Comparative efficacy of ivermectin and fenbendazole against ancylostomiasis in dogs

The present experiment was carried out to assess the comparative efficacy of ivermectin and fenbendazole individually for anthelmintic therapy for the hookworm infested dogs. Dogs presented to the Department of Veterinary Clinical Medicine or Veterinary Clinical Complex, Bhubaneswar were randomly screened for Ancylostoma caninum infection and the positive dogs were selected for the therapeutic trial Faecal samples were collected randomly from presented dogs immediately after defaecation or from the rectum directly using a faecal scoop. The collected sample was examined by floatation technique to detect the positive cases of Ancylostoma caninum infection. The dogs with normal clinical parameters and no eggs or ova in the faeces were included in in group 1 (n = 12). Dogs with faecal sample positive for Ancylostoma caninum ova were recruited for the comparative study (n = 24) which were grouped into two groups consisting of 12 dogs in each (group 2 and 3). Group 2 dogs (n = 12) were treated with ivermectin at 200 µg/kg body weight once orally repeated after 15 days with proper supportive therapy each time. Group 3 (n = 12) were treated with fenbendazole at 50 mg/kg body weight once orally repeated after 15 days with proper supportive therapy each time. Haematological examinations and serum biochemical tests were carried out in all groups each time on day 0, 15 and 30 of the experiment. The therapeutic efficacy of both the drugs was calculated on the basis of number of animals found free of Ancylostoma infection as determined by reduction in EPG count of the faeces of the group following the treatment. The reduction in eggs per gram (EPG) count on day 15 and day 30 was more significant in group 2 than group 3. The mean EPG count reduced significantly to 24.17 ± 11.44 on day 15 from day 0 level of 1650.00 ± 247.25 in fenbendazole-treated dogs. On day 30, the mean value further reduced to become nil.The 15th day after treatment, mean (± SE) value of protein, albumin and globulin was changed to 5.63 ± 0.12, 2.64 ± 0.12 and 2.99 ± 0.15 g/dl, respectively. The 30th day after treatment, the values were 6.23 ± 0.14, 3.20 ± 0.18 and 3.03 ± 0.21 g/dl, respectively. The total protein and albumin values were significantly changed from day 0 level in group 2 and 3 by 15th day and 30th day, respectively, at 1% level of significance. Following treatment with ivermectin, the 15th day haematological values increased significantly at 1% level (P < 0.01) of significance. There was significant increase in the values at 1% level on the 30th day compared to day 0 and the mean values were non-significantly comparable to the healthy control group except PCV and TEC.


Introduction
Tropical climate with high rainfall make the animals susceptible to diverse intestinal helminthes. Frequent infections by one or more species of endoparasites can cause high morbidity, weight loss, anaemia and lowered immune status. Among all helminthes infestations, Ancylostoma caninum is the most common and dangerous parasite in dogs which has global importance (Vaughan and Murphy 1962). This parasite is the most widespread of all hookworm species and parasitizes dogs throughout the tropics and subtropics, inflicting serious health hazards including blood loss. Due to its high prevalence and its zoonotic significance, especially in developing countries and socioeconomically disadvantaged communities with poor levels of hygiene and overcrowding, together with the lack of veterinary attention and zoonotic awareness, exacerbating the risk of disease transmission and becoming responsible to cause human gut disease, Ancylostoma caninum has gained major importance in the field of veterinary as well as public health research, and. has sparked off renewed interest in its study in recent years (Craig and Macpherson 2000;Obiukwu and Onyali 2006).This hookworm affects dogs of almost all age groups. However, it is life threatening for very young pups (Soulsby 1982). The worm gets entry into the pups through transmammary route (Miller 1965) or into the adults through skin penetration. Ancylostomiasis causes severe haemorrhagic gastroenteritis leading to severe iron deficient microcytic hypochromic anaemia (Layrisse et al. 1961). The adult worms attach to and feed on the intestinal mucosa causing intestinal hemorrhages (Georgi et al. 1968), presence of blood in faecal matter, severe anaemia and intestinal ulceration. The measured daily loss of blood is reported to be 0.013 ml per adult male worm and 0.043 ml per female adult worm (Wang et al. 1983). Hence, more severe blood loss is caused by the female adult worms. The blood picture reveals varying degree of anaemia and hypoproteinaemia (Miller 1974). The incidence of this disease is more in the stray dogs (Arle et al. 1992). Panigrahi et al. (2014) studied the presence of gastrointestinal helminthic parasites in clinically normal dogs in Bhubaneswar, Odisha and reported the incidence of ancylostomiasis to be the second highest after the mixed parasitic infection in stray dogs. However, routine deworming practice at regular intervals can minimize the ill health effects in dogs and reduce public health concerns. Many dog owners are unaware of mode of transmission and severity of the disease both in dogs and humans. Hence, they don't commonly follow strict deworming protocol. Various drugs like ivermectin, pyrantel pamoate, praziquintal, fenbendazole and other combination of anthelmintics are being used in India for the treatment of endoparasitic infestations in dogs. Studies have revealed high level of pyrantel resistance in canine ancylostomiasis (Kopp et al. 2008). Therefore, the present study was aimed at assessing the comparative efficacy of ivermectin and fenbendazole individually to ascertain the best anthelmintic therapy for the hookworm infected dogs.

