Epidemiological Molecular Study of Combined Severe Immune Deficiency in Arabian Horses in Tunisia

Severe Combined Immuno Deficiency (SCID) is a hereditary deficiency transmitted through autosomal recessive gene, carried on by purebred Arabian and crossbred horses (PerryMan et al., 1980). A deletion of five base pairs at the gene encoding the catalytic subunit of the Protein Kinase DNA-dependent (DNA-PKcs) is responsible for this disease (Shin et al., 1997; Bailey et al., 1997) and the affected animals have a deficiency in the number and function of lymphocytes T and B leading to a reduction in the immune response to infectious diseases, consequently, SCID-affected foals die in the first six months, regardless of the level of veterinary care administered. Therefore, it is important to carry out a molecular epidemiological study to estimate the frequency of the SCID allele in Arabian purebred horses in Tunisia.

This work was conducted at the Veterinary Research Institute of Tunis, laboratory of Genetic from December 2018 to June 2019.

Our study concerned 164 purebred Arabian horses belonging to the FNARC of Sidi Thabet in Tunisia. The crossings of these horses gave foals that died at an age that did not exceed 6 months. Considering that the cause of death remains unknown, it has been assumed that SCID exists in these foals. Therefore, parents have been examined to see if they were heterozygous carriers of the SCID allele. This check was performed during the period of 9 years.

The biological material used is a sample of blood taken on a tube with anticoagulant. This blood was found to have been already collected for a parentage test. The blood was kept at a freezing temperature of -20°C.
 
Extraction of the DNA
 
The extraction of the DNA is made from a blood sample taken on anticoagulant by the spin-column method using DNA extraction kit (Pure LinkTM Genomic DNA Mini Kit produced by the company « Invitrogen Thermo Fisher Scientific») by maintaining a sterile environment when handling DNA to avoid contamination by DNase.

In order to estimate the concentration as well as the quality of the DNA obtained after extraction, we have used UV spectrophotometer, UL-2000 Macy and wavelengths of 260 nm and 280 nm.

L’extraction de l’ADN se fait à partir d’un échantillon de sang prélevé sur anticoagulant par la méthode de membrane de silice grâce au kit d’extraction d’ADN Pure LinkTM Genomic DNA Mini Kit.
 
Amplification of the gene by the PCR technique
 
The primers used are the same used in Great Britain (Swinburne et al., 1999) and Morocco (Piro et al., 2008).
Forward primer: 5'-AAGTTGGTCTTGTCATTGAGC-3'-
Reverse primer: 5'-TTTGTGATGATGTCATCCCAG-3'

We have marked the forward primer with Fam or 6-carboxyfluorescein wich is a fluorochrome (chemical substance) able to emit fluorescence light after excitation.

The free water nuclease and a PCR or Master Mix (Promega) PCR kit including 3 mM of MgCl2, 50 units / ml of Taq polymerase and 400 μm of each dNTP were used in order to optimize the PCR reaction.

The PCR steps used were also as described by Swinburne et al., (1999). The first step at 95°C for 10 minutes for the primary denaturation of the matrix and 30 cycles at:

 

At the end, a step of one hour at 72°C was programmed to complete unfinished amplifications.

Then, we have added to each PCR sample a marker of standard size and formamide (standard ROX350 and 2 ml formamide) knowing that the fragment we wanted to amplify is formed of 163 base pairs. The PCR products were genotyped using an automatic sequencer, model ABI PRISM 3130 Genetic Analyzer (APPLIED Biosystem) with 4 capillaries of 36 cm long.

The amplified gene is detected by its migration in the automatic sequencer. This detection is done automatically due to the marking of the forward primer by the fluorochrome Fam. This fluorochrome is excited by a laser and the fluorescence emitted is detected by a sensor. The size standard (Rox 500) migrates at the same time as the DNA fragments. This standard contains a series of known size bands that will be used to determine the size of the amplified gene. The fluorochrome is converted into electric current by a camera and is then analyzed by Gene Mapper software version 4.1 (Applied Biosystem). This software transcribes the data recorded by the sequencer by calculating the size of the analyzed fragments in base pairs and transforms them into peaks in the form of an electrophoretic trace.

In order to verify the correct functioning of all the steps of the PCR and its analysis, we first tested the technique with a positive sample: a DNA sample from a horse recognized as a non-sick carrier of the SCID gene sent by our Moroccan counterparts.

The result of the positive temper evident heterozygous control is in the form of two peaks on the electrophoretic plot (Fig 1 and 2):
-  a peak for a 163 base pair DNA fragment which represents the non-mutated allele.
-  a peak for a 158 base pair DNA fragment which represents the mutated allele (deletion of 5 base pairs).
-  A negative result is in the form of a single peak at 163 base pairs as can be seen in Fig 2.





After verification we searched for the gene on the DNA samples of the horses selected during the sampling.

By analyzing the electropherograms, we noticed the absence of the SCID deletion in the group of 164 Arabian purebred horses studied with 100% negative results.

