Research for anti - Toxoplasma gondii antibodies in free-living ducks ( Cairina moschata )

Toxoplasmosis is a zoonosis determined by the protozoan Toxoplasma gondii with wide geographic distribution, which affects homeothermic animals, with felids being their definitive hosts and other mammals and birds being their intermediate hosts. Regarding the Latin American bird species Cairina moschata , there are few reports in Brazil of the exposure of these animals to this protozoan. Based on the above, this study aimed to report for the first time the exposure of C. moschata to infection by this parasite in Rio de Janeiro, Brazil, determining the serological frequency of anti-T. gondii antibodies in birds of this species. Sixty-seven specimens of C. moschata were captured in the vicinity of the Rio de Janeiro Zoo, and these animals at the time of capture were sorted by gender and type of feather. Blood samples from these animals were obtained by venipuncture to obtain the serum. The serum samples were subjected to IFAT for anti-T. gondii IgG. In general, the frequency of positive results was 58.21% (n=39), with a statistically significant association (p<0.05) between the animals with adult plumage and seropositivity. The exposure of ducks to T. gondii in the vicinity of the Zoo may indicate contamination of this area with protozoan oocysts.


INTRODUCTION
Toxoplasmosis is a parasitic zoonosis of worldwide distribution determined by the protozoan Toxoplasma gondii.This facultative heteroxene cycle protozoan has felids as its definitive hosts because they eliminate oocysts in their feces, while other mammals, including humans and birds, are their intermediate hosts (Amendoeira et al., 1999;Hill et al., 2005;Robert-Gangneux and Dardé, 2012).The transmission of this protozoan occurs from the ingestion of tissue cysts present in raw or undercooked meat, sporulated oocysts present in water and food contaminated by feline feces or by transplacental transmission of tachyzoites (Amendoeira, 1995;Tenter et al., 2000;Montoya and Liesenfeld, 2004).In Brazil, the seroprevalence of T. gondii infection in the human population can vary from 21.5% to 97.4% (Souza et al., 2010;Dubey et al., 2012).
Like other bird species, those belonging to the Anatidae family, which includes individuals popularly known as ducks, drakes' bird, geese, and swans, are also susceptible to infection by T. gondii.Frequencies of T. gondii infection in birds of the Anatidae Family from different countries, such as Mexico, Czech Republic, Egypt, China, and Brazil, ranged from 1 to 50% (Alvarado-Esquivel et al., 2011;Bártová et al., 2009;El -Massry et al., 2000;Literák and Hejlícek, 1993;Yang et al., 2012;Konell et al., 2019).Except for the outbreak in domestic ducks in the Argentine chaco in 1962, there are no reports of clinical toxoplasmosis in other duck populations (Boehringer et al., 1962;Dubey, 2010).Similar to chickens, these birds can also be considered bioindicators of environmental contamination by T. gondii oocysts due to their habit of revolving the soil with their beaks in search of food (AbouLaila et al., 2011;Ibrahim et al., 2018).In addition, viable forms of the protozoan were recovered from tissue samples from birds of the genus Anas naturally infected in Egypt, Anas platyrhynchos experimentally infected in the Czech Republic and in birds of the Anatidae family in China, indicating the possibility of transmission of T. gondii to the populations with the habit of consuming the meat of these animals (Dubey et al., 2003;Bártová et al., 2004;Zhao et al., 2015).
Little is known about the role of domestic anatids in the epidemiology and ecology of toxoplasmosis when compared to other bird species.Within this context, the species Cairina moschata is included, whose subspecies C. moschata domestica descends from the South American wild species found from Mexico to northern Argentina, including Brazil (Sick, 1997;Mattos Junior et al., 2008;WikiAves, 2015).In Brazil, there are several seroepidemiological surveys on T. gondii infection in different taxa of birds, domestic and wild (Gondim et al., 2010;Feitosa et al., 2017;Camillo et al., 2018;Gonçalves et al., 2018;Konell et al., 2019;Sato et al., 2020).There are few reports of the exposure of Cairina moschata to T. gondii infection in the national territory (Ferraroni and Marzochi, 1980;Witter et al., 2020).In view of the above, this study aimed to report for the first time the exposure of C. moschata to infection by this protozoan in Rio de Janeiro, Brazil, determining the serological frequency of anti-T.gondii antibodies in birds of this species.

