Bioscience Journal | 2023 | vol. 39, e39066 | ISSN 1981-3163 1 Naseem RAFIQ 1 , Sultan AYAZ 1 , Sadaf NIAZ 1 , Mustafa KAMAL 1 , Luqman KHAN 2 1 Department of Zoology, Faculty of Chemical and Life Sciences, Abdul Wali Khan University Mardan, 2300 Pakistan. 2 Department of Zoology, Government College Women University Sialkot, Pakistan. Corresponding author: Luqman Khan luqman.zoology@gmail.com How to cite: RAFIQ, N., et al. Epidemiological analysis of Paramphistomum species in Ruminants in selected Districts of Khyber Pakhtunkhwa (KPK), Pakistan. Bioscience Journal. 2023, 39, e39066. https://doi.org/10.14393/BJ-v39n0a2023-66497 Abstract This epidemiological study was conducted to compare the climatic conditions of four different districts of Khyber Pakhtunkhwa, Pakistan, in terms of the prevalence of parasitic infection Paramphistomum in cows and buffaloes. A total of 2400/cows and buffaloes (200/per month and 50/per district) fecal samples were collected. The samples were examined for Paramphistomum egg contamination by direct microscopic examination and sedimentation methods. The infectivity ratio was 17.5% in cows and 17% in buffaloes; the higher rate of infection was recorded in autumn with 41% in cows and 36% in buffaloes. Whereas the lowest ratio was 6.5% in cows and 7% in buffaloes recorded during spring. It was also noted that a high rate of prevalence was recorded in older animals (higher than 2 years of age) than younger (less than 2 years of age), while gender-wise, it was observed that male animals were more affected than females. Prevalence was higher in cows in Mardan district (19.30%) followed by Nowshera (19.10%) and Swabi (17.80%) districts while lowest in Charsadda district (13.80%) where in buffaloes the higher prevalence was recorded in Swabi (18.80%) followed by Mardan (18.60%) then Charsadda (16.80%) while lowest in Nowshera (13.80%). Keywords: Epidemiology. Paramphistomum. Parasitic. Prevalence. Sedimentation. 1. Introduction Paramphistomosis is caused by a Paramphistomum species (Rafiq et al. 2020; Qureshi et al. 2021) having a worldwide distribution, however widespread in tropical and subtropical regions, predominantly in Australia, Africa, Russia, Asia and Eastern Europe (Rolfe et al. 1991; Ayaz et al. 2013). The gastrointestinal parasites incidence in cattle in different areas of Pakistan has been stated from 25.1 to 92% at different periods (Iqbal et al. 1993, Ali et al. 2000; Raza et al. 2007; Ijaz et al. 2008; Al-Shaibani et al. 2008; Kakar et al. 2008). In the world, some countries measured this parasite as a key restriction on production. In the same way, Paramphistomum infections (e.g., P. cervi, P. microbothrium, and P. ichikawai) have the same allocation, mostly in Asia. The rates of prevalence in Asia (30-60%) are still noted in some areas (Raza et al. 2009). Paramphistomosis is a significant endoparasitic disease of ruminants with great economic loss to livestock (Sivajothi et al. 2014; Rafiq et al. 2020; Qureshi et al. 2021). The main source of severe injury to the lining of the intestine (enteritis) is immature flukes, and especially in young animals, which effect EPIDEMIOLOGICAL ANALYSIS OF Paramphistomum SPECIES IN RUMINANTS IN SELECTED DISTRICTS OF KHYBER PAKHTUNKHWA (KPK), PAKISTAN https://orcid.org/0000-0002-4840-3285 https://orcid.org/0000-0002-3479-0835 https://orcid.org/0000-0002-2452-8497 https://orcid.org/0000-0001-5729-8037 https://orcid.org/0000-0003-3412-9294 Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 2 Epidemiological analysis of Paramphistomum species in Ruminants in selected Districts of Khyber Pakhtunkhwa (KPK), Pakistan dehydration, maldigestion, and death. Paramphistomum causes low diet exchange, which consequences in weight loss reduce fertility, decrease milk production, necrosis, polydipsia, anorexia, which ultimately results in great finical loss (Horak et al. 1971; Rolfe et al. 1991; Rangel et al. 2003). The present study was designed to record the epidemiology of Paramphistomum in ruminants, especially in cows and buffaloes of Khyber Pakhtunkhwa, Pakistan. 2. Material and Methods Study area and sample collection Fecal samples were collected from ruminants especially cows and buffaloes in different Districts i.e., Nowshera, Mardan, Swabi, and Charsadda of Khyber Pakhtunkhwa Province, Pakistan, in the months of October 2017 to September 2018 (200 each of cow and buffaloes in each month). Fecal samples were collected from each area with a total number of 50 fecal samples (5-10 g) from cows and buffaloes month wise in plastic bottles clearly written gender, species, date & place. Analysis of Fecal Samples To determine the presence of Paramphistomum eggs, all samples were analyzed on the same day by direct microscopic examination and sedimentation methods. Samples which were not observed on the same day of collection were preserved in formalin 10% to prevent eggs development and hatching. Direct smear method Few drops of water were added to the fecal samples on microscopic slides. The lighter eggs were allowed to flow away from the heavier debris by tilting the slide. Cover slip was placed on the fluid and the prepared slide was then observed under the microscope (Urquhart et al. 1996). Sedimentation method Approximately 2 g of collected sample was mixed in 45 ml of saline water using a spatula and beaker. The fecal suspension was filtered through a filter paper (45 μm), then put in centrifuge tubes as described for centrifuging samples at 1500-2000 rpm. The supernatant was discarded carefully by pipetting (decantation) and the sediment was stained by adding one drop of ethylene blue and then transferred to a microscopic slide. The sample was covered with a coverslip and observed under the microscope (Jorgan 1990). Based on morphological details, the paramphistomosis eggs were identified by the same technique as described by (Soulsby 1982). The prevalence of Paramphistomosis was noted with the help of formulae described by (Abunna 2012). 3. Results Paramphistomosis in cows in the selected area of KPK Overall prevalence in cows An overall prevalence of 17.5% of Paramphistomosis was found in cows from October 2017 to September 2018. Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 3 RAFIQ, N., et al. Month-wise prevalence For month-wise prevalence, the analysed data showed a highest prevalence of 41% in September followed by 27.5 % in October. The lowest prevalence of 2.5 % of Paramphistomosis was recorded in March. Statistical analysis showed no significant difference in the prevalence of Paramphistomosis from October 2017 to September 2018, at P<0.05 (Table 1 and Figure 1). Table 1. Month-wise, season-wise, area-wise, age, and gender-wise prevalence of Paramphistomum in Cows at different Districts of Khyber Pakhtunkhwa. Factors Total No. of sample observed Total No. of sample infected % age of infection Month Oct-17 200 55 27.50% Nov-17 200 43 21.50% Dec-17 200 39 19.50% Jan-18 200 36 18% Feb-18 200 51 25.50% Mar-18 200 5 2.50% Apr-18 200 16 8% May-18 200 12 6% Jun-18 200 13 6.50% Jul-18 200 26 13% Aug-18 200 43 21.50% Sep-18 200 82 41% Season Winter 800 137 17.10% Spring 400 26 6.50% Summer 800 94 11.75% Autumn 400 164 41% Area Mardan 600 116 19.30% Charsadda 600 83 13.80% Swabi 600 115 19.10% Nowshera 600 107 17.80% Age 0-2 years 850 137 16.10% >2 years 1550 284 18.30% Gender Male 1450 281 19.30% Female 950 140 14.70% Total 2400 421 17.5% Area-wise prevalence Area-wise prevalence in cows in various Districts of KPK showed that the infection was highest in Mardan (19.3%) followed by Nowshera (19.1%). The lowest prevalence (13.8%) was observed in Charsadda. Statistical analysis (Student t-test) showed significant differences in all areas (Table 1, Table 2 and Figure 1). Season-wise prevalence It is cleared from the results that data collected during autumn season has the highest prevalence 41% compared to winter season which was 17.1% and spring season which was 6.5%. Statistical analysis further clarified our results having significant difference in winter season (P<0.01) compared to summer and spring (Table 1 and Figure 1) Gender- wise prevalence Gender-wise analysis of Paramphistomosis showed the highest prevalence in males (19.3%) followed by females (14.7%). This indicated that males were more susceptible than females to the disease. Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 4 Epidemiological analysis of Paramphistomum species in Ruminants in selected Districts of Khyber Pakhtunkhwa (KPK), Pakistan Female animals are found to be more resistance as compared to males. Males and females showed statistical difference at P<0.05 (Table 1 and Figure 1). Figure 1. Month Wise, Season wise, Area wise, Age and Gender Wise Prevalence of Paramphistomum in Cow in selected areas of Khyber Pakhtunkhwa. Age-wise prevalence It was observed that the overall prevalence in adult cows was significantly higher (18.3%) than younger ones (16.1%) (Table 1 and Figure 1). Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 5 RAFIQ, N., et al. Table 2. Month wise Prevalence of Paramphistomum in Cow in District Mardan, Charsadda, Nowshera and Swabi KPK. Factors Mardan Charsadda Nowshera Swabi Inf/total Prevalence (%) Inf/Total Prevalence (%) Inf/Total Prevalence (%) Inf/Total Prevalence (%) Month Oct-17 20/50 40 18/50 36 23/50 46 20/50 40 Nov-17 19/50 38 5/50 10 20/50 40 8/50 16 Dec-17 11/50 22 8/50 16 8/50 16 12/50 24 Jan-18 15/50 30 6/50 12 9/50 18 6/50 12 Feb-18 7/50 14 2/50 4 4/50 8 2/50 4 Mar- 18 0/50 0 3/50 6 0/50 0 2/50 4 Apr-18 7/50 14 2/50 4 3/50 6 4/50 8 May- 18 2/50 4 3/50 6 3/50 6 4/50 8 Jun-18 3/50 6 5/50 10 2/50 4 3/50 6 Jul-18 4/50 8 7/50 14 9/50 18 6/50 12 Aug-18 6/50 12 8/50 16 10/50 2 19/50 38 Sep-18 22/50 44 16/50 32 24/50 48 21/50 42 Overall Prevalence in Buffaloes Prevalence of Paramphistomosis in buffaloes was recorded in selected areas of KPK from October 2017 to September 2018. A total of 409 animal’s samples were found infected with overall 17% of infection. Month-wise prevalence The prevalence of Paramphistomosis showed highest prevalence in September (43.5%) and November (30.5%), followed by October (28.5%). The lowest prevalence has been seen in March (1.5%) (Table 3 and Figure 2). Statistical analysis showed the highest significant difference in the month of September compared to March 2018 (Table 3 and Figure 2). Area-wise prevalence Paramphistomosis prevalence was highest in Swabi (18.8%) while the lowest was seen in Nowshera (13.8%). However, there was no significant difference in prevalence between Swabi and Nowshera (Table 3, Table 4, Figure 2). Season-wise prevalence Highest prevalence of Paramphistomosis was recorded in autumn (36%) followed by winter (16.7%) and summer (12.8%), while in spring (7%). Statistical analysis also showed significant differences in different seasons (Table 3 and Figure 2). Gender-wise prevalence The Paramphistomosis prevalence was higher in male (19.5%) as compared to female (13.5%). No significant difference has been seen in male and female buffaloes (Table 3 and Figure 2). Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 6 Epidemiological analysis of Paramphistomum species in Ruminants in selected Districts of Khyber Pakhtunkhwa (KPK), Pakistan Table 3. Month Wise, Season wise, Area wise, Age and Gender Wise Prevalence of Paramphistomum in Buffalos in selected areas of Khyber Pakhtunkhwa. Factors Total No. of sample observed Total No. of sample infected % age of infection Month Oct-17 200 57 28.50% Nov-17 200 61 30.50% Dec-17 200 45 22.50% Jan-18 200 44 22% Feb-18 200 17 8.50% Mar-18 200 3 1.50% Apr-18 200 16 8% May-18 200 10 5% Jun-18 200 7 3.50% Jul-18 200 25 12.50% Aug-18 200 47 23.50% Sep-18 200 87 43.50% Season Autumn 400 144 36% Winter 800 134 16.70% Summer 800 103 12.80% Spring 400 28 7% Area Mardan 600 112 18.60% Charsadda 600 101 16.80% Swabi 600 83 13.80% Nowshera 600 113 18.80% Age 0-2 years 900 103 11.40% >2 years 1500 304 20.20% Gender Male 1450 283 19.50% Female 950 126 13.20% Total 2400 409 17% Table 4. Prevalence of Paramphistomum in Buffalos in District Mardan, Charsadda, Nowshera and Swabi KPK. Factors Mardan Charsadda Nowshera Swabi Inf/ Total Prevalence (%) Inf/ Total Prevalence (%) Inf/ Total Prevalence (%) Inf/ Total Prevalence (%) Month Oct-17 17/50 34 18/50 36 6/50 12 16/50 32 Nov-17 20/50 40 14/50 28 8/50 16 19/50 38 Dec-17 10/50 20 6/50 12 9/50 18 10/50 20 Jan-18 14/50 28 9/50 18 6/50 12 15/50 30 Feb-18 7/50 14 4/50 8 1/50 2 5/50 10 Mar-18 0/50 0 0/50 0 1/50 2 2/50 4 Apr-18 6/50 12 3/50 6 3/50 6 4/50 8 May-18 1/50 2 5/50 10 2/50 4 2/50 4 Jun-18 2/50 4 2/50 4 1/50 2 2/50 4 Jul-18 4/50 8 7/50 14 8/50 16 6/50 12 Aug-18 10/50 20 11/50 22 15/50 30 11/50 22 Sep-18 21/50 42 22/50 44 23 46 21/50 42 Total 112/600 18.6 101/600 16.8 83/60 13.8 113/600 18.8 Age-wise prevalence A higher prevalence of Paramphistomosis was observed in in adult buffaloes above age 2 years (20.2%) than those of young age below 2 years (11.4%). Statistical analysis showed a significant difference at P<0.05 between adult and young buffaloes (Table 3, Figure 2). Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 7 RAFIQ, N., et al. Figure 2. Month-Wise, Season-wise, Area-wise, Age and Gender-Wise Prevalence of Paramphistomum in Buffaloes in selected areas of Khyber Pakhtunkhwa. 4. Discussion In the present study, epidemiological data on Paramphistomosis were collected from clinically affected cows and buffaloes from four districts, i.e. “Mardan, Charsadda, Nowshera and Swabi” of the Khyber Pakhtunkhwa province. Bioscience Journal | 2023 | vol. 39, e39066 | https://doi.org/10.14393/BJ-v39n0a2023-66497 8 Epidemiological analysis of Paramphistomum species in Ruminants in selected Districts of Khyber Pakhtunkhwa (KPK), Pakistan Overall, a total of 4800 animals (2400 buffaloes and 2400 cows) were examined from October 2017 to September 2018. Of these, 409 (17%) buffaloes and 421 (17.5%) cows were found positive for Paramphistomosis. Similar findings of Paramphistomum species were also reported by Kotrla and Kotryl (1982), Chowdhry and Tada (1994), Kassai (1999), Urquhart et al. (1996), Shanila and Hafeez (2005), Dreyfuss et al. (2007) and Khan et al. (2008). Haridy et al. (2006) recorded the prevalence rates of infection were 20% in buffaloes and cattle, similarly 7.3% infection in cattle and 10% in buffaloes in Egypt, and 16.6% in Cattle reported by Jithendran (2000) from India, 25.20% in Buffaloes and 23.8% in Cattle by Juyal et al. (2003) and 5.94% in cattle by Shanila and Hafeez (2005) in India, 13.6% in cattle in Turkey by Sevimli et al. (2005), 7.1 % by Phiri et al. (2006) from Zambian, 28% in cattle from Thailand by Morakot and Sakchai (2006) where as 36% prevalence was observed in cattle from oceanic climatic areas of Lugo (NW Spain) by Diaz et al. (2007) Seasonal reproduction in Paramphistomum species showed striking changes in egg production, with the peak activity during the monsoon and post-monsoon months. Hanna et al. (1988), Gupta and Singh (1990), Chaudhri et al. (1993), Dutta et al. (1995), Georgi et al. (1999), Szmidt et al. (2000), Juyal et al. (2003), Shanila and Hafeez (2005), Keyyu et al. (2005), Pfukeni et at. (2005), Jithendran (2000), Morakot and Sakchai (2006) and Diaz et al. (2007) also recorded similar results. The highest prevalence in both buffaloes and cows were recorded in September (36.6% in cows and 46.6% in buffaloes) while the lowest (3.3%) in cows during January 2018. Szmidt et al. (2000), Rangel et al. (2003), Juyal et al. (2003), Morakot and Sakchai (2006) and Diaz et al. (2007) also recorded similar results, supporting the present findings. In the present study, the incidence of paramphistomosis was higher in adult animals compared to young. Maqbool et al. (2003), Khan et al. (2006) and Raza et al. (2007) also recorded that younger animals are considered to act as reservoirs of infection than adults, the reason may be that mostly farmers kept the young ones with care and adults mostly grazing freely while Khan et al. (2008) reported totally opposite results, it may be due to climatic conditions and period (year) of study. It was reported that infection was higher in males (19.5% in buffaloes and 19.3% in cows) compared to female animals (13.2% in buffaloes and 14.7%in cows). In the present study, it was found that infection was slightly lower in females than males, the reason seems to be related to the social practice of keeping females under better management and feeding conditions in comparison to males which are generally grazed freely in grasslands. Khan et al. (2008) and Chaudhri et al. (1993) found the same results. Sevimli et al. (2005) reported similar findings to the current study in which males with paramphistomosis were 3.05 % higher than female animals in comparison to horses Raza et al. (2007) found greater prevalence in males than females in buffaloes. Such findings are in contrast with Szmidt et al. (2000) reports that female cattle in France were substantially more contaminated than male animals due to free grazing by the farmers. 5. Conclusions The findings of the recorded data indicate that there are no significant differences between the rate of infection of Paramphistomum in cows and buffaloes during the study period. The highest prevalence was recorded in autumn and the lowest in spring. It was also observed that old animals are more likely to be infected then young ones due to low immunity at elder age. Female animals are found to be more resistance as compared to males. Authors' Contributions: RAFIQ, N.: conception and design, acquisition of data, analysis and interpretation of data, and drafting the article; AYAZ, S.: acquisition of data, analysis and interpretation of data, , and drafting the article; NIAZ, S.: acquisition of data, analysis and interpretation of data, drafting the article, and critical review of important intellectual content; KAMAL, M.: acquisition of data, analysis and interpretation of data, drafting the article, and critical review of important intellectual content; KHAN, L.: conception and design, acquisition of data, analysis and interpretation of data, drafting the article, and critical review of important intellectual content. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: The present study was approved by the advanced studies and research board, Abdul Wali Khan University Mardan. 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Black well Science limited, London, UK, 1996. Received: 28 July 2022 | Accepted: 18 January 2023 | Published: 6 April 2023 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1007/s00436-006-0148-5 https://doi.org/10.4102/ojvr.v72i1.224 https://doi.org/10.1016/j.sjbs.2021.05.079 https://doi.org/10.22456/1679-9216.107107 https://doi.org/10.1016/j.vetpar.2003.07.002 https://doi.org/10.1017/s0022149x07818554 http://dx.doi.org/10.18805/ag.D-5014 https://doi.org/10.1007/s12639-014-0491-x https://doi.org/10.1016/s0304-4017(99)00168-5