The original method used to differentiate between normal PCR products and those with SCID deletion was based on the Southern blot technique (Don-van Slot HP et al., 2000). Two probes; N (specific for the normal allele) and S (specific for the SCID allele) enabling the identification the expected genotypes (homozygous healthy, homozygous affected SCID, or heterozygous carrier SCID). Hybridization of both N and S probes at both the amplicon band level identifies the animals as healthy heterozygous carriers. Another, simpler and faster method was used by a team from Slovenia (Lunn DP et al., 1995). This method consists in using a high resolution 4% agarose gel to separate the deleted band (244 bp) from the normal band (249 bp) of an amplicon corresponding to a heterozygous healthy carrier. Since these bands differ by 5 base pairs, this team chose a gel with a resolving power of 4 base pairs. In this work the DNA test used is the technique offering the highest level of sensitivity and specificity. PCR reactions that seek to amplify the desired gene from a tiny amount of DNA to give hundreds of copies, explaining the high sensitivity of the method.

The primer pair chosen is specific for the gene encoding DNA-PKcs. The probability of finding the same DNA sequence in another gene is very low, especially since capillary electrophoresis genotyping offers even more specificity since it gives the precise length of the amplified gene fragment: 163 base pairs for the wild-type allele and 158 for the mutated allele (the mutation being a deletion of 5 base pairs). If the primers have hybridized to another gene, the length given by genotyping will be different.

An adult horse is necessarily carrying the wild allele, so the peak at 163 base pairs must always be present and it’s considered as the witness pic. Its presence proves that the PCR reactions have taken place and its length proves the specificity of the primers.

We have selected 164 horses to be tested. These 164 samples all gave the same result: a single peak at 163 base pairs on the electrophoretic plot: these horses are; therefore, homozygous for the wild-type allele.

The absence of a positive result on these samples shows a low prevalence of less than 0.8% (1 case out of 164) but does not exclude the presence of the mutated allele in Tunisian livestock and other studies in other sites in Tunisia are needed to accurately estimate this frequency and assess its economic impact. Indeed, a large number of breeders are imported from several countries (France, Great Britain etc.), whose prevalence rate of the SCID gene is respectively 1.12% (Acafrance., 2017) and 2.8% (Swinburne et al., 1999). The highest prevalence has been reported in the USA and estimated at 8.9% (Ding et al., 2002) and part of the imported seed in Tunisia comes from it.

This importing policy which aimed to limit the consanguinity rate and improve the genetic potential of Tunisian horses, could have allowed the introduction of the mutated allele, as was the case for Morocco and the US. Indeed, a study of the pedigree as well as the DNA analysis of Moroccan heterozygote horses contributed to trace the origin of the introduction of the SCID gene to 3 Arabian stallions: 2 were imported in 1978 and one in 1990 (Piro et al., 2008). In the US, a stallion imported in 1920 has been identified as a likely source for the introduction of the SCID gene (Bernoco and Bailey., 1998).

Our results are comparable to the results found in many other countries such as Poland (Terry et al., 1999), Romania (Goergescu et al., 2006), Slovenia (Jana Zavrtanik et al., 2005) and Turkey ( Cinar et al., 2014) who did not find any horses carrying the SCID gene.

Also, another study was done in Slovenia by Lunn DP et al (1995) on 128 horsepower PSAR. None of these horses proved to be carriers of the morbid allele. It is remarkable that a good part of these horses were imported from Tunisia which indirectly confirms the low frequency of the SCID allele in our horse population.

It is possible that this difference in frequency between countries is due to the breeding strategy followed in each country. Developed countries such as the USA, Canada, Australia and Great Britain rely primarily on their breeding on and selection. On the other hand, in Tunisia, or in other countries such as Slovenia and perhaps Poland and Hungary, there is a natural variation since the consanguinity rate is kept to a minimum by strategy or because of the importation of horses from different countries under strict control. The natural variation is an effective mechanism for keeping the frequency of the disease at a low level. In the USA or Europe, however, we are looking for quality so there is a selection of good breeders and only the best performing will be used for breeding. This same high-performance breeder can be phenotypically healthy but genotypically carrying the mutant allele and can therefore transmit the defective gene to successive generations. This results in an increase of the consanguinity rate and hence an increase in the frequency of the mutation within this restricted population.

The risk of the presence of the mutated allele is still very real and with it the risk of seeing cases of SCID in foals.

The lethal character of the disease which may affect foals of high economic value justifies the importance of establishing a system of prophylaxis which is based on the systematic screening of each breeding animal before admission to reproduction.

In this work we have tried to detect the SCID deletion by amplification and analysis of DNA in the region of the deletion of the DNA-PKcs gene using a population of 164 purebred Arabian Tunisian horses. This population was selected from the suspect pairs who had early deaths among their off springs, in order to promote the detection of the SCID allele. Despite this biased choice, the results showed the absence of the SCID allele in the horses studied. This does not mean that SCID disease is non-existent in Tunisia, but it is infrequent. It is therefore essential to carry out molecular screening of the SCID deletion in other sites in Tunisia in order to determine its prevalence in the country’s population of purebred Arabian horses.