Biomarkers for Pathogenic Clostridium perfringens in Small Ruminants of Khyber Pakhtunkhwa, Pakistan

Mumtaz Ali Khan1, Aneela Zameer Durrani1, Sher Bahadar Khan2, Naimat Ullah Khan3, Muhammad Asfandyar Khan2, Kashif Prince1,*, Mahboob Ali1, Ghazunfar Rashid1 and Azmat Ulah Khan2

1University of Veterinary and Animal Sciences, Lahore

2Department of Animal Health, the University of Agriculture, Peshawar

3College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan

ABSTRACT

Pathogenic isolates of Clostridium perfringens were identified in lambs and kids population from non-vaccinated dams of selected districts of Khyber Pakhtunkhwa province, Pakistan. Samples were obtained from non-vaccinated lambs and kids with the signs of enterotoxaemia. The isolates were initially identified by colony characteristics, Gram staining, biochemical tests and CFU/g. Clostridium perfringens was isolated from 43.45% of lambs and 50.59% of the kids. Obtained isolates were further analyzed for toxinotyping with conventional PCR. Sequence analysis of 73 strains from diseased lambs showed 13.10% type A, 9.52% type B and highest 20.83% proportion of Clostridium perfringens type D. Out of 108 strains from kids, 54.62% were type A, 9.25% were type B and 36.11% were type D. Clostridium perfringens type C and E were neither detected in lambs nor in kids. The association of various factors (i.e. area, housing, seasons, colostrum feeding, hygienic condition, body condition and animal sex) with each type of Clostridium perfringens infection were analyzed by Chi- square test of association (χ2). The results indicated that Colostrums feeding, hygienic condition and body condition scores of animals are significantly (P<0.05) associated with Clostridium perfringens type A, B and D in both lambs and kids. Besides that, Season with Clostridium perfringens type A in kids, Area and Season with Clostridium perfringens type D in lambs are also significantly (P<0.05) associated.

Article Information

Received 27 January 2018

Revised 23 August 2018

Accepted 02 April 2019

Available online 08 October 2019

Authors’ Contribution

MA conducted research. AZD supervised the project. SBK, NUK and MAK helped in sample collection and processing. KP wrote the manuscript. MA, GR, KP and AUK helped in conducting research.

Key words

C. perfringens, Kids, Lambs, PCR, Toxin genes.

DOI: https://dx.doi.org/10.17582/journal.pjz/2020.52.1.107.114

* Corresponding author: kashif_prince@live.com

0030-9923/2020/0107-0001 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

INTRoDUCTION

Goats and sheep are integral part of livestock enumerating 70.3 and 29.8 million, with 867 and 39 tons of milk production, respectively while mutton production from small ruminants has reached to 657 tones (Economic Survey, 2015-16).The surveys have reported 6.476 and 1.177 million of goat and sheep in Khyber Pakhtunkhwa (KPK) province while districts Mardan and Swat are well populated with small ruminants (Livestock Census, 2010).Clostridium perfringens causes enterotoxemia in small ruminants and other laboratory animals which cause huge economic losses in sheep and goat farming globally (Nillo, 1980). The host is vulnerable to C. perfringens infection at any age but younger age ismore susceptible (Uzal et al., 2004). C. perfringens is the normal GIT flora of various animals including sheep and goats (McClane et al., 2006).

Morphologically, the pathogen is anaerobic Gram-positive bacillus which produces spores in unfavourable environmental conditions in soil (Meer and Songer, 1997). C. perfringens infection is in endemic in goats and sheep in Pakistan (Khan et al., 2008; Tahir et al., 2013). This infection mostly occurs in acute form in young stock of sheep and goats when their mothers are not immunized (Javed et al., 2009; Nasir et al., 2013). The disease is classified into 5 types i.e. A-E on the production of 4 different toxins viz. α, β, ε and i (Petit et al., 1999). Polymerase chain reaction is being used as diagnostic tool for C. perfringens genotyping (Meer and Songer, 1997; Baums et al., 2004; Gkiourtzidis et al., 2001).

Keeping in view the mortality in young population of small ruminants the present study was planned to find out the common pathogenic types of the C. perfringens in lambs and kids not only to know the burden of this disease in young flock but also to develop effective toxoid to prevent and control future losses.

