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    Micoplasma Bovis

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    CARINA DOLORES HERNANDEZ PADILLA
    Micoplasma Bovis

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    gay SAUNDERS Research in Veterinary Seen 4 (2083) 108-112 swan conocer Review Mycoplasma bovis: disease, diagnosis, and control R.AJ. Nicholas", R.D. Ayling Mycoplasma Geagp, Deportent of Bacteriol Discoses, Veterinary LabaratresAgeney(Wevbide),Adeson,Sutey KTIS 3B, UK Abstract, Mycoplasma bovis is x major, bat often overlook, pahuen causing resitatory Usase, mastitis, and athits in eat, I is found workdwide and has spread into new area, inching Ireland and parts of South America, in te last decade, In Europe itis responsible fr atleast a quater ca tied ofall elf pneumonis although this may bean underestimate as ew laboratories regularly monitor for mycoplasmss. Like all molicutes, M. ows is inherently refractory to certain groups of antibotis because it doesnot ‘possess a cell wall furthermore evidence is accumulating that stains of A. bows are becoming resistant to antibiotics, cluding tetracycline, imicosin and spectinomyc 1M. bosisinfections. traditionally used for their control. No vaecines ae presently available forthe control oF ‘Crosun Consricht © 2047 Publiched hy Fleer Seem Fu A sights eserved 1. Toeroduetion It is litle known fact that mycoplasmas cause some of the most serious and economically most costly diseases Of eatue, Contagious dovine pleuropneumonta (CBPP), the only bacterial disease classified by the Office des Epizooties as alist A discase, is caused by Mycoplasma Imyenidessuhep. moroides mall enlony (SC) variant This mycoplasma was isolated just over a century ago but, while largely eradicated from Europe, it still presents immense problems in. Africa. Mycoplasma bovis is a ‘major, and often overlooked, cause of call preumonts, ‘mastitis, arthritis, and other conditions. Table I lists ‘some comparative properties of these two mycoplasmas land the disenses that they cause, In addition to theee respiratory pathogens, Mycoplasma dispar, Ureaplasmna diversum, Mycoplasma bovirkinis and, more recently, Mycoplasma canis have been isolated from the lungs of jpneumonic cattle though not all are believed to be pr mary causes of disease. M. mycoides subsp mycoides SC. has been the subject of a number of reviews (Nicholas ‘anu ashirudln, 1993; Wletolus et, 20003, Reel et al, 1996), The present paper focuses mainly on the practical aspects of the diseases caused by M. bovis M. boris was frst isolated in 1961 in the USA from a case of severe mastitis in cattle (Hale et al, 1962). 1 then ‘Comsponging author, te: 419525915195 fe: #1952978, mal odes 1 ichloviva dea (RAL, Nila) appears to have spread. via animal movements. to, amongst many countries: Israel (1964), Spain (1967), Australia (1970), France (1974), Britain (1975), Ceechoslovakia (1975), Germany (1977), Denmark (0951), Swivestand (1983), Moras (1988), Soul, Korea (1989), Brazil (1989) Northern Ireland (1993), Republic of Ireland (1994) and Chile (2000) (Nicholas, 200?) Today. infection has heen reported throuehout the world including most European countries ‘The prevalence of M, bovis is almost certainly un- derestimated as Mannheimia haemolytica, Pasteurella ‘multocida and. Haemophalus somnus are wwanably 180 lated first from pneumonic calves; in arthritis and mas- titis where M. bovis és a primary and uncomplicated ‘cause, itis often overlooked as fow laboratories rau tinely monitor for mycoplasmas. However unlike these conventional bacteria, the occurrence of M. bovis in a herd is invariably linked to eases of disease in particular pneumonia, mastitis and/or arthritis (Pfutener and Sachse, 1996). M, boris has also been associated with keratoconjuctivitis (Kirby and Nicholas, 1996), endo- Inciiis, salpingitis, Goplinitis, abortion avd semine= vesicultis, all of which have been reproduced experimentally (Ruhnke, 1994). 