The Prevalence of Cryptosporidium Infection in Neonatal Diarrhoeic Sheep Lambs with Observation of Resolution using Paromomycin Treatment

Osman A Hameed1*, Taj Elsir SA Abu-Zeid2, Ghulam Rasool3, Albadri Makki4, Mohamed Khidr Taha5 and Brigitte Duquesne6

1Central Veterinary Laboratory, Sudan

2Animal Production Research Centre, Sudan

3Sindh Agriculture University, Pakistan

4Sharge Elnile College of Laboratory Science, Sudan

5Department of Veterinary Medicine, Khartoum University, Sudan

6Huvepharma, NV, Belgium

*Corresponding Author:

Osman A Hameed
Central Veterinary Laboratory, Sudan
E-mail: [email protected]

Received: December 11, 2019; Accepted: December 19, 2019; Published: December 23, 2019

Citation: Hameed OA, Abu-Zeid TESA, Rasool G, Makki A, Taha MK, et al. (2019) The Prevalence of Cryptosporidium Infection in Neonatal Diarrhoeic Sheep Lambs with Observation of Resolution using Paromomycin Treatment. J Anim Sci Livest Prod Vol.3 No.2:7.

 
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Abstract

This study was conducted between May and August 2016. A total of 45 fecal specimens obtained from 6-37 day old lamb were examined for the presence and prevalence of Cryptosporidium by rapid Immunochromatographic (ICT). The prevalence of the infection was found in 33 specimens out of 45 (73.33%). The infected lambs were treated by using Paromomycin sulphate (Parofor®) and retested for the presence of Cryptosporidium infection after 5 days of consecutive treatment. We report in this study the successful resolution of Cryptosporidium with Parofor®.

Keywords

Cryptosporidium; Protozoan; Diarrheic; Neonatal; Resolution; Parofor-Paromomycin; Sulphate Immunochromatographic; BoviD4; Rotavirus; Coronavirus; E. coli K99; BoviD4; MZN; Aminoglycosides

Introduction

Cryptosporidium parvum, a protozoan parasite, is considered to be an important agent in the etiology of the neonatal diarrhoea syndrome of calves, lambs and goat kids, causing considerable direct and indirect economic losses [1-5]. Moreover, the zoonotic potential of cryptosporidiosis makes it a public health concern. Of the seven Cryptosporidium species identified in sheep, two are predominant: Cryptosporidium parvum and Cryptosporidium ubiquitin [6]. Cryptosporidiosis occurs in lambs and kids at an early age of life (5-10 days of age), [7]. The most prominent clinical signs of ovine cryptosporidiosis are diarrhoea lasting 2 to 12 days and this is sometimes accompanied by anorexia, stiffness, hyperpnoea, slow gait and depression [8-10].

Cryptosporidiosis causes high morbidity and it has been recorded in various domestic young animals such as lambs, kids, foals, and calves, leading sometimes to mortality. Huge economic losses due to Cryptosporidium infection were recorded by different researchers [11-15]. There are different methods used for the detection of Cryptosporidium in faecal samples. Generally, microscopic examination is used for the detection of Cryptosporidium oocyst in faecal samples. The most procedure has been widely used, the Modified Ziehl-Nielsen (MZN) acid-fast stain [16]. Different antigen detection methods such as ELISA, immunofluorescence, and genome detection methods such as PCR are used for the detection of the Cryptosporidium [17].

In recent years, the Immunonochromatic Dipstick Test (ICT) has been used for the rapid diagnosis of Cryptosporidium. According to the manufacturer, these tests are rapid and sensitive enough but provide only qualitative results for the presence of the pathogen in faecal samples [18]. To control cryptosporidiosis is a big global challenge for veterinary as well as for human medicine. Different therapeutic agents up to thousands had been tested in vivo as well as in vitro conditions to treat cryptosporidiosis. Some agents were active against Cryptosporidium infection in vitro condition but showed poor efficacy or no efficacy during treatment therapy under field conditions [19]. More recently, Huvepharma Bulgaria has developed Parofor® (Paromomycin sulphate). Paromomycin is a broad-spectrum antibiotic and belongs to the group of aminoglycosides. Paromomycin is poorly absorbed from gastro-intestinal tract and is not inactivated by organic material, remaining active form in the intestinal lumen. Two forms of Parofor® are produced by Huvepharma, water powder for use in drinking water or milk replacer. One gram of it contains 100 mg of paromomycin sulphate (=70 mg paromomycin as base). The second form is Parofor® 140 mg/ml OS, a solution used in drinking water, milk or milk replacer.

