mycoplasma infection in swine - an overview

Arlette Laval Ecole Nationale Vtrinaire de Nantes, Nantes, France

Introduction
Mycoplasmal infections are very common chronic diseases in pig herds. In spite of the continuing improvement of control strategies, they remain the most devastating agents in terms of economic losses in nursery-finishing pigs in the swine industry worldwide. The important Mycoplasma spp., in swine are Mycoplasma hyopneumoniae, M. hyorhinis and M. hyosynoviae. Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia and an important contributor of the composite infection known as the porcine respiratory disease complex (PRDC), that implies also viruses (Porcine Reproductive and Respiratory Syndrome Virus PRRSv, Swine Influenza SI, Porcine Circovirus type 2 PCV-2) and other bacteria (eg. Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus suis etc). Mycoplasma hyorhinis is also a very common inhabitant of respiratory tract and occasionally causes polyserositis and arthritis in young pigs. Mycoplasma hyosynoviae causes polyarthritis in finishing pigs.

days. The colony looks like the sunny side up egg (Quinn, 2002). Mycoplasma hyorhinis is easier to cultivate. Mycoplasma synoviae grows quickly, if it is cultivated in traditional media especially enriched with mucinous material from swine (Kobisch and Friis, 1996).

Mycoplasma hyopneumoniae infection

It is the causative agent of enzootic pneumonia, the most important and frequently occuring mycoplasmosis. Surveys conducted in different countries have indicated that typical pneumonic lesions occur in 30 - 80% of slaughter pigs.

Clinical signs vary according to the quantity and virulence of the bacteria, but other factors such as environmental and management conditions are important, as enzootic pneumonia is a multi-factorial disease. Several risk factors are involved: poor disease prevention measures, number of visitors, volume of pig trade, low general health status, poor feeding, poor housing system, (especially in the continuous flow system where contacts between young and older pigs in adjacent pens is allowed). Pen stocking density, airspace stocking density, ammonia level and the amount of dust are all important predisposing factors. In the case of non-infected farms, the distance to the nearest non-SPF herd is a very important factor for the risk of re-infection.

Epidemiology
Mycoplasma hyopneumoniae is fragile and cannot survive in the environment for long. However, long survival in aerosols is possible under adequate meteorological conditions. It is host-specific but passive human carriage is possible. Airborne transmission is possible on at least 5 km, with decreasing frequency to 3.2 km away (Godwin, 1985). However, carrier swine are the major source of infection, mainly by direct contact with infected animals or from respiratory tract secretions. Each cough may carry live organisms as far as 3.5 meters. Transmission occurs among penmate swine and from infected sows to their progeny in the first days of life. Infection is mainly transmitted by gilts and low parity sows. They have a lower immunity and excrete more microorganisms than older sows. Non-immune pigs of various ages seem to be equally susceptible to the disease. Under continuous production systems, spread occurs in the finishing sheds when old and young animals are placed in adjacent pens (Maes, 1996). The spread throughout a newly infected farm is fast. Then, when a farm is infected, there are always carrier pigs which ensure the persistence of the disease which explains why it is so prevalent in all countries. There is neither intrauterine nor lactogenic transmission. Transmission by contaminated tools is possible but not frequent.

Pathogenesis
The incubation period is approximately 10-16 days (Ross, 1999). The prerequisite for disease initiation is the adhesion to the ciliated epithelium of trachea, bronchi and bronchioles. Only few mycoplasmas reach the smaller bronchioles and alveoli. The organism remains at the apex of the cilia, in the intercilia space or in contact with the microvilli. At least four proteins of M. hyopneumoniae are involved in adhesion to target host cells. The next step is the reduction in cilia activity, loss of cilia, micro-colony formation and accumulation of bacteria. Finally, the loss of cilia is complete, and destruction and exfoliation of epithelial cells can be seen. Viscous exudates accumulate in airways, explained by reduced cilia activity, hypersecretion and altered glucoprotein production in goblet cells. The bacterial membrane contains a mitogenic protein, responsible for the massive lymphoid

Bacteriology
Mycoplasma spp., are in the family Mycoplasmataceae. They belong to the taxonomic group Mollicutes (Kobisch and Friis, 1996). They are the smallest autonomously replicating organisms, and differ structurally from other eubacteria in that they lack a cell wall, have a small genome and possess an elementary DNA. Their shapes vary from spherical (0.30.9 ) to filamentous. They can be cultivated in an artificial medium; however, culture and identification are tedious and time-consuming. The growth is low, especially for Mycoplasma hyopneumoniae. In primary broth cultures, Mycoplasma grows slowly, producing a faint turbidity and an acid color shift after 4 to 15 days. Cultures are passed several times in broth, then inoculated on agar medium and incubated in a 5-10% carbon dioxide atmosphere. Colonies become visible after 2-3 days incubation and they increase in size 0.25-1 mm diameter in about 10

hyperplasia around blood vessels and airways. They can obliterate the lumen of bronchioles and cause collapse of surrounding alveoli. M. hyopneumoniae has an immunedepressive effect both on humoral and cellular immunity. Reduction of humoral immune response is due to a reducted capacity of lymphocytes to produce antibodies. Cellular immunity is suppressed by inhibition of macrophage mediated phagocytosis. This suppression is most pronounced in the early post-infection period but can last for several weeks. Enhancement of pro-inflammatory cytokines (IL10, IL12 and interferon gamma) is another factor involved in M. hyo pathogenesis, especially in case of co-infection with PRRS virus (Thanawongnuwech et al., 2004). The presence of damaged epithelial cells and thick, viscous mucus as well as reducted immune response facilitate secondary bacterial infections. The most frequent are subsequent infection by Pasteurella multocida and, in case of an infected herd, Actinobacillus pleuropneumoniae. In addition, Actinomyces pyogenes, streptococci, staphylococci are also frequently implicated. Then, clinical symptoms and lesions are more severe and the mortality rate increases (Maes et al., 1996). Viral co-infections are very frequent. Lung lesions are more severe in case of co-infection by PRRSv and render pigs more susceptible to other pathogens. Additive effects between M. hyo and swine influenza virus or M. hyo and PCV-2 have been demonstrated (Thacker et al., 2001, Opriessnig et al., 2004). M. hyo enhances PMWS primarily induced by PCV-2.

Clinical signs
In conventional farrow-to-finish herds, under field conditions, enzootic pneumonia is characterised by high morbidity and low mortality. Symptoms begin when maternal antibodies disappear, usually between seven and 12 weeks of age. Coughing is the principal clinical sign. It is a dry cough with no sputum that starts at 2-4 weeks and peaks at 4 weeks post-infection. It can last for 8 weeks depending on the immune status and on herd management. There is little or no fever. Anorexia leads to poor growth rate. Mortality rate is unchanged or only slightly increased. If the infection is complicated by bacteria, mainly Pasteurella multocida, clinical disease is more severe, with a productive cough, high fever, anorexia, laboured breathing and prostration. The morbidity is high and the mortality rate increases. The groups of pigs are not homogeneous and contain unthrifty pigs with severe growth retardation and a rough hairy coat. In the case of young gilts purchased from high health status herd (Mycoplasma free) and introduced into a contamined herd, the clinical signs can be severe and very difficult to control. Mortality is not uncommon either nor is the necessity to cull most of these gilts. Therefore, it is necessary to be very careful in managing the quarantine of replacement gilts and to adopt an appropriate program of vaccination. This acute form is also seen when M. hyopneumoniae is introduced into a herd for the first time. For a period of 6 to 8 weeks after introduction, there may be severe acute

pneumonia, coughing, respiratory distress, fever and high mortality across all ages of stock. However, even in these cases, the disease may remain mild or inapparent.

Lesions
At the early and middle stages of infection, a catarrhal bronchopneumonia is found in the apical and cardiac lobes, the accessory lobe and the cranial portion of the caudal lobes of the lungs. This localisation reflects air flow patterns as the right apical lobe receives a stem bronchus directly from the trachea. Bronchial and mediastinal lymph nodes are enlarged. In the chronic phase of the infection, atelectatic lungs lesions are observed, with purple to grey areas of consolidation. Rarely, M. hyo can induce joint and pericardial lesions (Kobisch and Friis, 1996). At the early stage of infection, microscopic lesions are characterised by loss of cilia, exfoliated ciliated cells and small accumulations of neutrophils in lumina and around airways. At the middle stage, increased numbers of lymphocytes appear in peribronchial, peribronchiolar and perivascular spaces. At the chronic stage, there is lymphoid hyperplasia or cuffing and thickening of the interalveolar septa in the atelectatic areas. Secondary infections worsen the lesions, with more pronounced purulent inflammation.

Economic impact of M. hyopneumoniae infection

Losses are linked to reduced productivity and increased medical expenses. They are directly linked to the severity of the disease. According to Muirhead and Alexander (1997), the lesion score given by slaughterhouse data allow to the following estimation (refer Table 1).

