Mycoplasma mycoides subsp. capri associated with goat respiratory disease and high flock mortality Laura Hernandez, Jose Lopez, Marcel St-Jacques, Lourdes Ontiveros, Jorge Acosta, Katherine Handel Abstract — A high mortality outbreak of respiratory mycoplasmosis occurred in goats in Mexico. The clinicopathologic presentation resembled contagious caprine pleuropneumonia caused by Mycoplasma capricolum subspecies capripneumoniae. By using a battery of polymerase chain reaction assays, the mycoplasma associated with this outbreak was identified as Mycoplasma mycoides subsp. capri. Résumé — Mycoplasma mycoides subsp. capri associé à la maladie respiratoire de la chèvre et à un taux élevé de mortalité dans les troupeaux. Une flambée de mycoplasmose respiratoire présentant une mortalité élevée a atteint des chèvres au Mexique. Les signes clinicopathologiques ressemblaient à la pleuropneumonie caprine contagieuse causée par Mycoplasma capricolum subsp. capripneumoniae. Par une batterie de tests d’amplification en chaîne par polymérase, Mycoplasma mycoides subsp. capri a été identifié comme responsable de cette flambée. (Traduit par Docteur André Blouin) Can Vet J 2006;47:366–369
number of mycoplasma species have been associated A with caprine respiratory disease (1), but some of the most virulent ones are Mycoplasma capricolum subsp. capripneumoniae (Mccp), M. mycoides subsp. mycoides Large Colony Type (MmmLC), and M. mycoides subsp. capri (Mmc), all of which belong to a group of very closely related mycoplasmas known as the “Mycoplasma mycoides (Mm) cluster,” which also includes 3 other pathogens of ruminants: M. mycoides subsp. mycoides Small Colony Type (MmmSC), M. capricolum subsp. capricolum (Mcc), and the bovine group 7. All these mycoplasmas share genomic and antigenic features, which result in very closely similar biochemical and serologic properties, making precise identification of individual isolates a difficult task and contributing to confusion among diagnosticians (2). Mycoplasma capricolum subsp. capripneumoniae is the etiologic agent of contagious caprine pleuropneumonia (CCPP), a severe, acute, and highly contagious respiratory condition, prevalent in many countries of Africa and Asia. The disease affects all ages and is characterized by high fever, high morbidity, and high mortality. Respiration is accelerated and painful, coughing is frequent, and, in the terminal stages, the animal is unable to move, standing base wide and neck extended. The gross lesions of the disease are typically limited to the thoracic cavity and characterized by fibrinous pleuropneumonia, lung hepatization, and accumulation of Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Centro Nacional de Investigación Disciplinaria (CENID), Km 15.5 Carr. México Toluca, Col. Palo Alto, CP 05110 México DF (Hernandez, Ontiveros, Acosta); National Centre for Foreign Animal Disease (NCFAD), Canadian Food Inspection Agency (CFIA), 1015 Arlington Street, Winnipeg, Manitoba R3E 3M4 (Lopez, St-Jacques, Handel). Address all correspondence and reprint requests to Dr. Jose Lopez; e-mail:
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pleural fluid (3,4). Often, the pneumonia may be unilateral (2,3). Other infectious agents, specifically MmmLC and Mmc, can produce a similar clinical presentation. Because CCPP is not known to occur in the Americas, it is important to rule out Mccp as an agent of foreign animal disease when outbreaks of goat respiratory mycoplasmoses appear with similar clinical, pathological, and epidemiological features. Mycoplasma mycoides subsp. mycoides Large Colony Type and Mmc infections can have a systemic manifestation and are sometimes referred to as the “MAKePS” (mastitis, arthritis, keratitis, pneumonia, and septicemia) syndrome (2). An outbreak of respiratory disease occurred in a herd of 2000 Sannen dairy goats in the State of Durango, Mexico, in 2003. The first cases were seen in September, but the number of affected animals peaked during December. This was a closed, intensive milk production flock. Due