on detection of oocysts in faeces. Several chemical staining techniques such as Modified Ziehl-Neelsen (MZN) and immunofluorescence assays (IFA) have been reported (Fayer and Xiao, 2008; Grinberg et al., 2002; Holland, 1990; Johnston et al., 2003). However, among the microscopy techniques, IFA has provided improved sensitivity and specificity compared to other conventional staining techniques (Arrowood and Sterling, 1989; Quilez et al., 1996), particularly for detecting low numbers of oocysts, in the samples. Therefore, it has been recommended that prevalence investigations should benefit from immunofluorescence methods, as many asymptomatically infected animals may shed low numbers of oocysts (Quilez et al.,
16 1996). Furthermore, detection of Cryptosporidium antigens by enzyme-linked immunosorbent assays (ELISA) has also been documented as a diagnostic method for cryptosporidiosis (Fayer and Xiao, 2008; Grinberg et al., 2002; Holland, 1990). This method also showed less sensitivity and specificity, however, when compared with IFA. This could be due to the presence of soluble Cryptosporidium antigens ingested from the environment, rather than antigens from cycling parasites (Johnston et al., 2003; Van Zijll et al., 2010). Overall, IFA test for Cryptosporidium is in a way considered the ‘‘gold standard’’ (Chalmers et al., 2011; Van Zijll et al., 2010), as it detects entire oocysts rather than soluble antigens or naked DNA. Since oocysts might not be detectable in clinical samples in all cases, especially in the prepatent or the end of the patent period, PCR-based techniques might be more sensitive (Fayer and Xiao, 2008; Thompson et al., 2008), but these techniques are relatively expensive, and their ability to differentiate infections from naked Cryptosporidium DNA is not well understood.
The control of bovine cryptosporidiosis on farms is problematic, for a number of reasons. Unlike other coccidian parasites, C. parvum does not require particular environmental conditions to become infectious, as its oocysts are excreted sporulated and fully infectious (Smith et al., 2005). Calves can excrete hundreds of millions of oocysts in the faeces (Grinberg et al., 2002; Naciri et al., 1999), and considering there is a high probability of infection with a dose as low as 50 oocysts (Moore et al., 2003), an infected calf could produce enough oocysts to infect thousands of new animals. A number of compounds, such as halofuginone lactate (HL) (De Waele et al., 2010; Jarvie et al., 2005; Klein, 2008; Lefay et al., 2001; Trotz-Williams et al., 2011), paromomycin sulphate (Fayer and Ellis, 1993; Grinberg et al., 2002), nitazoxanide (Ollivett et al., 2009; Schnyder et al., 2009) and decoquinate (Lallemond et al., 2006; Moore et al., 2003) have been tested for the prevention of cryptosporidium infection in calves, with variable results. HL is a synthetic derivative of a quinazolinone alkaloid with cryptosporidiostatic activity, but its mode of action is poorly characterised. In naturally and experimentally infected calves, the oral administration of 60 μg/kg HL for seven consecutive days from the first day of life delays the onset of oocyst shedding, reduces the number of oocysts excreted, and lowers the severity of diarrhoea (Jarvie et al., 2005; Joachim et al., 2003; Klein, 2008; Lefay et al., 2001; Trotz-Williams et al., 2011; Villacorta et al., 1991). A
17 formulation containing HL (Halocur, Intervet Ltd., Republic of Ireland) is currently the only prescription drug registered for the prevention of cryptosporidiosis of calves in several countries, including New Zealand. The use of HL has a number of limitations, including a substantial market price and a narrow therapeutic index, with toxicity observable at approximately twice the recommended dose (Villacorta et al., 1991, Naciri et al., 1993 and Trotz-Williams et al., 2005; http://www.msd- animal- health. co.uk/ products_public/halocur/090_ product_ datasheet.aspx, accessed 15 July 2012). Furthermore, notwithstanding the frequent occurrence of co-infections with other enteropathogens in the field (De la Fuente et al., 1999; Naciri et al., 1999; Tzipori et al., 1980), the utility of HL in the presence of such co-infections is not well understood as, with some exceptions (Klein, 2008 and Lefay et al., 2001), most anti- Cryptosporidium efficacy studies of HL did not analyse or take into account the presence of co-infections. Co-infections might modify the anti- Cryptosporidium effect of HL in various ways. The increased fluid content and intestinal motility determined by the presence of the co-infecting pathogens may reduce the activity of HL by dilution, or by reducing the transit time of the drug in the intestinal tract. Furthermore, enteric infection with BRV or other agents may cause exfoliation of infected cells, altering cellular function (Ramig, 2004), potentially enhancing the toxicity of HL via systemic absorption. Chapter 3 reports a randomised-controlled field study of HL performed on a farm co-infected with BRV and Salmonella Typhimurium.
Vaccination has been also suggested for the prevention of calf cryptosporidiosis. As C. parvum infections are acquired perinatally, vaccination of calves does not seem a feasible option. Nonetheless, a vaccine against C. parvum would complement the already available range of vaccines against neonatal enteropathogens administered to cows during the last trimester of pregnancy, which include BRV, BCV and K99. Further, the use of a vaccine would prevent the development of drug resistance compared to the use of drugs (although such resistance has not been reported), and would be more environmentally friendly. Experimental vaccination trials against C. parvum have been performed using whole oocysts, subunit vaccines, or DNA vaccines (Burton et al., 2011; Harp and Goff, 1995, 1998; Jenkins et al., 2004; Perryman et al., 1999). For example, young calves fed colostrum obtained from dams vaccinated a few weeks before parturition using C. parvum oocysts showed partial
18 improvements in the clinical outcomes, but vaccination did not eliminate oocyst shedding (Fayer et al., 1989). In an active vaccination trial where newborn calves received an oral preparation of lyophilised inactivated C. parvum oocysts, a partial protection against experimental C. parvum infection was observed (Harp and Goff, 1995), with reduction of the diarrhoea and oocyst shedding periods in the vaccinated animals. However, the same vaccine failed to induce protection when tested on a large dairy farm with severe endemic C. parvum infection (Harp et al., 1996). To date, no effective commercial vaccine against C. parvum infection in calves is available, the main reason being the difficulty in in vitro cultivation of the parasite.