2. Economic losses In the UK, it ie estimated that up 10 1.9 million cattle are affected annually by respiratory disease which costs 4 sas8 «sz ont mater Crown Copysght ©2008 Polished by Elsie Science L1, All righs reserve Pit-soosd-s288(02)00185-8 106 RAL, Nicholas, R.D. Ayling |Research n Veterinary Science 74 (2008) 105-112 Table | Comparative propenies ofthe two most important cate myeoplasmas Props “M-mode sabap meer SC Me bow Discs ‘Contagious Bovine plewopnsumonia eal, ascasonally ‘Gall paeumona, mast, arin aor, artim enter ‘eraoconunetivtis Disation Subssharan Aca, probably in pars of Middle Fast, Censal Worldwide Asia Hosts Cat, goats ene) cae Histoloptholgial brine pleropneamonia with necro Inter pneumonia, ympho-histioestic ‘sion broach, catachl broncko-poeumonit Clic sgns Few sips, respiratory distess evident afer exercise Respiratory dsres, mastitis, avis Dragos thomon, arog. PLR, ator survsiance Serooqy, iain, FER Treatment (Chemotherapy not recommended Besse encourages Chemetberapy Conta ‘aceinaton, movement cont, slaughter fanagement eg, improved venation the cattle industry £54 million (Reeve-Johnson, 1999). Furthermore approximately 157,000 calves die annually as a result of pneumonia and related illnesses which would have a potential market value of £99 million Auoss Europe with appusimately 90 wuilion vate cis extrapolates to total losses of 576 million euros. It is likely that Mf, bovis is responsible for at least a quarter toa third of these losses although this is likely to he an underestimate (Nicholas et a., 2000). In the USA, the cost of M, hovis infections as a result of loss of weight again and carcass value have been estimated at $32 iillion per year (Rosengarten and Cit, 1999). The losses due to bovine mastitis caused by M. bovis may be higher than that for respiratory disease with estimates from the USA of up ta S108 million por yoar with in fection rates of up to 70% of a herd (Rosengarten and iti, 1999) 3. Epidemiology Me buvis ig wot ubiquitous but widely spread wit the bovine population in enzootically infected areas. The infection is usually introduced to M. bovisfree herds by clinically healthy calves or young cattle shedding the ‘mycoplasma and once established on multi-age sites it becomes very dificult to eradicate. Its appearance on some farms suffering low grade respiratory disease can lead 10 mereasea morbidity ana mortaity (Gourlay tal, 1989). Infected cattle shed the mycoplasma via the respiratory tract for many months and even years where they sot ne reesrvsire af infection (Pfotaner, 1000) Contact animals become infected via the respiratory tract, the teat canal or genital tract; artificial insemina- tion ‘with infected semen is another common route (Prutzner, 1990). The mate genital tract can become in- fected with M, bovis through contact with other animals or, possibly, via a heavily contaminated environment. Infection of the prepuce oF urethra by IM. bovis leads to ‘an ascending infection of the testes causing orchitis, sewed sacking Seoty vesicultis, decrease of semen quality and ultimately shedding in the semen (Kreusel etal, 1989). Ina study of eaves in the Netherlands, ter Laake al. (19922, 1992b) found M. boris in 20% of pneumonic Tugs fin Fisting hsads but vuly fn @ seal wumber of apparently healthy calves. Following its introduction into the North and South of Treland in 1994 from ‘mainland Europe. 1M. bovis has been consistently iso- Tated from 13 10 23% of pneumonic lungs (Brice ct al, 2000; Byrne et al., 2001). In France, ML. bovis was iso- lated from 30% of ealf herds with pneumonia (Le Grand et al, 2001) while in Britain, about 20-25% of pne monic herds contained animals with antibodies to M. ‘ovis (Nicholas etal, 2001). In the UK, significant an- tibody titoe to ML howe wines datantn in jet andor hall ‘of 55 pneumonic herds examined, of which only 7 herds, had rising titres to viral pathogens: respiratory syncytial virus, infectious bovine rhinotracheitis or bovine viral rhoea virus (Nicholas et al., 2001), Clearly