Prevalence and Risk Factors Analysis for Cryptosporidium in Apparently Healthy Lambs of Southern Khyber Pakhtunkhwa, Pakistan

Naimat Ullah Khan1,2, Muhammad Hassan Saleem1, Aneela Zameer Durrani1, Nisar Ahmad3, Ayesha Hassan1, Muhammad Kashif Prince1, Muhammad Luqman Sohail4,*, Mian Saeed Sarwar2, Hazrat Ali2, Tahir Usman2, Asadullah Khan2, Siffat Ullah Khan3 and Shahid Zaman5

1Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore

2College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan

3Department of Parasitology, University of Veterinary and Animal Sciences, Lahore

4University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur

5Department of Microbiology, Quaid e Azam University, Islamabad

ABSTRACT

Cryptosporidium is considered one of the major gastrointestinal pathogens in young ruminants and has been associated with diarrhea. The current study was conducted in three selected districts (Bannu, Lakki Marwat and Kohat) of southern Khyber Pakhtunkhwa (KPK) to determine the prevalence and risk factors responsible for the occurrence of Cryptosporidium in lambs and has been known to cause human infection. Three hundred and sixty fecal samples (n=360) were collected through convenient sampling from apparently healthy lambs and were examined using the modified Ziehl Neelsen acid fast staining. Prevalence was calculated along with different risk factors associated with the disease (month, age, sex and season). Results showed highest prevalence (P < 0.05) of the Cryptosporidium in Kohat (33.33%) followed by Lakki Marwat (25%), while the lowest percent prevalence was recorded in Bannu (23.33%). Highest month-wise prevalence was recorded in the month of August (46.6%), followed by July (40%), and the lowest during February and December (16.66%). Results showed highest prevalence in summer season (36.66%), while the lowest in the winter season (18.33%). The highest prevalence (P < 0.05) was recorded at the age of ≤1-15 days (38.09%) followed by 16-30 days (29.41%) while the lowest was at the age of ≥31-60 days or above (15.15%). In lambs, the females had higher (P < 0.05) prevalence (31.18%) then males (22.98%). This study addresses the prevalence of infection in lambs, responsible for the spread of etiological agent in environment.

Article Information

Received 26 July 2017

Revised 28 August 2017

Accepted 02 October 2017

Available online 10 April 2018

Authors’ Contribution

HS, AZD, NA, AH and SA designed the study project. All others collected samples, did lab work and executed the study. MLS statistically analyzed the data and wrote the article.

Key words

Cryptosporidium, Cryptosporidial infection, Risk factors, Fecal-oral route.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.3.863.868

* Corresponding author: vet.luqman@gmail.com

0030-9923/2018/0003-0863 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Cryptosporidial infection is caused by Cryptosporidium species, which are enteric protozoan parasites, having global distribution. It belongs to the phylum Apicomplexa and family Cryptosporidiidae (Bouzid et al., 2013). Cryptosporidium causes heavy economic losses by infecting humans and animals (Lendner et al., 2011; Yang et al., 2017). Cryptosporidium parvum is the main zoonotic species which principally infects small ruminants and have public health significance (Wang et al., 2014). The C. parvum oocysts were so minute that their presence in any acute diarrhea was not detectable until 1970s (Ryan et al., 2014). The transmission of the cryptosporidial infection mainly occurs through fecal-oral route. In lambs, the prevalence is mainly connected with certain risk factors such as early age, sex and seasonal effect (Taylor et al., 2007). Cryptosporidial infection in lambs is one of the main reason of diarrhea in lambs (Ahamed et al., 2015). It affects the epithelial cells of small intestines, however it rarely affects stomach, liver, gall bladder, trachea, and lungs (Hunter and Thompson, 2005). Infected animals can shed a huge number (108–109/g) of oocysts (Ortegamora and Wright, 1994), which cause infection in humans and animals by contaminating the surface water of routine usage (Maurya et al., 2013). Cryptosporidial infection causes diarrhea, weight loss and dehydration in humans and clinically characterized by dehydration and is highly fatal for immunocompromised patients (Checkley et al., 2015; Khan et al., 2017). In addition, the cryptosporidial infection has been found to be associated with cancer in humans (Benamrouze et al., 2012). However it is necessary to understand the different environmental risk factors that enhance the susceptibility to the infection (Collinet-Adler et al., 2015). To date, there is dearth of reports regarding prevalence of Cryptosporidium in lambs in southern KPK. Therefore, this study was conducted to find out the prevalence of the Cryptosporidium in lambs and its association with various risk factors such as age, sex, season and months that effects the occurrence of disease.

