk of infection.
Interestingly, a statistically significant positive association between the number of nestlings per nest and presence of Giardia spp. was observed. This association, however, was not detected in the case of Cryptosporidium spp., nor when both protozoa were analyzed together. A similar situation occurred in the case of sampling season and infection rates. It was possible to detect a statistically significant difference between the amount of Cryptosporidium spp. positive samples collected during the 2017 sampling season and the 2018 sampling season. This difference, however, was not detected in the case of positive samples to Giardia spp. Further investigation is warranted to determine if these findings are associated with specific environmental variables or are casual results, a consequence of the sampling limitations of the present study.
Due to the lack of statistical significance associated with the existence of infection clusters, it was not possible to confirm an association between the occurrence of infection with one of these two agents and a specific geographic area within the study site. Nonetheless, a borderline significant cluster was detected in the central area of the city, compromising the municipalities of Santiago, San Miguel, and San Joaquin. Further analyses would be required to confirm the existence of this cluster and to determine the factors underlying its existence.
Monk Parakeets were introduced in Chile during the early 1970s, as fueled by the international pet trade market [17]. It is unknown if parakeets carried these pathogens upon their arrival or whether they acquired them in Chile. No reports of these agents existing within Monk Parakeets in their original distribution range have been issued, and although several endoparasitic studies have been performed on different Chilean birds [66][67][68][69][70], and more specifically on birds located in the city of Santiago [71], only Cryptosporidium spp. has so far been reported in Monk Parakeets [5]. As previously mentioned, nestlings most likely became infected via contact with adult individuals, which in turn may have acquired the pathogens from the surrounding environment. Infection with Cryptosporidium spp. and/or Giardia spp. is most commonly associated with the consumption of water contaminated with (oo)cysts [22][23][24]. Further studies should evaluate the quality of water sources present in public parks and squares. Interestingly, Giardia spp. was detected in soil samples from public parks in Spain [28], evidencing this substrate as a possible additional source of pathogens.
A different factor that could possibly be contributing to the presence of these parasites in Monk Parakeets relates to the coexistence of these birds and other domestic animals in public squares or parks. A study performed in 2012 detected the presence of Cryptosporidium spp. and Giardia spp. in fecal samples of dogs that were collected in public squares of the city of Santiago, Chile [72]. A similar study conducted in Spain also detected the presence of Cryptosporidium spp. and Giardia spp. in fecal samples collected in public parks of Madrid [28]. A second investigation, also in Madrid (Spain), detected the presence of Giardia duodenalis in dogs from an animal shelter [73]. This is a relevant finding since G. duodenalis has been previously described in aquatic birds [74] and has been isolated from a parrot [75]. Considering that in Santiago Monk Parakeets and dogs make use of the same environment to forage, and where Monk Parakeets also nest [20], the possibility of these agents being transmitted back and forth between parakeets and other species is possible. This also opens the possibility of Monk Parakeets being infected with other species of endoparasites and would also explain the general ubiquity of these protozoa in the studied area. Lastly, the possibility of Monk Parakeets contracting these pathogens from other bird species with which they share a common niche should also be considered, as it has been previously reported that Monk Parakeets in Santiago are able to peacefully forage and share space with other birds [20].
Interestingly, other species of birds make use of parakeet's nests [20,21]. Such occurrence was recently registered in Santiago, Chile, where nine different species of birds, including Rock Doves and seven native species, were detected nesting inside Monk Parakeet's nests [20]. Monk Parakeets often abandon their nest constructions in order to build new ones [76]. Additionally, incidents in which other birds attack Monk Parakeets, expel them from their nest, and later proceed to occupy it have been registered previously [77], and instances in which other bird species have been observed using empty chambers of parakeet nests that are otherwise still being occupied by parakeets have also been documented in Chile and other countries [6,20]. Regardless of whether Monk Parakeets abandon their nests voluntarily, are expelled from them, or share them, the sanitary implications of finding other birds inside these structures should not be overlooked. Coexistence in nesting sites, can eventually increase the transmission of parasites and diseases between different species [78]. Cryptosporidium spp. and Giardia spp. are orofecally transmitted parasites [23], and considering that Monk Parakeets defecate within their nests [12], it is quite possible that bird species using abandoned parakeet's nests or making use of unoccupied chambers while parakeets still remain in the nest could eventually acquire these and other pathogenic agents, especially given that these protozoa may stay viable for weeks [79].