 

Materials and methods

The Animal Ethical Committee approved this study project via Ref. No. DAS/5121, dated March 9, 2016.

Selection of study area and population for sampling

This project was carried out in topographically different districts Swat and Mardan of Khyber Pakhtunkhwa, Pakistan. The samples were collected from lambs and kids brought to veterinary hospital from enterotoxaemia endemic areas.

Sampling

Fecal or intestinal samples were obtained by purposive convenient sampling technique from lambs and kids suffering from the clinical signs of enterotoxaemia or sudden dead animals with the history of the above signs from 168 lambs and 170 kids up to 4 months age irrespective of breed and sex. These animals or their mothers had not been previously vaccinated with C. perfringens. The ingesta or fecal samples collected with swab and sent to the laboratory in sterile bottles under refrigeration temperature (Nayel et al., 2013).

Identification of C. perfringens

All the samples were cultured on prepared plates containing Perfringens Agar Base TSC (HiMedia Laboratories India) and TSC Supplement (FD014, HiMedia). The cultures were incubated for 24 h in anaerobic jar with Anaerogen (Oxoid Ltd, Basingstoke, Hants) at 37°C. The colonies were identified by morphology and Gram staining followed by biochemical tests (remel Rapid ANA 2 kit, USA). Being a normal inhabitant of intestinal tract in animals, Clostridium perfringens produce bacterial concentration 104-107 CFU/g as normal range in fecal samples while the positive samples produces elevated level of CFU/g (>107) for the C. perfringens. Colony count was performed on blood agar and only positive ones were included in this study (Kalender et al., 2005).

Molecular identification of C. perfringens

The identified isolates were cultured in Robertson cooked meat brothunder incubation at37°Cin shaking incubator for twelve hours.

DNA extraction kits (GeneAll Bldg, South Korea) was used for DNA extraction. For this purpose the cultured broth was centrifuged at full speed (1300-20000×g) in 1.5 mL micro centrifuge tube. The pellets of all samples were treated with 180µL Lysozyme 30mg/mL mixture (LYS702, Bioshop, Canada) and incubated 30 min at 37oC. 20µL proteinase K solution (20mg/mL) and 200uL Buffer BL were mixed. These samples were incubated for half an hour at 56oC and then for a further 30 min at 70oC after vertexing. 200µL absolute Ethanol was added and vertexed for proper mixing of the samples. The mixture was transferred the mixture into the SV column and centrifuged at (6000×g) for 60 sec. Washed with 600µL buffer BW and then 700µL buffer TW and centrifuged similarly after the addition of each buffer. At the end centrifuged at full speed for 60 sec to remove buffers and changed 1.5 mL micro centrifuge tube and added 200µl buffer AE to elute DNA.DNA’s quantity and quality was calculated through NanoDrop (NanoDrop 2000, USA). For long term use DNA was stored at -20oC.

PCR assays were performed in thermocycler (BIO RAD T 100) with described primers in Table I. Their actions were performed in micro amplification tubes in 25µl volumes containing 10µL 2X AmpmasterTM Taq (Gene All Biotechnology Co., Ltd), 1.5µL forward and reverse primers (10 pmol/uL) alpha, beta, epsilon, and iota toxin primers, 5uL DNA and 7µL DNA free distilled water.

DNAs were amplified under initial denaturation at 95°C for five min, 35 cycles of denaturation at 94°C for 1 min, annealing at α=60°C, β=64°C, ε=53.4°C for 1/2 min and extension at 72°C for 1 min while final extension at 72.0°C for five min. PCR products were run on 1.50% agrose gel and stained with ethidium bromide. Optimized positive DNA, negative control product and 1 Kb DNA ladder of (GenestaTM) as a marker were used. Visualized bands were compared and photographed in UV light (Greco et al., 2005; Khan et al., 2017).

 

Table I.- Primers used for PCR amplification of gene for C. perfringens toxins α, β, ε and i.