Materials and methods
The present study was conducted on dogs presented to the Department of Veterinary Clinical Medicine and Veterinary Clinical Complex, Bhubaneswar. The presented dogs were randomly screened for Ancylostoma caninum infection and the positive dogs were selected for the present study.

Faecal sample
Faecal samples were collected immediately after defaecation, or from the rectum directly using a faecal scoop. The collected sample was examined by floatation technique to detect the positive cases of Ancylostoma caninum infection. Identification was made according to the morphological characters of the eggs.

Experimental protocol
The dogs with normal clinical parameters and no eggs or ova in the faeces were included in group 1 (n = 12). Dogs with faecal sample positive for Ancylostoma caninum ova were recruited for the comparative study (n = 24) which were grouped into two groups consisting of 12 dogs in each (group 2 and 3). Group 2 dogs (n = 12) were treated with ivermectin at the dose rate of 200 µg/kg body weight orally, repeated after 15 days with proper supportive therapy such as hepatoprotective drugs, hematinics. Group 3 (n = 12) were treated with fenbendazole at 50 mg/kg body weight orally, and repeated after 15 days with supportive therapy as required.
Hookworm positive samples were then analyzed qualitatively by Mc Master Technique for EPG (Eggs per Gram) count before treatment (day 0) and thereafter on 15th and 30th day post treatment (Soulsby 1982). Hematological examinations and serum biochemical tests were carried out in all groups on day 0, 15 and 30 of the experiment, and the dog owners were advised to keep the dogs indoors and in hygienic management conditions. Analysis of faecal sample Microscopic examination: The samples were examined microscopically adopting qualitative techniques using flotation method for detection of parasitic material).
Direct smear: A pinch of faecal sample was placed on one end of a slide and mixed with a drop of water. After mixing and spreading, smears were covered with cover slip, and examined directly under microscope. At least 3 slides from different parts of the faecal sample were examined.
Flotation method: Saturated salt or sugar solution was used to prepare the faecal suspension. Faecal sample (1-2 g) was taken in a mortar and pestle and triturated after adding a little quantity of saturated salt or sugar solution. Then the faecal suspension was allowed to settle for twenty minutes. After this from The superficial layer (10-15 ml) was collected in a test tube and a clean cover slip was placed on the top of the tube in such a way that the upper meniscus of the faecal suspension present in the test tube touches the lower surface of the cover slip. The test tube was then allowed to stand undisturbed for twenty minutes after which the cover slip was lifted gently and placed over a glass slide for microscopic examination under 10X and 40X.

Haematological studies
Blood sample (2 ml) was collected from saphenous or cephalic vein of dogs in EDTA coated vial for haematological examinations. Haemoglobin was estimated by Sahli's acid haematin method using N/10 HCl using a haemoglobinometer. The haemoglobin concentration was expressed in g/dl.PCV (Packed cell volume) was estimated by microhematocrit method. The Wintrobe haematocrit tube was filled with EDTA containing blood and centrifuged for 40 min at 2500 rpm. Total Leukocyte Count (TLC) was estimated using haemocytometer method and the values were expressed /µl of blood. Similarly, Total Erythrocyte Count (TEC) was done by Haemocytometer method and the values were expressed in 10 6 / mm 3 . Differential Leukocyte Count (DLC) was estimated after staining thin blood smear on a glass slide with diluted Giemsa stain. Hundred cells were counted in high power oil immersion and different white blood cells were calculated basing on their morphology. The values were expressed as percentage of cells. The erythrocytic indices such as MCV (Mean Corpuscular volume), MCH ((Mean Corpuscular Haemoglobin) and MCHC (Mean Corpuscular Haemoglobin Concentration) were calculated.

