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Sampling instruction for whole herd sampling

The following instructions have been developed as an aid for samplers in order to reduce the risk of cross contamination on the farm or in the laboratory. 

The risk of contamination is high when sampling for salmonella. First, there is a risk of contamination between different animals within the same herd. Secondly, there is a risk that the package is contaminated on the outside, which can cause a risk of cross contamination, at transport or in the laboratory, with packages from other herds. The latter may lead to restriction and sampling of herds that do not have salmonella and hence incur unnecessary costs. It is no less important that the test results are reliable on herd level.   Contamination between animals within the same herd can make the effort to rid the herd from salmonella more difficult and actions may be taken concerning the wrong animals/animal groups.
Here follows a suggestion for a sampling routine to reduce the risk of contamination.
Careful planning prior to sampling is essential for the sampling work to run smoothly and for the results to be reliable. Take your time; it is worth it!

Sampling principle:

The basic principle of sampling is that individual samples are taken from an individual animal when possible. Pooling is best done at the laboratory, where the exact same amount of faeces is taken from usually five different individual samples. The individual faeces samples can be stored in the laboratory, and if the pool turns out positive for salmonella, culture from the individual samples that were included in the pool can be done. In this way, the positive animal or animals in the pool are identified. If it does not matter which individual is positive, the laboratory should be notified that no individual tracing needs to be done.  The laboratory can then discard the individual faeces samples immediately after the pooling.
When individual sampling is not possible, faecal samples are collected so that the samples represent an animal unit, e.g. a box. Faeces are taken from at least as many “faecal piles” as there are animals in the box/pen but in the same jar a maximum of ten manure clots  can be collected. The aim is that all the manure clots collected in a jar are of the same size.

Sampling of individual pigs:

During sampling there needs to be at least three people present:
• Two persons taking the samples.
• One person handling the samples (assistant)
Equipment:
• Disposable gloves (vinyl), one per animal sampled.
• Sample jars (50 ml) with lids.
• A pen for marking the jars. Please note that the handling in the laboratory is facilitated if the lids are marked!
• Plastic bags for packing of jars, such as robust 5-litre bags.
• Spray-paint (for marking sampled individuals).
• Garbage bags to put used material in.
• Bag sealers.
• Referral forms and pen.
Preparation:
• The assistant attaches a belt around the waist and on the belt hangs four plastic bags (5 litres) (through a hole in the upper edge of the plastic bag). In one of the plastic bags jars are placed upside-down, in another lids and a marking pen is kept, in the third bag extra disposable gloves are stored and the fourth one is used for litter.
• The samplers each need a clean bag and extra disposable gloves.

The assistant provides the samplers with one jar at a time. The samplers use a new vinyl glove for each animal and take samples directly from the rectum. In a small group of sows, there is often no problem to take samples from free-range sows without fixating them. If there is no faeces in the rectum it usually works with a moment of “rectal massage” = two fingers massage the “rectum ceiling”. The faecal sample is placed in the jar the sampler got from the assistant.  The jars may be filled to a maximum of 2/3 otherwise there is a risk that the fermentation process in the jar will pop the lid off during transport. The jars are handed to the assistant who puts a lid on and labels the lid with the sow´s number. The jar is placed in a dedicated bag for samples taken.  The assistant must avoid getting faecal contamination on the outside of jars and gloved hands. If that happens the contaminated jar and/or the glove must be replaced.  Finally,  on a clean surface with clean hands/gloves the jars are put in 5-litre plastic bags. Samples from animals in the same group/establishment shall not be mixed with samples from other groups. Once the sampling is completed, the plastic bags are placed in an extra outer plastic bag so that the jars are packed in double bags, and the referral form is inside the outer bag.  The bags are finally placed in a box. It is important that neither jars, plastic bags nor the box is contaminated. The referral form shall state whether the individual samples should be pooled or not and if individual tracing is needed or not.

Collection of herd based fecal samples from cattle or swine:

During sampling there needs to be at least three people present:
• Two or more persons taking the samples.
• One person handling the samples (assistant). The assistant can serve up to four persons collecting the samples.
Equipment:
• Disposable gloves.
• Plastic teaspoons (disposable), one per animal sampled.
• Sample jars (100 ml) with lids.
• Pre-written labels or a pen for marking the jar/jar lids.
• Plastic bags for packing of jars, such as robust 5-litre bags.
• Bag sealers.
• Garbage bags to put used material in.
• Referral forms and pen.
Preparation:
• Label the jars and place them in the same order as boxes or pens.
• The assistant attaches a belt around the waist and on the belt hangs four plastic bags (5 litres) (through a hole in the upper edge of the plastic bag). In one of the plastic bags jars are placed upside-down, in another lids and a marking pen is kept, in the third bag samples are stored and the fourth one is used for litter. Some extra disposable gloves are also useful if a glove becomes contaminated.
• The samplers need disposable spoons with the handle upwards in a clean bag and extra disposable gloves.