Toxin/gene	Oligonucleotide sequence	Fragment length (bp)
α/cpa	5′ -TGC TAA TGTTAC TGC CGT TGA TAG-3′	247	Daube et al. (1994)
	5′-TGC TAA TGTTAC TGC CGT TGA TAG-3′
β/cpb	5′-AAC TTA ACT GGA TTT ATG TCT TCA-3′	317	Kadra et al. (1999)
	5′-ATA GTA GAA AAA TCA GGT TGG ACA-3′
ε/etx	5′-ATT AAA ATC ACA ATC ATT CAC TTG-3′	206	Daube et al. (1994)
	5′-CTT GTG AAG GGA CAT TAT GAG TAA-3′
i/iap	5′ -TTT TAA CTA GTT CAT TTC CTA GTT A-3′	298	Daube et al. (1994)
	5′-TTT TTG TAT TCT TTT TCT CTA GGA TT-3′

 

Table II.- Percentage of C. perfringens type A, type B and type D in C. perfringens infected lambs and kids in two districts of Khyber Pakhtunkhwa, Pakistan.

Bacter ial strains	Anim als	District Swat		District Mardan		Overall
		Infected /total	Distrib ution (%)	Infected /total	Distrib ution (%)	Infected /total	Distribu tion (%)
Type A	Lambs	12/73	16.44	10/73	13.70	22/73	30.14
	Kids	21/108	19.44	38/108	35.18	59/108	54.62
Type B	Lambs	10/73	13.70	6/73	8.00	16/73	21.92
	Kids	4/108	3.70	6/108	5.55	10/108	9.25
Type D	Lambs	24/73	32.88	11/73	15.07	35/73	47.95
	Kids	17/108	15.74	22/108	20.37	39/108	36.11

 

Risk factor analysis

Association of various risk factors i.e. area, season, housing for animal, colostrum feeding to animals, body condition of animal, hygienic condition and sex were studied by interviewing each owner at the retail shop selected for sampling. Each factor like area (Swat, Mardan), housing (farm, house hold, pastoral flock), season (summer, autumn, winter, spring), colostrum fed (least, moderate, excessive), hygienic condition (hygienic, moderate, poor), body condition (fatty, moderate, lean), and sex (female and male) was categorized accordingly.

Statistical analysis

Chi-square test (χ2) was used for comparison of associations between risk factors to the prevalence of the disease at 95% confidence interval (P<0.05) while prevalence of each type of C. perfringens was analyzed through descriptive statistics.

 

RESULTS

Of 168 samples obtained from lambs, 95 (56.54%) were identified as C. perfringens by colony characteristics, Gram staining and biochemical tests. 73 isolates (76.84%) were found pathogenic by CFU/g out of 95 isolates. Genotyping of 73 pathogenic isolates from diseased lambs indicated 22 (30.14%) type A, 16 (21.92%) type B and 35 (47.95%) were type D. In total 22 (30.14%). Out of C. perfringens type A pathogenic isolates, 16.44% was obtained from district Swat and 13.70% from district Mardan. Of 16 (21.92%) C. perfringens type B pathogenic isolates 13.70% was obtained from district Swat and 8.00% from district Mardan. Of 35 (47.95%) C. perfringens type D pathogenic isolates, 32.88% were obtained from district Swat and 15.07% were obtained from district Mardan.

Of 170 samples obtained from kids, 130 (76.47%) were initially identified by colony morphology, Gram staining, and with biochemical tests. Total, 108 isolates (83.07%) were found pathogenic by CFU/g. Genotyping of 108 pathogenic isolates showed 59 (54.62%) type A, 10 (9.25%) type B and 39 (36.11%) type D. Types C and E were not identified in lambs and kids. Out of total 59 (54.62%) C. perfringens type A pathogenic isolates, 19.44% were obtained from district Swat and 35.18% from district Mardan, Out of 10 (9.25%) C. perfringens type B pathogenic isolates, 3.70% were obtained from district Swat and 5.55% from district Mardan and of 39 (36.11%) C. perfringens type D pathogenic isolates, 15.74% were obtained from district Swat and 20.37% from district Mardan.