Table 1: A guide to the effects of enzootic pneumonia lesions on daily weight gain (muirhead and Alexander, 1997)
Total score maximum 55 Zero 1-10 11-20 21-30 31-40 40-55 % loss in daily gain 0 0 6 18 26 50

Diagnosis
Clinical signs that lead to suspected enzootic pneumonia include a chronic non-productive cough, retarded growth, stunting, with low mortality, slow onset and spread, and repeated occurrence of the disease. Lesions are not specific in that similar lesions may be seen in pneumonias caused by other agents, but they give a good orientation on the prevalence of M. hyopneumoniae and its pattern of circulation in a given herd.

Atelectatic lesions in the cranioventral areas of lungs will be checked and histopathology may be used to help make a differential diagnosis. Periodic examination of lung lesions at the slaughterhouse allows practitioners the opportunity to track the evolution of the disease and the efficacy of the control measures. A specific notation must be adopted to allow standardisation of reporting which is essential when comparing results between checks performed by different operators. Each lobe is scored from 0 to 4 (France) or 0 to 10 with a maximal score of 5 for both diaphragmatic and azygos lobes (USA, UK, Australia). The final notation takes into account the average score on tested pigs and the percentage of non-affected lungs. Specific laboratory testing may be necessary to confirm the diagnosis or to perform profiles in order to know when exactly the bacteria begin to spread in a given herd. Serology tests for specific antibodies are currently used. Complement fixation test, agglutination, latex agglutination, indirect IF test and ELISA tests are available. Not all of them are widely available. ELISA is the most convenient test which can easily be automated. The indirect test is very sensitive, but may give crossreaction with Mycoplasma floculare, a nonpathogenic species. The blocking ELISA is more specific to M. hyopneumoniae. Serum sampling can be performed using a cross-sectional method by sampling pigs at various ages or at the same time. Results may be used for monitoring disease status and planning disease prevention strategies. Antibodies are first detected 3 to 5 weeks post-exposure, they peak at 5-7 weeks postexposure and can be detected during 3 to 52 weeks depending on the type of test. The ELISA test can be adapted for using colostrum as a sample for diagnosis. Direct research of M. hyopneumoniae is performed using nasal swabs from live animals or lung samples from necropsy. Culture and identification are difficult and time consuming. M. hyo is often masked by the presence of M. hyorhinis because the detection of M. hyo antigen is performed by direct or indirect ImmunoFluorescence (IF), on frozen thin sections of lung. However IF is not very specific, due to cross-reactions with M. floculare or M. hyorhinis. The most reliable and quick technique is PCR, now widely used in most laboratories. Real time PCR, the advantage of which is that it is quantitative,

has been recently developed. All these tests are required to differentiate M. hyo infection from other respiratory pathogens able to induce consolidation of the anterior lobes of the lungs: SI, PRRSv, PCV-2, mild Haemophilus parasuis infection and other mycoplasma.

Diagnosis
Gross lesions of polyserositis in 3 to 10 week old pigs are very evocative of M. hyorhinis infection, but similar lesions can be caused by Haemophilus parasuis, Streptococcus suis and Mycoplasma hyosynoviae. M. hyorhinis can be isolated from acute and subacute phase lesions, before treatment. Serum antibodies can be detected by different techniques, such as complement fixation test and indirect hemagglutination. After experimental infection, they can be detected 6 weeks post-infection.

Mycoplasma hyorhinis infection

Mycoplasma hyorhinis is a very common in host nasal cavities and tonsils of pigs. Usually, it is not pathogenic. However, it may easily descend to the pigs lungs and become a causative agent of pneumonia. It is also frequent in cases of polyserositis, causing the systemic disease named Mycoplasmal Polyserositis-Arthritis of pigs. M. hyorhinis is transmitted to young piglets shortly after birth by sows or older pigs. It has been isolated from pneumonic lung tissue of SPF piglets (20%) and conventional piglets (66%) (Kobisch and Friis, 1996). In case of clinical infection, M. hyorhinis spreads hematogenously from respiratory tract and multiplies in the pericardial, pleural, peritoneal and synovial cavities.

Mycoplasma hyosynoviae infection

Mycoplasma hyosynoviae is a porcine species with a special affinity for joint tissue. It may be the cause of serious and troublesome arthritic diseases with a high infection rate and great economic losses for pig producers. However, this infection is not common in the field. It can be isolated from arthritic joints, but also respiratory tract and tonsils. Tonsils remain infected throughout the life of the host and play a critical role in transmission of the bacteria. Usually, piglets become infected from their mother after approximately 4 to 6 weeks of life, later than for other mycoplasmas.