other factors play a role in bovine respiratory disease such as concurrent viral and bacterial diseases as > environmental factors but itis increasingly be Tieved that Af. bocis is the predisposing factor in the infectious process leading to invasion by other bacterial pathogens possibly by compromising host defences (Poumarat etal, 2001; Rebhun et al, 1995). In a study of ealves which had died of pneumonia, Buchvarova and Vesselinova (1989) showed that over a third of lungs \wore infected only with, boots white Wie rest contained fa combination of M. hovis with P. multocida andlor H, somnus. These workers concluded that alterations in the lunge mora hiefly de tr myraplacma infection with the remaining bacteria contributing to complications in the pneumonie process. ‘Tschopp et al. (2001) confirmed the importance of -M. bovis as an agent of respiratory disease in a recent field study in which 50% of over 400 calves introduced to infected fattening sites developed respiratory disease attributable to AC bovis; they also showed an 8% reduetion in weight gain in the 55% of calves which RAL, Nicholas, R.D. Ayling | Resorch in Veterinary Science 74 (2003) 108-112 w became seropositive seven weeks after introduction, with these calves requiring twice as many antibiotics as seronegative calves, 'M. bovis is highly adapted to cattle but occasional isolations have been made in buffaloes, small ruminants and even humans. In one case, M. bovis was isolated from the throat of a woman who had developed bron chopneumonia after heavy exposure to cow manure (Madoff et al, 1979). The disease ameliorated following tetracycline therapy with the development of antibodies 10 M. pneumoniae but not M. bovis during convalescence suggesting that M, bovis may not have been the primary aetiological agent. In a second case M. bovis was iso- lated fiom w patient with respiratory disease wh only successfully responded to tetracycline treatment (Ball, personal communication), 4. Characteristics of the mycoplasma M, bovis, once regarded as a subspectes of the closely related small ruminant mycoplasma, M. agalactiae, was elevated to species rank and given its present name in 1076 (Ackaa and Eeno, 1976). Howover, in opto of the fact that the two mycoplasmas cause different diseases in different animal species and have a DNA-DNA reas- sociation value of only 40%, they show a very close 16S IRNA similarity of 99.8%. Mf. bovis is also biochemi- cally similar to M. agalactiae as neither ferment glucose ‘or hydrolyse arginine but instead use organic acids, auiaie wid pyruvate for dieit eneray source for growl (Miles ct l., 1988); they also give film and spot reactions in media indicating the possession of lipolytic activity Table 2 shows comparative biochemical characteristics of bovine mycoplasmas. Like all mollicutes, IM. bovis is small and pleomor- hie; it has a genome size of 1,080 kbp, lacks a cell wall dnd has. low G-+C ratio of 2/.8-529 mol’ (Hermann, 1992), Despite the fragility of most mycoplasmas in the environment, M. boris can survive at 4°C for nearly 2 months in sponges and milk and for over 2 wooks in water; at higher temperatures survival drops consider~ ably (PTutzner and Sachse, 1996). However it is unlikely that the environment provides a major source of infoc~ tion compared with that posed by mfected animals. On the other hand, mycoplasma contaminated frozen sperm can remain infectious for years and probably represents fan overlooked infection source, ‘The virulence factors of Af. bovis and mechanisms of pathogenicity are still largely unknown but the ability ff the agent to vary the expression of a family of ‘membrane surface proteins (ryp) with high frequency is elucidating this area (Pfutzner and Sachse, 1996). M. ovis has been reported to possess 13 sp genes involved in antigenic variation which alter the antigenic character of its surface components, and may act 10 enhance colonisation and/or adherence or evade the host's immune defence systems (Lysnyansky et