Previous studies have shown the efficacy of Paromomycin against Cryptosporidium [20,21]. Parofor® was first only registered for the treatment of diarrhoea caused by Escherichia coli but the recognized efficacy against Cryptosporidium leads Huvepharma to obtain an antiprotozoal registration (Parofor Crypto®, UK, July 2018, following by several European countries). The aim of the present study was to determine the prevalence of Cryptosporidium infection in lambs using rapid Immunochromatographic Test (Bovi D4) manufactured by Bionote Inc, South Korea. The second aim was to investigate the effect of Parofor®, water-soluble powder, in treating positive cases of Cryptosporidium infection in lambs.

Materials and Methods

This study was conducted between May and August 2016.

Samples

A total of 45 faecal specimens were obtained from 45 lambs, from 6-37 days old. They were grouped into 5 groups. The first group is from 6-14 day old lamb, the second group is from 8-14 day old, the third group is from 18-37 day old; the fourth group is from 6-11 day old and the fifth group is from 6-9 day old. The fecal samples were detected by Bovi 4 Ag test Kit (Bionote). This kit detects Cryptosporidium, Rotavirus, Coronavirus and E. coli K 99. Faecal samples were processed according to the manufacture's recommendations. Briefly collect the sample from diarrhoeic lamb using the swab. Insert the swab into the sample tube containing assay diluent and mix the swab until the sample has been dissolved into the assay diluent. Squeeze the swab against the well of the tube and then discard it. Wait 30 seconds for sedimentation and take the supernatant with the disposable dropper provided. Add 4 drops into the sample hole with the disposable dropper and wait for 5-10 minutes.

Interpretation of the result

The appearance of red or purple color in the control line (C) indicates that the sample is negative for any of the 4 pathogens (Cryptosporidium, Rotavirus, Coronavirus and E. coli K99). The appearance of red or purple color in control line (C) and test line (T), indicates that the sample positive for Cryptosporidium, Rota, Corona and E. coli K99 or there is a mixed infection. The results are invalid when there is no purple colour in both control or test line, or it is only appearing in the test line.

Parofor® treatment

Parofor® (Paromomycin sulphate) manufactured by Huvepharma=70 mg Paromomycin as a base, is a water-soluble powder. Each of 33 Cryptosporidium positive cases was treated with Parofor®. Each lamb is given 5 gm Parofor®/10 Kg of live bodyweight for 5 consecutive days and retested for the presence of Cryptosporidium pathogen using the BoviD4 Ag Test Kit of Bionote.

Results

Cryptosporidium is detected in 33 faecal samples, while 12 samples were negative by BoviD4 Ag test. The prevalence of infection is 73.33%. Those gave negative Cryptosporidium results represented 26.67% Tables 1-5. Two of them were positive for Rotavirus infection. All the 33 positive animals gave negative results to Cryptosporidium after treatment with Parofor for 5 days and they had responded to this treatment Tables 6-10.

S. No. Tag No. D.O.B. Result Date
Crypto Rotavirus E. coli Giardiasis
1 Y-3136 07-06-2016 Positive Negative Negative Negative 20-06-2016
2 Y-3031 07-06-2016 Negative Negative Negative Negative 20-06-2016
3 Y-3044 08-06-2016 Negative Negative Negative Negative 20-06-2016
4 B-1053 08-06-2016 Positive Negative Negative Negative 20-06-2016
5 Y-3072 10-06-2016 Positive Negative Negative Negative 20-06-2016
6 B-1120 12-06-2016 Positive Negative Negative Negative 20-06-2016
7 Y-3096 12-06-2016 Positive Negative Negative Negative 20-06-2016
8 B-1119 12-06-2016 Positive Negative Negative Negative 20-06-2016
9 Y-3088 12-06-2016 Positive Negative Negative Negative 20-06-2016
10 Y-3094 12-06-2016 Positive Negative Negative Negative 20-06-2016
11 B-1118 12-06-2016 Positive Negative Negative Negative 20-06-2016
12 Y-3114 13-06-2016 Positive Negative Negative Negative 20-06-2016
13 B-1151 13-06-2016 Positive Negative Negative Negative 20-06-2016
14 B-1156 14-06-2016 Positive Negative Negative Negative 20-06-2016
15 Y-3141 14-06-2016 Negative Negative Negative Negative 20-06-2016
16 B-1208 17-06-2016 Positive Negative Negative Negative 20-06-2016

Table 1: Diarrhoea test kit result, Group 1.