to increased demand for goat milk, animals of different breeds were imported from Canada and the USA, approximately 6 mo before the start of the outbreak. Initially, 200 animals died over a period of 15 d and, after 6 mo, the flock mortality had reached 40%. Animals of all ages were affected, although they were mostly adults. Kids usually died in less than 1 wk after the onset of clinical signs, whereas adults survived longer, up to 2 mo. The clinical picture was characterized by fever, abundant nasal secretion, difficult breathing (extended neck), prostration, ear drop, and low milk production. Breathing difficulty was exacerbated by forced exercise. Although there were some animals with clinical mastitis, it was not a common feature of the outbreak. Arthritis was not observed. A number of antibiotics, including tylosin, enrofloxacin, oxytetracycline, kanamycin, and penicillin were used at different times in therapeutic attempts, without success. The extreme weather conditions prevalent in the region might have contributed to the high mortality observed in the outbreak. Mycoplasmas are Can Vet J Volume 47, April 2006
Figure 1. Polymerase chain reaction products obtained after amplification of template DNA from mycoplasma isolates recovered from Mexican goats with respiratory disease (A) and from reference mycoplasma strains (B), using the MmF-MmR primer pair, which target sequences of the Mycoplasma mycoides cluster (7); and the corresponding PstI digestion products of the 548 bp amplicons seen in A (C). MW: Molecular weight ladder, 50 bp; 1–7: Mycoplasma isolates from Mexican goats; F38 and GL102: M. capricolum subsp. capripneumoniae; USM: Unidentified sheep mycoplasma; PG3: M. mycoides subsp. capri; YG: M. mycoides subsp. mycoides Large Colony Type; Cal. Kid: M. capricolum subsp. capricolum.
osmotically very fragile, because they lack a protective cell wall and are bound by the cell membrane only; therefore, transmission is achieved through inhalation of microdroplets containing the microorganisms by animals in close proximity. Fourteen animals were necropsied. Grey, red, or both, areas of consolidation were seen in the lungs, affecting 1 or more lobes. All animals showed marked pleuritis and pleural effusion. The pleural fluid was not thick and was yellowish. The cut surface of some affected lungs revealed a fine granular texture with hepatization. The joints were not affected. These findings are similar to those described for CCPP (3). No significant gross lesions were observed in other organs, except for hemorrhagic small and large intestine in some animals. The histopathologic findings were consistent with proliferative interstitial pneumonia. There was thickening of the interlobular septa, fibrin, and increased numbers of mononuclear cells. There was a marked vascular response, characterized by congestion, hemorrhagic zones, and occasional thrombosis. In all instances, there was proliferation of the bronchus-associated lymphoid tissue (BALT) and alveolar macrophage activation. Polymorphonuclear leukocytes in alveolar and bronchiolar spaces were seen in 7 animals only. All animals had considerable pleural thickening, fibrin accumulation, and marked dilatation of local lymphatics; similar lesions were seen in the interlobular septa. There were multifocal hemorrhages and neutrophil infiltration in the liver and spleen, as well as hepatic multifocal necrosis. Can Vet J Volume 47, April 2006
Pleural fluid, lung, synovial fluid, ocular secretion, and milk samples from 14 animals were inoculated onto 5% sheep blood agar and in Friis medium (5), as modified by Rose et al (6), enriched with horse serum instead of pig serum. Both solid and liquid media were used. Friis broth and blood agar plates that had been inoculated were incubated at 37ºC in a humid aerobic atmosphere. Friis agar plates that had been inoculated were incubated in a 5% CO2 atmosphere. Mycoplasma growth from pleural fluid and lung samples was evident after 48 to 72 h incubation. The colonies had the typical fried egg appearance. Broth