The assistant and the samplers are recommended to use gloves since they can easily be replaced if they are contaminated. With a teaspoon, faeces are taken from the box floor into a 100 ml sample jar. Clots of manure should be collected from as many piles as there are individuals in the group. If there are more than ten individuals in a box, several pooled samples have to be taken. The aim is that all the collected clots of manure are the same size. Do not mix faeces from the various boxes and take a new spoon for each sample. The jars may be filled to a maximum of 2/3 otherwise there is a risk that the fermentation process in the jar will pop the lid off during transport. The jar is handed to the assistant who puts a lid on and places it in a dedicated bag, with samples from animals in the same group/pen. There is room for 30-35 jars in a 5-litre plastic bag, so if there are more than 35-40 jars, several plastic bags may be used from the same pen. If the jar, gloves or plastic bag is contaminated they must be replaced by new clean ones. Once the sampling is completed, the plastic bags are placed in an extra outer plastic bag so that the jars are packed in double bags, and the referral form is inside the outer bag.  The bags are finally placed in a carton. It is important that neither jars, plastic bags nor the carton is contaminated.

The referral form

The referral form shall always state the reason for sampling. It shall also state whether the individual samples should be pooled or not and if there should be a possibility to make an individual tracing or not. Correct labelling on jars/jar lids and on referral forms is very important to make sure that positive samples will be traced back to the right animal! It has to be made clear from what animal and from which animal group/pen the samples come from. Also, note which animal group you expect to be free of salmonella and in which animal group you think there is a low respectively high presence of salmonella. If the laboratory receives that information, the samples from the groups expected to be free of salmonella can be analysed first thus reducing the risks of cross-contamination in the lab.

Please contact the laboratory you intend to send the samples to if there are more than 50 samples so the laboratory staff can plan their work.

Sampling of individual cattle: 

 During sampling there needs to be at least three people present:
• One person holding the animal.
• One person taking the samples.
• One person handling the samples (assistant)
Equipment:
• Rectal gloves (cattle), one per animal sampled and paraffin oil.
• Plastic tablespoons (disposable), one per animal sampled.
• Sample bottles (100 ml) with lids.
• Pre-written labels or a pen for marking the jar lids.
• Plastic bags for packing of jars, such as robust 5-litre bags.
• Spray-paint (for marking sampled individuals in loose housing). 
• Clean bucket to place the jars in after sampling. The bucket may well be provided with a garbage bag so the sample jars can be protected from splashes.
• Disposable gloves.
• Garbage bag to put used material in.
• Bag sealers.
• Referral forms and pen.
Preparation:
• To make it easier, decide which animals to sample and prepare labels accordingly. The assistant attaches a belt around the waist and on the belt hangs four plastic bags (5 litres) (through a hole in the upper edge of the plastic bag). In one of the plastic bags jars are placed upside-down, in another lids and a marking pen is kept, in the third tablespoons with the handle upwards are stored and the fourth one is used for litter, mainly used tablespoons. Some extra disposable gloves may be useful if a glove becomes contaminated.

Calves are sampled according to the instruction for individual sampling of pigs. When adult animals are sampled a new rectal glove is used for each new animal. When the faecal sample is taken the assistant immediately takes a clean spoon and scoops up an amount the size of a walnut in a disposable jar (100 ml), after which the spoon is disposed of. The jar is sealed, labelled and placed in a clean bucket. The jars may be filled to a maximum of 2/3, otherwise, there is a risk that the fermentation process in the jar will pop off the lid. The assistant must avoid getting faecal contamination on the outside of jars and gloved hands. If that happens the contaminated jar and/or the glove must be replaced. Finally,  on a clean surface with clean hands/gloves, the jars are put in 5-litre plastic bags. Samples from animals in the same group/establishment shall not be mixed with samples from other groups. Once the sampling is completed, the plastic bags are put in an outer extra plastic bag so that the jars are packed in double bags, and the referral form is inside the outer bag.  The bags are finally placed in a box. It is important that neither jars, plastic bags nor the box is contaminated. The referral form shall state whether the individual samples should be pooled or not and if there should be a possibility to make an individual tracing or not.


 

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