Current Research Journal of Biological Sciences 3(1): 12-16, 2011
ISSN: 2041-0778
© M axwell Scientific Organization, 2011
Received: September 23, 2010
Accepted: November 01, 2010
Published: January 15, 2011
Enzyme-Linked Immunosorbent Assay (Elisa) Based Detection of Antibodies
to Mycoplasma bovis in Cattle Naturally Infected with Haemoparasites
in Institutional farms in Sokoto State, Nigeria
1
F.M . Tam buw al, 2 L. Stipko vits, 1 G.O . Egwu, 3 A.U . Junaidu, 1 M.B. Abubakar and 4 U.A. Turaki
1
Department of Veterinary Pathology and Microbiology, Usmanu Danfodiyo University,
Sokoto , Nigeria
2
Mycop lasma L aboratory, Veterinary M edical Research Institute, Budapest Hungary
3
Department of Veterinary Public Health and Animal Production, Usmanu Danfodiyo University,
Sokoto , Nigeria
4
National Veterinary Research Institute, V om, Nigeria
Abstract: This was a cross-sectional study involving cattle from four (4) institutional farms (Prison farm,
Livestock Investigation and Breeding Centre (LIBC), Usmanu Danfodiyo University Teaching and Research
(UDUTRF) and K ebbe Ca ttle Ran ch (K CR ) in Sok oto state, Nigeria. A total of 62 cattle comprising 49 females
and 13 males were randomly selected and bled from a total population of 205. The cattle sampled were local
breeds comprising Gudali, Rahaji, White-Fulani and their crosses. They were aged 1-10 years and are managed
semi-intensively. The enzyme-linked immunosorbent assay was used for the detection M. b ovis antibody. Of
the 62 cattle screened, M. b ovis antibody was detected in 41(66%). Also, 24 out of the 41 M. b ovis positive
cattle were found infected with haemoparasites. Similarly, 11 out of the 21 serologically negative cattle were
infected with one or more haemoparasites. Seven (17%), 3 (7.3%) and 7 (17%) of the M. b ovis positive cattle
were infected with Babesia bigemina, Anaplasma marginale, and or B. bigemina and A. m arginale respectively.
In the overall, 27 of the 62 screened cattle w ere infected w ith one of blood parasites or a combination of both.
How ever, there is no significant statistical relationship (p> 0.05) between the number of cattle positive for
M. b ovis and the presence of haemoparasites among the examined cattle.
Key w ords: Antibodies, cattle, ELISA , haem oparasitism, institutional farms, My coplasm a bovis, Nige ria
INTRODUCTION
The two most important mycoplasmal disease agen ts
in cattle are My coplasm a mycoid es sub species mycoid es
small colony type (Mm mSC) and My coplasm a bovis
(Bashirud din et al., 2001; Thomson, 2005). Whilst the
former is known to cause Contagious Bovine
Pleuropneum onia (CBPP), the later is known to be
involved in a variety of bovine diseases but neglected in
most African countries (Nicholas and Ayling, 2003,
Nicholas, 2004). M. bovis is widely spread and causes
major economic losses in cattle in Europe and N orth
America (Boothby et al., 1983; Rebhun et al., 1995). It is
a proven cause of contagious mastitis in dairy cattle,
pneumonia, septic arthritis (Pfutzner and Sachse, 1996)
and otitis media (Walz et al., 1997). It is also rep orted to
cause keratocon juctivitis (Kirby and N icholas, 1996 ),
men ingitis (Stipkovits et al., 1993) and abscesses
(Kinde et al., 1993). M. b ovis infection was also
incriminated in repro ductive disorders of cows
(endometritis, salphingitis, reduction of conception rate,
abortion) (Hirth et al., 1966 as well as seminal vesiculitis,
epididymitis, orchitis and impaired sperm atozo a mo tility
in bulls (Lein, 1974; Jurmanova and Sterbova, 1977;
Kissi et al., 1985 ). Furthe rmore, M. b ovis is one of the
mycoplasmas that colonizes bovine respiratory tract
(Mu esster et al., 1979 ).
Diagnosis of M bovis infection can be performed by
several methods including isolation of the agent
(Sachse et al., 1993; Stipkovits et al., 2001),
immunohistochemical staining (Adegboye et al., 1995;
Knudtson et al., 1996) and the use of specific PCR probe
in the lung sam ples (G hadersoh i et al., 1997; Hayman and
Hirst, 2003) as well as detection of specific antibo dies in
the serum (Byrne et al., 2000; Le Grand et al., 2001).
