Spotty liver disease in poultry

What do we know about spotty liver disease (Campylobacter hepaticus) in poultry?

In 2019 the first infection of Campylobacter hepaticus responsible for spotty liver disease (SLD) has been proven in a flock of Dutch laying hens (Molenaar 2019). However, the disease is not only emerging in the Netherlands, a fact proven by the publication of several articles on this subject in the recent years. This news article provides you with the practical information available so far.

History

The disease has already been described over 60 years ago and was mainly seen in the USA, UK and Germany since then. It gained increased interest when the number of outbreaks increased, starting in Australia. It was noticed that the incidence increased in line with an increase in birds in kept free range and in barns, instead of in cages (Van et al. 2017a).

Crawshaw et al. published about the identification of a novel Campylobacter strain in 2015. They described the isolation, biochemical, structural and molecular characteristics of the bacterium, but did not determine a name yet (Crawshaw et al. 2015).

Van et al. later also identified the same unknown Campylobacter species causing spotty liver disease in commercial chickens in Australia. Phylogenetic analyses based on the 16S rRNA gene and the heat shock protein 60 (hsp60) gene sequences indicated that the strains formed a uniform cluster that was clearly distinct from recognized Campylobacter species. When the average nucleotide identity was calculated, the new strains had a 99% concordance, but showed less than 84% similarity with the nearest sequenced species. A similarity of less than 95% indicates that the bacteria are from a different species, so Van et al. then proposed Campylobacter (C.) hepaticus as new name for this bacterium (Van et al. 2016).

The same group later proved that this bacterium is much more invasive to LMH cells (a chicken liver cell line) than other Campylobacter species. Besides, they showed that SLD could be induced by infecting mature layers orally with C. hepaticus and that the bacterium could be isolated from the liver and bile of these animals. They hereby fulfilled the postulates of Koch and proved C. hepaticus is the causative agent of SLD (Van et al. 2017a).

The bacterium

The most important characteristics of C. hepaticus are (Van et al. 2016):

• bacterial morphology:
  • S-shaped;
  • contains long flagella at both poles;
  • motile;
  • 3-0.4 μm wide and 1.0–1.2 μm long after 3 days of incubation on HBA (horse blood agar) in a microaerophilic atmosphere at 37 °C;
  • Gram-negative;
• colony morphology:
  • wet;
  • cream colored;
  • convex or flat and spreading;
• biochemical characteristics:
  • non-hemolytic;
  • catalase positive;
  • oxidase positive;
  • urease negative.

In figure 1 you can see the morphology of C. hepaticus (Van et al. 2016).

A whole genome sequence has also been performed on C. hepaticus. This confirmed that this bacterium is most closely related to C. jejuni and C. coli, but still a distinct group (Petrovska et al. 2017). The phylogenetic tree is shown in figure 2.

Infection

SLD occurs mainly in laying hens in free range systems, but has also been found in laying and breeding hens in barns and cages (Molenaar 2019; Van et al. 2016).

It is expected that birds get SLD after they get infected via the fecal-oral route with C. hepaticus. The bacterium is present in the gastro-intestinal tract of infected birds, and viable bacteria can be isolated from chicken faeces (Phung et al. 2020; Van et al. 2017a; 2017b).

C. hepaticus DNA has been identified in wild birds, rats, mites and flies. It has also been shown in water and soil from infected farms. It is however unknown if these are also vectors for viable C. hepaticus organisms. This means that more research is needed to determine their possible role in transmission and introduction on the farm (Phung et al. 2020).

Australian research has showed that the moment of infection does not have to correlate with the moment of mortality or lesions found at necropsy. Chickens can be infected with C. hepaticus up to eight weeks before SLD manifests itself clinically. A case has been described were birds were already infected during rearing (week 12). This implicates that an infection with C. hepaticus is not enough to cause SLD, but that other predisposing factors should also be present. Proposed predisposing factors are liver metabolism during peak production or changes in the gastro-intestinal microbiota (Phung et al. 2020). More research is needed to determine these factors.

Infections occur mainly during peak production, but are not limited to this period. Besides, after the initial infection at the peak of lay, further outbreaks can occur in the same flock at later ages (Phung et al. 2020).

Clinical disease

Flocks are often affected during peak production, but this can occur all year round (Molenaar 2019; Phung et al. 2020).
Flocks with SLD have higher mortality with acute deaths (Molenaar 2019). The mortality can be increased with more than 1% per day (Van et al 2017a) and can reach up to 10% in total (Phung et al. 2020).
In some flocks the egg production is decreased, with maximum decreases of 25% (Molenaar 2019; Phung et al. 2020). Sick animals are not always observed, due to the rapid death of affected animals (Molenaar 2019).

Necropsy

The disease is characterized by a large amount of small grey / white necrotic foci in the liver (spotty liver), as can be seen in figure 3 (Molenaar 2019; Van et al. 2016).
From the inoculation trials done by Van et al., it can be concluded that chickens with SLD can recover and that the liver lesions will then also disappear of the course of a couple of weeks (Van et al. 2017a).