Prevalence of C. perfringens infection in relation to risk factors in lambs and kids

In lambs the highest prevalence (23.81%) of C. perfringens type A was recorded during the spring followed by winter (14.29%) and autumn (11.90%) while the lowest prevalence (4.76%) was recorded during summer. The statistical differences were found nonsignificant (P=0.085). In kids the highest prevalence (52.38%) of C. perfringens type A was recorded during the spring followed by winter (35.71%) while the lowest prevalence (30.95%) was recorded both in summer and autumn. The statistical differences were found nonsignificant (P=0.134) among seasons and significant difference (P=0.000) between districts. Farm animals indicated highest prevalence (5.95%) in lambs and (14.70%) kids followed by house hold animals in lambs (4.16%) and kids (11.76) while the lowest prevalence (2.97%) in lambs (2.97%) and kids (8.23%) was recorded in pastoral animals. Nonsignificant difference in lambs (P=0.405) and kids (P=0.174) were observed among different groups of housing. Least colostrum fed animals indicated highest prevalence in lambs (7.73%) and kids (16.46%) followed by colostrum fed ad libitum animals in lambs (3.57%) and kids (11.76%) while the lowest prevalence in lambs (1.78%) and kids (6.47%) was recorded in moderate colostrum fed animals. Significant differences in ‘colostrum feeding in lambs (P=0.023) and kids (P=0.015) were observed among different groups. Highest prevalence in lambs (7.73%) and kids (16.46%) was recorded followed by lean animals in lambs (3.57%) and kids (11.17%) while the lowest prevalence in lambs (1.78%) and in kids (7.05%) was recorded in moderate animals. Significant differences in lambs (P=0.023) and kids (P=0.024) were recorded in various groups of body condition. Highest prevalence (7.73%) in lambs and (15.88%) in kids was observed in poor hygienic housed animals followed by animals housed with moderate hygienic conditions in lambs (3.57%) and kids (11.76%) while the lowest prevalence in lambs (1.78%) and kids (7.05%) were observed in hygienic housed animals. Significant difference in hygienic condition in lambs (P=0.023) and kids (P=0.039) were noted among various groups. Male indicated higher prevalence (15.62%) in lambs and (35.71%) kids than the female in lambs (14.91%) and kids (34.50%). Nonsignificant differences in lambs (P=0.931) and kids (P=0.902) were indicated in both groups of sex in lambs and kids infected with C. perfringens type A.

In lambs the highest prevalence of C. perfringens type B was recorded during the winter (21.43%) followed by autumn (11.90%) and spring (7.14%) while the lowest prevalence (4.76%) was recorded in summer season. The statistical differences were found nonsignificant among seasons (P=0.077) and between districts (P=0.223) in lambs. In kids the highest prevalence of C. perfringens type B was recorded during the winter (11.90%) followed by autumn (7.14%) and summer (4.76%) while the no prevalence was recorded in spring. The statistical differences were found nonsignificant among seasons (P=0.136) and between districts (P=0.514) in kids. The highest prevalence of C. perfringens type B was recorded in House hold animals in lambs (5.35%) and kids (3.52%) followed by farm animals in lambs (2.38%) and kids (1.76%) while the lowest prevalence in lambs (1.78%) and in kids (0.58%) was recorded in pastoral animals. The statistical differences were found non-significant in lambs (P=0.135) and in kids (P=0.143) in various groups of housing. Highest prevalence in lambs (5.95%) and kids (4.11%) were noted in animal fed on colostrum ad-libitum followed by animals fed on least colostrum (2.38%) in lambs and (1.17%) in kids while the animals fed on moderate colostrum fed animals indicated lowest prevalence in lambs (1.19%) and in kids (0.58%). The statistical differences were found significant in lambs (P=0.034) and in kids (P=0.042) in various groups of colostrum feeding. Highest prevalence in lambs (5.95%) and in kids (4.11%) were noted in fatty animals followed by lean animals in lambs (2.38%) and kids (1.17%) while the moderate animals indicated lowest prevalence in lambs (1.19%) and in kids (0.58%). The statistical differences were found significant in lambs (P=0.034) and in kids (P=0.042) in various groups of body condition. Highest prevalence in lambs (5.95%) and in kids (4.11%) were noted in animals housed in poor hygienic condition followed by animals housed in moderate hygienic conditions in lambs (2.38%) and in kids (1.17%) while the lowest prevalence in lambs (1.19%) and in kids (0.58%) were noted in animals housed in hygienic condition. The statistical differences were found significant in lambs (P=0.034) and in kids (P=0.042) with various groups of ‘hygienic condition. Higher prevalence was noted in the female (9.56%) than in the male (9.09%) in lambs while in kids higher prevalence was noted males (6.16%) than in female (5.7%). The statistical differences were found nonsignificant in lambs (P=0.701) and in kids (P=0.899) in various groups of sex.