MATERIAL AND METHODS
This study was carried out at the Rio de Janeiro Zoo Foundation (RIOZOO) from December 2015 to June 2016.At the time of the study, this institution had an area of 138,000 m 2 and approximately 1300 animals under human care composed of various species, including mammals, birds, reptiles, and amphibians of national and exotic fauna.The zoo in Rio is the oldest in Brazil (Barbosa et al., 2020).In addition to the animals under human care in the domains of this institution, straying domestic cats, rodents, marsupials and birds circulated, including anseriformes of the Anatidae family standing out individuals from Cairina moschata, popularly known in Brazil as duck-of-the-bush or Muscovy duck (Pato-do-mato (Cairina moschata).WikiAves, 2015; International Union for Conservation Nature.2018).
During this period, 67 specimens of ducks that lived freely around the Zoo were captured and kept in a temporary enclosure bounded by bars and tiles, without access to the public, for approximately ten months.Blood samples were collected by venipuncture of the ulnar vein by the responsible veterinarians at the Zoo and transferred to tubes without anticoagulant.A maximum volume of 2 mL of blood was collected, depending on the size and weight of the animal.During the collection of blood samples, the birds were inspected to categorize them by gender and type of feather.In this study, adult plumage or young feathers presence was used as a longevity parameter, since they are free-living animals of unknown age.For that, the body morphology was observed, as well as the commitment from each individual.This information was stored in the laboratory's database.
Blood samples were sent to the Toxoplasmosis and other Protozoan Diseases Laboratory at the Oswaldo Cruz Institute/Fiocruz in isothermal boxes.To obtain the serum, the blood samples were centrifuged at 3000 rpm for 10 minutes, transferred to previously identified 1.5 mL microtubes and stored at -20ºC until the serological test was performed.Subsequently, the samples were subjected to the indirect fluorescent antibody test (IFAT) according to Camargo (1964).Tachyzoites of T. gondii strain RH kept in Swiss Webster outbred mice were used as antigens.For the detection of anti-T.gondii IgG, the commercial conjugate anti-duck IgG (H + L) antibody was used; Kirkegaard and Perry Laboratories -KPL® diluted in Evans Blue solution.Positive and negative control serum samples stored in the laboratory were used for each reaction.The reactions that showed total fluorescence of the surface of the tachyzoite at dilutions equal to or greater than 1:16 were considered positive (Millar et al., 2012).The statistical analysis of the data was performed using the statistical program GraphPad Prism 7.
To check the association between two categorical variables, Pearson's χ² test was performed.In assessing the impact between the variables gender and type of feather, the odds ratio (OR) values with their respective 95% confidence intervals (CI) were described.Associations where p≤0.05 were considered significant.
This study was approved by the Ethics Committee on the Use of Animals -CEUA / IOC-Fiocruz under license L-045/2016 and by the Biodiversity Authorization and Information System (SISBIO), Chico Mendes Institute for Biodiversity Conservation (ICMBio), Ministry of the Environment, under number 54797.

RESULTS
Of the total sera analyzed, 58.2% (39/67) were reagents for anti-T.gondii IgG on IFAT.Antibody titers ranged from 1:16 to 1:1024, with 20.5% (8/39) positive in 1:16 titration, 35.9% (14/39) in 1:64, 35.9% (14/39) in 1:256 and 7.7% (3/39) at 1:1024.Regarding the gender of birds, 59.5% of the females and 56.7% of the males had anti-T.gondii antibodies, with no significant difference between genders and the frequency of birds seropositive for T. gondii included in the study.For type of feather, the frequency of reactive individuals with adult plumage was significantly higher than that ducks with young feather.Ducks with young feather had a 0.71 times lower chance of exposure to T. gondii than adult plumage (Table 1).The high frequency observed in the population of C. moschata evaluated was already expected, considering that they were animals in free life.This correlation between free-living anatids and the high frequency of birds exposed to T. gondii has also been reported in China (Yang et al., 2012;Zhao et al., 2015).It is known that freeliving birds, such as those included in the present study, end up presenting a greater chance of ingesting T. gondii oocysts in different environments (Millar et al., 2012).It is important to note that C. moschata individuals cohabited the Zoo's surroundings with stray populations of domestic cats, which may have favored the parasite's prey-predator biological cycle.Even so, it cannot be ruled out that the small sample number of birds included may have contributed to the detection of high seropositivity.It is worth mentioning that the animals evaluated in the present study were free-living birds; thus, obtaining blood samples was dependent on the success of the capture, a situation not always achieved regularly, highlighting the importance and rarity of the information obtained with this biological material.
