Study on prevalence and liver function test enzymes of differently plumaged peafowls (Pavo cristatus) infected with Toxoplasma gondii in captivity

Department of Zoology, The Islamia University of Bahawalpur ˗ Bahawalpur Pakistan Department of Zoology, Virtual University of Pakistan Department of Chemistry, The Women University Multan Pakistan Sub-Campus Toba-Tek Singh, University of Agriculture Faisalabad Pakistan Department of Forestry and Range Management B. Z. University Multan Pakistan University College of Veterinary and Animal Sciences,The Islamia University of Bahawalpur ˗Bahawalpur, Pakistan Department of Botany. Ghazi University ˗ D.G. Khan, Pakistan Department of Environmental Sciences, B.Z. University ˗ Multan, Pakistan Institute of Pure & Applied Biology ˗ B.Z. University ˗ Multan, Pakistan School of Energy and Power Engineering, Xi’an Jiaotong University, 28 West Xianning Road, Xi’an 710049, Shaanxi, PR. China

The birds in captivity in Pakistan and elsewhere face many infectious disease problems such as toxoplasmosis caused by Toxoplasma gondii (T. gondii). The T. gondii is an obligate intracellular parasite which causes toxoplasmosis in birds and mammalia (Tenter et al., 2000). Felids or cats are its definitive hosts while birds and mammals are intermediate hosts (Dubey et al., 2010). Cats shed oocysts in their stool that contaminate the environment and remain infective for more than one year, which permits the transmission of infection to intermediate hosts like birds, mammals, sheep, goats and humans.
In birds, seroprevalence of T. gondii is best indicator to access the soil contamination with sporulated oocytes. In birds, it causes loss of appetite, yellowing of mucous, maceration, diarrhea and abnormal functioning of the central nervous system. Like other birds, peafowl is also an intermediate host of Toxoplasma gondii. The infection rate of Toxoplasma gondii varies by country.
Recebido em 21 de setembro de 2019 Aceito em 22 de junho de 2020 Autor para correspondência (corresponding author) *E-mail: mushtaqlashary@gmail.com; raoimranishaq@gmail.com Studies on toxoplasmosis in peafowl are limited in Pakistan. Similarly, hematological and biochemical parameters show the health and metabolic status of the body, but no information is available about biochemical parameters altered due to the infestation of T. gondii in captive peafowls.
Considering the role of birds in infection of Toxoplasma gondii to humans and also according to our knowledge, there is no documented report about the prevalence of toxoplasmosis in captive birds in Pakistan. Therefore, the present survey was conducted to examine the effect of toxoplasmosis in peafowl in captivity and its effects on serum activity of liver enzymes of both seropositive and seronegative peafowls. Blood samples were collected from the peafowls (n=100) (45 male and 55 females) from differently plumaged peafowls including blue (n=28), white (n=10), pied (n=10) and black shoulder (n=52). Information about age, sex and physical condition of birds was gathered during blood sampling. Age-wise peafowls were divided into two groups viz. adults (24-36 months) and young (7-9 months). The blood samples were allowed to clot for nearly one hour and then subjected to centrifuge for 15min. Serum was harvested and kept at -20ºC until further analyzed for serum chemistry attributes at the Parasitology Research Laboratory of Department of Zoology, IUB.
The commercially available Toxo Latex Kit (Atlas Medical, Blankenfelde, Germany) was used to detect specific antibodies in serum of birds that consisted of a positive control, a negative control and a Latex Reagent. The reagent contained suspension of polystyrene particles coated with antigen of T. gondii. Positive control shows agglutination when added to serum and negative control does not show agglutination. Antigen-antibody reaction could take place when serums which contain antibodies against T. gondii were tested and this reaction can be easily visualized because of agglutination. Both the reagents and serum were brought at the room temperature prior to use. A serum sample of bird was serially diluted twofold in phosphate buffered saline from 1:2 to 1:8.
A sample found positive at 1:2 to 1:8 serial dilutions was tested at higher, doubling dilutions 1:16, 1:32, 1:64. One drop of diluted serum sample was placed onto the black area of the slide. The latex reagent was mixed well, and one drop was added to each serum drop. Both drops were mixed with the help of a stirrer and the slide was tilted. A clear positive reaction indicated the presence of T. gondii antibodies, which reflected either a past infection or an evolving infection. A negative reaction indicated the absence of T. gondii antibodies.
