Strategic Diagnosis of Diarrhoea in Suckling Piglets

Which piglets should be selected for diagnostic sampling?

The first step of diagnosis consists of an appropriate selection of the animals that we are going to sample. The natural tendency is usually to select those animals that present more serious symptoms of the disease, which can lead us to make two mistakes.

The first is that these animals with more serious symptoms of the disease may have been with clinical symptoms for several days, so there is a probability that they have been treated recently, which may partly mask the final result of the diagnostic tests that we are going to perform. Hence the first factor to take into account: it is essential to take samples from piglets or litters that have not been previously treated.

The second mistake may arise from the fact that, after several days of being affected, the final result we obtain will be a sum of the pathogens that started the process, together with those opportunistic pathogens that take advantage of the weakness of the animals or the dysbiosis generated at the intestinal level, to produce a pathogenic effect, with which we run the risk of underestimating the initial or primary causal agent and overestimating the secondary problems or opportunistic agents. Therefore, the best strategy is to choose animals to sample, in an initial phase of clinical symptomatology.

What type of sample should I take?

The next question to ask is: What type of sample am I going to take? In the case of enteric problems, faecal samples are usually the main ally for a good diagnosis. Faecal samples can be taken in several ways, depending on our objective: individual faecal samples taken at the rectal level, either by swab (more common in animals of a few days, the sample quantity is smaller), or by rectal stimulation (the faeces are placed in a sample bottle, in this case a larger amount of sample is obtained). In both cases we will obtain a sample from a specific animal, with regard to the quantity, in the case of enteric problems in lactating piglets, the amount of faeces is only relevant when our objective is to perform flotation techniques for counting oocysts of Cystoisospora suis, for which a minimum amount of faeces is necessary.

On other occasions, we may not be interested in obtaining samples from individual animals, but from specific litters, that is, we need a representative sample of a litter, for which sponges can be used to rub the faeces of the farrowing pen in which the litter is housed, socks for sampling, with which the farrowing pen is stepped, or simply samples are taken from 4-5 piglets, which are introduced into a sample jar. The relevant thing in this case is that we will have a composite or aggregate sample, which will be representative of the entire litter.

Historically, we have always started talking about culture, for several reasons: when we did not have molecular techniques at our disposal, it was the first step in diagnosis, and on the other hand, and this is still in force, if we want to perform an antibiogram, it will be essential to have the previous culture of the bacteria.

Culture, on the other hand, has a great disadvantage, the fact that a bacterium grows, does not necessarily indicate that it is responsible for the clinical picture that we are observing in animals, even less in the context of enteric diseases. In this case, the strange thing would be that nothing grows in a stool sample, and on the other hand not all laboratories have the capacity to culture potentially pathogenic bacteria such as those of the genus Clostridium, and obviously viruses and/or parasites do not grow in culture media for bacteria, this in itself shows that a culture of the sample is insufficient to obtain a good diagnosis of the process.

Luckily, molecular techniques have changed the rules of laboratory diagnosis today. In a very efficient way (results in 24 hours) and at an acceptable cost, we can perform a whole battery of molecular techniques (PCR) against the different pathogens or their virulence factors.

Molecular techniques have made it much easier for us to detect potential pathogens in a sample and differentiate between the different pathotypes or virotypes of a bacterium.

How can pathogenic Escherichia coli be identified?

In this way, using PCR techniques, we can detect the presence of different genes of the bacterium Escherichia coli (E. coli) associated with virulence (virulence factors), and thus differentiate between pathotypes:

• Enteropathogenic E. coli (EPEC): strains with the presence of the intimin gene (EAE).

• Enterotoxigenic E. coli (ETEC): strains with the presence of genes encoding adhesion fimbriae and different enterotoxins:

+Adhesion fimbriae: F4, F5, F6, F41

+Toxins: STa, STb, LT, EAST1

How can Clostridium perfringens be differentiated?

In a similar way happens with Clostridium perfringens, the detection of the genes that code for the different toxins will allow us to perform the toxinotyping, and thus determine which type of bacteria is present in the sample:

• Clostridium perfringens Type A (CpA): alpha and beta-2 toxin

• Clostridium perfringens Type C (CpC): alpha and beta toxin

Which viruses should be considered?

In the case of viruses potentially involved in a diarrhoea process in lactating piglets, these molecular techniques will allow the detection of the genome of the different types of Rotavirus, mainly A and C (RVA and RVC), as well as the porcine epidemic diarrhoea virus (PEDv).

Which parasites should be considered?

Similarly, the genome of parasites such as Cystoisospora suis, the causative agent of porcine coccidiosis, which affects piglets from the second week of life, can also be detected.

Are there other bacteria that should be evaluated?

Other bacteria such as Enterococcus hirae or Clostridioides difficile (by detecting CdA and CdB toxins), could also be involved in the diarrheal process, so it is advisable to evaluate them.

Although molecular techniques have made it easier for us to detect potential pathogens in a sample, it happens that, in certain cases, the problem can be just the opposite, we detect so many pathogens or virulence factors, that the difficulty may be to establish which of them are relevant in the process we are observing.

Key Take-Home Messages

When faced with the diagnosis of a case of neonatal diarrhea, it is advisable to follow a sequence that helps us to reach the most accurate diagnosis possible:

1. Selects the animals or litters with the process in the initial or acute phase
2. Choose a type of sample that is practical to take and to send and that offers us an optimal result, if there are doubts, call the laboratory.
3. Think carefully about the determinations to be requested, depending on the decisions you are going to make: "if the results of a technique or diagnosis are not going to make you change your strategy, you may not need it".
4. Interpret the results well, and if they are not clear enough, you may need additional sampling or techniques.

Frequently asked questions

How can qPCR help determine pathogen relevance?

On the one hand, using qPCR techniques we can know the amount of genome detected in the sample. In this way, if the presence of a potential pathogen is very high in the samples, it will suggest its possible involvement in the process, while if the amount is very small, it can indicate that it is a chance finding.

We also have to consider that with PCR, we are detecting the genome sequence that codes for the specific protein. This does not mean that the pathogen is alive or that the virulence factor is being produced.

When should histopathology be used?

In addition, in cases where we have detected the presence of several pathogens simultaneously, or the presence of pathogens that may be present in both healthy and sick animals (their detection alone is not an indication that they are involved in the case), it is highly recommended to take samples from different sections of intestine, to perform histopathology. So, through the type of microscopic lesion, we can determine if we are dealing with a process with a mainly bacterial or viral component, or if we find lesions indicative of a pathogen such as the volcano eruptions present in cases of Clostridioides difficile.