Diagnosis

All C. hepaticus isolates described in literature have been isolated from liver or bile samples, where it can often be found as monoculture (Phung et al. 2020; Van et al. 2017).
C. hepaticus is also present in the gastro-intestinal tract, with increasing concentrations along the gastro-intestinal tract (duodenum < jejunum < ileum < cecum). Despite the fact that gastro-intestinal concentrations are higher than liver concentrations, isolation from the gastro-intestinal tract has so far not been described (Phung et al. 2020; Van et al. 2017).

Cultivation of C. hepaticus is very difficult and it will not be successful by using standard cultivation methods (Molenaar 2019). C. hepaticus grows on nutrient agar with blood, but most don’t grow on MacConkey or Karmali agar (Van et al. 2016).

A PCR is available to determine if samples contain C. hepaticus DNA (Van et al 2017b). The analyses of cloacal swabs with PCR seems to be a reliable method to determine if C. hepaticus is present in live birds (Van et al. 2017).

Treatment

Several antibiotics can be used to treat Campylobacter infections. Tetracyclines are generally first choice antibiotics. In the literature oxytetracycline is reported as main treatment option for SLD in Australia, but plasmid-borne resistance has already been reported (Phung et al. 2020).
Macrolides can also be used, but due to the risk of resistance in zoonotic Campylobacter species on public health, these are considered second choice antibiotics.
Lastly, also fluoroquinolones can be used.

Prevention

Since C. hepaticus DNA has been shown in several materials incl. rats and wild birds, biosecurity seems to be a very important preventive tool to prevent infection of a farm, and also to prevent spread between stables.

There is no registered vaccine available. The use of herd specific (autogenous) vaccines is possible, but more experience will be needed to evaluate the efficacy.

Due to the fact that C. hepaticus was only recently identified as causative agent, little information is known about other preventive measures, such as the use of certain feed ingredients. Research did however already show some promising results for the use of biochar (Wilson et al. 2019). More research is however needed before this can be put into practice.

Zoonosis

The whole genome sequencing showed that it is most closely related to Campylobacter jejuni and C. coli, which are both zoonotic bacteria. The bacterium however has not been detected in humans. More information is needed before a conclusion can be drawn on the zoonotic potential of Campylobacter hepaticus (Crawshaw 2019; Petrovska et al. 2017).

RIPAC-LABOR

With RIPAC-LABOR we have a partner who is specialized in the isolation and cultivation of bacterial pathogens. RIPAC-LABOR invested in the culturing method of C. hepaticus and can now say that they can cultivate and identify C. hepaticus with MALDI-TOF. RIPAC-LABOR can also perform a PCR to detect C. hepaticus DNA in tissues.

The laboratory of RIPAC-LABOR is also possible and allowed to produce herd specific vaccines against C. hepaticus.

In case of a flock of which you suspect they are infected with C. hepaticus, you can always contact our Technical Support department to discuss the possibilities and further steps.

Literature

1. Crawshaw, T. (2019) A review of the novel thermophilic Campylobacter, Campylobacter hepaticus, a pathogen of poultry. Transboundary and Emerging Diseases 66(4): 1481-1492.
2. Crawshaw, T., Chanter, J., Young, S.C.L., Cawthraw, S., Whatmore, A.M., Koylass, M.S., Vidal, A.B., Salugero, F.J., Irvine, R.M. (2015) Isolation of a novel thermophilic Campylobacter from cases of spotty liver disease in laying hens and experimental reproduction of infection and microscopic pathology. Veterinary microbiology 179 (3-4): 315-321.
3. Molenaar, Robert Jan (2019) Nieuws uit de monitoring – Spotty Liver Disease. Tijdschrift voor Diergeneeskunde.
4. Petrovska, L., Tang, Y., Jansen van Rensbrug, M.J., Cawthraw, S., Nunez, J., Sheppard, S.K., Ellis, R.J., Whatmore, A.M., Crawshaw, T.R., Irvine, R.M. (2017) Genome reduction for niche associated in Campylobacter hepaticus, a cause of spotty liver disease in poultry. Frontiers in cellular and infection microbiology 7: 354.
5. Phung, C., Vezina, B., Anwar, A., Wilson, t., Scott, P.C., Moore, R.J., Van, T.T.H. (2020) Campylobacter hepaticus, the cause of spotty liver disease in chickens: transmission and routes of infection. Infection. Frontiers in Veterinary Science 6:505.
6. Van T.T.H., Elshagmani E., Gor M.C., Scott P.C., Moore R.J. (2016) Campylobacter hepaticus nov., isolated from chickens with spotty liver disease. International Journal of Systematic and Evolutionary Microbiology 66, 4518–4524.
7. Van T.T.H., Elshagmani, E., Gor, M.C., Anwar, A., Scott, P.C., Moore, R.J. (2017a) Induction of spotty liver disease in layer hens by infection with Campylobacter hepaticus. Veterinary Microbiology 199: 85-90.
8. Van, T.T.H., Gor, M.C., Anwar, A., Scott, P.C., Moore, R.J. (2017b) Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds. Veterinary microbiology 207: 226-230.
9. Willson, N. L., Van, T., Bhattarai, S. P., Courtice, J. M., McIntyre, J. R., Prasai, T. P., Moore, R. J., Walsh, K., & Stanley, D. (2019) Feed supplementation with biochar may reduce poultry pathogens, including Campylobacter hepaticus, the causative agent of Spotty Liver Disease. PloS one, 14(4) e0214471.