Abstract: The control of cryptosporidiosis relies mainly on hygienic measures and good management. Preventive hygienic measures are by far the most effective approach to control this parasite in animals, the objective being to destroy the external forms of the parasite (infective mature oocysts) and to prevent their transmission among animals. The destruction of oocysts on surfaces of housing facilities, pens and parturition buildings is possible using effective disinfectants such as 50% ammonia, 3% hydrogen peroxide or 10% formalin. Measures to reduce transmission between animals should be encouraged. Limiting the number of animals enclosed in the same facilities and avoidance of high stocking rates in the parturition area, maintaining a short calving period, administration of appropriated supplies of colostrum especially hyper immune colostrum from immunized dams, isolation and treatment of diarrheic infected animals; all help to prevent outbreaks of cryptosporidiosis, reduce the spread of infection within a herd and minimize mortality and morbidity in infected herds. Chemotherapeutic agents such as paromomycin, decoquinate, lasalocid, halofuginone lactate, nitazoxanide, dinitrooryzalin, β-cyclodextrin and probioties have proved a potential therapeutic effect against cryptosporidiosis in the form of reduction the duration and numbers of oocysts shedding and the incidence and severity of the diarrhea. Immunization of ruminants during pregnancy with either recombinant C. parvum sporozoites surface antigens or plasmid DNA encoding the CP15 or CP23 antigens appear to be a valuable approach for producing colostrum for the passive immunotherapy of cryptosporidiosis. Hyper immune colostrum prevented diarrhea and reduced oocysts shedding in newly born calves, lambs and goat kids exposed to Cryptosporidium infections.
INTRODUCTION
Cryptosporidiosis is a common gastrointestinal disorder in humans and animals caused by various Cryptosporidium species. The disease is self-limiting in the immune competent host but poses a significant threat in the immune deficient individuals1,2. Cryptosporidium parvum is the most common zoonotic species affecting a wide variety of mammals and this species accounts for the majority of water borne outbreaks3,4. The infectious stages of the organism (oocysts) are shed in the feces of affected individuals, survive in adverse environmental conditions and spread by direct contact or through contaminants (food, water). Due to the robustness of the oocysts, their tenacity, tiny size and resistance to common disinfectants, the parasite is difficult to eradicate from contaminated environments. To obtain sufficient control, both treatment of infected hosts and inactivation of oocysts are necessary5. Several drugs are commonly used to treat cryptosporidiosis in man and very few in animals but none of them are completely effective in terms of both clinical and parasitological response. Only a few chemical agents are able to inactivate oocysts in the environment including water treatment plants but their application has certain limitations. Therefore, control of cryptosporidiosis remains a global challenge in both veterinary and human medicine. Extensive research has been performed on suitable drugs and disinfectants. Thousands of agents have been tested both in vivo and in vitro. Some are excitingly active in vitro but exhibit poor or no response in clinical trials5.
Many chemotherapeutic antimicrobial compounds have been tested, but most have only a partial effectiveness in the treatment and prophylaxis of cryptosporidiosis in animals and humans. There is no clearly recognized, widely accepted and immediately available compound that can be used as a prophylactic or therapeutic agent6, despite some encouraging recent advances including the use of β-cyclodextrin7, nitazoxanide8, halofuginonelactate9 and premomycin10. Because of the limited availability of effective drugs, hygienic measures and good management are currently the most valid weapons in controlling this disease. However, a number of studies have demonstrated the efficacy of oral administration of hyper immune, bovine colostrum in reducing the shedding of oocysts and the clinical signs of disease in lambs and calves11.
Therefore, the objective of the current review article was to highlight and evaluate the new trends for control of cryptosporidiosis in animals and humans.
