Journal of Animal Sciences and Livestock Production Open Access

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.

Table 1: Diarrhoea test kit result, Group 1.

Table 2: Diarrhoea test kit results, Group 2.

Table 3: Diarrhoea test kit results, Group 3.

Table 4: Diarrhoea Kit test results, Group 4.

Table 5: Diarrhoea Kit test results, Group 5.

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

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

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

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

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.

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