Updated: Information of Disinfectants:  17 July 2017

Introduction:

Numbers of reviews state that Cryptosporidium is difficult to kill. This applies to municipal water treatment for human risk management and the farm shed to protect and prevent clinical cryptosporidiosis for calves. Cryptosporidial oocysts are at least 15 x more resistant than Giardia to disinfectants. In mild infections without scouring, the output of infective stage oocysts is still far above an infective dose. In fact there is still a possibility that non-detectable and inapparent infections are occurring because of the insensitivity of the tests systems. With a scouring calf there are massive numbers of oocysts produced.
Therefore the activity of any active ingredient of a disinfectant requires to be

  • very high in the face of such a challenge when applied for control.
  • ideally it should be safe to use, and
  • environmentally ‘friendly’.

Assessments of Performance

Assessment of effectiveness against the infective oocyst may be made by a number of in vivo, and/or in vitro tests. Different results in performance can arise by using different techniques.

Establishing a relative performance for each ingredient, leads to studies on

  • each concentration recommended,
  • a comparison of the time of contact for the relevant concentration.
Brand names Ref Active ingredient Availability Comments
Neopredisan 2-4% Keidel & Daugschies 2013 p-chloro-m cresol Not available in New Zealand
KenoTMCox 2-3% Naciri et al 2011 Amine based formulation Not available in New Zealand
Ox-Virin 10% Quilez et al 2005 25% hydrogen peroxide, 5% peracetic acid An alternative brand formulated with the same active ingredients is available in New Zealand Dangerous goods with strict guidelines on Protective Clothing
Peroxide 3% (No brand) 6% is recommended for higher performance

Comparison of Disinfectants and Methods of Decontamination:

Chemical Disinfection

Against this background there are a number of brands which have been assessed for efficacy in the laboratory using in vivo and in vitro methods. A number of active ingredients have not performed to give any assurance of effective disinfection when faced with the challenge of very high numbers of oocysts in a calf shed.
One independent group which has checked research and reached some conclusions is Moredun Research Institute in Scotland (Hotchkiss et al 2015). Their nominated brands do not relate to what is available in New Zealand, but it does emphasise how difficult it is to transfer benchtesting to actual live field performances in the face of an outbreak.
None of the products above are recommended be used in a shed with livestock, but they are used for shed disinfection. All require protective clothing including masks.
Others options are based on ammonia and hydrogen peroxide provided they are used at the correct concentration and given the appropriate contact time. Safety measures will be required for these products as well.

Physical methods:
  1. Steam cleaning is safe and effective.
  2. Decontamination with water blasting can be an important first step in disinfection, but in a calf shed this is not suitable if the bedding becomes wet. It may be suitable for grated or concreted areas on a regular basis e.g. daily or every second day when calves are present. Though mists created by high pressure hosing may create a problem of vaporisation and spread of oocysts over the surrounding areas.
  3. Drying of calf faeces (spread out thinly) for over 3-4 days is effective in preventing oocyst survival (Anderson 1986).
  4. Temperature effects are overridden by moisture, moisture being protective esp with colder temperatures, with months recorded of survival in damp and cool conditions. Warmer temperatures over 15 degrees reduces infectivity to over 2 weeks, or faster with higher temperatures.
Other (NZ) active Ingredients
Brand names Supplier Claim
Vetsan Vetpak “Stabilised” chlorine dioxide Amount used and contact time varies. Has recommendations for use in calf shed. Stabilised activity claims has yet to be measured against the gold standard of mice infectivity testing.
Swift Ethical Agents Chlorine dioxide (2 pack) Must be used fresh, and is extremely pungent. Unable to be used in shed with calves present Traditional source of chlorine dioxide.

Other active ingredients such as chlorine dioxide are sometimes recommended than those above but these fail to be listed as preferred products. There are varying opinions on their usefulness.

Below is a summary of research reported in the literature:
Chauret et al 2001 reports for example on tests completed on purified oocysts originating from three different suppliers. Results showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Contact time values of 75, 550, and 1,000 mg . min/liter were required to achieve approximately 2.0 log10 units (lowering by a hundredth) of inactivation with oocysts from different sources.