Despite the high frequency detected of T. gondii seropositive C. moschata, the birds evaluated in the present study did not show clinical signs suggestive of toxoplasmosis at the time of containment.A similar panorama was found with A. platyrhynchos in the Czech Republic, infected with T. gondii oocysts orally, who did not develop any clinical signs after 28 days of infection, although they were seroconverted (Bártová et al., 2004).The findings of the present study, added to what was exposed in the literature, indicate that these anatids, although susceptible to infection by T. gondii, can develop infections with an asymptomatic profile.
Most seroepidemiological surveys that assessed the exposure of anatids to T. gondii are restricted to countries where these animals are consumed by the human population, such as Southeast Asia and Egypt (El-Massry et al., 2000;Dubey et al., 2003, Yan et al., 2009;Yang et al., 2012;Zhao et al., 2015;Ibrahim et al., 2018).Similar to other homoeothermic animals intended for human consumption, anatids can harbor tissue forms of T. gondii in their meat.Thus, the consumption of raw or undercooked meat from these animals can represent an important risk factor for the human population that consumes them.In their free lives, anatids such as C. moschata in the present study can be prey for countless species of mammals and carnivorous birds.In addition, these anatids are generally territorial, but they fly in need of food and shelter, which may favor the exchange of genetic lines of T. gondii when chronically infected birds are preyed upon by felids.
Most of the studies that evaluated the frequency of anti-T.gondii antibodies was performed with anatids of the genus Anas, mainly in the species A. platyrhynchos (Dubey et al., 2003;Bártová et al., 2004;Alvarado-Esquivel et al., 2011;Maksimov et al., 2011;Ibrahim et al., 2018) In the present study, high titers of anti-T.gondii IgG were detected by IFAT, up to 1:1024.In the study by Bártová et al. (2009), anti-T.gondii antibody titers ranged from 1:40 to 1:320 in Anas platyrhynchos in the Czech Republic using the same technique.However, most other reports used the modified agglutination test (MAT) as a method for the detection of anti-T.gondii IgG (Dubey et al., 2003;Yan et al., 2009;Alvarado-Esquivel et al., 2011;AbouLaila et al., 2011;Yang et al., 2012;Puvanesuaran et al., 2013;Zhao et al., 2015).The absence of a gold standard technique for the diagnosis of avian toxoplasmosis makes comparisons between the few studies carried out with these birds difficult.
From the results obtained, both genders of C. moschata that circulate around the RIOZOO Foundation are equally exposed to infection by T. gondii.A similar result was observed in Anas platyrhyncos from different provinces in Egypt (Ibrahim et al., 2018).Regarding of adult plumage or young feather presence, the greatest exposure was found in birds with adult plumage.This result was already expected, considering that birds with adult plumage have a higher presumed age and, consequently, a greater chance of exposure to the possibly contaminated environment throughout their lives compared to individuals with young feather.Brasil. Rev. Bras. Cienc. Vet., v.15, p.140-142, 2008.MILLAR, P.R.; ALVES, F.M.X.; TEIXEIRA, V.Q.et al.Occurrence of infection with Toxoplasma gondii and factors associated with transmission in broiler chickens and laying hens in different raising systems.Pesqui.Vet. Bras., v.32, p.231-236, 2012. MONTOYA, J.G.;LIESENFELD, O. Toxoplasmosis. Lancet., v.363, p.1965-1976, 2004.PATO-do-mato (Cairina moschata).Wiki Aves, 2015.