Haematological alterations under the anti-helminthic application in Pavo cristatus

ABSTRACT Anti-helminthic drugs (Albendazole and Levamisole) trials were conducted on Indian Peafowls (n = 20) kept at Jallo Wildlife Park, Lahore, Pakistan for 15. Sampling was conducted on days 7 and 15 of treatments. The results showed that the WBC count was significantly (P < 0.05) higher in both samples in response to Levamisole treatment. Haemoglobin (HGB) and Mean Corpuscular Haemoglobin Concentration (MCHC) concentration increased significantly (P < 0.05) in response to both treatments; however, Mean Corpuscular Haemoglobin (MCH) decreased significantly (P < 0.05) in Albendazole-treated birds on both the sampling days compared to the control. Red Blood Cell Distribution Width-Standard Deviation (RDW-SD) decreased significantly (P < 0.05) in all treatment groups. On the other hand, the prevalence of eggs per gram (EPG) was reduced by 20% after the application of Levamisole on days 7 and 15. It was concluded that both drugs had significant effects on WBCs, HGB, MCH, MCHC and RDW-SD.


Introduction
Indian Peafowl (Pavo cristatus) is a large fancy bird with males having a distinct long colourful train of feathers (Arooj et al. 2023).It belongs to Order Galliformes and Family Phasianidae (Kushwaha and Kumar 2016).Indian Peafowl is a unique bird in the poultry industry and captivity.It originated in India and was later introduced to Europe through Italy and Greece.It is widely distributed in the wilderness of the Indian sub-continent including Pakistan, Jammu and Kashmir (Qamar et al. 2013).Globally, it is listed as the least concern species in the IUCN Red List, 2019.But in Pakistan, its wild population is under threat and hunting is prohibited under Punjab Wildlife Act-1974 (Protection, Preservation, Conservation and Management) (Mushtaq et al. 2012).Threats in the wild include predation by tigers, leopards, eagle owls and crested hark eagles (Qamar et al. 2013).Severe threats include degradation and loss of habitat, intensive agricultural practices, illegal poaching and hunting for feathers, eggs and other body parts for medicinal use (Anwar et al. 2015).
P. cristatus is also found in captivity where they are kept for recreation, farming research, conservation and protection.A captive environment affects the health and performance of these birds.Overcrowding in cages, unnatural habitat, suboptimal management practices, poor hygiene, improper use of anti-helminthic drugs and environmental conditions are some stress which results in changed behavioural patterns and reduced immunity (Athar et al. 2001;Khursheed et al. 2014).These factors reduce the immunity of birds; thus, they become more susceptible to gastrointestinal parasites (Khursheed et al. 2014;Pradeep et al. 2017).Parasites compete for nutrients in the digestive tract (Colditz 2008;Maizels et al. 2012) and make birds sluggish and lethargic due to undernourishment (Badran and Lukesova 2006).They also damage gut epithelium, reduce egg production, cause diarrhoea and reduce body weight (Basit et al. 2014).Apart from that, defense mechanisms and immune reactions in birds against parasitism can add to the metabolic cost resulting in higher metabolic rate and immunopathology (Lochmiller and Deerenberg 2000;Maizels et al. 2012).So, the infected birds are challenged to divert their metabolic resources to fight off parasites and compete for nutrition which they could have used for important functions like growth and reproduction (Lochmiller and Deerenberg 2000).
There are numerous ways to assess the health status of birds.Analysis of various haematological parameters provides reliable information regarding the health status of birds and animals.It is a widely used health analysis tool in domestic animals but not so common in birds.(Kral and Suchý 2000).Haematological parameters, such as packed cell volume, haemoglobin levels, white blood cell count, blood coagulation time and total protein levels in the blood, are crucial indicators of an animal's health and can provide valuable information for diagnosis and monitoring of various diseases and conditions (Tibbo et al. 2004).White blood cells offer immunity to the body and fight against pathogens to keep the body healthy (Meseguer et al. 2002;Gauri et al. 2012).Red blood cells and haemoglobin are important cells that carry oxygen from the lungs and transport it to all cells of the body.Oxygen is needed to extract energy by oxidative metabolism to maintain the biochemical activities of the body (Roberson and Bennett-Guerrero 2012).When there is a physical or vascular injury, platelets are activated by exposed collagen to prevent any blood loss and platelets accumulate at the injury site and seal the site with clotting factors (Meseguer et al. 2002;Gauri et al. 2012).High values of haematocrit indicate that the body of the animal is dehydrated.On the other hand, lower values of haematocrit point towards the presence of parasitic infections (Dimitrijević et al. 2016).
Haemoglobin and erythrocytes are indicators of the oxygen uptake capacity of birds.They give information about the average cell size, the amount of haemoglobin and the proportion of haemoglobin content in erythrocytes.Mean Corpuscular Haemoglobin Concentration is a precise red blood cell index.Its higher levels in the blood indicate haemolysis.Platelets play an important role in the healing process of injury.All these haematological parameters help to elaborate the metabolic status of birds.They also highlight nutritional deficiencies of birds (Dimitrijević et al. 2016).Many studies have been done to profile haematological parameters of pheasants (Hauptmanova et al. 2006;Masek et al. 2007;Kececi and ÇÖL 2011;Hrabcakova et al. 2014).Few studies have focused on peafowls (Samour et al. 2010;Xiao-xia et al., 2015;Kumar et al. 2017).All the studies have focused on either providing reference haematological values or checking the effect of sex and age on these values.Anti-helminthic drug studies have been mostly done to check the comparative efficacy of one or more drugs against gastrointestinal parasites in peafowls (Ashraf et al. 2002;Basit et al. 2014), pheasants (Draycott et al. 2006) and other birds (Schou and Permin 2003;Grandemange et al. 2007;Tucker et al. 2007;Khan et al. 2010).
The present research was designed to investigate the combined effect of parasite load and drugs (Albendazole and Levamisole) use on some haemotlogical parameters of peafowls.Results of this study have been compared with the taxonomically related and other avian species wherever possible.For the sake of equal comparison, units of haematological parameters in the literature were converted to units used in the current study.