Biochemical studies
Another 3 ml of blood sample was collected in clot activator vials on day 0 and thereafter on day 15 and 30 post treatment for harvesting serum. Total serum protein levels were determined by Biuret method using colorimeter and Biuret kit manufactured by Coral clinical systems. Serum albumin was determined by BCG (Bromo Cresol Green) method using colorimeter and BCG kit manufactured by Coral Clinical Systems. The serum globulin was estimated by subtracting serum albumin from serum total protein.

Therapeutic efficacy
The therapeutic efficacy of both the drugs was calculated on the basis of number of animals found free of Ancylostoma infection as determined by faecal sample examination and reduction in EPG count of the faeces of the group following the treatment by applying the following formula:

Statistical analysis
The data collected during the present study in respect of different parameters were statistically analysed for calculation of mean value, standard error of mean and two -way analysis of variance to compare the means of different treatment groups over different periods using SPSS version 16.0 (SPSS Inc, IBM, USA).

Results
The hookworms infested dogs exhibited clinical signs of diarrhoea, red or black colour faeces, pale mucous membrane, vomition, inappetence, anorexia, and dermatitis ( Table 1). The mean (± SE) haematological values of the healthy control and therapeutic groups on day 0, 15 and 30 of the experiment are given in Table 2. The day 0 mean ± S.E of Hb (g/dl), PCV (%), TEC (× 10 6 /µl) were 7.32 ± 0.37, 27.33 ± 0.98, and 4.13 ± 0.18, respectively. However, the 15th day values following treatment, increased significantly at 1% level (P < 0.01) of significance to reach 10.00 ± 0.37, 32.75 ± 1.06 and 4.95 ± 0.22, respectively. On the 30th day, there was significant increase in the mean values at 1% level in group 2 compared to day 0 level. The mean values of haematological parameters except PCV and TEC were nonsignificantly comparable to the healthy control group on the 30th day. The day 0 mean (± S.E.) level of Hb (g/dl), PCV (%) and TEC (× 10 6 /µl) in group 3 dogs treated with fenbendazole were 9.68 ± 0.28, 31.92 ± 0.87 and 4.82 ± 0.13, respectively. The MCV, MCH, and MCHC value on day 0 were 66.38 ± 1.70, 20.13 ± 0.60 and 30.55 ± 1.17, respectively. However, the treatment with fenbendazole, the values of PCV and TEC increased significantly at 5% level to reach 35.25 ± 0.93 and 5.21 ± 0.14 on day 15 but the Hb, MCV, MCH, MCHC values did not increase significantly (P < 0.05). The values of Hb, PCV, TEC increased significantly from day 0 on day 30 at 5% level. The TLC values were 11,625 ± 962.25 on day 0, 11,466 ± 586.76 on day 15 Mean EPG before treatment -Mean EPG after treatment Mean EPG of the group before treatment × 100.
and 11,466 ± 586.76 on day 30, and the variation was nonsignificant (P < 0.05) over the periods.
The erythrocytic indices such as MCV (fl), MCH (pg), MCHC (g/dl) on day 0 were 66.87 ± 2.49, 18.11 ± 1.18 and 27.03 ± 1.37, respectively in group 2 dogs. The mean values changed to 66.92 ± 2.18, 20.58 ± 1.08 and 30.70 ± 1.07, respectively on 15th day after treatment with ivermectin. On 30th day, the values increased to 67.09 ± 1.99, 21.59 ± 0.93 and 32.12 ± 0.84, respectively. There was non-significant change in MCV noted on 15th and 30th day after treatment compared to day 0 mean value. The MCH on day 0 was below reference range and on 15th and 30th day after treatment it was improved significantly at 5% level of significance. The MCHC values also increased significantly from day 0 to day 15 and day 30. The mean ± SE of TLC (/µl) on 0 day, 15th and 30th day after treatment were 11,791 ± 918.80, 11,550 ± 667.02 and 11,725 ± 482.75, respectively in group 2 dogs. There was apparent decrease in TLC from day 0 by day 15 and apparent increase from day 15 to day 30. The change was not significant at 5% level of significance. The neutrophil (/µl) count on day 0, 15 and 30 were 61.83 ± 1.99, 62.42 ± 1.50 and 62.75 ± 0.96, respectively in group 2 dogs. There was absolute neutrophilia throughout the treatment but it was not significant statistically at 5% level of significance. The lymphocyte count did not change significantly. The values remained almost in a constant range throughout the periods. The eosinophil count reduced significantly at 1% level from 6.83 ± 0.53 on day 0 to 3.58 ± 0.48 on day 15 and 3.07 ± 0.26 on day 30.
The biochemical parameters of healthy control group and therapeutic groups are given in Table 3. On day 0, the mean (± SE) of total protein (g/dl), albumin (g/dl) and globulin (g/ dl) in negative control group were 6.39 ± 0.20, 3.21 ± 0.17 and 2.93 ± 0.27, respectively. The 0 day values of total protein (g/dl), albumin (g/dl) and globulin (g/dl) in hook worm positive group 2 dogs were 5.01 ± 0.12, 2.25 ± 0.15 and 2.75 ± 0.25 g/dl, respectively. The 15th day after treatment with ivermectin, mean ± SE values changed to 5.63 ± 0.12, 2.64 ± 0.12 and 2.99 ± 0.15 g/dl, respectively. On 30th day after treatment, the values were 6.23 ± 0.14, 3.20 ± 0.18 and 3.03 ± 0.21 g/dl, respectively. The total protein and albumin values were significantly changed by day 15th and 30th at 1% level of significance as compared to that of day 0. On day 0, the mean total protein, albumin, globulin in group 3 dogs were 4.94 ± 0.27, 2.48 ± 0.17 and 2.45 ± 0.21, respectively (Table 3). On 15th day after treatment with fenbendazole, the values increased to 5.00 ± 0.26, 2.51 ± 0.19 and The mean EPG counts in infected dogs were observed to be too high (1725 ± 331.23 and 1650 ± 247.25 in group 2 and 3, respectively) before treatment which drastically fell down following treatment (Table 4). The reduction in EPG count on day 15 and day 30 was more significant in group 2 than group 3. Eggs per gram of faeces (EPG) after treatment with ivermectin significantly reduced to 0 on day 15 and day 30 from the mean count of 1725 ± 331.23 on day 0 ( Table 4). The mean EPG count in fenbendazoletreated dogs reduced significantly to 24.17 ± 11.44 on day 15 from day 0 level of 1650 ± 247.25. The value further reduced to nil on day 30th (Table 4).