PREVENTION AND CONTROL
Treatment: Oral or intravenous fluid therapy remains the single most important treatment to diminish clinical signs of disease in humans and animals. Although more than 150 antimicrobial agents have been studied and a few have been demonstrated to reduce the magnitude of the symptoms, none eliminates the disease completely and none has received regulatory approval for the treatment of animal cryptosporidiosis. Trials evaluating different drugs, novel classes of compounds and immune therapy are currently in progress. Recently, the US FDA12 approved the drug nitazoxanide (AliniaTM) for the treatment of pediatric diarrhea caused by C. parvum in children 1-11 years of age. The efficacy of nitazoxanide (a synthetic antiprotozoal agent) in children was studied in double-blind placebo-controlled trials which showed reduced duration of diarrhea and oocyst excretion13,14. Nitazoxanide (NTZ) has been tested in animal models but showed only partial efficacy in the gnotobiotic pig model (with high doses, 150-250 mg kg–1 day–1, reducing oocyst shedding) and no efficacy in the mouse model, bringing into question the true efficacy of the drug15,16. Aminoglycoside antibiotics, such as paromomycin have proven to decrease the patent period, oocyst shedding and clinical symptoms in mice and calves infected with C. parvum (Table 1)15,17. A recent report determined that dinitroanilineoryzalin reduced oocyst numbers and pathologic manifestations in the gut of mice infected with C. parvum18. The short term feeding (2-3 days) of relatively high levels (6-15 mg kg–1 day–1) of the ionophore polyether antibiotic, lasalocid was reported to be an effective treatment for acute cryptosporidiosis in young calves19 but was highly toxic when fed (at 8 mg kg–1 day–1) for 2 weeks as a prophylaxis. Lower doses of Lasalocid have not demonstrated any efficacy against C. parvum infections. Awadalla20 used sulphadimidine powder at a dose rate of 0.4 g Kg–1 b.wt., for treatment of cryptosporidiosis in diarrheic buffalo-calves and reported stoppage of diarrhea and improvement in the general health condition.
Alternative therapies such as passive immunotherapy using hyper immune, serum and hyper immune, bovine colostrum containing antibodies against C. parvum surface proteins as well as anti sporozoites monoclonal antibodies have also been tested with promising but inconclusive results21,22. Several colostrum preparations have been used to treat infections, the best results being obtained with hyper immune, bovine colostrum harvested from dairy cows vaccinated during gestation with C. parvum oocyst/sporozoites antigens.
| Table 1: | Efficacy of different drugs against cryptosporidiosis in ruminants29 |
| *Per kilogram body weight | |
Treatment with hyper immune colostrum containing high levels of antibodies against Cryptosporidium (titers ranging from 1:3200-1:200000) has been associated with the symptomatic improvement or resolution of infection23. Laboratory studies have shown that prophylactic treatment with hyper immune colostrum partially protected calves and mice against experimental infection and clinical disease and incubation in hyper immune, colostrum has been found to neutralize sporozoites and reduced their infectivity24. Colostrum harvested from cows vaccinated by a combination of intramuscular injection and intra-mammary infusion was found to be effective in human and animal trials25, whereas colostrum harvested from cows vaccinated by intramuscular injection alone did not protect calves against experimental infection despite the presence of specific antibodies at titer up to 1:10240. Another study, however, has found that colostrum harvested from cows following repeated parenteral immunization was effective in protecting calves against infection24.
The administration of probiotics (live bacterial cell supplements) has been shown to reduce the duration and number of oocysts shed by experimentally infected mice26,27. In addition, in vitro studies have demonstrated that lactic acid bacteria supernatants significantly reduced oocyst viability28. Although bacterial mechanisms involved in protection against cryptosporidiosis are not identified, the studies suggest that probiotic bacteria may have potential for therapeutic use against Cryptosporidium.
Many other chemotherapeutic agents have been tested in vitro and in vivo for the treatment of cryptosporidiosis, but few agents have shown promise. Drugs such as decoquinate decrease oocyst shedding and improve the frequency and severity of diarrhea in calves, lambs and goat kids10,29. Halofuginone lactate (Halocur, Intervet) has recently been registered in Europe as a chemotherapeutic agent for cryptosporidiosis in domestic cattle, which has been shown to reduce incidence and severity of diarrhea30. It has also been reported to markedly reduce oocyst output in experimentally infected lambs and naturally infected calves (Table 1). The drug prevents reinfection of the gut by sporozoites and recycling merozoites. The preliminary studies suggest that an oral dose of 60-125 μg kg–1 b.wt., daily for 7 days will protect against clinical disease and will markedly reduce oocyst excretion but will allow some intestinal infections and thus the development of immunity31. The effectiveness of β-cyclodextrin in treatment of natural infection by C. parvum in suckling calves was also evaluated7. Administration of the drug at a dose of 50 mg kg–1 b.wt. for 3 consecutive days from birth (prophylactically) or following confirmation of the infection (therapeutically) decreased the severity of diarrhoea and shortened the duration of oocyst shedding. The efficacy of β-cyclodextrin demonstrated in this study suggests that it could be suitable for the use in treatment of cryptosporidiosis in dairy calves.