Sampling site
P. cristatus were sampled from the Jallo Wildlife Park in Punjab, Pakistan.The park was selected for the availability of Indian Peafowls in ample numbers, permission to hold drug trials and permission for blood sampling.The selected Park is in a semi-arid plain area of Punjab at an elevation of 217 m.Its average temperature ranges 10-45°C with an average annual rainfall of about 628 mm (Figure 1).

Selection of Birds and trail set-p
Twenty Indian Peafowls aged between 3 and 6 years and weighed 3.5-5 kg were selected.These peafowls represented no signs of illness or disease.Sampling was done in the nonbreeding season from September to October 2022.Feed and water were given as per routine.Peafowls were not shifted to new cages for trial to avoid additional stress.For the control group birds from each cage were randomly sampled.Since birds were never moved to new cages, there was no need for an acclimatization period.It was made sure that all factors (season, nutrition and cages) remained the same so that the comparative effect of both drugs on haematological parameters could be observed.

Sampling plan and drug trial
For drug treatment, on day 1, when no drug was given twenty birds were sampled.This was considered as the control group.After first sampling all selected Peafowls were divided into two groups.One group was given Albendazole and the other group was given Levamisole.They were housed in separate cages for convenience.Peafowls were given single doses of Albendazole (20 mg/kg body weight) and Levamisole (25 mg/kg body weight) in drinking water as prescribed by the label.To make peafowls thirsty, water was removed from the cages one day before the administration of drugs.Drug-added water was changed twice a day to ensure efficient consumption of drugs.Fecal and blood samples were collected thrice, i.e. once from the control group on Day 1 before dividing into two treatment groups and twice (Day 7 and Day 15) after the administration of anti-helminthic drugs.

Collection and transportation of blood sample
A total of 180 blood samples were taken.Peafowls were captured and restrained.A brachial vein was exposed by spreading the wing.About 3 ml of blood was drawn, and pressure was applied on the puncture site for 30 s to stop bleeding.Blood was transferred to a vacutainer containing EDTA.Samples were placed in a handling pack for safe transport to the Research Laboratory of Zoology Department, Government College University Faisalabad for analysis.Analysis of blood samples was done within 24 h of collection.