The Toxoplasma IgM ELISA kit (Calbiotech Inc. CA, USA) was used to examine T. gondii antibodies in birds as per manufacturer's instructions. Serum samples, positive and negative controls were added to the microwells. T. gondii antibodies if present in serum samples showed agglutination by forming an antibodyantigen complex. All unbound antibodies were washed away, and enzyme conjugate was added which bind to the antibody-antigen complex. Excess enzyme conjugate was washed away and the substrate (TMB) was added. The plate was allowed to incubate, and the color was developed. A blue solution appeared in the presence of antibodies, which turned yellow after the addition of the stop solution. There was no coloration in the absence of antibodies. The development of color was stopped by adding stop solution. The optical density (OD) of the microplate was read at 450nm with microplate absorbance reader 800 TS, BIOTK, UK. Results were written as the % age of the mean absorbance values of the sample (S) to the mean absorbance value of positive (P) control given with the diagnostic kit. According to manufacturer's reference, sera with S/P ≤ 40% = Negative, 40% > 50% Doubtful, 50%≤S/P < 200% = Positive, S/P ≥ 200% = Strong positive.
Among birds, peafowls also serve as intermediate hosts which cause significant health problems to the public (Tian et al., 2012). The results attained through LAT were re-tested by ELISA in order to enhance diagnostic accuracy in the present study. Previous researches have reported a higher seroprevalence of T. gondii in birds in different regions of the world including Iran (32.3%), China (31.8%), and Arq. Bras. Med. Vet. Zootec., v.73, n.2, p.529-533, 2021 Iraq (31%, 67% and 56%) in geese, peafowls, ducks and chicken, respectively (Tian et al., 2012, Lashari et al., 2018. Lower prevalence rate has been recorded in Colorado (3.9%), Portugal (4.6%), and China (8.36%) by Dubey et al., 2010, Waap et al., 2008and Zhang et al., 2014 for pigeons, parrots and pet birds, respectively. The difference in prevalence could be attributed to difference in breed and species of birds.
In the present study, according to LAT and ELISA, a higher prevalence rate was recorded in males as compared to females. Zhang et al. (2104) in China reported higher prevalence of T. gondii in male (10.43%) parrots as compare to females (6.08%). Same results have been reported by Lashari et al. (2018). The relation of gender with susceptibility of host to toxoplasmosis might be due to genetic predisposition and difference in hormones. Testosterone is known for its immunosuppressive activity (Seli and Arici, 2002). The females can be immune because of different factors e.g. diet, age and surrounding conditions.
Males are less susceptible to protozoan parasites than females. Causes of higher prevalence of T. gondii antibodies in males in present study may be due to steroid hormonal difference.
In the present study, a significant difference (P<0.05) was detected in seropositive adult hosts as compared to young hosts. Similar results have been reported by Zhang et al. (2104). Present and previous studies showed that adults have more chance to get an infection by ingestion of oocysts with contaminated food because they have a wide range of feeding area than young ones.
The present study showed significant biochemical changes with statistically high level of bilirubin, Albumin and ALP in the infected host as compared to non-infected hosts. ALT increased in infected blue and pied plumaged peafowls whereas level of AST elevated in infected white and pied plumaged peafowls. In general, the analysis revealed that there was a significant effect of toxoplasmosis on liver function test enzymes of hosts. Similar results were reported by Amany et al. (2010) from Egypt, Dawood and Mahmood (2012). In contrast to the current study, a decreased level of albumin and elevated level of ALT and AST have also been recorded in T. gondii infected hosts (Amany et al., 2010). In serum, biochemical profile level of AST and ALT indicate the working condition of liver cells.
These enzymes play a significant role in metabolic transmission of amino acid mostly present in the liver and some other tissues. In case of any cellular damage, AST and ALT spread into the surrounding tissues and its level of activity increased. In case of any hepatic injury activity level of AST and ALT increases (Adeyemi and Akanji, 2011). Toxoplasmosis causes infiltration of liver cells and in portal area, damage endothelial cells and necrosis in hepatocytes. Moreover, the elevated level of AST and ALT also provide information about infection and inflammation induced by T. gondii. Toxoplasmosis causes significant and permanent destruction to the liver, noteworthy developments of organisms arising in alterations in the metabolism of the liver (Amany et al., 2010).
In a nutshell, toxoplasmosis is widespread in peafowls in Bahawalpur Zoo, Pakistan which indicates an environmental contamination with oocysts of T. gondii. According to LAT and ELISA highest prevalence was recorded in males as compared to females. Age-wise higher prevalence was recorded in adult peafowls. Toxoplasmosis also affects the liver function test enzymes of the hosts. The present study may provide preliminary data for the control of T. gondii in other zoo birds to minimize the load of T. gondii. We recommend the zookeepers, veterinary staff and allied stake holders to devise a directional strategy towards control and eradication of T. gondii.