Materials and Methods

The present study was conducted in three selected districts of southern Khyber Pakhtunkhwa (KPK), Pakistan viz.; Bannu, Lakki Marwat and Kohat.

A total of 360 fecal samples were collected directly from the rectum of the lambs with the help of cotton swab using convenient sampling technique, for a period of one year i.e., January-December, 2016. Observational study was conducted where basic information was entered on a questionnaire to collect useful indistinguishable data such as species, age, sex and season (winter, autumn, spring, summer). All the collected samples were preserved in 10% formalin.

Ethical concerns

Samples were collected after the written formal permission from owners at the time of visit and free adequate veterinary services were provided to all animals.

Laboratory analysis of fecal samples

Fecal samples were analyzed using a Faust modified centrifuge- flotation technique (Leventhal and Cheadle, 1992). Weighed (1-2 grams) fecal material was dissolved in distilled water to make homogenized solution. After homogenization, the solution was centrifuged at 1500 rpm for 60 seconds. As a result of centrifugation, the supernatant was discarded and the sediment was re-suspended in the flotation solution (ZnSO4, 44%). The solution was again centrifuged at 1500 rpm for 60 seconds. Finally, the sediment was examined under microscope. The Cryptosporidium oocysts were stained by modified Ziehl-Neelsen (MZN) staining technique (Casemore et al., 1985). All the stained slides were examined with the help of calibrated light microscope for the detection of Cryptosporidium oocysts at 100X magnification using oil immersion as reported by Bakiret (2003). Cryptosporidium oocysts/eggs were identified on the basis of morphology, size and the key as reported by Wantanbe et al. (2005). Cryptosporidium oocysts appeared as bright red granules on a blue-green background in MZN stained fecal smears. A fecal sample was considered positive if at least one, clearly identifiable Cryptosporidium oocyst was recognized.

Statistical analysis

The data collected were analyzed using Statistical Product and Service Solutions (SPSS) version 20.0 (IBM Corp., Armonk, NY, USA). The prevalence was the proportion of positive animals out of the total animals analyzed and was presented in percentage (%). Statistical differences in the prevalence and other variables were determined using Chi-square test (X2). All values at statistical difference P < 0.05 were considered significant (CI; 95%).

Results

In the present study, overall prevalence of Cryptosporidium infection was 27.22% in lambs in three selected zones of Southern KPK. The highest prevalence of was recorded in Kohat (33.33%), followed by Lakki Marwat (25%) while the lowest prevalence was recorded in Bannu (23.3%). On the basis of statistical analysis of the data, overall there was no significant difference (P > 0.05) in three selected zones of study.

Month-wise prevalence showed highest prevalence in the month of August (46.6%), followed by July (40%), April, May and June (30%), September and October (26.66%), March (23.33%), November and January (20%) while the lowest prevalence was recorded in the months of February and December (16.66%) (P < 0.05) (Table I).

Overall highest prevalence of Cryptosporidium was recorded in the summer season (36.66%), followed by spring and autumn (26.66%) while the lowest prevalence was recorded in winter season (18.33%). On the basis of statistical analysis, significant difference (P < 0.05) was recorded in prevalence of Cryptosporidium in during different seasons (Table II).