cultures were subcultured onto solid medium, and isolated colonies on Friis agar plates were subcultured 3 times to obtain pure cultures of the isolates. All isolates were digitonin sensitive, glucose positive, did not produce “film and spots,” and digested casein. No other bacterial pathogens were isolated on blood agar. Only 1 eye secretion sample yielded mycoplasma growth in culture. To rule out the possibility that the outbreak might have been caused by the exotic Mccp, the PCR-PstI digestion protocol of Bascunana et al (7) was followed, using DNA templates obtained from all the mycoplasma isolates and directly from the pleural fluid and lung tissue. Primers MmF and MmR resulted in the amplification of a 548 bp fragment, common to all mycoplasmas of the Mm cluster, thus identifying all isolates as members of this cluster (Figure 1A, 1B). Digestion of the 548 bp amplicons obtained from all the field isolates with the enzyme PstI resulted in 2 fragments only, 420 bp and 128 bp. The 367
Figure 2. Polymerase chain reaction products obtained after amplification of template DNA from mycoplasma isolates recovered from Mexican goats with respiratory disease, using the following primer pairs: P4/P5, specific for M. mycoides subsp. mycoides Large Colony Type (A), P4/P6, specific for M. mycoides subsp. capri (B), P4/P8, specific for M. capricolum subsp. capricolum (C), P4/P9, specific for M. capricolum subsp. capripneumoniae (D), P4/P10, specific for bovine group 7 mycoplasmas (E), and MSC1/MSC2, specific for M. mycoides subsp. mycoides Small Colony Type (F). MW: Molecular weight ladder, 50 bp; 950010 and YG: M. mycoides subsp. mycoides Large Colony Type; USM: Unidentified sheep mycoplasma; 1, 3 and 7: Mycoplasma isolates from Mexican goats; PG3: M. mycoides subsp. capri; 7714 and Cal. Kid: M. capricolum subsp. capricolum; F38 and GL102: M. capricolum subsp. capripneumoniae; 8859-291 and PG50: Bovine Group 7 mycoplasmas; PG1 and Afade: M. mycoides subsp. mycoides Small Colony Type.
same amplification and digestion patterns were obtained with DNA templates prepared directly from pleural fluid and lung tissue. The control digestion reaction containing the amplicon from the Mccp type strain F38 yielded the anticipated 3 fragments, 548 bp (undigested), 420 bp, and 128 bp, characteristic of this microorganism (Figure 1C). The possibility that Mccp was associated with this outbreak was therefore ruled out. The identification of the Mm cluster subgroup to which the field isolates belonged was done according to the PCR scheme of Hotzel et al (8), using several primer pairs. Since it was already known that the goat mycoplasma isolates belonged to the Mm cluster, the 1st stage PCR of the scheme (cluster-specific) was not necessary; the 2nd stage PCR is subgroup-specific. The PCR assay used to rule out MmmSC was as described by Dedieu et al (9). Amplification of the template DNA from field goat mycoplasma isolates was observed only when prim368
ers P4/P6 were used, which recognize a sequence specific for Mmc, resulting in an amplicon of 194 bp (Figure 2B). Polymerase chain reaction assays in which all the other primer pair combinations specific for the other subgroups of the Mm cluster were used did not yield any amplification products with DNA templates obtained from the goat field isolates (Figure 2A, 2C, 2D, 2E, 2F). The isolates were therefore identified as Mmc. The P4/P6 amplicons were sequenced and a comparison search of nucleotide databases, using a basic alignment search tool (BLAST), was consistent with the identification. The sequencing data matched a highly conserved region of the Mm genome, top rankings corresponding to Mmc and Mmm. The prevalence and relative importance of Mmc infection in Mexico is not known. There is only 1 report of this condition in the 60s and, at the time, it was thought to be the first report of CCPP in the Americas (10). CVJ Can Vet J Volume 47, April 2006
References 1. Matthews J. Diseases of the Goat, 2nd ed. Oxford: Blackwell Sci, 1999:258–265. 2. Thiaucourt F, Bölske G. