Identification of BLAD, DUMPS, Citrullinamia and Factor XI Deficiency in Holstein Cattle in Turkey
In this study 170 Holstein cows reared in the Bursa Region of Turkey were screened in order to detect four autosomal recessive genetic diseases, i.e., BLAD, DUMPS, citrullinamia and FXI deficiency by Polymerase Chain Reaction (PCR) based methods. We found that none of the animals were carried BLAD, citrullinamia and DUMPS, but two cows were detected as carriers of FXI deficiency. After the PCR applications, FXI deficiency free individuals produced one bands were length of 244 bp and carriers individuals for FXI deficiency gave two bands length of 244 and 320 bp. The frequency of the FXI mutant allele and the FXI carrier prevalence were 0.06 and 1.17%, respectively. The results of present study showed that, the situation regarding to the investigated recessive disorders of BLAD, DUMPS, Citrullinamia and FXI deficiency seems to be good.
Inherited disorders affect all kinds of farm animals. Functional and physiological
defects arising from inherited disorders have negative impact on health and
productivity of farm animals. Autosomal recessive disorders lead to economic
loss in the dairy cattle industry which is kept on with Holstein cattle, due
to difficulty in detection of carrier individuals. BLAD (Bovine Leukocyte Adhesion
Deficiency), DUMPS (Deficiency of Uridine Monophosphate Synthase), Citrullinamia
and FXI (Factor XI) deficiency are four of Holstein-specific autosomal recessive
disorders. Increased use of artificial insemination and worldwide use of service
bull cause to widespread of this kind of disorders via carriers seem to be normal
(Patel et al., 2006). Polymerase chain reaction-based
techniques are very useful tools to detect autosomal recessive disorders and
it can be used for eradication programme of these kinds of disease in dairy
BLAD is an autosomal recessive disease caused by reduced expression synthesis
of C11/C18 protein family on the leucocytes surface (Kehrli
et al., 1990; Shuster et al., 1992).
It is known that BLAD is caused by the point mutation (A→G) at the position
383 of CD18 gene located on the first chromosome of bovine (Shuster
et al., 1992). This disease leads to calf death in the early life
stage (before 1 to 3 month) from chronic diarrhea, pneumonia, high fever and
other infections (Kehrli et al., 1990; Nagahata
et al., 1993). It is very important to detect carrier animals for
this mutation in order to avoid spread of this disease across the dairy herds.
The occurrence of the mutant allele leads to BLAD when homozygote situation
has been reported in different countries. The existence of this mutation among
Holstein populations is reported in previous studies (Nagahata
et al., 1997; Ribeiro et al., 2000;
Ertugrul et al., 2004; Rahimi
et al., 2006; Patel et al., 2007;
Meydan et al., 2007).
Citrullinamia is a rare metabolic disorder due to lack of argininosuccinate
synthetase (ASS) and is critical for urea cycle and was firstly reported by
Mcmurray et al. (1962) in humans. This disorder
is characterized by serious neurologic symptoms in newborn calves (Harper
et al., 1986, 1989). The reason of the disorder
is a C→T transition in the position of 86 on ASS gene (Dennis
et al., 1989). There are few studies aimed to determination of existence
this mutant allele. The allele frequency of the mutant allele was found to be
high in Australia (Healy et al., 1991). Robinson
et al. (1993) detected only one heterozygote among 367 Holstein bulls
tested in USA. In other countries, such as Germany and India, the mutant allel
didnt detect in studied populations (Grupe et al.,
1996; Patel et al., 2006).
The UMPS is an enzyme which has a key role on the pyrimidine nucleotide synthesis
which is essential for normal growth and development for several ruminant and
nonruminant species (Healy and Shanks, 1987). Inactivation
of this enzyme is caused by an autosomal-recessive heredity mutation which occurs
in the gene of UMPS. The mutation (C→T) leads to the loss of the restriction
site of AvaI site in codon 405 of the gene (Schwenger
et al., 1993). This disorder is named as DUMPS in the Holstein cattle
and characterized by lowered blood activity of enzyme UMPS (Healy
and Shanks, 1987). DUMPS leads to embryonic death in early stage of pregnancy
(Ghanem et al., 2006). So some serious reproductive
problems take place in dairy herds. Several investigations were carried out
in different countries. No carrier animals were found among Holstein populations
in Poland (Kaminski et al., 2005), Iran (Rahimi
et al., 2006), India (Patel et al., 2006)
and Turkey (Meydan et al., 2006; Akyuz
and Ertugrul, 2008), but the mutant allele was detected in the studies carried
out in U.S.A (Shanks et al., 1987) and Argentina
(Patel et al., 2006).
FXI is a plasma serine protease acts in the transformation of insoluble fibrin
clot from soluble fibrinogen (Brush et al., 1987).
As other three disorders investigated with present study, FXI deficiency is
inherited autosomal-recessive. It is caused by an insertion of 76 bp within
the exon 12 of the gene of FXI (Marron et al., 2004).
Heterozygote individuals for FXI deficiency have serious health problems (Brush
et al., 1987). On the other hand both heterozygote and homozygote
animals for this mutation have susceptibility to some diseases such as mastitis,
metritis and pneumonia and also they have lower calving and survival rates (Liptrap
et al., 1995). The frequency of mutant allele has been reported at
very low in America and Japan (Marron et al., 2004;
Ghanem et al., 2005; Citek
et al., 2008; Meydan et al., 2009).