al. 2001). These asp genes operate by various mechanisms including DNA transposition and intrachromosomal recombination within the nsp locus (Nussbaum et al. 2002), 5. Dizcase course A clinical study of endemic pneumonia, from which M. boris and P. multocida were frequently isolated from. the herd, showed that nearly half of dairy calves were shedding mycoplasmas at § days of age and over 90% by 4 weeks (Stipkovits et al., 2001). Clinical disease in the walves, which Gauses up 40 1079 woHtality ay a sesull of severe serofibrinous pneumonia, was highest between 10-15 days. Surviving ealves showed very poor weight, again and remained retarded: other signs included fever. depression, hyperpnoea, dysnpnoca, nasal discharge, mild (0 continuous coughing and loss of appetite. In the UK, calf pneumonia usually begins in November and peaiis around January but deatns duc to 1. ports con- finue to occur in some herds in the spring at pasture representing relapses because of unresolved lung lesions (Nicholas etal, 2001). “Table? Comparative biochemical properties of bovine mycoplasmas incading ME bus Mycoplsma ——Gusoxe Arginine Urea —Flm-—Castio” Phosphatase Terai ‘erobio Anaerobic fone we me we ae he He ve M Boothe ae He Ue fe Ne Movigentalian ve oe we ee oe te bos ee oe oe ove te he M mis te = we “ve ove tye Malorne oe o* ee “e Most tig +90 M.canaiewe =e re tk wate oe the Mr ae ore 1 we fe M. mide SC te oe we the ne Tk ow Arthritis associated with M. hovis infection may be a sequel to either the respiratory or mastitic form of the disease (Pfutzner, 1990). There is lameness, swelling of joints, slight elevation of temperature, allure of antibi- otic treatment, and if severe, reduced consumption of feed and debilitation. This condition often arises within 23 socks of howsing and may also follow transporta tion of calves over long distances, A case of severe ke- ratoconjunctivitis, impairing vision in a number of bullocks, involving M. bovis may also have been exac- erbated by the stress of transport (Kirby and Nicholas, 1996), Pulmonary lesions in naturally infected calves com= prise un exudative bronchopneumonia and extensive Foci of congulative necrosis surrounded by inflammatory cells, In studies involving experimental infections of gnotabiotic calves with M. boris, significant pneumonia \was induced involving up to 30% ofthe lung surface and was of sufficient severity 10 cause clinical respiratory disease in some calves (Thomas et al., 1986). Distinctive areas of coagulative necrosis were prominent within the lesions. Chronic infections are often associated with a ymphocytic “cuffing” pneumonia where there is marked hyperplasia of peribronchial lymphoid tows causing stenosis of airway lumina and compression and collapse ‘of adjacent pulmonary parenchyma, M. bovis antigen is mainly detected at the periphery of the areas of coagu- lative necrosis, in necrotic exudates and in close assoc! ation with infiltrating macrophages and neutrophils (Rodriguez et al, 1996). 6. Identification Clinical and pathological signs are not characteristic for M. bovis $0 laboratory diagnosis is necessary for identification. Samples of choice are listed in Table 3. In A recent study, Thomas et al (2002) showed that sam- pling by bronchoalveolar lavage was more predictive of | lower respiratory airway pathogens, including M. bovis, than nacal swabs although clearly not ag convenient. M. ovis grows well in a variety of media and produces ‘centred colonies within 3-5 days (Nicholas and Baker. 