Analysis of blood samples
Haematological parameters were analysed by running the whole blood samples in the Beckman Coulter AcT Diff Hematology Analyzer (Manufacturer: Beckman Coulter, Tokyo, Japan).Protocol of the kit was followed.Blood samples were brought to room temperature before analysis.Whole blood mode was opted for

Collection of fecal samples
Faecal analysis was performed to evaluate the prevalence of gastrointestinal parasites in each treatment group.Freshly dropped faeces were collected in cups using a sterilized spatula (Keatts et al. 2016).To avoid contamination separate cups and spatulas were used for each sample.Cup lids were tightly closed.Each sample was labelled for the date, site of collection and treatment group.They were stored in a handling pack for safe transport to a laboratory (Kathiravan et al. 2017).Fecal samples were analysed within 24 h.
After calculating eggs per gram for each sample, the prevalence percentage was calculated by following the formula Prevalence% = Number of infected birds Number of examined birds × 100 (Basit et al.2014).

Statistical analysis
Results were tabulated and statistically analysed using SPSS software.Normality distribution was found by the Kolmogorov-Smirnov test.Since data followed a normal distribution, a Oneway Analysis of Variance was applied.The significance level was set at P < 0.05.When the whole model was significant, Duncan's multiple range test was performed as a post hoc test to find statistically significant differences between various treatment groups.Mean and standard deviation (S.D) were also calculated.

Results
The effect of different antiparasitic drugs on leucocyte count is given in Table 1.Except for day 15 of Albendazole treatment, a significantly (P < 0.05) higher value of WBC was noted on the rest of the days in both the treatments compared to the control.However, the LYM count was significantly (P < 0.05) higher on day 7 in response to Albendazole treatment compared to the control.The count of MID and GRA did not change significantly between the treatments.Table 2 shows a comparative analysis of red blood cell and haemoglobin parameters of P. cristatus kept at Jallo Wildlife Park.The results showed that HGB and RDW-SD count significantly (P < 0.05) reduced on days 7 and 15 in response to Albendazole and Levamisole treatments.The MCH count decreased significantly (P < 0.05) on the day both intervals of sampling in response to Albendazole treatment; however, it decreased significantly (P < 0.05) on day 15 of Levamisole treatment.The count of MCHC increased significantly (P < 0.05) on both sampling days in response to antiparasitic drug treatments.However, RBC, HCT and MCV count did not change significantly (P > 0.05) on both sampling days in response to the treatments.
Table 3 provides information on comparative analysis of platelet-derived haematological parameters of P. cristatus kept at Jallo Wildlife Park.No significant (P > 0.05) change was observed in PLT, MPV, PDWs and PDWc on days 7 and 15 in response to Albendazole and Levamisole treatments compared to the control.
Table 4 shows treatment-wise incidence of gastrointestinal parasites in P. cristatus at three Wildlife Parks in Pakistan.The EPG level was higher on day 7 of Albendazole treatment in P. cristatus; however, in response to Levamisole on days 7 and 15, EPG was lower showing the lethal effects against the parasites.