Serological detection of M. b ovis antibody is a more
reliable diagnostic method for evidence of previous or
recent infections, as antibody level detected by ELISA
remains high for many mon ths (N icholas, 2004). This
diagn ostic technique is also suitable for detection of
Corresponding Author: F.M. Tambuwal, Department of Veterinary Pathology and Microbiology, Usmanu Danfodiyo University,
Sokoto, Nigeria
12
Curr. Res. J. Biol. Sci., 3(1): 12-16, 2011
M. bovis antibody in milk (Byrne et al., 2000). Awareness
of serological status of M. b ovis in the herd/farm is
important for preventing economic losses and instituting
control measures (Pfutzner and Sachse, 1996). On the
other hand, haemoparasites of cattle such as Bab esia
bigemina and A. marginale are known to cause serious
anae mia and debilitations in this species (Jongejan et al.,
1988). Concurrent infections of M. b ovis with some
haemoparasites can be more devastating in cattle than
single infection. Cattle exposed to M. bovis
natura lly or experimentally are immunosuppressive
(Thomas
et
al.,
1991,
Vanden Bush and
Rosenbusch, 2004). This could precipitate other coexisting latent infection(s) in debilitated cattle w ith
delitarious consequences. The present study therefore
examines the presence of M. b ovis infection in cattle
concurrently infected with some haem oparasites.
Switzerland (Stipkovits et al., 2001). The test was carried
out according to the manufacturer’s instruction. Serum
samples as well as positive and negative controls were
diluted 1:100 with CHECK IT diluting solution. 200 :L of
the diluted sera were distributed into the wells of
sensitized plates and incubated at room temperature for 90
min. Then the plates were washed three times w ith
CHECKIT washing solution. 200 :L of the included
peroxidase-labelled anti-ruminant IgG conjugate was
added to the wells in a dilution of 1: 600 and incubated as
earlier described. After washing off the excessive
conjugate, the reaction was visualized with 200 :L of
CHECKIT chromogen solution. The absorbance values
were read at 405 n m w ith a Titertek M ultiscan MS plate
reader. The OD (optical density) value was calculated
from the measured OD values of the samples and the
negative and positive sera as shown:
OD sample – OD negative serum
/ OD positive serum – OD negative serum × 100 = OD%
MATERIALS AND METHODS
This study was conducted in 2008 (January to
Septem ber) involving registered farms (only) in So koto
metropolis and it’s environ of Sokoto state, Nigeria.
Based on the protocol of the original kit, cattle having
OD% values between 60 and 80% are classified as
suspicious for M. b ovis infection and a value above 80%
is a clear sign of infection. H owever, since our aim was to
detect any trace of M. b ovis antibodies, O D% values
above 60% were regarded as positive
Study area: Sokoto state is located between the longitude
11º 30 ! to 13º 30! East and latitude 4º to 6º 40 ! North. The
state is semi-arid area of Nigeria w ith tropica l climate
whe re the temperature reaches 10 - 15ºC from November
to January, and 38-42ºC from M arch to May w ith scan ty
rainfall that starts late in A pril and ends early in
September with a mean range between 500-1300 mm
(Abdullahi, 1985). The state is one of the leading
producers of livestock in the co untry (RIM , 1992).
Parasitological investigation: Five ml of blood was
collected from the same animal population and transferred
into bijou bottles containing 1 mg/mL of Ethylene
Diam inetetra Acetatate (EDTA) and analysed for
haem oparasites as de scribed by F AO (1986).
Statistics: Data was analysed by descriptive and
inferential statistics using Graph Pad InStat (2000)
Version 3.05, 32bit for Windows 95/NT.
Sampling: A total of 62 cattle comprising 49 females and
13 males were randomly selected from four institutional
farms with total population of 205 . Sam ples co llected
were only from registere d (Institutional) farm s with
update records of their stock in the state. Other farms
short of these were not included in the studies. The
number of samples collected from each farm was 17, 15,
15, and 15 from cattle population of 66, 51, 42 and 46,
respectively. The an imals sampled were local breeds that
comprised Gudali, Rahaji, White Fulani and their crosses.
They were aged 1-10 years and m anag ed semiintensively, fed dry legume hay/pasture and supplemented
with local concentrate (W heat offals). Water was
provided ad libiton (ad-lib) in the farms.
RESULTS
Details of results of serology and haemoparasites
examination are shown in Table 1. O ut of the 62 cattle
examined, 41 (66% ) were po sitive for M. bovis
antibodies, while 21 (34%) cattle were found to be
negative. More females (65.3%) showed antibodies to M.
bovis than males (69.2%) although the difference was not
statistically significant (p> 0.05). Similarly, 24 (58.3%) of
the 41 M. b ovis positive cattle were infected w ith
haemoparasites. Eleven (52.3%) out of 21 M. b ovis
negative cattle were infected with one or mixed
haemoparasites.
Seven (17% ), 3 (7.3%) and 7 (17%) of the 41
M. bovis positive cattle were infected with B. bigemina,
A. marginale and B. bigemina and A. m arginale infection,
Co llection of sera: Blood was collected in vacutainers
tubes from jugular vein, allow ed to clot and sera separated
and stored at -20ºC until ana lysed.
Sera were examined with CHECKIT Mycoplasma
bovis Sero ELISA kit (Bommeli AG, Liebefeld-Bern,
13
Curr. Res. J. Biol. Sci., 3(1): 12-16, 2011
Table 1: N umber o f M . bov is positive cattle with corresponding number of positive single and multiple haemoparasitic infection
Parasitological examination
---------------------------------------------------------------------------------------------------------------ELISA assay n = 62
B. bigemina
A. marginale
B. big em ina a nd A . ma rgin ale
Positive
4 1 (6 6% )
7 (1 7% )
3 (7.3% )
7 (1 7% )
Negative
2 1 (3 4% )
7 (3 3.3 % )
1 (4 .7 % )
2 (9 .5 % )
Total
6 2 (1 00 % )
1 4 (3 4% )
4 (1 2% )
9 (2 6.5 % )
n = No. of samples
Total
41.4%
47.6%
27
Agb onlahor, 1980; Dogo, 2001; Ameh et al., 2004 ) with
t r e m e n d o u s i m p a c t o n li v e s to c k p r o d u c ti o n.
Immunosuppression which is a com mon characteristictic
among some of these diseases will exacerbate other
intercurrent infections (Ajuwape et al., 2004). The
concurrent detection of M. b ovis antibody and
haemoparasites in more than 50% serologically positive
cattle may have been due to stress induced
immunosuppression. It may as well be linked by system
of husbandry practice of the farms which exposes them to
health threat thro ugh contact with other potentially
infected animals. However, there is no statistical
correlation (p>0.05) between the occurrences of the two
diseases.
respectively. In the same vein, 7 (33.3%), 1(4.7%) and
2(9.5%) of M. b ovis nega tive cattle were infected with B.
bigemina, A. m arginale and B. bigemina and A. m arginale
respectively. Overall, 27 out of the 62 screened cattle
were infected by one o f haem oparasite or a combination
of both. However, there is n o significant relationship
between the number of cattle positive for M. b ovis and the
presence of haemoparasites amongst the examin ed cattle
(p>0.05).
DISCUSSION
M. bovis in spite of its importance is under estimated
in Nigeria (Tambuwal et al., 2009). Co nseque ntly, there
is pauc ity of information on the sero-prevalence of
M. bovis in Nigerian cattle except for the restricted studies
in the N orth eastern N igeria (Egwu et al., 1999 ). Prior to
this investigation, no serological examination has been
performed on the prevalenc e of M. b ovis in Sokoto state.
Therefore, it is likely that some of the previo usly
diagnosed cases of bovine respiratory problems may have
been associated with M. b ovis either singly or in
combination with other infectious agents incriminated
with the disease. M oreover, Manheimma haem olytica has
been reported to act synergistically with M. bovis,
producing very severe exudative pneumonia in calves
and aggravates the disease caused by the latter
(Doherty et al., 1994). This sometimes creates confusion
in the accura te aetiological diagno ses in the herd s with
persistent disease problem as common with the
myco plasma infec tions (Adegboye , 1986).
M. bovis antibody was detec ted in 66% of the cattle
examined. Cattle used in these farms were managed semiintensively; a practice which predisposes them to
nutritional deficiencies and diseases (RIM, 1992). This
probably explains the high rate of M. b ovis infection
recorded in this study. It also probably explains its causal
role in endemic respiratory diseases and mastitis as
indicated by the respective farms record. The figure 66%
is a true position of serological prevalence rate since the
disease (M. b ovis) is not va ccinated against in the state
and Nigeria as a w hole. Thu s, the recorded prevalence
rate in the study provides a reliable sero-prevalence
baseline data for M. b ovis in the state.
Haemo parasitic diseases are endem ic in Nigeria
(Dipeolu,
1975;
Ajayi,
1978;
Osiyemi
and
CONCLUSION
Paradoxically, the presen ce of M. b ovis infection
singly or with other concurrent infections in these cattle
may have some synergistic roles in precipitating
Contagious Bovine Pleuropneumonia (CB PP) disease
which is a con stant threat to cattle industry in Nig eria
(Egwu et al., 1996; Egwu, 2001; PAC E, 2004 ).
Importantly, the results obtained from this study may
indicate the importance of M. b ovis in cattle especially as
a respirato ry disease (R odriguez et al., 1996; Le
Grand et al., 2001), arthritis (Romvary et al., 1977;
Adegboye et al., 1996), mastitis (Tao udi et al., 1988;
Abbas, 1996) an d reproductive disorders frequently
observed in herds in Nigeria (Tambuw al, 2009). It as well
sugg ests a neglect of simple management practice such as
vaccination, deworming, dipping/spraying and proper
housing in these farms.
ACKNOWLEDGMENT
The authors wish to acknowledge the help and
support of Prof. (Dr) L. Stipkovits of the Mycoplasma
Laboratory, Veterinary Medical Research Institute of
Hungarian Academy of Science Budapest, Hungary for
providing the test kit and laboratory space for the assay.
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