In lambs the highest prevalence of C. perfringens type D was recorded during spring (35.17%) followed by winter (23.71%) and summer (21.41%) while the lowest prevalence was recorded in autumn (11.9%) season. Statistically these differences were non-significant among seasons (P=0.000) and between districts (P=0.017) in lambs. In kids the highest prevalence of C. perfringens type D was recorded during spring (16.66%) followed by summer and winter (9.52%) each while the lowest prevalence was recorded in autumn season (4.76%). These statistical differences were found nonsignificant among seasons (P=0.342) and between districts (P=0.442) in kids. Farm animals indicated highest prevalence in lambs (6.54%) and in kids (11.17%) followed by house hold animals in lambs (4.76%) and in kids (7.05%) while the lowest prevalence in lambs (3.57%) and in kids (4.70%) were observed in pastoral animals. The statistical differences were found nonsignificant in lambs (P=0.222) and in kids (P=0.075) in various groups of housing. Highest prevalence in lambs (11.31%) and in kids (11.76%) was noted in animals fed with ad-libitum colostrum followed by animal in lambs (5.35%) and in kids (7.05%) fed with least colostrum while the lowest prevalence in lambs (4.16%) and in kids (4.11%) was noted in animals fed with moderate colostrum. The statistical differences were found significant in lambs (P=0.022) and in kids (P=0.027) in various groups of colostrum feeding. Highest prevalence in lambs (11.30%) and in kids (11.17%) was noted in fatty animals followed by lean animals in lambs (5.95%) and in kids (8.23%) while the lowest prevalence in lambs (7.73%) and in kids (3.52%) was observed in animals with moderate body score. The statistically significant differences were observed in lambs (P=0.016) and in kids (0.027) in various groups of body condition scores. Highest prevalence (10.31%) in lambs and (11.76%) in kids was noted in animals in poor hygienic condition followed in lambs (7.73%) and in kids (7.05%) in moderate hygienic condition while the lowest prevalence in lambs (6.54%) and in kids (4.11%) was observed in hygienically housed animals. The statistically significant differences were observed in lambs (P=0.035) and in kids (P=0.027) were observed in various groups of hygienic condition. Higher prevalence in lambs (28.84%) and in kids (29.33%) was observed in the males as compared to the females in lambs (17.84%) and in kids (17.89%). Nonsignificant differences in lambs (P=0.086) and kids (P=0.078) were note in various groups of sex.

 

DISCUSSION

Clostridium perfringens is the causative agent of enterotoxaemia. It is an important disease in sheep and goats causes great economic losses in these animals throughout the world due to high prevalence and fatality rates (Uzal and Songer, 2008). Our results of indicated that C. perfringens type A, type B and type Din lambs and kids are the most predominant causes of enterotoxaemia in Khyber Pakhtunkhwa, Pakistan. Overall C. perfringens infection in present study is in line with the finding of Raana (2007) who reported about 12% C. perfringens infection in sheep and about 57% C. perfringens infection in goats from Punjab area of Pakistan. The results of our study are in close accordance with the findings of indicated by Kalender et al. (2005) from Turkey who reported 50% prevalence of C. perfringens infection in sheep with Enterotoxaemia signs; Aschfalk et al. (2002) from West Africa who reported C. perfringens prevalence 71% healthy sheep; Greco et al. (2005) from Italy who reported C. perfringens prevalence 25% in healthy lambs; and kids and Uzal and Songer (2008) from California who reported prevalence of C. perfringens in goats. Some discrepancy might be because of different geographical area, climate, breed, and system of rearing. The percentages of bacterial isolates of present study remained agreed with the outcomes of researchers.