Discussion
Usually studies on birds in captivity have been done to estimate the prevalence of endoparasites and to find out the load of different endoparasitic species in the gut, and use of various anti-helminthic drugs against the endoparasites to evaluate the efficacy of these anti-helminthic drugs (Ashraf et al. 2002;Villanua et al. 2007;Khan et al. 2010;Basit et al. 2014).In all studies, drugs were effective in eliminating gastrointestinal parasites.Very little research work has been done on the impact of endo-parasitic load on blood biochemistry and evaluation of different blood cells before and after the applications of anti-helminthic drugs.
The evaluation of haematology in captive birds can play a crucial role in wildlife conservation and reintroduction projects, as well as endangered species breeding programmes.Analysis of haematological parameters gives reliable information regarding the health status of animals (Kral and Suchý 2000).Haematological analysis is indeed a valuable tool in veterinary medicine as it provides important insights into an animal's overall health and how it responds to various internal and external factors.(Tibbo et al. 2004).
The aim of the present study was not only to check the efficacy of drugs but also to observe drug effects on haematology.The occurrence of parasites in treatment groups were only studied to confirm that drugs did reduce parasite burden and whether parasite load could be a factor in altering haematological values.That is why the discussion focuses on changes in haematology in lieu of drug usage rather than a comparative discussion about the efficacies of drugs or the prevalence of parasitic species.
Values of WBCs increased after treatment with anti-helminthic drugs.In control groups, it ranged from 70.7 ± 17.9 10 9 /L to 91.7 ± 3.64b.The rise in WBC values with drug administration in birds may be due to the peafowl body's immune response to drugs.White blood cells circulate in blood fight parasites as claimed by D'Amico et al. ( 2017) in a study on penguin chicks.Generally, the WBC count should drop upon the use of drugs because drugs eliminate parasites.Howard et al. (2002) investigated the toxicity of Fenbendazole and Albendazole in pigeons and doves.They reported a drop in WBC values (leukopenia) and weight loss after drug 13.2 ± 6.5 14.5 ± 6.9 15.8 ± 6.9 14.5 ± 7.0 13.8 ± 8.0 GRA (10 9 /L) 9.6 ± 6.1 8.3 ± 5.5 9.6 ± 2.6 15.1 ± 14.8 13.1 ± 8.9 Means having similar letters do not differ significantly at P < 0.05.WBC: White Blood Cells, LYP: Lymphocytes, MID: Other White Blood Cells, GRA: Granulocytes.
Table 2. Comparative analysis of red blood cell and haemoglobin parameters of P. cristatus kept at Jallo Wildlife Park.administration.On the other hand, another study on commercial broilers had different results where Levamisole's effect on cellular immunity was investigated.After treating broilers with levamisole, their cellular immunity, WBCs and LYM increased and these results are similar to the present study where WBCs and LYM increased after treatment.Oladele et al. (2012) implied that Levamisole is a good anti-helminthic drug and can be used in veterinary medicine and the present studies also confirm that birds do not show resistance against it compared to that of Albendazole.The WBC count observed for the current study was higher compared to the results of Samour et al. (2010), where the mean WBC value of captive peafowls was 9.52 ± 3.54 10 9 /L.Its range was 3.80 ± 18.90 10 9 /L.Bounous et al. (2000) did a study on juvenile turkeys and proposed that the rise in haematocrit, leukocytes and LYM is due to the excitement in birds and animals.Campbell (1995) also concluded the same.In another study conducted by Kumar et al. (2017) on wild populations of Indian peafowls, higher (Male = 210.38 ± 7.17 10 9 /L, Female = 218.47± 5.54 10 9 /L) WBC values were more noted than the values in the present study.Similarly, higher lymphocytes (Male = 197.86± 11.42 10 9 /L, Female = 151.34± 18.29 10 9 /L) were reported in wild populations of Peafowl by Kumar et al. (2017) compared to captive Peafowls (4.86 ± 1.94 10 9 /L) by Samour et al. (2010).