Affected calves should be treated with fluids and electrolytes both orally and parenterally as necessary until spontaneous recovery occurs. Cow’s whole milk should be given in small quantities several times daily to optimize digestion and to minimize loss of body weight. It is important to continue to feed milk to the full level of requirement despite the presence of diarrhea, as a reduction in intake may lead to death from inanition. Several days of intensive care and feeding may be required before recovery is apparent. Parenteral nutrition could be considered for valuable calves31.
Advances and prospects for subunit vaccines against cryptosporidiosis: Cryptosporidiosis causes self-limiting diarrhoea in humans and animals that resolves within 1-2 weeks post infection23,32. The disease is prevalent in the young and in immunocompromised animals and persons due to immature or impaired T cell immunity33. Prior exposure to C. parvum leads to increased, but not complete resistance to subsequent infection34. Protective immunity appears, based on studies in humans and mice to involve CD4+T lymphocytes and associated the cytokines, IFN-γ and IL-1235. Although C. parvum specific IgG, IgA and IgM responses have been demonstrated, the role of these antibodies in resolution, parasite clearance and prevention of clinical signs remains unclear36,37. Cellular immunity could be more important for prevention of clinical signs and elimination of the parasite30. Vaccination has been proposed as a method to control cryptosporidiosis in animal populations29,38. The importance of colostrum in protecting neonatal ruminants against infection by C. parvum is a very valuable point to address. In field conditions, passively acquired antibodies did not protect calves29,39 and lambs against naturally acquired infection40. However, both calves and lambs fed with colostrum from immunized mothers with high titres of specific antibodies were partially protected against infection41. Several studies have shown that passive immunotherapy with hyper immune, serum or colostrum against C. parvum antigens can ameliorate clinical signs of disease38,42. Vaccination against cryptosporidiosis has focused on antigens that may be used to generate colostrum for passive immunotherapy rather than stimulating protective immunity29. There are several reasons for this approach. First, vaccination of vulnerable individuals (the young) would probably not be effective during the period of peak susceptibility. Second, due to the sporadic occurrence of cryptosporidiosis, the age-related resistance to infection and since disease symptoms are rarely life-threatening, there is little perceived need for widespread vaccination. Vaccination of young animals with an immature immune system may not be effective and widespread vaccination may not be economical, as the disease is rarely lethal when good preventive measures are in place19.
Immunodominant Cryptosporidium antigens have been identified from natural infections and subunit vaccines have been prepared and vaccination trials have been conducted in calves30,38. Using active and passive immunization approaches, vaccines have shown to reduce clinical signs but in most cases, have not eliminated or reduced oocyst shedding. Indeed, selection of immunodominant antigen-subunit vaccine candidates is based upon the premise that the T helper cell type 2 (Th 2) responses is responsible for eliminating the parasite and for protection from further infection30.
Thus, cloning of C. parvum DNA has concentrated on expressing epitopes on the surface of invasive stages (sporozoites, merozoites) which may be involved in attachment to host cells of the intestinal epithelium. Although a number of recombinant C. parvum antigens have been demonstrated43-47. The greatest effort has been described at sequences coding for antigens related to sporozoites 15 or 23 kDa surface proteins48-52. Plasmid DNA expressing The CP 15/60 protein has been used to elicit serum and colostrum antibodies in sheep42 and cows53 against recombinant and native CP15 antigens. Treatment of immunosuppressed adult inbred mice with this anti-CP 15/60 hyper immune, bovine colostrum prior to and during experimental C. parvum oocyst infection hindered parasite development in vivo53. Although it is unknown whether direct plasmid DNA injection is required, a recent study showed that expression of CP15/60 in an eukaryotic vector (baculovirus) was superior to a prokaryotic system (E. coli) in terms of humoral and cellular responses elicited54. Nasal immunization of goats and mice with a second CP15 DNA sequence also led to anti-CP15 antibodies in serum that in the latter host species, remained high for at least 1 year post-immunization55. In mice, CP15-specific T cell proliferative responses were also demonstrated in both the spleen and the mesenteric lymph nodes56. Furthermore, kids challenged with a high number of C. parvum oocysts (106 oocysts per kid) and fed colostrum from CP15-immunized goats shed fewer oocysts and displayed less severe clinical effects compared to kids fed normal colostrum55. Another study showed that administration of monoclonal antibodies (mAbs) specific for a 23 kDa C. parvum sporozoites protein reduced the severity of C. parvum oocyst infection in mice50. Hyper immune, bovine colostrum prepared against recombinant CP23 antigen prevented diarrhea and reduced oocysts shedding in calves challenged with 106 C. parvum oocysts36. Thus, immunization of ruminants with either recombinant C. parvum sporozoites surface antigens or plasmid DNA encoding the CP15 or CP23 antigens appears to be a viable approach to produce colostrum for the passive immunotherapy of cryptosporidiosis.