Clinical signs and lesions

Outbreaks of polyserositis generally occur in piglets of 3 to 10 weeks of age, but the disease can also occur in young adults. Symptoms start 3 to 10 days after exposure or following stress. They consist of a moderate hyperthermia, lameness, inappetence, difficulty in moving and swollen joints. Then, laboured breathing, stretching movements and lying in sternal decumbency characterise the diseases evolution. The disease then becomes chronic with less severe lameness and joint swelling. In the acute stage, lesions consist in serofibrinous pericarditis, pleuritis and peritonitis. Synovial membranes are hypertrophied, synovial fluid is sanguineous and the amount of fluid increased. In the chronic stage, organised fibrous adhesions and articular erosions are observed. Pneumonia is inconstant. M. hyorhinis is commonly found in the Eustachian tube of young pigs and can be the primary cause of porcine otitis media. Microscopic changes are characterised by fibrinous inflammation of most serosal membranes and mononuclear cell infiltration.

Clinical signs and lesions

Symptoms occur in the young, growing pigs, from approximately 8 to 30 weeks of age especially in gilts recently introduced onto a farm or in the early stages of pregnancy. Disease is sudden in onset, the first signs being a reluctance to rise at feeding time. The pain is considerable and the affected pigs will only stand for short periods of time. The temperature is normal or slightly elevated. Joints are more or less swollen and painful at palpation.

Macroscopic examination of joints reveals a yellow non-suppurative viscous fluid, of increased volume. In chronic cases, the joint capsule is distended. Microscopically, hypertrophy and hyperplasia of the synovial membrane is more prominent and erosion of the articular cartilage may be seen. Diagnosis must be made by isolation of the bacteria or PCR from the joints and a positive response to macrolide or tiamulin injections. Serology is not really helpful due to the large number of healthy carriers.

References
GOODWIN R.F. 1985. Apparent reinfection of enzootic pneumonia-free pig herds: search for possible causes.Vet. Rec. 116, (26), 690-694. KOBISCH M. and FRIIS N.F. 1996. Swine mycoplasmoses. Rev. Sci. Tech. Off. Int. Epiz. 15, 1569-1605. MAES D., VERDONCK M., DELUYKER H., De KRUIF A. 1996. Enzootic pneumonia in pigs. Vet. Quart. 18, 104-109. MUIRHEAD M., ALEXANDER T.J. 1997. Management pig health and the treatment of disease. 5M Enterprises Limited, PO Box 233, Sheffield S35 0BP, UK, 608 pp. OPRIESSING T., THACKER E.L., FENAUX M., MENG X.J., HALBUR P. 2004. Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet Path. 41, 624-640. QUINN P.J., MARKEY B.K., CARTER M.E., DONNELLY W.J., LEONARD F.C. 2002. Mycoplasma. In: Veterinary Microbiology and Microbial Disease. Blackwell Science Ltd. English. p. 189-195. ROSS R.F. Mycoplasmal Diseases. In: Diseases of Swine. 8th Edition. Barbara Straw, Sylvie DAllaire, William L. Mengeling and David J. Taylor (Ed.). Iowa State University Press. Ames. Iowa. USA. p 495-509.

THACKER E.L., HALBUR P.G., ROSS R.F., THANAWONGNUWECH R., THACKER B.J. 1999. Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia. J. Clin. Microbiol. 79, 115-127. THANAWONGNUWECH R., THACKER B.J., HALBUR P.G., THACKER E.L. 2004. Increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumonia. Diagn. Lab. Immunol. 11, 910-908.

Conclusion
Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma hyosynoviae are
three major pathogens in pigs. They cannot be dissociated for they share epidemiological particularities and bacteriological properties. However, Mycoplasma hyopneumonia, the causative agent of enzootic pneumonia, is from far the most important. Economic losses, through retarded growth, poor feed conversion and predisposition to other bacterial infections are considerable. As an uncomplicated infection in well-housed and managed pigs, it is not a very severe disease. However, if there are other infections present, particularly App, PRRS, SI, PCV-2 or, most often Pasteurella multocida, the pneumonia becomes more complex with serious effects on the pig health and on the herd productivity. The appropriate use of laboratory tests is necessary to optimise the control of the disease.

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