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DIAGNOSTIC APPLICATION OF ELISA AND IS900 PCR USING
BUFFY COAT AS A SOURCE SAMPLE FOR THE DETECTION
OF Mycobacterium avium SUBSPECIES Paratuberculosis IN
SUBCLINICAL CASES OF BOVINE PARATUBERCULOSIS.
Kawther K. J., Fawziah A. A., Rasha M. O.
Department of Microbiology, College of Veterinary Medicine, University
Of Basrah ,Basrah,Iraq
Keywords;Buffy coat,paratuberculosis ,Bovine.
Corresponding Author; rashamunther2014@yahoo.com
ABSTRACT
John's disease or paratuberculosis is a chronic mycobacterial infection that affects
ruminants, adversely, leading to huge economic losses throughout the world. The
estimation of sero-prevalence and molecular confirmation of this disease in the cattle
population of south-Iraq were the objectives of this study. One of the diagnostic tools
used to detect an antibody in plasma samples was the Enzyme Linked Immuno-
Sorbent Assay, indirect-ELISA was used to diagnose and estimate the sero-prevalence
of paratuberculosis in cattle. Out of 156 bovine plasma samples, 81 (51.9%) were
positive and this MAP-seroprevalence is not significantly connected to age or breed
of cows (P>0.05). In this study, a PCR-based detection of IS900, distinct insertion
sequences of MAP from the buffy coat of seropositive cattle (n = 81) were used as a
confirmative diagnosis. The positive PCR-based detection of IS900 was observed in
animals having high S:P% ELISA values (n=29 :35.8%).
INTRODUCTION
Mycobacterium avium subsp. paratuberculosis (MAP) is etiological cause of
Johne´s disease, a chronic inflammatory enteric disease affecting cattle, sheep, goats
and other ruminants. Bovine JD has been recognized as a major disease causing high
economic losses to cattle globally, India (1, 2), Mexico (3); Colombia (4); Pakistan (5,
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6). A great individual variability of subclinical and clinical symptoms was detected.
The infection is typically characterized by a long incubation period followed by
chronic progressive diarrhea, decrease in milk production, edema, anemia, loss of
weight leading to cachexia and finally to death (7,8). Initial intermittent and later
persistent fecal excretion of MAP starts several months up to 2.5 years before the
onset of clinical signs and leads to the contamination of both the infected animal and
the environment. Following the previous way, the transmission of the infection mainly
occurs following ingestion of MAP by offspring during the first month of life.
Intrauterine transmission and direct excretion with milk are also described as
additional routes of infection but their epidemiological importance is hardly to
estimate (8, 9). Therefore, the essential precondition for eradication of
paratuberculosis from herds is the identification and elimination of MAP-shedders. In
control programs, ELISAs were widely used as screening assays for the detection of
antibodies against MAP in blood or milk because of their advantages like easy
performance, high capacity and low costs. Due to the late formation of antibodies,
ELISAs are characterized by low sensitivity especially in animals without or with
moderate shedding of MAP in their faeces (10, 11).
PCR-assays provide a rapid alternative and sensitive detection of MAP in clinical
samples including blood. The insertion element 900 (IS900) is the mostly used target
for identification and differentiation of MAP from other mycobacteria. The IS900 is
1.451 bp in length and is found in 15-20 copies in the MAP genome (12, 13, 14). The
main goal of this study was paratuberculosis diagnosis through the objectives,
estimation of sero-prevalence and molecular confirmation of this disease in the cattle
population of south-Iraq.
MATERIALS AND METHODS
Animals and Samples
Apparently healthy cows (N = 156) from south of Iraq were selected for case and
control study for autoimmune disease (John's disease). These cows aged from 2 to 14
years (two groups; < 7 years verses, ≥ 7 years). A minimum of 10 ml blood sample
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was collected from the jugular vein of each animal into K2-EDTA-blood tubes by
using 18 gauge needles. The K2-EDTA-blood tubes were transported to the laboratory
cold within 24 hours.
Isolation of peripheral leukocytes (buffy coat)
The K2- EDTA-blood tubes was processed by centrifugation at 3000 × g for 10
minutes at room temperature. A volume of 1 mL of plasma was transferred to a new
sterile tube and stored at −20°C for future testing for anti-MAP IgG, The leukocyte
containing buffy coat layer was carefully transferred to a new sterile tube. Leukocytes
were then mixed with two volumes of red blood cell lysis buffer (Roche Applied
Sciences, IN, USA). The hemolyzed samples were then centrifuged at 2500 × g rpm
for 5 minutes at room temperature. The supernatant was discarded and the leukocyte
pellet was stored at −20°C for further use in PCR analysis (15).
DNA Extraction
DNA was extracted from isolated leukocytes by using Geneaid gSYNC™DNA
Extraction (Korea) as per recommended protocols. The concentration of DNA was
determined using (Nanoodrop Quawell USA) l).
ELISA Methodology
The IDvet.310.rue ELISA (France) was performed according to the manufacturer’s
instructions. Briefly, plasma samples, including positive and negative controls, were
diluted (1:12) in a Dilution r and incubated for 45 minutes at room temperature (RT).
The previously diluted samples and controls (100 μl) were dispensed to each well of
an ELISA plate pre-coated with antigen, mixed with gentle tapping on the sides and
incubated for overnight at RT. The plates were hand-washed three times with wash
buffer, and a peroxidase-labelled monoclonal anti-ruminant IgG conjugate (100 μl)
diluted with dilution buffer (1:25) was added to each well and incubated for 30 min at
RT. The plates were washed as above and 100 μl of substrate was added and
incubated for 15 min in dark. The chromogenic reaction was stopped by adding 100
μl of stop solution, and the optical density (OD) values were read at 450 nm using a
plate reader (Wellkang Ltd., London, UK). The ELISA results were interpreted as the
signal of the test sample as a proportion of the positive control, corrected for the
negative control (sample-to-positive; SP %), according to the formula: SP% =
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[(OD450 nm of Test sera _ OD450 nm of Negative control) / (OD450 nm of
Positive control _ OD450 nm of Negative control)] × 100. SP% cut point for test
plasma ≥70% were considered positive, according to the manufacturer’s instructions.
Test sera with an SP% (60%< S/P% <70%)were considered as suspect and re-tested.
The re-test values were used for analysis. Test sera with an SP% ≤60% were
considered negative.
Detection of MAP DNA by PCR
M. avium subsp. Paratuberculosis infection further identified by the presence of
the IS900 elements in the peripheral leukocytes of 81 seropositive cows. PCR
employing IS900 gene specific primers of Mycobacterium avium subsp.
parartuberculosis (MAP) was used for diagnosis of Map DNA .The primers (MAP5:
5’-CTG GCTACC AAA CTC CCG A-3’, BA6:5’-GAA CTC AGC GCC CAG GAT-
3’) (314 bp) were designed from the IS900 sequence of MAP (16). The solated DNA
was amplified in 50 μl reaction mixture containing PCR buffer, mM MgCl2, dNTPs,
Taq polymerase (Promega / USAl), 1 μM of primers (MAP5 and MAP6) and 1 μl of
purified genomic DNA solution. The PCR conditions consisted of initial denaturation
at 94°C for 4 min, 40 cycles each of denaturation at 94°C for 1 min, annealing at 58°C
for 1 min and synthesis at 72°C for 1 min, and final elongation at 72°C for 4 min. The
PCR product was analyzed on 2% agarose gel.
Statistical analysis
The data obtained from the IS900 gene PCR and ELISA were analysed by Fisher's
exact test (SPSS software version 11).
RESULTS
S/P percentage and status of paratuberculosis disease
Table 1 , display the concentration of anti- MAP antibodies in different categories
of S/P ratios ( Strong positive (SP), Positive (P), Low Positive (LP) , Negative (N)
with respect to status of MAP infection in ‘tested cattle plasma. The positive category
appeared in higher percentage (33.3%) of tested plasma samples followed by Strong
positive (18.6%) category.
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Influence of cows age on ELISA
Table (2) display the influence of age on ELISA results of sero prevalence among
the cows screened by indirect ELISA. The higher seropositive prevalence (31.4%)
was observed in the age group ≤7 compared with age group > 7 years (20.5%).
However the difference between the two tested age groups is considered to be not
statistically significant between the two tested age groups (P=0.4195).
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Influence of cows breed on ELISA results
The seroprevalence of paratuberculosis in Indigenous breed (32 %) was higher than
cross breed cattle ELISA prevalence (19.9 %), however the difference between the
two tested breeds with respect to ELISA positivity is considered to be not statistically
significant (P=0.2010; Table3).
Table3: Distribution of ELISA results according to cows breed
Detection of MAP by IS900 PCR
IS900 PCR confirmed the presence of MAP in tested samples by using BA5: BA6
primers. The amplified product IS900 sequence (314 bp) were detected in 29 (35.8%)
out of 81 cow peripheral leukocytes (buffy coat) samples (Table 4; Figure,1).
IS900 PCR detected 29 (35.8%) positive, cows of which 19 (23.5%) were at age
group ≤ 7 year and 10(12.3%) were at age group > 7year (Table 4; Figure-1.) There
was no significant difference in the detection rate of MAP genome between the two
age groups( P=0.6361).
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Figure 1: Mycobacterium avium subsp. paratuberculosis specific amplicons (314 bp) by PCR using IS900 specific
primers. Lane M: 100 bp DNA ladder, Lane 2, 3,7,11, 17, 22: tested DNA samples
In table ( 5 ) Indigenous breed showed higher percentage of IS900 PCR
positivity(24.7%) compare to cross breed but this difference was not considered to
be statistically significant (P>0.05).
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DISCUSSION
Due to insidious nature of MAP infection in animals, prolonged incubation period
and detection of disease only in adult animals, the control and eradication of Johne’s
disease has been difficult throughout the world. In early stages of the infection, where
excretion of the bacilli is too low to be detected by culture or PCR, serological
screening of anti-Map antibodies and subsequent confirmation by genetic test appear
to be a logical option for detecting MAP infection in the livestock. an accurate
estimation of the prevalence of Map in cattle is difficult since most infected animals
are asymptomatic, the diagnosis in the early stages of disease is difficult and the
animals with clinical signs of decreased milk production can be slaughtered without
confirmation of Map infection (17). To date, only one Iraqi study (18) had tried to
assess the prevalence of paratuberculosis in Iraq, namely in the cattle population,
where this disease probably runs under-diagnosed. ELISA can detect specific
antibodies in blood or milk with a high sensitivity, and antibodies generally develop
late in the infection (19). Accordingly, an indirect ELISA for detection of antibodies
against Mycobacterium paratuberculosis was performed on plasma samples obtained
from 156 Iraqi apparently healthy cattle representing 110 local and 46 crossbreed. The
present results revealed that seropositive ELISA results (51.9%) was observed in
tested plasma.
In the present study, the sero-prevalence was found ( 51.9%) in south of Iaq which
can be considered high when compared with the Abdulrasool 2016 who reported
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2.64% sero prevalence in 6 animals out of 227 apparently healthy cows in south and
middle of Iraq .Separately 50% herd level prevalence in European countries (20,21)
other studies in different parts of the world have shown a varying range of cattle lower
level sero-prevalence: as 15.14% in India (1) 5% in Mexico (3); 3.125% in India
(2);8% in Colombia(4 ).; 5.88% in Pakistan. (5) and 2.31% in Bhutan (6). Many
factors probably contribute to the differences in prevalences between present results
and other countries results, such as animal characteristics management, climate, and
environment effects.
As Iraq depends on importation of livestock and livestock products from many
countries such as india where JD is prevalent in cattle, importation of large volumes
of dairy and meat products may act as potential sources of MAP exposure for humans
as reported by other studies in other parts of the world (22) There is obvious risk of
spreading MAP in ruminant population and possibly also to human.