Monk Parakeets could also be spreading these parasites to other animals when being consumed by predators. American Kestrel (Falco sparverius) and Harris's Hawk (Parabuteo unicinctus) have been reported as parakeet predators in Chile [20,80]. Recently, the role of Monk Parakeets as potential ecosystem engineers, providing a novel nesting resource in urban areas, was determined [20]. The present report shows that their role as ecosystem engineers could extend beyond the addition of a new nesting resources and include the potential role of Monk Parakeets as pathogens disseminators. This is supported by recent findings in Spain where Monk Parakeets were found to be infected with the beak and feather disease virus and the parasite Leucocytozoon [18,19].
As a synanthropic species, this may be of high health impact if some of these parasites are zoonotic, representing a major risk upon urbanized metropolises as Santiago, where these parakeets seem to be successful invaders.
Further, Monk Parakeet pathogen transmission risk is increased as this species tend to be favored by the public and are coveted as pets. In 2014, a domiciliary outbreak of psittacosis in the city of Dom Pedrito (Rio Grande do Sul State, Brazil) was attributed to Monk Parakeets after eight members of a family presented psittacosis-associated symptoms. Two parakeets illegally purchased by this family were identified as the source of Chlamydophila spp. [81]. Incidents such as this one could eventually increase and include the transmission of other pathogens.
For the time being, Monk Parakeets have remained confined to urban environments, but if they reach rural areas they could eventually encounter native Chilean parrots and transmit pathogens to them or to other bird species (e.g., beak and feather disease). This possibility is supported by a recent report issued in Spain that describes how Monk Parakeets from Madrid have spread into rural areas where they have been detected nesting in association with White Storks (Ciconia ciconia). This type of behavior could allow parakeets to increase their invasive potential by avoiding biotic resistance in the form of predator pressure, for example [82]. Although White Storks are not present in Chile, parakeets could eventually benefit from the presence of other bird species and expand their range of distribution.
Current environmental variations associated with climate change and agricultural land use can, generally, be positively associated with higher risks of contagion with Cryptosporidium spp. and Giardia spp. [83]. In addition to that, since infection can occur by consumption of contaminated water, control efforts for these pathogens usually focus on the sanitary management of water sources [84]. The present findings could contribute to highlight the importance of biological invasions in the emergence and control of diseases, especially those of urban densely populated areas.
It must be noted that despite exhibiting a wide variety of potential hosts, the Cryptosporidium and Giardia genus contain many different species and genotypes that differ in their ability to infect specific host species. This translates to a high level of species-specific infections. Since the identification of the parasites detected in this study was restricted to the genus identity of the pathogens, further analyses should be performed in order to determine the specific species infecting Monk Parakeets from the city of Santiago. Determining the species-and the genotypes-of the Cryptosporidium spp. and Giardia spp. (oo)cysts found in this study could help in assessing the real potential of these birds as possible transmitters of zoonotic agents to human and animal populations.
It should also be considered that even though the detection of Cryptosporidium spp. and Giardia spp. is important and the finding of pruned trees as a possible protective factor is interesting, further studies should be conducted in order to draw proper ecological and epidemiological conclusions about the presence of these pathogens and the environmental factors that influence their presence or absence. The sampling process carried out in this study was one of convenience, meaning that several arbitrary factors influenced the selection of the sampled trees. These ranged from nest accessibility to time and resources availability to permits granted by municipalities. For this reason, even though the statistical analyses performed in this work can reflect reality, they should still be considered with caution. Future studies on the sanitary state of Monk Parakeets should aim at a more thorough sampling design, ideally with a larger sample size or using a random sampling process not influenced by the previously mentioned factors.

Conclusions
This study corresponds with the second report of Cryptosporidium spp. in Chile and the first worldwide report of Giardia spp. in free-ranging parakeets from Santiago. Further studies focused on determining the specific identity of these protozoa, the presence of other pathogens in this invasive bird, and the existence of environmental variables favoring or impeding infections are of great importance, not only because of the potential transmission of pathogens to human beings, but also because, by coexisting with other bird species, this invader may by affecting the health of native birds in central Chile, one of the world's 25 biodiversity hotspots and a priority for conservation measures [85].