Discussion
Ancylostomiasis is a very serious nematode disease in the dogs which exerts deleterious effects and cause serious health hazards resulting in generalized ill health due to loss of blood and body tissues, lowered body resistance to other infectious diseases, reduced efficiency etc. The haematological parameters like haemoglobin, PCV and TEC in the  (Areekul et al. 1974) resulting in a state of anaemia with lowered levels of haemoglobin, PCV and TEC on day 0, indicating hemorrhagic anaemia. In group 1, there was no significant change in haematological values between day 0 and day 15 or 30. However, in group 2 and 3, the haemoglobin, PCV and TEC were seen to be below normal level on day 0. There was improvement in mean level in subsequent observations in these two therapeutic groups. Certain degree of anaemia has earlier been reported in ancylostomiasis as evidenced by decreased RBC (3.95 + 1.24 m/c.mm), Hb (6.3 ± 2.5 g/dl) and PCV (20.3 ± 8.8%) (Ramakrishnan et al. 1972) In very severe cases, haemoglobin was as low as 2.5 g/dl in pups. Mitra and Sasmal (1985) also demonstrated haematological changes in pups experimentally infected with 500 infective larva of Ancylostoma caninum by different routes on day 21, and all the erythrocytic values reduced correspondingly with the age of infection. Srivastava et al. (1988) reported that hookworm infestation leads to reduction in blood Hb, PCV and TEC levels, and these parameters return to normal after deparasitization. Udonsi and Agunama (1991) reported progressive decreases in both PCV and Hb in 20 puppies of 8 weeks age, with negative correlation noted between these parameters and faecal egg counts. Kalkofen (1987) reported Hb levels commonly below 10 mg/dl and TEC lower than 4 million/cu mm from 14th day post inoculation (D.P.I) and concluded hook worm infection to be synonymous with iron deficiency anaemia. The parasitized animals had significantly (P < 0.01) lower levels of haemoglobin, packed cell volume and total erythrocyte counts than non-parasitized animals (Qadir et al. 2011). In the present study, the values of haemoglobin, PCV and TEC gradually increased, and became significantly different from day 0 mean level on day 15 and 30 subsequent to treatment with anthelmintic and haematinics / hepatoprotective drugs.
The erythrocytic indices such as MCV, MCH, and MCHC in group 1 on day 0, 15 and 30 were within normal range. The mean values were significantly (P < 0.01) lower on day 0 in group 2 and 3. The drastic reduction in MCH and MCHC values in hook worm infected dogs indicates the decrease in mean Hb content. On day 15 and 30, the MCH and MCHC values increased significantly from day 0 in treatment group 2 whereas they did not vary significantly in treatment group 3. The MCV value also changed significantly (P < 0.01) in group 2 dogs treated with ivermectin but the changes were apparent in group 3. There was moderate eosinophilia in both the infected groups. Migasena et al. (1971) reported eosinophilia in all hookworm infected dogs irrespective of the severity of infection. Meeusen and Balic (2000) also documented eosinophilia as a prominent leukocytic response to hookworm infection, because of the reason that they attack and kill the infective larvae. Ogunkoya et al. (2006) demonstrated eosinophilia as the most common in dogs infected with gastrointestinal parasites. The increase in eosinophil count in hookworm infection may be due to allergic reactions to proteins or to metabolic products of the worm. The hookworm infested patients are repeatedly being sensitized by antigens present in the metabolic products or arising from the bone marrow parenchyma into the sinusoids, thereby inducing eosinophilia in the peripheral blood (Hiraki and Inoue 1959). There was significant fall in eosinophil count in group 2 and group 3 after treatment on day 15 and 30. Table 3 Serum biochemical parameters (Mean ± S.E.) in healthy controls (n = 12) and treatment groups before treatment and thereafter on day 15 and 30 *Significant at 1% level of significance compared to day 0 in a row. Mean value with similar superscript in a column within a parameter does not differ significantly (P ≤ 0.05) Mean values with similar superscript in row does not vary between the periods at P< 0.05

Parameters
Group Observation day 0 15 30 Total protein (g/ dl) 1 6.39 ± 0.20 b 6.29 ± 0.21 b 6.30 ± 0.20 b 2 5.01 ± 0.12 a 5.63 ± 0.12* a 6.23 ± 0.14* b 3 4.94 ± 0.27 a 5.00 ± 0.26 a 5.05 ± 0.28 a Albumin (g/dl) 1 3.21 ± 0.17 b 3.13 ± 0.17 b 3.17 ± 0.20 b 2 2.25 ± 0.15 a 2.64 ± 0.12* a 3.20 ± 0.18*  The mean level of protein, albumin and globulin was significantly lower in the infected population on day 0 compared to resp[ective mean level of group 1. In group 2, the total protein and albumin values increased significantly after treatment at 5% level of significance from 0 day mean level but globulin level did not improve significantly. Sushma Rachel and Suryanarayana (2001) recorded the total serum protein in dogs with Ancylostomiasis as 5.47 ± 0.09 and 6.89 ± 0.07 g/dl before and after therapy, respectively. Nwoha et al. (2013) reported decreased levels of albumin (hypoalbuminaemia) in dogs experimentally infected with Ancylostoma caninum. The reduction in total serum protein might be due to chronic internal haemorrhage during worm infestation and loss of serum via exudation or leakage in to the lumen of gut causing enteropathy. The altered rate of intestinal absorption of nutrients especially protein from the ulcerated intestinal tract during infection might have also contributed to the drop in the total serum protein (Dargie and Allonby 1975). Hypoproteinemia may also be attributed to the interference with the efficacy of digestion and absorption by damaged intestinal mucosa and diarrhoea due to mechanical obstruction and irritation (Vanbeers et al. 1983) Miller (1974 documented caused varying degree of anaemia and hypoproteinemia in dogs infested with A. caninum. In the present study,, ivermectin was seen to be 100% effective as anthelmintic at oral doses of 200 µg/kg body weight taking into consideration the significant changes in haematological and serological parameters. Ivermectin is a derivative of avermectin B1, one of the naturally occurring substances produced by Actinomycetes, Streptomyces avermectins (Wang et al. 1989). It removed all the adult worms in its primary dose bringing down the faecal count to 0 on day 15th and 30th mean EPG of 1725.00 ± 331.23. Ramisz (1984) recorded ivermectin as 97-100% effective against Ancylostoma spp. when given at a dose rate of 200 µg/kg body weight in canines. Bagherwal (1992) in a study on the efficacy of single dose of ivermectin against A. caninum at the rate of 0.1 mg/kg body weight subcutaneously in 48 domesticated dogs of either sex aged between 3 months to six years reported that out of 48 treated dogs, 29 (60.5%) were found negative for A. caninum eggs on 3 rd day post treatment while 13 (27%) and 6 (12.5%) dogs got cured by 5th and 7th day post treatment, respectively and concluded that ivermectin was 100% effective against A. caninum in dogs without any side effects. Chhabra et al. (2001) reported complete elimination of Ancylostoma caninum in dogs treated with single dose of ivermectin at 200 µg/kg body weight one week after its administration. Daurio et al. (1993) reported the efficacy of chewable formulation of ivermectin to be 52%, 98%, 95% and 97% at 6, 12, 18 and 24 µg/kg body weights respectively in 35 young dogs with induced infections of A. caninum and U. stenocephala.