Table I.- Month wise (Jan- Dec. 2016) prevalence (%) of Cryptosporidium in lambs of Southern KPK.

Table II.- Season wise prevalence (%) of Cryptosporidium in lambs of Southern KPK.

In the current study, the highest percent prevalence was recorded at the age of ≤1-15 days (38.09%), followed by at the age of 16-30 days (29.41%) while the lowest prevalence (15.15% ) was recorded at the age of ≥ 31-60 days (P < 0.05) (Table III).

Results showed significantly higher (P < 0.05) prevalence in female lambs (31.18%) as compared to male lambs (22.98%) (Table III).

Table III.- Age and sex wise prevalence (%) of Cryptosporidium in lambs of Southern KPK.

The cryptosporidiosis mostly occurs in lambs as compared to the adult sheep and likewise the severity and intensity of the infection in lambs is more than adult sheep (Majewska et al., 2000). A significant difference in prevalence of the Cryptosporidium infection has already been reported in young and older lambs (Noordeen et al., 2000).

In the present study, overall prevalence of Cryptosporidium was 27.22% in lambs, in three selected zones of study area. Similar study was also conducted in Spain to find out the prevalence of different enteric pathogens in lambs and goat kids which showed higher prevalence in lambs (45%) as compared to kids (42%) (Munoz et al., 1996). There are certain variations in prevalence in our study and other researchers that might be due to some factors such as study design, breed variation, immune status of the animal, area, hygienic conditions and other environmental conditions such as humidity, temperature and rain fall (Majewska et al., 2000).

The Cryptosporidium infection is distributed worldwide and is prevalent in wetter and warmer months of the year with higher ambient temperature, relative humidity and rainfall (Jafari et al., 2013), which is in agreement with the findings of current study. Higher prevalence of Cryptosporidium was reported in monsoon season in India due to higher humidity and overcrowding of animals in shelters, which is also in line with the findings of this study (Maurya et al., 2013)

Highest prevalence was observed in summer season and least in winter which is correlated with the temperature and humidity. Similar results were recorded in the autumn season (79.3% and 75%) by Causape et al. (2002), which buttress the results of this study. It is the fact that the age related resistance to cryptosporidial infection exists naturally in lambs and kids. During the first two weeks of age, infection is more severe and obvious decrease has been reported in the severity of the clinical signs as the age increases (Giadinis et al., 2007). A study was conducted in Spain by Ortege-Mora et al. (1999); and it was estimated that 20,000-444000 oocysts/day can be excreted by an asymptomatic ewe. It was observed that 1 oocyst/lamb can cause the infection which is the minimum infective dose whereas the average numbers of oocysts were 5/lamb to cause infection. Therefore, it was recorded that 4,000-110,000 infective doses per day were shed by ewe without any clinical symptoms (Ortega-Mora et al., 1999). Age is the main risk factor responsible for the spread of cryptosporidiosis in lambs and goat kids (Sari et al., 2009); and high morbidity mainly occurs in neonatal animals such as lambs and kids (Panousis et al., 2008).

Our results are coherent with the findings of Sari et al. (2009), where the highest prevalence was recorded at the age of <7 days (44.4%), followed by two weeks of the age (37.5%), 3 weeks (32.3%), while the lowest prevalence was recorded at the age of 4 weeks(22%) in lambs. Ahamed et al. (2013) also reported similar findings with highest prevalence in lambs of less than one month of age and decreased infection rate in older animas.

It might be due to certain factors as reported by Fasihi-Harandi and Fotohi-Ardakani (2008); that at early age, high prevalence of the Cryptosporidium infection might be due to poorly developed immune system and their maximum sensitivity to the Cryptosporidium infection.

Conclusion

The Cryptosporidium is widespread in lambs in spite of the age of animals, breed type, and farm location. The presence of Cryptosporidium parvum and Cryptosporidium hominis in small ruminants enhance and highlights the importance of lambs because the parasite circulates between humans and animal hosts.

Statement of conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article

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