Preventive hygienic measures: From a perspective of disease control, preventive hygienic measures are the most important tools in the struggle against cryptosporidiosis in farm animals, the objective being to destroy external forms of the parasite and to prevent their transmission among animals and from the environment to the host. Preventive measures are by far the most effective approach to control this parasite19. This was proven by the marked decline in human and animal cases of cryptosporidiosis during the 2001 epidemic of Foot and Mouth Disease (FMD) in UK. Limiting human access to the countryside, containment of animals and restriction of livestock movement for trade or to pasture and extensive slaughtering of FMD-affected animals were the immediate actions taken that not only ended the FMD epidemic but also resulted in a significant reduction (81.8%) in reported cryptosporidiosis cases as compared to the previous year57,58 2000. These reports also provide clear evidence that zoonotic transmission is indeed a major route of human Cryptosporidium infection.
Because the major source of infection is contaminated water supplies, implementation of measures to decrease the spread of the parasitic oocysts in the environment is critical. Thus, identification of the risk factors for infection in livestock will allow for the logical development of oocyst shedding management strategies. Prevention of cryptosporidiosis transmissions is dependent on hygienic measures in any setting. The destruction of oocysts on surfaces of housing facilities is possible by cleaning with 5% ammonia solutions30, especially if heat can also be applied. Isolation of ill animals and ensuring that newborns have received adequate colostrum are always good preventive actions to control infectious diseases. Although passively acquired antibodies have not been effective in protecting calves against Cryptosporidium infections, calves fed with hyper immune, , colostrum from immunized dams developed less severe diarrhea and shed fewer oocysts than calves fed "nonhyper immune, " colostrum19.
Measures that reduce transmission between animals should be encouraged. Limiting the number of animals enclosed in the same facilities (i.e. reduced stocking density), keeping young animals separated from adults, minimizing contact between personnel and calves and maintaining a short calving period may assure reduced opportunities for the parasite to spread within a herd. Reproductive management such as increasing herd fertility and increasing the bull: cow ratio, limiting the time of cow and bull exposure may help reduce the calving period while maintaining calving rates59. In ruminant husbandry, the destruction of oocysts in the pens and building used for parturition by applying moist heat and/or chemical disinfection, the use of abundant clean straw beds, avoidance of high stocking rates in the parturition area and the separation of healthy and ill animals during outbreaks of diarrhoea, in addition to the administration of appropriated supplies of colostrum to neonates, all help to prevent outbreaks of cryptosporidiosis and to minimize mortality and morbidity in infected herds29.
Many recommendations have been made for the prevention and control of infections in specific locations; such as hospitals, laboratories, households, zoos and farms. These recommendations have basically involved managerial practice designed to minimize further host contact with sources of infection and the use of different disinfection procedures to destroy infective oocysts. Infected individuals should be identified and isolated to confine infections to particular areas which can be regularly cleaned and disinfected. Susceptible individuals should avoid contact with contaminated areas or to be removed to safe locations. Care should be exercised in the handling and disposal of biohazardous waste and suspected contaminated water should be boiled prior to consumption or use. Cryptosporidium species can be removed from drinking water by either boiling for 1 min or filtering through a filter with a pore size of less than 1 μm60. Immunosuppressed persons should avoid contact with animals with diarrhea, dogs or cats younger than 6 months of age and stray animals61. The examination of an animal’s stool by the veterinarian before the person has contact with the animal is critical for those at risk of infection.
Laboratory studies have shown that oocysts stored at aqueous solutions have remained viable for up to 3 months at ambient temperatures (15-20°C) and for up to one year when refrigerated (4-6°C). Infectivity was lost after oocysts had been heated to 65°C for at least 30 min or when desiccated for at least 4 h (Table 2). Snap-freezing has been shown to kill oocysts, whereas slow freezing was less effective and some oocycts have survived freezing at -22°C for up to 1 month62.