1998). In an appropriate medium (such as Table RAG Nickles, LD. Ayling} Rescarch in Veterinary Selene 74 (2008) 108-112 Eaton's) M. bovis produces films and spots and gives an ‘orange colour to the broth. Other biochemical charac teristics are shown in Table 2 alongside other bovine ‘mycoplasmas. Growth inhibition, film inhibition, fo rescent antibody, or metabolic inhibitions tests can be used to identify the mycoplasma using hyperimmune rabbit corum (Poveda and Nicholas, 1999) Ball and Findlay (1998) described a sandwich ELISA for M. bovis in which specific monoclonal antibod fixed to the microplate, captured M. bovis antigen from the medium; the sensitivity ofthe test, similar to that of conventional culture diagnosis, can be improved by a short enrichment stage. This test is now available in a ‘wommcicial format (Bio X, Luxembourg). M. hocis can be easily outgrown by opportunistic mycoplasmas like M. boviphinis and acholeplasmas and occasionally anti- ‘genic variability of strains may make serological tests unreliable (Ayling et al, 1997), For these situations, polymerase chain reaction (PCR) tests are very conve- nient. Barly PCRs, based on 16S rRNA genes, also am- plied M, agalactiae DNA (Ayling etal, 199/; Prutzner and Sachse, 1996) but recent PCR developments based fon the uirC gene are more specific (Frey et al., 1999), PCRs havo been woed to datook AM. bowie diewotly in mill and nasal samples (Hotzel et a, 1996) and even in pre- servative treated milk (Pinnow et al, 2001). ‘Serological detection of M. hovis antibody is often a more reliable diagnostic method as antibody levels de tected by ELISA remain high for many months. This is, particularly true where antibiotics have besn used ex- eusively vir ie Head while evesely hampers notations M, bovis is also difficult to isolate from chronically af= fected cattle (Nicholas, 1997). The presence of specific antihodies also indicates thatthe infection is invasive as animals in which M, bovis is found only in the nasal passages rarely seroconvert. While a number of sero- logical tests including indirect haemagglutination and fim inmpition nave teen reported, these nave vee mostly superseded by the indirect ELISA using whole call or chemically treated antigens (Nicholas et al., 20006); commercial tests are also available (Rommel ‘Switzerland; Biovet, Canada), The use of the ELISA for ‘accurately detecting antibodies inthe milk has also been described and was capable of identifying infected indi Sump of choi for deans cased by M. bois Disco Nas abe! ik Joi yevwate Semanigeialdacaree Sera for aibady DALfalce ang Ayn Prep washings dton Pacimona v Mase v ¥ Artis v y Ke v v Infrty ‘ y "Hronhial aol age > Kemtocanjnetvis. RAG Nicholas RD. Ayling | Rescorch in Veterinary Science 14 (2003) 105-112 19 vidual udder quarters (Byrne et al, 2000). The ELISA has been used successfully to select M. bovisefree cattle following BSE depopulation in Ireland (O'Farrell et al., 2001); its use in maintaining Af. doris free herds by testing newly bought-in animals before introduction to the herd should also be seriously considered. Re- combinant antigenc, eancieting af variahla snefare pr tein A, expressed in Escherichia coli as a fusion protein, were incorporated into an ELISA and were capable of detecting specific Mf. bovis antibodies in naturally in- feeted eatte (Brank etal, 1999) promising more specific diagnostic tests. Isolation of 4. bovis from lungs is not always pos- sible particularly in chronically affected animals: an biotic treatment, poor lung condition or gross bacterial contamination also inhibit isolation. Here, immunohis- tochemical techniques preferably using monoclonal an- tibodies may be valuable to visualise the mycoplasma antigens in the affected tissue (Adegboye etal, 1995), 7. Treatment of disease Pfiatsner (1000) stated that dicoasee de to. ME han were resistant to. any chemotherapy. In spite of this antibiotics are widely used sometimes correctly to re- dduce secondary bacterial infections but often ineffec- lively to treat the mycoplasma infections. Many in vitro studies have compared the susceptibility of M. bovis to a range of antibiotics (Table 4). While it is clear that an- tibietion which aro inoffootive in vitro are unllely to Be effective in vivo, those with strong activities in vitro will not necessarily perform well in the field for reasons which are unclear (Ayling etal, 2000). Recent evidence suggests that M. bovis strains in Europe are becoming resistant to antibiotics traditionally used for treatment of mycoplasma infections in particular oxytetracyclines, vos, aun spectinomyein (AgHing et al, 2000). The Tables fluoroquinolones are still effective but their use in ani- imal is controversial (Nicholas et a., 2000b). However, in one study in the UK monthly treatment with fluor- oguinolones for three months given at the outset of disease did not prevent respiratory disease in treated calves on a site persistently affected with M. bocis (Nicholas unpblished rrsnlts) Further field evidence oF the ineffectiveness of antibiotics was provided by Haines ct al (2001) who found M. bovis in the lungs and joints cof 80% of cases in feedlot cattle that had failed to re- spond to antibiotic therapy; bovine viral diarrhoea virus and M, haemolytica were found in only 40 and 23%, respectively, of these cases ‘Some success against M. hovis pneumonia and ar thritis in ealves was reported by Stipkovits etal. (2001) using valnemulin added to the milk from four days of ‘age for 3 weeks. Animals in the treated group had fewer clinical signs and reduced clinical scores though the ‘number of animals with nasal discharge was similar. In spite of this continuous chemotherapy, animals required A conslaerable number oF individual treaunents. 8 Diageo protontion and cantral ‘The inability of chemotherapy to control M. horis infections has focused attention on vaccination. Sur- prisingly there are no vaccines currently available al- ‘though a quadrivalent inactivated vaccine containing respiratory syncytial virus, parainfluenza type 3 and 2 protection against respiratory disease in the field (Howard et al., 1987). A vaccine prepared with formalin inactivated strains of M, bovis and M. haemolytica taken from the target herd reduced losses from pneumonia and cost of treatment in newly introduced feedlot calves, (Urbaneck et al, 2000). More recently a saponised in- sacle vaccine was Show 10 De SATE, Iihly [Eleivenes of anos in vt against eld strains of M. fois (inimem inkibiory concentrations yg) (CouuryGouree) No.of Envofouaciny’ —Lincomycin—Specinompein ‘Tylon Tlinkodn Owen strains danofoacin Reiberanas ier osuNb OT a Tuer ND wise Laak etal 1983) NeTeland (Bal 23 tao. ons 46 ND 006-05 ND eval, 195), (9 teins) 8 sean Tely(Marzotini 23 06-0290 028-1 ona ors wood etal, 19) BiaincAsing = 2 NBOIZS-2—-ND Losin) ND 4th a8 sta 200), 12s t0 = 138 (2 sean) Taipan (er Taal eal, 1oDa,by © Tyan mycoplasmas vasptes > appa, myophama show inert sep, > wa mye esi IND: not determined 10 RAL Nicholas, RD. Ayling |Research tm Veterinary Scene 74 (2003) 105-112 rogenic and protective against a strong experimental challenge of virulent M. bovis (Nicholas et al., 2002). ‘Vaccinated calves showed few respiratory signs while all tunvacemated calves developed signs of pneumonia, ‘There was a statistically significant decrease in body ‘weight gain in unvaccinated calves compared with vac- sinates and a cignifcant increase in hing lesions and rectal temperatures in unvaccinated calves, The vaccine also reduced the spread of IM. hotis to internal organs including the joints. However, attempts to vaccinate against M. bovis ar- thritis have been less successful against experimental infection although high levels of antibody were detected Defore challenge (Poumarat et a, 1999). Experimental vaccines against mastitis have not been successful and indeed may make the condition worse (Ross, 1993). Mastitis caused by M. bonis responds poorly to an- tibioties, therefore itis best 10 segregate or cull carrier cows and to instigate rigid sanitation procedures 10, prevent transmission from infected to non-infected cows (Plutzner, 1990), Identiication of meted cows m the carly stages of mastitis may be detected conveniently using the indirect ELISA for specific antibody in the mill moreover the tect oam identify the infacted quarter of an individual cow (Byrne et al, 2000), ‘The prophylatic use of antibiotic is generally unde- sirable but its use may be justified when calves are in- troduced to a site heavily contaminated with M. bovis infection in which high levels of mortality are being sustained. Nagatomo et al. (1996) treated one group of | nuoduced calves wile leuwomyein prior wy die devel- ‘opment of clinical signs. Untreated groups of calves showed mortality rates of up to 41% while all the calves in the treated group survived although coughing and nasal discharges were evident. Interestingly, while M. bovis antibodies were detected in non-treated groups after 2 months following introduction, antibody devel- fopment was significantly delayed untit over ¥ months in the antibiotic treated groups. This suggests that antibody responses are rarely protective in mycoplasma infections. ‘Control of calf pneumonia should also include mea- sures to reduce environmental stress and to ensure ad~ ‘equate housing with good circulation of air. Wherever possible consideration should be given to “all in, all out practices” to prevent older animals infecting younger ‘ones. If this isnot possible separation of calves from the fadulls is advisabie at die caulleat passible uppur tunity ‘where endemic disease exists and where the particular farm husbandry allows this. 9. Conclusion Work carried out (0-20 years ago at the Institute of ‘Animal Health, Compton, UK and the former Institut fur Bakterielle Tierseuchenforschung, Jena, Germany, clearly demonstrated a major pathogenic rote for M. bovis in respiratory disease, arthritis, and mastitis. De~ spite this there #8 Still a pervasive view amongst many Veterinarians that the role of M. Bovis in the respiratory disease complex is not proven (Step and Kirkpatrick, 2001), Ite importance has bon shown conclusively fal. lowing ts introduction into both northern and the southern Ireland in the early 1990s where it rapidly be- ‘came a major cause of respiratory disease as well as frequent cause of mastitis and arthritis. Major outbreaks have also been seen in Israel and Grevce following im- portation of affected cattle from westem and central Europe. The mycoplasma should be recognised by the Office International des Epizooties as causing a list B disease because of its serious socio-economic effects and the impact it has on international trade. In view of the large amount of evidence showing the ineflectiveness of antibiotics to control respiratory disease caused by MF bovis, research efforts should now be directed toward developing ellecive vaccines which could be used with ‘oF incorporated into those already existing for the bac- terial and viral respiratory pathogens. Acknowledgments We wish to acknowledge the financial support of the Department for the Environment, Food and Rural At fairs (defta) for aspects of this work. References, Adeghoye, DS, Rasberry, U, Halbur, PG, Andrews, 1, Rosen busch, RF 1995. Monoclonal antibody-bise snmenohisachen ial techie forthe detction of Aopen born forma Disgnoai Inventions 7, 241-26, Aska, G., Ero, Ha. 1916, Evaluation of Mjeoplasms agate Susp. bot to speses rank Meoplamna hone. Tnleratons Ayling, RD, Nichols, R.A, Jokunison, KE. 1997. Applicaton of ‘the polmerase chain reaction forthe routine detcation of Myeloma bonis. Vetnary Reed 141, 307-308, hla: Comparison of in vivo activity of danotloxac, Roreica, ‘ovgttseeting,specinomycin and timicoin against recent eld isles of Maplaons boi, Veterinary Record 146, 748-74 Ral HT Cag Rally GA. Arn. DG. 1995, Antibc "uscepibilty of Mjrpfowma hor seis in Noten land Trish Verna Jornal 4.316 318 Dal, H, Findlay, D, 1998. Diagnostic applications of monoclonal ‘nibody-hased sandwich ELISAs. fm Mile Ru, Nichols RAT. (Eds), Mycoplasma Prococols. Humana Pies, Totowa, USA, pp 127132 ‘rank, My, Le Grand, D.,Poumarat,F, Beil, P, Rosengren, R. ‘Ca, C, 199, Development of recombinant antigen or ansbody bas dgooss of Mpcoplamna bor sft ia cate lineal and Diagnostic Laboratory Immunology 6, 8-867 RAL Nicholas, RD, Ayling Reseurch in Veterinary Science 74 (2002) 15-112 mn Trice, N, Finlay. 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