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 losses1-5. 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 ubiquitin6. 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 depression8-10. 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 researchers11-15.
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 widely used procedure is the Modified Ziehl-Nielsen (MZN) acid-fast stain16. Different antigen detection methods such as ELISA, immunofluorescence, and genome detection methods such as PCR are used for the detection of the Cryptosporidium17.
In recent years, the Immunochromatic 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 samples18. To control cryptosporidiosis is a big global challenge for veterinary as well as for human medicine. Different therapeutic agents up to thousands have 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 conditions19. 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 product contains 100 mg of paromomycin sulphate (equivalent to 70 mg paromomycin as a 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 Cryptosporidium20,21. Parofor® was first only registered for the treatment of diarrhoea caused by Escherichia coli but the recognized efficacy against Cryptosporidium led Huvepharma® to obtain an antiprotozoal registration (Parofor® Crypto, UK, July 2018, followed by several other 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.
A total of 45 faecal specimens were obtained from 45 lambs, from 6-37 days old. They were grouped into 5 groups.
- Group 1 = 6-14 days old
- Group 2 = 8-14 days old
- Group 3 = 18-37 days old
- Group 4 = 6-11 days old
- Group 5 = 6-9 days old
The faecal samples were detected by Bovi 4 Ag test kit (Bionote). This ket detects Cryptosporidium, Rotavirus, Coronavirus and E. coli K99. Faecal samples were processed according to the manufactures recommendations. Shortly after collecting the samples from diarrhoeic lambs using a swab, the samples were inserted into the sample tube containing the assay diluent, and mixed until the sample was fully dissolved in the assay diluent. The swab was then squeezed against the side of the sample tube before being discarded. After waiting 30 seconds for sedminentation, a sample of the supernatant was taken using a disposable dropper. 4 drops of the sample were added into the sample tube via the sample hole and the results were read after 10 minutes.
Interpretation of the result
The appearance of red or purple colour in the control line (C) indicates that the sample is negative for any of the four pathogens (Cryptosporidium, Rotavirus, Coronavirus and E. coli K99). The appearance of red or purple colour in the C line and the test line (T), indicates that the sample positive for Cryptosporidium, Rotavirus, Coronavirus or E. coli K99 or there is a mixed infection. The results are invalid when there is no purple colour in both C and T lines, or the colour only appears in the T line.
Parofor® (Paromomycin sulphate) manufactured by Huvepharma® water soluble powder contains 70 mg of Paromomycin as a base. Each of 33 Cryptosporidium positive cases was treated with Parofor®. Each lamb was given 5 gm Parofor® /10kg live bodyweight for five consecutive days and retested for the presence of Cryptosporidium pathogen using the Bovi D4 Ag test kit of Bionote.
Cryptosporidium was detected in 33 faecal samples, while 12 samples were negative according to the BoviD4 Ag test. The prevalence of infection was 73.33%. The samples that gave negative Cryptosporidium results represented 26.67%.
Two of the samples were positive for Rotavirus infection. All the 33 positive animals gave negative results to Cryptosporidium after treatment with Parofor® for five consecutive days and they had responded to this treatment.
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, providing 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 the 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 conditions). 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.
References are available on request.
This article originally appeared in the Journal of Animal Sciences and Livestock Production, Vol 3 Number 2:7, 2019.