Like many other coccidian oocysts, those of Cryptosporidium have proven remarkably resistant to chemical disinfection. Laboratory studies have shown that many commercial disinfectants (based on aldehyde, ammonia, alcohol, chlorine or alkaline compounds) are ineffective when used according to the manufacturer’s instructions.
| Table 2: | Disinfection procedures reportedly effective against Cryptosporidium oocysts32 |
| *Chlorine dioxide-based cold sterilant (Alcide Co.), **Two-phase product producing ammonia (Antec International Ltd) | |
In some cases, higher concentrations and longer exposure periods have been found to kill oocysts but the prolonged use of concentrated disinfectants in confined areas is inadvisable or impractical in many situations. To date, over 35 disinfectants have been tested but only 5 have been found to be effective following relatively short exposure periods; 50% ammonia, 3% hydrogen peroxide, 10% formalin, Exspor and Oo-cide (Table 2). Steam heat sterilization and fumiogation with formaldehyde or ammonia gas have also been recommended as appropriate forms of decontamination23,63.
Recent studies have indicated that most conventional methods of water treatment do not effectively remove or kill all Cryptosporidium oocysts from contaminated water. Laboratory simulation of different water treatment processes has shown that some oocysts can survive treatment with chlorine, monochloramine, lime, polyelectrolytes, alum and ferric sulphate64. Because chlorination does not inactivate Cryptosporidium, alternative disinfectants such as ozone and UV radiation have being evaluated. Low doses of UV (1-9 mJ cm‾2) can inactivate 2-4 log10 (99-99.9%) of C. parvum oocysts65 and low doses (2 mg L–1) of ozone for short times (1 min) are able to inactive up to 99% of oocysts66. The percent inactivation rate of free chlorine dioxide disinfection for Cryptosporidium is 90-97% using higher level (60-80 mg for 1 min L–1)64. Quantitative studies on the treatment of wastewater and sewage have shown that 74-84% of oocysts are removed by activated-sludge systems and 87-99% by subsequent sand filtration67. Sand filters used to treat drinking water and swimming pools have been found to remove 91% of oocysts68. Alternative to conventional water treatment, pressure-driven membrane processes, microfiltration (0.1-10 μM pore membrane), ultrafiltration (0.002-0.1 μM pore membrane), nano filtration and Reverse Osmosis (RO) have proven complete removal of all protozoan cysts69,70. As a result, these membrane technologies are playing a major role in drinking water production in the US and Europe71. In the US, the Centers for Disease Control and prevention (CDC) reported that only RO or less than 1 micron filters are effective for the removal of Cryptosporidium oocysts and advise the exclusive use of bottled water processed under those treatments72.
CONCLUSION
Preventive hygienic measures are by far the most effective approach to control this parasite in farm animals, the objective being to destroy the external forms of the parasite (infective mature oocysts) and to prevent their transmission among animals. The destruction of oocysts on surfaces of housing facilities, pens and parturition buildings is possible using effective disinfectants such as 50% ammonia, 3% hydrogen peroxide or 10% formalin. Measures to reduce transmission between animals should be encouraged. Limiting the number of animals enclosed in the same facilities and avoidance of high stocking rates in the parturition area, maintaining a short calving period, administration of appropriated supplies of colostrum especially hyper immune, colostrum from immunized dams, isolation and treatment of diarrheic infected animals; all help to prevent outbreaks of cryptosporidiosis, reduce the spread of infection within a herd and minimize mortality and morbidity in infected herds. Chemotherapeutic agents such as paromomycin, decoquinate, lasalocid, halofuginone lactate, nitazoxanide, dinitrooryzalin, β-cyclodextrin and probioties have proved a potential therapeutic effect against cryptosporidiosis in the form of reduction the duration and numbers of oocysts shedding and the incidence and severity of the diarrhea. Immunization of ruminants during pregnancy with either recombinant C. parvum sporozoites surface antigens or plasmid DNA encoding the CP15 or CP23 antigens appear to be a valuable approach for producing colostrum for the passive immunotherapy of cryptosporidiosis. Hyper immune, colostrum prevented diarrhea and reduced oocysts shedding in newly born calves, lambs and goat kids exposed to Cryptosporidium infections.