Rapid penside detection of salmonella from calves with scour (PhD)

To reduce illnesses associated with food products, a multifaceted approach from farm to table is needed. Salmonella sp. causes gastroenteritis in humans, impacting public health. Salmonella sp. have a high impact on economics and animal welfare, causing a high level of sickness in infected cattle, and a high death rate amongst infected calves. As asymptomatic S. Dublin carriers can excrete bacteria in milk and faeces, herd environment is contaminated which, if not effectively controlled for, can result in persistent intra-herd infection with the potential to spread inter-herd, to wildlife, farm hands and the public. A rapid and inexpensive diagnostic kit would be a useful in this situation ensuring Salmonella infections are controlled.

Despite diarrhoea a common symptom of salmonellosis, scour can also be caused by viruses, such as BVD, and parasites, such as lung worm. Salmonellosis can kill calves within 48 hours, which with current methods is quicker than a diagnosis. Thus, when presented with newly born calves suffering from scour, prophylactic treatment with antibiotics to stave off potential salmonellosis is common, despite a variety of potential causal agents. With the increase in antimicrobial resistance, this is a cause for concern, that governments worldwide are acting upon.

The emergence of antimicrobial resistance (AMR) is a prominent concern, Salmonella sp. are adapted to invade the gut, with AMR salmonellosis will become harder to treat, resulting in increased morbidity and mortality, already the emergence of MDR Salmonella strains are beginning to limit treatment options within cattle herds. Current antimicrobials need to be safeguarded and the spread of MDR strains needs to be controlled, targeted treatment is needed to confirm that antimicrobials are only administered in the presence of a bacterial infection.

To ensure this, quicker methods of Salmonella sp. detection is needed to determine the cause of scour in ailing calves. To aid this, two rapid detection methods have been developed to target Salmonella sp.:

• A potentiometric immunoassay for the detection of Salmonella serovars developed on the Vantix Reader 2 (VR2) is rapid, completed and read in under an hour, and is robust through calf scour. The biosensor immunoassay has the potential for cheap, easy mass production, as well as the ability to simply adapt established ELISA techniques utilising commercially available antibodies, the VR2 shows great promise as a rapid detection system that could easily be immediately utilised.

  - The VR2 be an excellent system for veterinarians, who already possess basic laboratory skills, as either a point of care system, or as a clinic detection system, allowing veterinarians to forgo sending samples to centralised labs and speeding up diagnostic and treatment intervals.

  - Without the lengthy sample preparation steps associated with Salmonella diagnostics, the Vantix is a reliable, robust biosensor that can detect multiple Salmonella serovars through calf scour. 65

• A fluorometric LAMP assay for the detection of Salmonella sp. that is quick and simple, with visible results generated in 40 minutes. Future research is needed to finalise protocol and ensure robustness through scour, LAMP shows promise as a detection method for Salmonella serovars on site, pen-side to infected cattle due to high specificity and sensitivity.

  - Pan-Salmonella fluorometric LAMP assay would be well suited to point of care testing, particularly on farm or in low resource settings, such as in developing countries.

  - Due to the potential for simple sample addition and easily interpreted results, a skilled professional would not be needed to operate this assay for pan-Salmonella detection.

By utilising either of the rapid detection methods developed within this study at the point of sample delivery, time and resources could be significantly reduced by screening out Salmonella negative samples and only culturing presumptive samples for confirmation. In the case of negative samples, this would allow Salmonella infection to be ruled out immediately, allowing for quicker diagnosis of other causal agents. Additionally, use of these diagnostics would facilitate the screening and sampling of Salmonella sp. to enable disease control, allowing monitoring of Salmonella sp. intra-herd, inter-herd, and on a national level.

By utilising on site diagnostics, continuous herd screening and quick counter measures could be employed to avoid the following contamination of the production site, quicker that sending samples to centralised laboratories. Controlling the spread of Salmonella sp. would protect herds, increasing animal welfare and reducing the economic impacts of salmonellosis. The potential for food contamination would be reduced, preserving public health. By targeting treatment with quick reliable diagnostics, cattle can receive the correct treatment for the correct disease, safeguarding antimicrobials and staying ahead of government legislation.

Overall two promising, rapid detection methods, capable of detecting multiple Salmonella serovars under 45 minutes have been developed both with advantages as point of care tests, including being simple to use, with easily interpretable results.

The challenge

Controlling and treating Salmonella species effectively is essential in cattle farming. Salmonellosis in cattle can result in diarrhoea, loss of appetite and depression and can lead to lameness, arthritis and bacteraemia, which can result in the death of the animal. Salmonella Dublin is host adapted to cattle and is the leading cause of cattle salmonellosis in the UK. This organism can cause abortion in cows and calves often die as a result of infection due to their immature (naive) immune systems. Infections with salmonella strains can also pose a risk to human health should the organism enter the food chain.

The project

I am developing a rapid detection method for Salmonella species that will be simple, robust, and cheap. The suite should be portable, enabling penside (point of decision making) detection of Salmonellosis. This will allow targeted treatment of the sick animal, increasing animal welfare and reducing the economic impact Salmonella sp. can have on cattle. To do this I am developing a multiplex system comprising a molecular LAMP (Loop mediated isothermal amplification) assay, and a serological test, on the Vantix system to detect both the genome and the physical presence of the organism during infection in calves.

Student

Fredericka Mitchell, Kingston University