A Standardised Blend of Plant-Derived Isoquinoline Alkaloids (IQA) in Laying Hens Mitigates the Impact of Campylobacter Hepaticus (Spotty Liver Disease) Challenge

Spotty Liver Disease (SLD) is characterised by increased mortality, particularly around the time of peak egg production, the occurrence of multiple grey/white lesions in the liver, and reduction in egg output. It is caused by Campylobacter hepaticus (Van et al., 2016), which responds to therapeutic antibiotics, although resistance has been reported (Grimes and Reece, 2011). In this study, the ability of a feed-additive containing a standardised blend of isoquinoline alkaloids to modify the progression of SLD in hens exposed to C. hepaticus was assessed. Before and after exposure of the treated birds to C. hepaticus, production parameters were measured. A necropsy examination of the hens was performed to assess the degree of liver damage as an indicator of disease caused by C. hepaticus.

Ethics approval: WSIAEC – 19.17. One hundred and thirty-two 21-week-old Hy-Line laying hens were distributed into: Negative control group (NC, 28), not treated and not exposed; positive control group (PC, 36), not treated and exposed; low dose IQA (LD-IQA, 100 mg of product/kg of feed or 1.4 ppm of IQA, 32) and high dose IQA (HD-IQA, 200 mg of product/kg of feed, equivalent to 2.8 ppm of IQA, 36). Hens were free of C. hepaticus. Each group was subdivided into “Short” and “Long”, determining the time of autopsy post-exposure; 6 days for the Short and 29 days for the Long groups. Short groups were used to assess the liver lesions of the hens, while Long groups were used to compare production parameters between groups (χ2 and two-sided Fisher’s exact test). Hens were fed with the phytobiotic supplemented diets for a period of 28 days. After that period, hens were orally exposed to a broth containing 1 × 10⁹ cfu of the C. hepaticus strain HV10^T, as described by Van et al. (2017), or sterile broth in the case of the NC group.

Egg weights are presented in Table 1. The egg-weights increased over time in the NC group. The egg-weights in the PC group decreased significantly between 3 and 6 days after exposure (DAE; P = 0.02). There was a recovery in the egg-weight at 14 DAE (4.12%). However, eggweights were not significantly higher compared with 3 and 6 DAE (P = 0.86 and 0.07, respectively). In the LD-IQA group, the egg-weight had no significant change between 3, 6 and 14 DAE. HD-IQA group egg weights increased numerically between 3 and 6 DAE and increased significantly by 14 DAE compared with 3 DAE (P = 0.01) and 6 DAE (P = 0.03), respectively. Diets with 2.8 ppm IQA prevented the egg-weight loss in SLD challenged birds.

Table 1 - Average egg weights per group and their percentage of change between different sampling days.

Egg production was significantly affected by SLD (PC versus NC groups, Table 2). Production was significantly higher in the HD-IQA group but not different in the LD-IQA group compared with PC group. The high dose of IQA was able improve egg production after a SLD challenge.

Table 2 – Number of eggs expected for each group to be produced after exposure calculated using the egg production data before exposure.

During postmortem examination, an estimation of the number of liver lesions and lesions score was made. Results are summarised in Figure 1. There were no lesions present in the NC group. The average numbers of lesions ± SD in the PC and LD-IQA groups were 201.1 ± 327.6 and 183.6 ± 318.4, respectively, being significantly higher compared to the NC group. The average number of lesions in the HD-IQA group was 41.7 ± 67.3, which was not significantly higher than the NC (P = 0.22), but not significantly lower than the average number in the PC group. Similarly, for lesion scores the median liver lesion score of the hens from the HD-IQA group was not significantly higher than the NC group, although not significantly lower compared with the PC group. The median scores of both the PC and LD-IQA groups were statistically higher compared to the NC. However, to include the negative control in the comparison is misleading, as the number of liver lesions in that group was zero. When the NC and LD-IQA groups are excluded from the calculations, and only the HD-IQA and PC groups are compared, the difference between the number of lesions was statistically significant (P = 0.04), but not the SLD liver scores (P = 0.11, Figure 1).

These results demonstrate that the phytobiotic produced a reduction of the pathological changes in the liver induced by SLD. Previous field reports describe the use of two phytobiotics (oregano and isoquinoline alkaloids) on multiple free-range operations which led to a trend of reduction in the incidence, the age of onset and severity of SLD clinical signs (Scott et al., 2020). The positive effect of diets containing isoquinoline alkaloid extracts in reducing the negative impact of gut bacteria has been previously described (Xue et al., 2017).

Figure 1 - Summary of the analysis of the livers from the post mortem analysis of the hens of the Short group, euthanised 7 DAE to C. hepaticus (PC and HD-IQA groups only). A, Columns, average number of lesions with SD (error bars). B, Central line on each rectangle, median score; margins of the box, interquartile range; external lines, minimum and maximum values. *, P < 0.05; ns, not significant

All hens had a modest increase in the average feed consumption during the first week after the exposure (Figure 2A). However, the increase was more prominent in the NC compared with the other three groups. In all Long groups, there was an increase in the average feed consumption post-exposure. HD-IQA was the only group where the increase in feed consumption at 29 DAE was significantly higher (Figure 2B). This could indicate an increase in the palatability of diets containing the phytobiotic.

Figure 2 - Average feed consumption by group and sampling period. Each column represents the average feed consumption from each group, and the black lines represent the SD. BE, Before exposure; DAE, days after exposure. Ns, not significant. **, P < 0.01.

Hens were blocked by weight at the start of the study so there were no differences between treatment groups. Hens fed with the higher dose of IQA exhibited a significant increase in feed consumption between pre-exposure and 29 days post exposure (not seen in the NC and PC groups), and a significantly higher final body weight compared with the NC and PC groups (Figure 3). It can be concluded that IQA produced an increase in the appetite of the hens. A previous study related some beneficial effects obtained by phytobiotics with an enhancement of feed intake, improved nutrient digestion, increases in digestive enzymes and a greater absorption of nutrients in the intestines (Abudabos et al., 2018).

Figure 3 - Weights of the hens from the Long groups at the beginning and the end of the study. *, P < 0.05.

Some dose-response to IQA concentration was observed in the present study with egg-weights immediately post-challenge, lesion scores and lesion numbers improved in the HD-IQA group compared to the LD-IQA group, indicating that inclusion rates may be critical in obtaining the benefits seen in this study when this phytobiotic is included in commercial layer-feed.

Feed rations containing the IQA at a dose rate of 200 mg/kg of feed or 2.8 ppm (1 ppm of sanguinarine and 1.8 ppm of other alkaloids) was capable of reducing the negative impact of SLD in layer hens, including higher egg weights and production, a reduction in the number of liver lesions, and an improvement in the weight of the hens and feed consumption. Further field and laboratory exposure studies should be undertaken to better define the benefits of this feed additive in ameliorating SLD and the mechanisms of action.