In the present study seroprevalence of paratuberculosis in local breed (32 %)was
higher than cross breed cattle seroprevalence (19.9 %), however the difference
between the two tested breeds with respect to ELISA positivity is considered to be
not statistically significant ( P=0.2010) difference between the two tested breeds
with respect to seropositivity. The finding of Benavides (5) .that MAP-seroprevalence
is not connected to age, race, location and clinical status supported the present results,
but it is linked to body condition.
Molecular methods, especially PCR, real-time PCR and multiplex PCR are the
most promising methods for the rapid and specific diagnosis of JD (23) to date;
studies have focused on the PCR-based detection of MAP from feces, milk or culture.
IS900-PCR-based MAP detection directly from peripheral blood of animal was
investigated by few studies (24, 25, 26). In this study, the presence of MAP was
investigate in buffy coat of cattle .The introduction of IS900-dependent PCR has
reduced the time and labor required for the Because of the extremely slow
progression of John's disease, infected animals appear healthy, without shedding MAP
in milk or feces, while harboring potential infection in phagocytic cells, such as
macrophages. Such animals pose a real threat for the herd.. The present study,
successfully detected MAP with the help of the IS900-PCR technique from peripheral
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blood leucocytes of cattle. The prevalence of MAP in cattle was 35.8%, reflected in
the risk of MAP infection in younger animals. This observation was not correlated
with the exceptionally long incubation period of John's disease. The higher occurrence
of MAP positive cases in apparently healthy cattle indicates the chances of either
mixed infections or increased susceptibility to MAP infection in stressed animals.
In contrast with the present prevalence (35.8%) Bhide et al (24) mentioned that
prevalence of MAP in cattle was 11.45% and revealed the risk of MAP infection in
older animals. A total of 81 samples were found positive by indirect ELISA, twenty
nine of these seropositive samples were confirmed positive for MAP infection by
IS900.The current finding disagreed with Bhide et al .,2006 who reported that all
(n=13) seropositive samples were confirmed positive for MAP infection by IS900. In
conclusion, results of the present study indicated the utility of ELISA and
conventional PCR assay in rapid, sensitive and specific detection of MAP in in cattle
plasma and buffy coat.
Mycobacterium avium فی تشخیص جرثومة IS900 PCR و ELISA تطبیق
باستخدام طبقة الخلایا البیضاء کمصدر لعینات الکشف Subspecies Paratuberculosis
عن الحالات تحت السریریھ لمرض نضیر السل البقری
کوثر کاظم جبر، فوزیھ علی عبدلله، رشا منذر عثمان
الخلاصة
مرض جونز او نظیر السل ھو مرض مزمن تسببھ بکتریا نظیر السل الطیری یصیب المجترات بشکل واسع
مؤدیا الى خسائر اقتصادیة ھائلة فی ارجاء مختلفة من العالم. ھدفت الدراسة ھو تحدید او تقدیر انتشار المرض
فی ابقار جنوب العراق سیرولوجیا وجزیئا. أحد الطرق التشخیصیة التی استخدمت لتحدید مستوى الاجسام
المناعیة الخاصة بالبکتریا ھواختبار الالیزا غیر المباشر حیث استخدم لتشخیص وتقدیر انتشار المرض
٥١,٩ %) من مجموع ١٥٦ عینة بلازما ابقار أظھرت نتیجة موجبة ل فحص ) سیرولوجیا فی الابقار. ٨١
الالیزا ولم تظھر النتائج ارتباط م بعمر او نوع الابقار( مستوى المعنویة اکبر من ٠,٠٥ ) .کأختبار تأکیدی تم
اجراء تفاعل البلمرة التسلسلی المعتمد على تحدید القطعة الممیزة لبکتریا نظیر السل الطیری ٩٠٠ على عینات
الحامض النووی المستخلصة من طبقة خلایا الدم البیضاء للأبقار التی أظھرت نتائج الیزا موجبة
(عدد ٨١ ).النتائج الموجبة لتفاعل البلمرة التسلسلی فی الابقار ذات النسب المئویة السیرولوجیة العالیة لاختبار
. (% الالیزا(عدد ٢٩ نسبة ٣٥,٨
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