Parameters
The results of the current study lie in the middle of these two studies where the least value was 38.9 ± 21.4 10 9 /L and the highest was 71.8 ± 5.1 10 9 /L.Packed cell volume is a good indicator of the value of haemoglobin and red blood cells.In birds, the severity of disease is reflected by changing levels of plasma globulin.They serve as a valuable tool for prognosis.They also help in differentiating the stress state from the normal state.Stress can be due to nutritional, physical or environmental factors.Haemoglobin and erythrocytes are indicators of the oxygen uptake capacity of birds.Platelets play an important role in the healing process of injury.All these haematological parameters help to elaborate the metabolic status of birds.They also highlight the nutritional deficiencies of birds.Parasitic infections cause dilation of the gut, enteritis, emaciation, damaged gut epithelium and blood loss.They also affect the health of birds by using host vitamins and nutrients.Diarrohea, loss of appetite, weight loss, lowered egg production and lethargy are among other symptoms caused by gastrointestinal parasites (Urquhart 1987;McSorley and Maizels 2012).In the present study, birds were apparently healthy despite being infected with parasites.Red blood cell count and haemoglobin either remained the same or got higher after drug administration.An increase in RBC and HGB could be due to less blood loss as the parasite burden decreases.
In the current study, there was no significant effect of both drugs on RBC count.On the contrary, Saha et al. (2015) observed a significant increase in RBC in chickens upon the administration of Levamisole.Hassan et al. (2012) reported a significant increase in RBC in Bengal goats treated with Levamisole and Ivermectin drugs.Begum et al. (2010) and Hoque et al. (2006) also reported an increase in RBC count in Levamisoletreated groups in chicken.Selected birds at Jallo Wildlife Park had HGB values that ranged from 14.0 ± 4.8 g/dL to 16.6 ± 1.5 g/dL.In the present study, HGB levels were higher than reported by Samour et al. (2010) and Kumar et al. (2017).Similar findings were reported by Saha et al. (2015) in poultry where they observed a significant increase in HGB upon treatment with Levamisole.Hoque et al. (2006) and Begum et al. (2010) also reported the same findings.Hassan et al. (2012) studied the effects of Levamisole and Ivermectin drugs on Bengal goats and found a significant increase in HGB in treated groups.
Mean HCT values were noted with the ranges of 33.1 ± 2.0% in the pretreated group and 30.7 ± 1.4% on day 15 of the Levamisole-treated group.These results coincide with the mean HCT values reported by Samour et al. (2010) and Kumar et al. (2017) where the values were 32.0-46.0%and 25.79 ± 0.46%, respectively.In the case of high-dose poisoning of dogs by Levamisole, a decrease in RBC and HGB values was observed by Gokce et al. (2004).
The overall prevalence of endo-parasites in peafowls was 70%, 56.66%.Previously, Shahbaz (2020) conducted a study on the prevalence of GI parasites at Jallo Wild Life Park of Lahore, they found a 59.1% prevalence rate of GI parasites that was lower than the current results.Similarly, Basit et al. (2014) reported an overall prevalence of 58.3%, in birds of prey and owls in Germany.The same trend was observed in the study by Akram et al. (2019) having a prevalence of 54.32%.Wojcik et al. reported a 69% prevalence in captive birds.At Jallo Wild Life Park, Lahore, the EPG of endoparasites was reported as 9150 in the present study in the pretreatment group.Ingestion of contaminated droppings or intermediary hosts such as cockroaches, beetles, earthworms, flies and grasshoppers in poorly managed aviaries could explain the high prevalence of GI parasitism in captive birds.The cohort effect and the stress of captivity could explain the greater incidence of endoparasites in adults (Radfar et al. 2012). Similarly, Akram et al. (2019) reported that there is a notable correlation between gastrointestinal parasites and two factors: age and the type of rearing system in which the captive birds are kept.
In the present study, it was found that Levamisole was more effective against intestinal parasites than Albandazole in peafowl.These results correlate with the results of Sharma et al. (1989) who reported that Levamisole was 100% effective against endo-parasites as it inhibits the activity of malate dehydrogenase.However, the results of Khan et al. (2010) were not matched with those of the current study, their study concluded that Albandazole was more effective than Levamisole.

Conclusion
In the present study the administration of anthelminthic drugs caused significant changes in certain haematological parameters in peafowls.In addition, Levamisole was more effective than Albandazole in eliminating intestinal parasites in peafowls.
sample identification and a range was set.The probe was dipped in the sample to get values.Readings displayed on the screen were noted on data sheets.Standard measuring units were used for all parameters.The following haematological parameters were measured: White Blood Cell (WBC), Red Blood Cell (RBC), Red Cell Distribution Width -Coefficient of Variation (RDW-CV) and the Red Cell Distribution Width -Standard Deviation (RDW-SD) haemoglobin (HBG), Haematocrit (HCT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH), mean Cell Haemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), Platelet count (PLT) and Mean Platelet Volume (MPV).A Blood smear was also performed for manual WBC cell differentiation and determination of percentages and absolute values of Lymphocytes, neutrophils, eosinophils, basophils and monocytes (GRA) and other white blood cells (MID).
). Identification keys by Jaiswal et al. (2013) were used for identification.McMaster technique was used to count the number of eggs for each species (Titilincu et al. 2009; Basit et al. 2014; Kathiravan et al. 2017).Eggs per gram (EPG) for each faecal sample was calculated by using the formula: EPG = (eggs in Chamber 1 + eggs in Chamber 2)

Figure 1 .
Figure 1.Map of Punjab showing study sites.

Table 1 .
Comparative analysis of leukocyte counts of P. cristatus kept at Jallo Wildlife Park.

Table 3 .
Comparative analysis of platelet-derived haematological parameters of P. cristatus kept at Jallo Wildlife Park.

Table 4 .
Treatment wise incidence of gastrointestinal parasites in P. cristatus at Jallo Wildlife Park.