S. No. Tag No. D.O.B. Result Date
Crypto Rotavirus E. coli Giardiasis
1 B-1076 06-09-2016 Positive Negative Negative Negative 6/21/2016
2 B-1142 06-13-2016 Positive Negative Negative Negative 6/21/2016
3 Y-3053 06-09-2016 Positive Negative Negative Negative 6/21/2016
4 B-1067 06-09-2016 Positive Negative Negative Negative 6/21/2016
5 Y- 3038 06-07-2016 Positive Negative Negative Negative 6/21/2016
6 B-1005 06-06-2016 Negative Negative Negative Negative 6/21/2016
7 B-1107 06-12-2016 Positive Negative Negative Negative 6/21/2016
8 B- 1108 06-12-2016 Positive Negative Negative Negative 6/21/2016
9 Y-3057 06-10-2016 Negative Negative Negative Negative 6/21/2016
10 Y-3067 06-10-2016 Positive Negative Negative Negative 6/21/2016

Table 2: Diarrhoea test kit results, Group 2.

S. No. Tag No. D.O.B. Result Date
Crypto Rotavirus E. coli Giardiasis
1 G-979 04-06-2016 Positive Negative Negative Negative 22-06-2016
2 O-451 02-06-2016 Positive Negative Negative Negative 22-06-2016
3 G-916 01-06-2016 Positive Negative Negative Negative 22-06-2016
4 G-848 24-05-2016 Negative Positive Negative Negative 22-06-2016
5 O-203 16-05-2016 Negative Positive Negative Negative 22-06-2016
6 O-1988 28-05-2016 Negative Negative Negative Negative 22-06-2016
7 O-500 04-06-2016 Negative Negative Negative Negative 22-06-2016
8 No Tag 25-05-2016 Negative Negative Negative Negative 22-06-2016
9 G-855 25-05-2016 Negative Negative Negative Negative 22-06-2016

Table 3: Diarrhoea test kit results, Group 3.

S. No. Tag No. D.O.B. Result Date
Crypto Rotavirus E. coli Giardiasis
1 G-1404 7/28/2016 Positive Negative Negative Negative 08-06-2016
2 O-1417 7/26/2016 Positive Negative Negative Negative 08-06-2016
3 O-1450 7/30/2016 Positive Negative Negative Negative 08-06-2016
4 O-1447 7/28/2016 Positive Negative Negative Negative 08-06-2016
5 O-1416 7/31/2016 Negative Negative Negative Negative 08-06-2016

Table 4: Diarrhoea Kit test results, Group 4.

1 O-1464 7/31/2016 Positive Negative Negative Negative 08-06-2016
2 O-1438 7/28/2016 Positive Negative Negative Negative 08-06-2016
3 O-1469 7/31/2016 Positive Negative Negative Negative 08-06-2016
4 O-1942 7/31/2016 Positive Negative Negative Negative 08-06-2016
5 G-1415 7/31/2016 Positive Negative Negative Negative 08-06-2016

Table 5: Diarrhoea Kit test results, Group 5.

S. No. Tag No. D.O.B. Result Date Treatment results
Crypto Rotavirus E. coli Giardiasis    
1 Y-3136 07-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
2 Y-3031 07-06-2016 Negative Negative Negative Negative 20-06-2016 Not treated
3 Y-3044 08-06-2016 Negative Negative Negative Negative 20-06-2016 Not treated
4 B-1053 08-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
5 Y-3072 10-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
6 B-1120 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
7 Y-3096 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
8 B-1119 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
9 Y-3088 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
10 Y-3094 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
11 B-1118 12-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
12 Y-3114 13-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
13 B-1151 13-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
14 B-1156 14-06-2016 Positive Negative Negative Negative 20-06-2016 Negative
15 Y-3141 14-06-2016 Negative Negative Negative Negative 20-06-2016 Not treated
16 B-1208 17-06-2018 positive Negative Negative Negative 20-06-2016 Negative

Table 6: Diarrhoea Test kit Results after Parofor treatment Group 1.