Toxinotypes in our results were in accordance with findings of Gkiourtzidiset al. (2001), Bueschel et al. (2003) and Khan et al. (2017). However, in contrast Kalender et al. (2005) and Greco et al. (2005) did not identify C. perfringens type B in their studies in Turkey and Italy, respectively. Their studies are in agreement with our data where types A and D were the leading cause of C. perfringens infection. Similarly types C and E were not prevalent in lambs and kids. Type E of C. perfringens was not isolated in our study agreed with previous data of Songer (1996). However Kurtkaya and Alver (1969) and Ozcan and Gurcay (2000) in Turkey isolated C. perfringens type A, type B, type C and D from sheep with enterotoxaemia, although the role of type A in disease production was considered doubtful by some researchers (Nillo, 1980). From the results of our study, in lambs were the most prevalent type was C. perfringens type D than type A and type B. However, in kids the prevalence of pathogenic C. perfringens type A was maximum of as compared to type D and type A. Our results are in line with the findings of most conducted researches in various countries where highest infection was recorded in poor hygienic animals. The pathogens find their way to animal body through contaminated feed and water. C. perfringens also found dust, fomites, air and beddings which transfer the infection from diseased to healthy animals (Schultz et al., 2003; Kobayashi et al., 2009). The multiplication and survival of pathogens are triggered by poor digestion or absorption in GIT in animals thus results increased toxin production. Poor hygienic environment and unconscious feeding might be the reason of high C. perfringens type A and type D prevalence in farms animals. High proteins and carbohydrates amounts trigger the growth of C. perfringens in gut (Roberfroid et al., 2010; Riddell and Kong, 1992; Katyal et al., 1999). Highest ratio of enterotoxaemia recorded in ad libitum colostrum fed animals in our study was in line with that of Quinn et al. (2011) who described various risk factors like change in diet, low proteolytic efficiency in neonates, trypsin inhibitors of colostrum, malnutrition, inefficient pancreatic secretion, no established intestinal flora, carbohydrates engorgement and intestinal hypomotility in enterotoxaemia (Sato et al., 1978). Increased quantity of anti-trypsin factor or protease inhibitors like sweet potatoes, soybeans and colostrum are the risk factors for enterotoxaemia (Palliyeguru et al., 2010; Palliyeguru et al., 2011). Highest prevalence in fatty animals in our project might be due to greedy nature. Our results are in line with the results of many researchers (Van Immerseel et al., 2004; Beaugerie and Petit, 2004). C. perfringens is known to be the main proteolytic species in the gut and it requires regular supply of amino acids. Any deficiency in the amino acids reduces its number rapidly because it cannot synthesize some amino acids (Roberfroid et al., 2010; Shimizu et al., 2002).

Animals, like cattle, horses, goats and sheep, fed on greater amount of carbohydrates develop enteritis (Waggett et al., 2010). C. perfringens cannot produce some essential amino acids (Roberfroid et al., 2010; Shimizu et al., 2002). The production of alpha, beta, and epsilon toxins are increased by dextrin or glucose feeding in vitro (Sakurai and Duncan, 1979). Starch injected to the abomasum of sheep and goats and dextrin into the duodenum in cattle was used in the challenge of infection of type D of C. perfringens (Uzal et al., 2004; Layana et al., 2006). Carbohydrates overfeeding increase the C. perfringens intensity in the rumen and caecum in cow (Allison et al., 1975).

Highest prevalence of C. perfringens infection was noted in animals on ad libitum colostrum feeding which is in line with the findings of Quinn et al. (2011), who described that the presence of trypsin inhibitors in colostrum is the main risk factor for enterotoxaemia. The activity of trypsin in the gut is also affected by malnutrition (Sato et al., 1978; Lawrence and Walker, 1976).

 

CONCLUSION

Large number of non-vaccinated lambs and kids were observed to have C. perfringens infection. C. perfringens type A, type B and type D were observed in many animals which emphasizes the role of α, β and ε-toxins in the pathogenesis. C. perfringens isolates were precisely and quickly characterized for toxinotyping with PCR technique. The identification of the prevalent C. perfringens types facilitated the development of adequate vaccines in epidemiological situation for protection and in control of the disease in small ruminants.

 

Acknowledgements

The authors are highly obliged to the teaching and laboratories staff of University of Veterinary and Animal Sciences, Lahore, Pakistan.

 

Statement of conflict of interest

The authors declared no conflict of interest.

 

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