The aim of this study was genotyping of these four autosomal recessive disorders
by using PCR-based techniques on Holstein cows reared in the Bursa district
located on South Marmara region of Turkey and show applicability of these PCR-based
techniques in the breeding systems for avoiding from autosomal recessive disorders.
MATERIALS AND METHODS
Whole blood samples were collected from one hundred seventy (170) Holstein
cows which are reared in different animal farms from Bursa region (in the Northwest
of Turkey) in 2008. Blood samples were collected from jugular vein into EDTA
containing test tubes. DNA isolation was performed shortly after the collection
of the blood samples. Total DNA was extracted using a genomic DNA purification
kit (K0512, Fermentas, Lithuania).
||Primer sequences and restriction enzymes (R.E) were used for
PCR and RFLP
||Polymerase Chain Reaction(PCR) genotyping of FXI deficiency.
Line 1 is FXI -carrier individuals(heterozygotes genotypes) has two fragments
of 244 and 230 bp. Line 2-6 is FXI deficiency-free animals(homozygotes genotypes)
produced only one 244 bp fragment. M is 50 bp DNA ladder (Fermentas, Lithuania)
Agarose gel electrophoresis and spectrophotometric methods were used to determine
DNA quality, purity and quantity prior to PCR amplifications. The genomic DNA
was stored at 4°C. While FXI deficiency was genotyped by polymerase chain
reaction, BLAD, DUMPS and citrullinamia were genotyped by restriction fragment
length polymorphism (PCR/RFLP). The sequences of primers for PCR and names of
restriction enzymes used for RFLP application are shown in Table
1. PCR products and RFLP fragments were visualized on agarose gels stained
with ethidium bromide (Fig. 1). The gene frequency of FXI
locus was estimated according to Nei (1987) by counting
the number of genes.
The primers were used to amplify 343 bp DNA fragment to detect studied population
for BLAD. The TagI restriction enzymes digested to PCR product into two
fragments of 152 and 191 bp in normal homozygote animals. After the analyses
all animals showed two fragments of 152 and 191 bp. For screening of possible
mutation in a gene coding for UMP synthase as described by Schwenger
et al. (1994), the 108 bp DNA fragment was amplified by PCR. The
digestion of PCR product gave three bands of 53 , 36 and 19 bp for normal animals.
Similarly, the 185 bp PCR products were digested by AvaII to reveal mutation
in a gene responsible for citrullinemia disease produced two bands of 103 and
82 bp for normal animals. None of the animals showed three bands of 185, 103
and 82 bp, so no animals found to be as carrier of citrullinemia disease. As
a result of electrophoretic analysis no carrier animal has been detected for
BLAD, citrullinemia and DUMPS. It was found that two carrier cows were FXI deficiency
carrier in the group of 170 animals. After the PCR applications, FXI deficiency
free individuals produced one bands were length of 244 bp and carriers individuals
for FXI deficiency gave two bands length of 244 and 320 bp (Fig.
1). The frequency of the FXI mutant allele and the FXI carrier prevalence
were 0.06 and 1.17%, respectively.
Although, some studies have reported that carrier animals of BLAD among Holstein
populations (Nagahata et al., 1997; Ribeiro
et al., 2000; Ertugrul et al., 2004;
Rahimi et al., 2006; Meydan
et al., 2007), in our study we did not find any carrier individual
for this disease. In Turkey two studies were carried out in order to identify
BLAD genotypes in Holstein populations (Ertugrul et al.,
2004; Meydan et al., 2007) and the frequency
of mutant allele was found as 0.0084 (Ertugrul et al.,
2004) and 0.035 (Meydan et al., 2007).
We didnt find any carrier individuals of citrullinemia and DUMPS. In
this respect results of present study are similar to the other results from
the different countries for citrullinemia (Grupe et al.,
1996; Patel et al., 2006) and DUMPS (Patel
et al., 2006; Kaminski et al., 2005;
Meydan et al., 2006; Akyuz
and Ertugrul, 2008). There are two studies of Holstein cows reared in Turkey
for UMPS and no mutant allele was found leads to DUMPS (Meydan
et al., 2006; Akyuz and Ertugrul, 2008) in
these studies. On the other hand, there have not been any studies carried out
in order to identify genotypes of citrullinemia until now. So, results on citrullinemia
were the first record in Turkey.
Undesirable allele of FXI deficiency has been determined in previous studies
in Holstein cattle (Marron et al., 2004; Ghanem
et al., 2005; Citek et al., 2008;
Meydan et al., 2009). Kunieda
et al. (2005) also reported another mutation located in exon 9 in
Japanese black cattle and their results were confirmed by Citek
et al. (2008). In Turkish Holstein cattle it was found four carrier
animals in the group of 225 animals (Meydan et al.,
2009). According to our results, mutant allele arising from an insertional
mutation at the exon 12 of FXI has been found in Holstein cattle populations
in Bursa district of Turkey at low frequency (0.06%). Large scale investigations
are needed in order to determinate carrier animals for eliminate them from whole
Based on the results of present study, the situation regarding to the investigated
recessive disorders of BLAD, DUMPS, Citrullinamia and FXI deficiency seems to
be good. But all of results need to be confirmed by further analyses in more
cattle in Turkey. This study showed that the frequency of carrier individuals
for this genetic disorder is very low among studied Turkish Holstein populations.
At the same time, it is not possible to say there are no carrier individuals
of BLAD, citrullinemia and DUMPS in Holstein population of Turkey.
This study was supported by grants from the Scientific Research Projects Council
of Uludag University (Project No. 2008-15), Bursa, Turkey.
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