Diarrhoea in piglets after weaning is both a welfare and economic problem. The use of antibiotics and high doses of zinc to prevent diarrhea is no longer allowed in Denmark due to the risk of resistance in the pathogenic bacteria and environmental pollution. Therefore, there is an intensive search for other ways to reduce the occurrence of diarrhoea.

Binding proteins in the feed

One approach may be to add certain binding proteins, ‘nanobodies’, to the feed. These are small antibody fragments termed VHH-constructs, that target toxin-producing E. coli.

Researchers at Aarhus University, together with colleagues from DTU, the University of Parma, and the biotech company Bactolife, found that adding a combination of two types of VHH constructs (BL1.2 and BL2.2) to the feed inhibited diarrhea in pigs inoculated with E. coli F4+. They showed that this reduced the number of coli bacteria in the feces and decreased the ability of the coli bacteria to adhere to the intestine.

At the same time, the binding proteins reduced the damaging effect of the pathogen on  the integrity of the intestine. An E. coli infection makes the intestine more permeable, which makes the pig vulnerable to the transfer of bacteria, viruses, and harmful substances to the blood. Feed containing VHH constructs improved the integrity of the intestinal barrier compared to pigs that were infected with E. coli but did not receive VHH constructs in the feed.

Finally, the researchers found that it also affected the pigs’ response to the inflammatory state that arises in the wake of an E. coli infection: the VHH constructs  maintained the same level of granulocytes in the blood as a control group that was not infected with E. coli. At the same time, the VHH constructs mitigated the changes in acute phase proteins, which are the animal’s first response to an infection or tissue damage.

Must withstand heat treatment

VHH constructs are quite stable compared to conventional antibodies. Still, it is important to ensure that they remain effective even when the feed is heated to at least 81 °C. This is due to Danish requirements for heat treatment of finished feed.

The researchers showed that BL1.2 and BL2.2 had the same binding ability after freeze-drying and after heating to 75°C. At 95°C, BL1.2’s binding ability disappeared completely, while BL2.2’s binding ability was slightly reduced. This suggests that the VHH constructs can withstand the heating used in practice.

Even more important, the addition of VHH constructs reduced the occurrence of diarrhea in the pigs, even when the feed was pelleted. “This is clearly one of our most important findings,” says Jiajia Xu, who is a postdoc at the institute but worked on the experiment during her PhD. “It means that the applicability of binding proteins in the feed is much more realistic.”.

Targeted effect

Usually, E. coli F4+ and F18+ are the cause of diarrhea when piglets are weaned. F4+ and F18+ refer to hair-like protein structures, fimbriae, which the bacteria have on their surface. These help the bacteria attach to the intestine and cause diarrhoea. This also means that the VHH constructs must fit like a key in a lock -they only work if they are targeted precisely at the structure (virulence factor) that causes the diarrhoea.

“We would very much like to see VHH constructs developed against F18+ as well,” says Jiajia Xu. “It would make it safer to target the bacteria that cause diarrhea if there were binding proteins against both F4+ and F18+.”

Attack on several fronts

The researchers chose to study a combination of two VHH constructs against F4+, because they work differently. In this way, the enemy is attacked on several fronts.

BL1.2 binds directly to the F4+ fimbriae, while BL2.2 binds to some heat-sensitive toxins that E. coli bacteria secrete and that make the piglets sick.

It was already known that the VHH-constructs work individually and in combination. That was shown before by giving the constructs directly into the mouths of piglets in an earlier collaboration with Bactolife. The new thing is that the constructs are now mixed into pelleted feed and are therefore more relevant for practice.

A llama is the key to success

There is no obvious connection between llamas living on the high plains of the Andes in Peru, and piglets in Danish pig barns – yet there is.

Unlike humans and pigs, whose antibodies consist of both light and heavy chains, llamas and other camelids produce only heavy-chain antibodies. This makes it much easier to isolate the active domain (VHH). It is also quite simple to cut out the genes that code for VHH and insert them into the genome of a microorganism. In this way, microorganisms can be made to produce VHH in large quantities.

The technique is new but widespread

It is only about three decades since the unique antibodies of the camel family were discovered and the biotechnology for purification and production of VHH took off. Since then, the method has become widespread in research, diagnostics, and therapy - for example against viruses, bacteria, or cancer). VHH constructs are not yet used routinely in commercial pig production. However, the study, which was recently published in Journal of Animal Science and Biotechnology, suggests that it could also soon become a reality in pig production.

On the way to Danish pig barns?

As it stands, legislation is preventing VHH constructs from being used as a feed additive. This requires approval in the EU based on thorough documentation of their effect and an assessment of the possible harmful effects of the substances.

The authors of this article also point out that the results from their study should be confirmed in a larger study under practical conditions. SEGES Innovation, who also participated in the GUDP project, has conducted a farm trial at Grønhøj Experimental Station. Here, it was also found that VHH constructs in the feed reduced the occurrence of diarrhea.

However, the protection only lasted as long as the pigs received the special feed. This is because treatment with VHH constructs is not a vaccination that actively stimulates the animal’s own immune system. Instead, it is a passive immunotherapy that supplies active antibodies capable of binding and neutralizing a specific target – in this case, infection with E. coli.

Although VHH constructs appear to be an effective weapon against diarrhea, it is still not a tool that farmers can take from their toolbox to mitigate weaning diarrhoea. But it could become a reality soon.