References in the literature often refer to them performing well in the face of lower numbers of challenge oocysts. Their performance when there are higher levels of oocysts challenging doses as found in a contaminated calf shed with endemic cryptosporidial scours may not be the same however.

Liyanage et al., (1997) determined that it is the un-dissociated chlorine dioxide that is the main anticryptosporidial component. The component parts do not have any significant effect, against the cryptosporidial oocyst e.g. chlorite, chlorates or chlorides. Given the instability of the chlorine dioxide and the narrow pH at which it is present/active, the speed at which it becomes inactivated, claims of stability need to be substantiated in a critical manner, using the gold standard of mice infectivity studies, and at varying concentrations and over variable contact times with varying number of oocysts of known infectivity in mice.
One report on chlorine dioxide by Chauret (2001) summarises a number of trials (11) and assesses the contact time, the degree of inactivation, and the test method used. It shows that for a lowering by a hundredth or a thousand, a contact time of more than 2 – 6 hours was generally required especially when the mouse infectivity gold standard was used. Cell culture tests used as an alternative test method demonstrated a wider variety of contact times. Note: it was not possible to lower oocysts to a useful degree of over 1000 fold reduction, which is the useful measure of disinfection for cryptosporidial oocyst control. A threshold of 99.5 % inactivation is proposed to evaluate disinfectants in vitro using C. parvum as model organism (Dresely et al, 2014). On this basis chlorine dioxide would fail to be classed as a disinfectant.
Trial work takes into account a large amount of information and for each comparison, the same conditions may not be able to be compared with a second trial result. For completeness the comparisons made include the following as reported by Casemore & Watson

Physical Variables Chemical Variables Study Design Variables
Temperature during test exposure Disinfectant type and test concentration; method of measuring concentration (e.g. start, end, integrated) Oocyst source and strain characterisation
Test system pH Disinfectant neutralisation prior to viability/infectivity testing Oocyst clean up method, age and storage conditions.
Contact time Composition of disinfectant (e.g. presence of surfactants or other agents in test product) Oocyst test concentration used; accuracy of counts
Effect of physical degradation and environmental stress on oocysts. Denaturing/dissociation of disinfectant during contact period; method of measuring Method of assessing efficacy – in vitro (excystation, sporozoite ratio, cell culture), infectivity in vivo.
Nature of test medium and organic load*. Method and timing of measuring disinfectant concentration (batch vs continuous, etc).

Conclusions made by such summaries include

*Size of the oocyst inoculum: the CT (Contact Time) value may be greatly affected by the number of oocysts present at the beginning of exposure to a disinfectant. This appears to have been orders of magnitude higher in some studies, e.g. ozone was reported to be the least effective of all the studies. Numbers of oocysts present may markedly affect the dynamics of disinfection.

Measuring Oocysts Viability and Infectivity

Background:

The load of oocysts is highly variable in an infected calf shed. Identifying how much ‘risk’ actually present is difficult.
The oocysts identified as present also vary in viability. The number required to infect and lead to clinical scours also varies with genotype strain and it also changes as oocysts age and lose infectivity. This is partly from their exposure to the environment. Temperature and moisture are critical conditions determining survivability and infectivity. Faeces dried to the air require only 4 days to be become non-viable. Wet and colder conditions lead to extended periods of infectivity of over 6 months.
The gold standard to assessment of viability is infecting newborn mice and measuring infection intensity in the intestine.
In vitro (without using mice) have also been reported and have been compared with mice infectivity studies. In vitro tests underestimates the loss from disinfection procedures when compared with the gold standard (mice testing).
Because numbers of oocysts in the calf shed are going to be extremely high – the efficacy of any disinfection needs to be at an extremely high level of over 99.9%, otherwise there will be sufficient levels of oocysts left to provide an infective dose.
Expectations that this can be achieved while calves are in the shed are unrealistic with the known chemicals available that can achieve this efficacy.

