ELISA-based serological survey of Mycoplasma bovis in cattle in three local government areas in Adamawa State , Nigeria

A serological survey for the detection of antibodies to Mycoplasma bovis was conducted in Adamawa State, Nigeria during the year 2012. A total of 400 serum samples were collected from three local government areas (LGA) namely Yola (n=140), Mubi (n=130) and Ganye (n=130), and the samples were examined for the presence of M. bovis antibodies using BIO-X M. bovis antibody ELISA Kit. The overall seroprevalence of M. bovis was recorded as 19.5% (n=78/400). The highest seroprevalence was recorded in Ganye LGA (27.7%; n=36/130), followed by Yola LGA (20.0%; n=28/140) and Mubi LGA (10.8%; n=14/130). The cattle aging <1-year had the highest prevalence (25.8%), followed by 4-year-old cattle (22.4%). Based on sex distribution, 20.4% of the cows and 17.4% of the bulls were seropositive to M. bovis. Breed susceptibility to M. bovis infection showed that White Fulani had the highest prevalence (21.8%), and Sokoto Gudali had the lowest (11.9%). No statistical significant association was found between M. bovis infection and age, sex and breed of the cattle. In conclusion, the above findings are indicative for the presence of M. bovis in the study area.


INTRODUCTION
Mycoplasma bovis is a common inhabitant of the upper and lower respiratory tracts of healthy and pneumonic cattle (Thomas et al., 2002).M. bovis is a significant but sometimes neglected bacterial pathogen of adult dairy cattle, intensively reared beef, and dairy calves (Maunsell et al., 2011;Rérat et al., 2012).In 1961, the bacterium was first isolated in the United States from a mastitis affected cow (Hale et al., 1962).The organism is belonging to the Class Mollicutes, Order Mycoplasmatales, Family Mycoplasmataceae, and the Genus Mycoplasma (Razin et al., 1998).M. bovis is considered as a significant cause of several diseases like mastitis (Byrne et al., 2000), arthritis (Stipkovits et al., 1993), genital disorders and abortion (Byrne et al., 1999), bovine pneumonia (Pfützner and Sachse, 1996), and reduction of in vitro fertility (Eaglesome and Garcia, 1990).
Mycoplasmas cause several economically costly diseases in cattle.Approximately 157,000 calves die each year due to pneumonia and related diseases, which have a approximate market value of about €99 million (Vanden-Bush and Rosenbusch, 2003).M. bovis may be responsible for at least one quarter to a third of these losses, although this may be underestimated (Nicholas et al., 2000).
Diagnosis of M. bovis organism can be performed through several methods including immunohistochemical staining (Adegboye et al., 1995), isolation of the agent (Stipkovits et al., 2001), and use of specific PCR probe on lung tissues (Hayman and Hirst, 2003).Besieds, detection of specific antibodies in the serum can be performed (Le Grand et al., 2001).Serologybased detection of antibody against M. bovis by ELISA is considered as a reliable method for herd diagnosis for evidence of previous or recent infections (Ball and Nicholas, 2010;Maunsell et al., 2011).
Because the status of M. bovis infection in cattle in Adamawa State remains unknown, awareness of serological status is important in instituting control measures and preventing economic losses.Cattle are considered as one of the most important farm animals for meat production in Adamawa State.The objective of this study was to assess the current status of M. bovis antibodies in serum samples obtained from cattle in Adamawa State.The results would provide baseline data for the implementation of effective strategies for the control of M. bovis infection in cattle in Adamawa State.The State has an estimated cattle population of 2.8 million (Anonymous, 1994), comprising of White Fulani (Bunaji), Red Bororo (Rahaji), Sokoto Gudali (Bokoloji) breeds and their crosses, which together constitutes 88% of cattle breeds in Nigeria (Ngere, 1983).

Sample collection:
The cattle were selected using systematic random sampling method.Each animal was properly restrained and its age, sex and breed were recorded before sample collection.About 6 mL of blood sample was aseptically collected from the jugular vein of the animal, using a sterile 10 mL syringe and 18 G hypodermic needle.The blood was dispensed into sample bottles free from anticoagulant and centrifuged at 1500 g for 10 min.The serum portions were decanted into serum vials and stored at -20 0 C until analyzed.
Serological examination: Serum samples were examined with BIO-X M. bovis antibody ELISA Kit (Bio-X Diagnostics, Jemelle-Belgium).The test was carried out according to the manufacturer's protocol.All the reagents were brought to a temperature of 21 0 C +/-3 0 C before use. 1 mL aliquot of the dilution buffer was prepared in 5 or 10 mL hemolysis tubes.10 µL serum samples were added in each tube (dilution 1/100) and were shaken briefly on mechanical agitator.Positive and negative control sera were diluted as 1/100 in a dilution buffer.Sera samples and the positive and negative sera were distributed to the wells (100 µL/well).The plates were incubated at 21 0 C +/-3 0 C for 1 h.Then the plates were rinsed with the washing solution, emptying the contents by flipping it over sharply above a sink.The washing step was repeated two more times.The conjugate was diluted as 1:50 in the dilution buffer and 100 µL of the conjugate solution was added to each well and incubated for 1 h at 21 0 C +/-3 0 C, and the the plates were washed as mentioned earlier.After washing, 100 µL of the chromagen solution was added to each well on the plate.The plates were incubated for 10 min at 21 0 C +/-3 0 C. Then 50 µL of stop solution was added to each well.The optical density (OD) at 450 nm in the microwell were read using APPAR Plate Reader (UniEquip GmbH ® , Martinsried, Munich, Germany).
The OD was calculated from the measured OD values, and negative and positive serum samples using the following formula-× 100 A sample was considered negative if its coefficient was less than 37%.A sample was taken as positive if its coefficient was greater than or equal to 37%.
Statistical Analysis: Differences in M. bovis prevalence in cattle of different age groups, breed and sex were performed using statistical package for social science (SPSS) version 17.0 (SPSS Inc.Chicago, IL, USA).The difference was considered statistically significant when p value was <0.05.

RESULTS AND DISCUSSION
Serum samples (n=400) of cattle from three LGAs in Adamawa State were examined using BIO K260 ELISA kits [(BIO-X Diagnostics, Belgium) for M. bovis antibodies.
Out of the 400 serum samples, 19.5% (n=78/400) were found to be seropositive for M. bovis (Table 1).Out of the positive samples, 36 (27.7%), 28 (20.0%) and 14 (10.8%) were collected from Ganye LGA, Yola LGA and Mubi LGA, respectively.From the results, the highest seroprevalence was recorded in Ganye (27.7%), followed by Yola (20.0%) and Mubi (10.8%)LGAs, these showed a statistical significant difference (p=0.003;p<0.05).The ages of the cattle ranged between <1-year and >5year.Seroprevalence in different age groups varied from 2.9 to 25.8% (Table 2).The cattle aging <1-yearold had the highest seroprevalence (25.8%; n=8/31), followed by 4-year-old cattle (22.4%; n=22/98) and 1year-old cattle (2.9%; n=1/35), although there were no statistically significant differences among the age groups (p=0.690;p>0.05).The findings from the seroprevalence indicated that M. bovis antibodies could be detected in 19.5% (n=78/400) cattle.The overall seroprevalence was lower than the earlier report of Tambuwal et al. (2011) who reported a prevalence of 66%.The cattle sampled in this study were extensively managed (free range), a practice which predisposes them to nutritional deficiencies and diseases leading to immuno-suppression and vulnerability to infection.The lower seroprevalence reported in this study might be attributed to inadequate storage of samples and distance in transporting the sample.This might explain the causes of low rate of M. bovis infection found in this study.This finding was in agreement with the previous report of Byrne et al. (2000) in Ireland and Ayling et al. (2004) in Great Britain who reported a prevalence of 13-23% and 22%, respectively, eventhough cattle sampled by both authors were intensively managed.The seroprevalence obtained was higher than the report of Fu et al. (2011) and Zhao et al. (2012) in China who reported a prevalence of 7.69% and 5.95%, respectively.This might be attributed to samples from different regions and adequate management and biosecurity practices.
The seroprevalence of M. bovis varied in different age groups (2.9 to 25.8%), with cattle of <1-year-old having the highest seroprevalence (25.8%) followed by cattle of 4 years old (22.4%).This agreed with the earlier report of Zhao et al. (2012) who also reported the highest seroprevalence in dairy cattle of <1-year-old with no statistical difference between the age groups.The varied seroprevalence in different age groups suggested the possibility of horizontal transmission.The highest M. bovis seroprevalence in calves <1-yearold might be due to ingestion of infected milk which could be an important means of M. bovis transmission.
Cows might become intermittent shedders of M. bovis as it had been shown that as many as 40% of cows could shed <100 cfu/mL M. bovis in the milk, since contaminated milk could be the source of infection to young calves.Higher M. bovis seroprevalence in cattle of 4-year-old might increase the risk of milk contamination with M. bovis.This was because the cattle of 3 to 4 years old cattle were the bedrock of herds that were of breeding age.They were in their first or second stage of gestation (calving), they had potential of the spread of M. bovis, harboring the organism sub-clinically or chronically.The milking system of herdsmen also had a potential for contaminating udders as hand milking encouraged mastitis.Therefore, carrier cows were the most likely the source for the infection for naive calves in herds.
In this study, female cattle had higher prevalence (20.4%) as compared to males (17.4%).This agreed with the earlier report of Tambuwal et al. (2011) who reported that females showed antibodies to M. bovis more than the males.This was because more females were kept in a herd in comparison to the males for the purpose of reproduction and milk production.The fact that female cattle predominates pastoral herds meaning that any maintenance of M. bovis in such hers would be done by these female cattle.They could play important roles in the spread/epidemiology of M. bovis because they remained for longer periods in herds, passed through more stress of reproduction, calving and nursing, had greater chance of coming in contact with contaminated water, pasture, fomites and environment.For this reason, they could transmit the infection from one generation to another.
Breed distribution showed White Fulani (21.8%),Red Bororo (16.4%),Adamawa Gudali (11.9%), and Cross breed (18.5%) even though there was no statistical significant difference.The higher prevalence was observed in White Fulani.However, M. bovis infection had not been reported to have breed specificity.Therefore, all cattle irrespective of the breed had equal chance of contracting the infection.
The presence of M. bovis infection as immunomodulator and/or co-factor might have an important role in the development of contagious bovine pleuropneumonia (CBPP) due to M. mycoides subspecies mycoides small colony (Mmm SC), being very much endemic in Nigeria.Certain similarity in pathogenesis of M. bovis infection (Stipkovits et al., 2001) with that of Mmm SC infection might contribute to inefficiency of vaccination against CBPP of cattle in the study area.Thus, the presence of M. bovis in these animals might account for the endemicity of CBPP in Nigeria especially that no vaccination campaigns were vigorously pursued in Nigeria.
The result obtained from this study might indicate the importance of M. bovis in cattle as a respiratory disease (Le Grand et al., 2001).Other workers had demonstrated the organism to be involved in arthritis, mastitis and reproductive diseases (Adegboye et al., 1996;Abbas, 1996;Le Grand et al., 2001) as frequently observed in cattle herds in Nigeria.Therefore, it was likely that some of the previously diagnosed cases of bovine respiratory, reproductive problems as well as other pathologic conditions of the udder (mastitis), otitis and arthritis might have been associated with M. bovis either singly or in combination with other infectious agents incriminated with the disease.

CONCLUSION
This study reports the occurrence of Mycoplasma bovis infection with a prevalence rate of 19.5%.Cattle of all ages, sexes and breeds are at risk with the organism.To the best of our knowledge, this is the first time serological survey of M. bovis conducted in this transboundary State in Nigeria.Therefore, baseline data for M. bovis provided in this study may help in taking an effective control strategy of the disease in Nigeria.
This study was conducted during August to November 2012 in three Local Government Areas (LGA; Ganye, Mubi and Yola) representing the three senatorial zones of Adamawa State, Nigeria.Study Area: Adamawa State is located in the north eastern part of Nigeria, roughly where the River Benue enters Nigeria from the Cameroun Republic.It lies between longitude 11°E of the Greenwich Meridian and latitude 10°N of the Equator.The State shares boundaries with Taraba State in the south and west; Gombe State to its north-west; and Borno State to the north.Adamawa State has an international boundary with the Cameroun Republic up along its eastern side.

Table 1 :
Seroprevalence of M. bovis in Cattle in three Local Government Areas in Adamawa State.

Table 2 :
Seroprevalence of M. bovis in Cattle of different ages in Adamawa State.

Table 3 :
Seroprevalence of M. bovis in Cattle based on Sex in Adamawa State.
Seroprevalence of M. bovis varied in different breeds of cattle, ranging from 11.9 to 21.8% (Table4).

Table 4 :
Seroprevalence of M. bovis in Cattle based on breed in Adamawa State.