The Fenbendazole treated group was not able to remove all the parasites in one oral dose of 50 mg/kg body weight. But as observed, treatment with fenbendazole at the dose rate of 50 mg/kg body weight was efficacious only on day 30. In group 3, the EPG count was 1650.00 ± 247.25 on day 0 which significantly reduced at 5% level of significance to reach 24.17 ± 11.44 on day 15 and further reduced to reach nil on day 30. Singh et al. (1977) conducted postmortem of 22 dogs 14 days after treatment with different anthelmintics and reported that 6 of those treated with 50 mg/kg fenbendazole eliminated 71% of hookworms. Bruke et al. (1978) used 10% suspension of fenbendazole against experimental infections of Toxocara canis and Ancylostoma caninum in Beagle pups at a dose level of 50 mg/kg Body weight for 3 days and obtained 93% efficacy against hookworms. The use of fenbendazole @ 50 mg/kg body weight led to disappearance of previously diagnosed Ancylostoma caninum eggs at 2 weeks post treatment (Dakhly and Soliman 2008). The mean EPG count on day15th in this study became 24.17 ± 11.44. It may be due to the entrance of parasites from the dormant tissue to the intestinal lumen and/or the production of eggs by the female worms may be erratic in moderate to low worm burden (Kopp et al. 2008).
Ivermectin is a chemical derivative of naturally occurring fermentation product, avermectin B, which now bears the nonproprietary name abamectin. This substance is one of a series of compound avermitilis, which was originally isolated from soil in Japan. It kills the parasite by opening glutamate gated chloride channels and increasing Cl-conductance, by binding to allosteric site on the acetylcholine nicotinic receptor to cause an increase in transmission, leading to motor paralysis or by binding to aminobutyric acid receptor. It is similar to the macrolide antibiotic, but is virtually devoid of antibacterial activity. The ivermectin is active against arrested and developing larvae and adults of important nematode, including those showing resistance to existing anthelmintic families. In nematodes, the neurotransmitter that sends inhibitory signals from inter neurons to motor neurons is Gama aminobutyric acid (GABA). A number of studies have shown that vermectin potentiates the inhibitory effect of GABA. Signals from CNS are therefore not received by motor neurons and a state of flaccid paralysis of parasite develops (Jones 1984).Fenbendazole is a broad spectrum antiparasitic drug. It belongs to benzimidazole group which acts by binding to tubulin, an essential structural protein of the microtubules. This blocks the microtubules in the worms so that the uptake of glucose is blocked which eventually depletes the glycogen reserves. This disrupts the energy management system of the worms resulting in paralysis and leading to death of the worms. Author contributions Applicable for submissions with multiple authors. RB: Project execution, Data analysis, writing. PRC: Planning of project, supervision, data analysis and editing of manuscript. DP: Writing. RS: Data analysis, writing, editing. DPD: pathological analysis writing, editing. BNM: Parasitological analysis and photography.
Funding Fund support from College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture and Technology, Bhubaneswar-751003.
Availability of data and materials None other than Research results output has been used to prepare the manuscript. (a statement on how any datasets used can be accessed). If any of these declarations listed are not relevant to the content of your submission please state that this declaration is "not applicable".

Conflict of interest No conflicts of interest.
Ethical approval The research work was conducted in clinical cases presented to Veterinary Clinical Complex with the consent of the owners to publish the research outcome after use of anthelmentic and supportive therapy in affected dogs.