S. No. Tag No. D.O.B. Result Date Treatment results
Cipro Rotavirus E. coli Giardiasis    
1 B-1076 06-09-2016 Positive Negative Negative Negative 6/21/2016 Negative
2 B-1142 06-13-2016 Positive Negative Negative Negative 6/21/2016 Negative
3 Y-3053 06-09-2016 Positive Negative Negative Negative 6/21/2016 Negative
4 B-1067 06-09-2016 Positive Negative Negative Negative 6/21/2016 Negative
5 Y- 3038 06-07-2016 Positive Negative Negative Negative 6/21/2016 Negative
6 B-1005 06-06-2016 Negative Negative Negative Negative 6/21/2016 Not treated 
7 B-1107 06-12-2016 Positive Negative Negative Negative 6/21/2016 Negative
8 B- 1108 06-12-2016 Positive Negative Negative Negative 6/21/2016 Negative
9 Y-3057 06-10-2016 Negative Negative Negative Negative 6/21/2016 Not treated 
10 Y-3067 06-10-2016 Positive Negative Negative Negative 6/21/2016 Negative

Table 7: Diarrhoea test kit results after Parofor treatment, Group 2.

S. No. Tag No. D.O.B. Result Date Treatment results
Crypto Rota Virus E. coli Giardiasis    
1 G-979 04-06-2016 Positive Negative Negative Negative 22-06-2016 Negative
2 O-451 02-06-2016 Positive Negative Negative Negative 22-06-2016 Negative
3 G-916 01-06-2016 Positive Negative Negative Negative 22-06-2016 Negative
4 G-848 24-05-2016 Negative Positive Negative Negative 22-06-2016 Not treated
5 O-203 16-05-2016 Negative Positive Negative Negative 22-06-2016 Not treated
6 O-1988 28-05-2016 Negative Negative Negative Negative 22-06-2016 Not treated
7 O-500 04-06-2016 Negative Negative Negative Negative 22-06-2016 Not treated 
8 No Tag 25-05-2016 Negative Negative Negative Negative 22-06-2016 Not treated 
9 G-855 25-05-2016 Negative Negative Negative Negative 22-06-2016 Not treated

Table 8: Diarrhoea Test kit Result after Parofor treatment, Group 3.

S. No. Tag No. D.O.B. Result Date Treatment
Crypto Rota Virus E. coli Giardiasis Result
1 G-1404 7/28/2016 Positive Negative Negative Negative 08-06-2016 Negative
2 O-1417 7/26/2016 Positive Negative Negative Negative 08-06-2016 Negative
3 O-1450 7/30/2016 Positive Negative Negative Negative 08-06-2016 Negative
4 O-1447 7/28/2016 Positive Negative Negative Negative 08-06-2016 Negative
5 O-1416 7/31/2016 Negative Negative Negative Negative 08-06-2016 Not treated 

Table 9: Diarrhoea test kit after Parofor treatment, Group 4.

1 O-1464 7/31/2016 Positive Negative Negative Negative 08-06-2016 Negative
2 O-1438 7/28/2016 Positive Negative Negative Negative 08-06-2016 Negative
3 O-1469 7/31/2016 Positive Negative Negative Negative 08-06-2016 Negative
4 O-1942 7/31/2016 Positive Negative Negative Negative 08-06-2016 Negative
5 G-1415 7/31/2016 Positive Negative Negative Negative 08-06-2016 Negative

Table 10: Diarrhoea test kit after Parofor treatment, Group 4.

Discussion and Conclusion

In recent years, the number of commercially diagnostic immunoassays for the presence of the antigen to Cryptosporidium spp. increased rapidly, focusing especially on speed, ease and sufficient sensitivity of testing. However, these tests are used as screening tests, provide qualitative results and are suitable only for detecting the presence/absence of infection in large groups of animals. BoviD4, a rapid Immunochromatographic Test is used in this study to detect the prevalence of Cryptosporidium in lambs. The test is simple, rapid with high specificity (99%) and high sensitivity (98.2%) versus PCR as claimed by the manufacturer (Bionote, Inc). Observations and investigations on the presence of Cryptosporidium infection in neonatal lambs were studied and the prevalence rate of infection was 73.33%. In the present study, all the diarrhoeic lambs were 6-37 days old. The chemotherapy of the cryptosporidiosis remains a major challenge. The main goal of modern and parasitic chemotherapy must bring the drug as directly to target pathogen as possible and minimize potential side effects.

Also, to study the efficacy of the therapeutic agent in vitro as well as in vivo conditions (under field condition). Different therapeutics were used but, up to now, only two molecules have shown satisfactory results to control Cryptosporidiosis: Halofuginone and Paromomycin. In this study, Parofor® showed good efficacy during treatment therapy under field conditions and proof as a convenient and promising therapeutic agent for Cryptosporidium infection.