Young calves are highly susceptible to becoming infected and expressing the clinical signs of scouring with loss of appetite. This is emphasised when considering the fact that some young calves get infected with as few as 10 oocysts (50% of a group of calves: 60 oocysts). The level at which cows are excreting is at a level which is lower than the sensitivity of the commercial laboratories to detect oocysts. Less orthodox test protocols are required to concentrate the oocysts in adult cow pats (dung) before counting them.
Part of the variability in the level of oocyst infection required to infect a calf is thought to be related to the genotypic strains that exists between Cryptosporidium parvum isolates.
The very young calf is a very sensitivity indicator that oocysts are present, being infected with doses that are lower than the sensitivity of the tests used to measure the presence of oocysts.

Doing some numbers:

This is especially relevant when assessing how effective a disinfectant or hygiene programme is. In the calf infection with scouring, their multiplication in the gut leads to massive numbers and output of greater than 1 x 106/g (and higher) of dung. The level of output in the cow at parturition with a partial post-parturient rise is about 400-500 oocysts per gram of dung. This is still below the sensitivity of the test (microscopic examination) which is about 1000 oocysts per gm of dung. Other tests can go as low as 100 oocysts (using immunofluorescent methods).  The calf in theory requires about 1/7th g of its dam’s dung to become infected. Detecting the excreting cow is currrently not available commercially.

Conclusions

  • It is unrealistic to expect disinfection to lower the level of infective oocysts in an infected calf shed. The transition shed may be the most suitable for change.

a) Physical removal of dung requires concrete surfaces to be sprayed (physical decontamination) down daily or twice daily, before signs of scours shows.

b) Concentrate efforts in the first 5-10 days especially the first 2 – 3 days in the calf rearing environment.

c) Early detection and separation into a hospital concreted/grated area. Get them when they are depressed and before they scour. Spend time watching for calves separating and becoming isolated. Swiss studies with one strain showed that only about 45% of infected scouring calves will lose their appetite. All of them require to be identified and treated with KRYPTADE for 3 days as on the label.

d.) If there is no transition area for calves EXAGEN is recommended to ensure numbers of oocysts excreted are capped at very low levels. It is not an alternative to low standards of hygiene and cleanliness of feeding utensils. Use EXAGEN twice daily for a minimum of 3 days at the start of the season. If scours becomes established in the calf shed e.g later in the season, this should be increased to up to 10 days or longer.

e) While twice daily split dosing of 25 g with EXAGEN is routinely recommended as optimal, there is another option for higher level control. This is aimed to further decrease cross infection and more effective prevention of new infections by splitting the daily dose into three; i.e EXAGEN three times (5 g for very small calves, or 8 g for larger calves.) eight hours apart if management can be adjusted

  • Because the cow is being recognised as important source of calf infections, the first calf case each season may be an important indicator of cow-calf transmission, keeping good records is aimed to determining which cow may have been responsible for the first infection (and the start of the scouring).
  • Have a broad understanding that disinfection and hygiene standards will include keeping out all visitors/strangers who may carry crypto into your unit. It includes your own risk factors such as clothing and footware as carrying infection/contaminants. If you practice limiting visitors, then enact protocols that address your own risks of transmission within your own unit. Create ‘barriers’ that identify where extra precautions and isolation start applying. Foot baths with scrubbing including footware changes, protective clothing. Clothing and footware spelling 3 – 4 days in sunlight/in warm conditions (winter allowing) before re-using  allows for natural disinfection and reduces any oocysts surviving on footware and clothing. Be critical of your own standards, and be prepared to be strict to ensure that your protocol reducing transmission is being implemented. Just because it doesn’t appear to be practical don’t discard an option. It may be the critical factor making your risks lower.

Watch for developments in cow detection methods to identify carriers.

Be prepared to consider EXAGEN from the first born calf coming into the calf unit especially through the transmission area, to maintain lower levels of oocycst output from inapparent infections. This product is oocystocidal meaning that there is no bounce back excretion of oocysts once treatment is stopped as with halofuginone lactate

Bruce Pauling
4 July 2017

Disinfectants for Practical Cryptosporidium Management