For various reasons, the bTB and E. coli studies were under-powered. There were also limitations relating to the sensitivity and interpretation of the diagnostic tests used.
Consideration of some of the difficulties encountered may increase the likelihood of gaining useful results in future studies.
7.4.1 Reaching the required sample size
The bTB case control and cross sectional studies both failed to reach the required sample size, in spite of considerable efforts made to recruit participants (chapter 3). A previous case control study on naturally exposed animals in the UK also failed to reach the required sample size (Claridge, 2012). The restrictions applied under the bTB control programme and the stigma surrounding this made recruitment of farmers difficult. Other research on co- infection with liver fluke and bTB in individuals has either been performed in laboratory animals (Flynn et al., 2009, 2007; Garza-Cuartero et al., 2016) or in settings where
restrictions do not apply such as in developing countries (Ameni et al., 2000; Munyeme et al., 2012) or prior to the introductions of bTB restriction in Europe (e.g. Ljesevic, 1957; Meyer, 1963). The exception was a study performed by DEFRA (2005), where access to information on bTB infected herds and samples from bTB test reactor animals were available due to the fact that the study was performed by the state animal health service. The E. coli study was under powered due to the very low levels of fluke seen. This was despite a sample size calculation having been conducted and published (Hickey et al., 2015). Difficulties resulting from adding our study onto the FSA study meant that resources were not ready in time, meaning that 35% of the samples were not available to us.
Secondly, fluke prevalence in the tested samples was much lower than anticipated, at least partly because the feasibility study did not take into consideration the low sensitivity of the copro-antigen ELISA compared to the antibody ELISAs on which prevalence estimates were based (Duscher et al., 2011; Salimi-Bejestani et al., 2005a). Another reason was that the study population was finishing cattle, which as a group are less likely to graze outside than other groups, and may be more likely to be proactively treated for parasites to maximise weight gain, and therefore may be less likely to have fluke than the populations of dairy cattle on which prevalence studies have been done (Howell et al., 2015; McCann et al., 2010). This study was intended to be a pilot study, using samples obtained through another study in order to save resources. However, the problems encountered demonstrate the importance of designing a study to answer the particular question of interest.
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7.4.2 Diagnostic test sensitivity
In chapter 3 and 5, diagnosis of fluke, MAP and bTB was via the immune response rather than direct identification of the pathogen: antibodies in the case of MAP and fluke, and the cellular response in the bTB skin test. The antibody titres give an indication of the burden of infection, but vary widely between individuals (Graham-Brown, 2016). They may give a more accurate indication of the immune response of the animal than absolute parasite numbers, but it may not be straightforward to tell the difference between protection and exposure (Bradley and Jackson, 2008). Direct identification of bTB and fluke is difficult in live animals, as faecal egg count (FEC) has low sensitivity and bTB infected cattle, in the early stages, are often not infectious and therefore the diagnostic methods used in humans, such as examination of a sputum smear, are not useful (DEFRA, 2005).
In general, error in the independent variable is likely to bias the effect size seen in a regression model towards zero, so determining the true infection status of an animal is important to accurately estimate the effect of risk factors (Schennach and Schennach, 2012). The relatively poor sensitivity of the bTB skin test can be mitigated to some extent by performing different tests in parallel, but as bTB testing in the UK is controlled by DEFRA, this was not possible in our study.
The main limitation of the MAP study concerns the poor sensitivity of the test used to detect MAP, although this is considered to be the most sensitive test available (Caldow et al., 2009). The ELISA is only suitable for detecting cattle in the later, clinical stages of disease. In the clinical situation, the test is used alongside other information such as age and clinical signs, and tests are interpreted as one of a sequence, with the animal coded red, orange or green, depending on the results of the entire series . In our study, follow-up was only possible for 1 year, but given the long incubation period of MAP, a longer follow period would be preferable.
In the E. coli study, direct diagnosis of both pathogens was performed, with no measures of immune response. Due to the fact that the study was an add-on to another study, the samples available were faecal samples, which were tested for fluke using the copro-antigen ELISA. This has a similar, or according to some studies, lower sensitivity than FEC (Brockwell et al., 2014, 2013; Duscher et al., 2011). Both only detect current, patent infections, which accounts for some of the reduced sensitivity compared to antibody tests that become positive early during infection and remain positive for some weeks afterwards (Brockwell et al., 2013). However, even when used on cows with patent infections, test sensitivity is low.
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One study reported that only 30% of seropositive animals were identified, compared to 45% of animals that were detected by faecal egg count (Duscher et al., 2011). There has also been debate about the cut-offs used. Some studies reported a higher sensitivity for the copro-antigen ELISA but used lower cut-offs than recommended by the manufacturer (Brockwell et al., 2013; Charlier et al., 2008). In our study, in the absence of any alternative method of fluke diagnosis, we did not alter the cut-off.
Whilst use of a poorly sensitive test may acceptable in clinical cases, where results are interpreted in conjunction with farm history and clinical signs, a sensitivity this low is probably not appropriate for epidemiological studies where no supporting information is available, as the increased error is likely to cause an unacceptable loss of power.
The pattern of E. coli O157 shedding is known to be intermittent, and in groups of animals where one is found to be shedding, one or most are thought to be infected (Robinson et al., 2009). The original FSA study was designed to determine whether a group was infected, and thus the intermittent nature of shedding was not a problem, but a single negative E.
coli test does not determine that an animal in uninfected.
7.4.3 Other confounding factors
In these studies, the amount of data available on confounding factors was limited. This was due to the anonymous nature of the sample collection in the MAP study and the bTB case control study. For the E. coli study, information was available but only at the group level. For the bTB cross sectional study, information was collected but the dataset was not big enough to allow its inclusion in the model.