In conclusion, Immunochromatographic Test (ICT) has been used as a simple, rapid and sensitive qualitative test to confirm the presence and prevalence of Cryptosporidium. Parofor® efficacy was studied and it showed a convenient, promising therapeutic drug for Cryptosporidium infection.

Acknowledgment

The author’s thanks Jinho Moon, Bionote, South Korea for providing BoviD4 used in this study. In addition, the authors thank Stephane Rousseau, Huvepharma for support and providing of Parofor® used in this study. Thanks is also extended to Ali Amro, Huvepharma Brand manager at Artat Enterprise for his continuous assistance and Support.

References

  1. Dubey JP, Fayer CA (1990) Cryptosporidiosis of man and animals. CRC Press, Boca Raton, Florida 2009.
  2. Current WL (1985) Cryptosporidiosis. J A Vet Med Assoc 187: 1334-1338.
  3. Foreyt WJ (1990) Coccidiosis and cryptosporidiosis in sheep and goats. Vet Clin North Am Food Anim Pract 6: 655-670.
  4. Snodgrass DR, Angus KW, Gray EW (1984) Experimental cryptosporidiosis in germfree lambs. J Comp Pathol 94: 141-152.
  5. Xiao L, Herd RP, Rings DM (1993) Diagnosis of Cryptosporidium on a sheep farm with neonatal diarrhoea by immunofluorescence assay. Vet Parasitol 47: 17-23.
  6. Xiao L (2010) Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124: 80-89.
  7. Radostits O, Gay C, Hinchciff K, Constable P (2008) Diseases associated with protozoa. 10th End In: Veterinary Medicine: A Textbook of cattle, horse, sheep, pigs and goats. Saunders Elsevier, pp: 1483-1540.
  8. Angus KW, Appleyard WT, Menzies JD, Campbell I, Sherwood D (1982) An outbreak of diarrhoea associated with cryptosporidiosis in naturally reared lambs. Vet Rec 110: 129-130.
  9. de Graaf DC, Vanopdenbosch E, Ortega-Mora LM, Abbassi H, Peeters JE (1999) A review of importance of cryptosporidiosis in farm animals. Int J Parasitol 29: 1269-1287.
  10. Tizpori S, Angus KW, Clerihew LW, Campbell I (1981) Diarrhoea due to Cryptosporidium infection in artificially reared lambs. J Clin Microbiol 14: 100-105.
  11. Degerli S, Celiksoz A, Kalkan K, Ozcelik S (2005) Prevalence of Cryptosporidium spp. and Giardia spp. in cow and calves in Sivas. Turk J Vet Anim Sci 29: 995-999.
  12. Nasir A, Avais M, Khan MS, Ahmed N (2009) Prevalence of Cryptosporidium parvum infection in (Pakistan) and its association with diarrhoea in dairy calves. Int J Agri Biol 11: 221-224.
  13. Prakash S, Prabu K, Palanivel KM (2009) Prevalence of cryptosporidiosis in dairy claves in Chennai. J Vet AnI Sci 5: 41-46.
  14. Potter I, Esbroeck VM (2010) Negative staining of Heine foe the detection of Cryptosporidium spp. A fast-simple technique. Open Para J 4: 1-4.
  15. Ayinmode FB, Fagbemi BO (2011) Cross-reactivity of some Cryptosporidium species with Cryptosporidium parvum coproantigen Commercial Elisa kit. Nig Vet J 10: 1-4.
  16. Fayer R, Ungar BLP (1986) Cryptosporidium spp and Cryptosporidiosis. Microbial Rev 50: 458- 483.
  17. Areeshi MY, Beeching NJ, Hart CA (2007) Cryptosporidiosis in Saudi Arabia and neighbouring countries. Ann Saudi Med 27: 325-332.
  18. Agnamey P, Sarfati C, Pinel C, Rabodoniriina M, Kapel N, et al. (2011) Evaluation of four commercial rapid immunochromatographic assays for detection of Cryptosporidium antigen in stool samples: a blind multicentre trial. J Clin Microbiol 49: 1605-1607.
  19. Shahiduzzaman M, Daugschies A (2012) Therapy and prevention of cryptosporidiosis in Animals. Vet Parasitol 188: 203-214.
  20. Viu M, Quílez J, Sánchez-Acedo C, del Cacho E, López-Bernad F, et al. (2000) Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs. Vet Parasitol 90: 163-170.
  21. Paraud C, Chartier C (2012) Cryptosporidiosis in small ruminants. Small Ruminant Research 103: 93-97
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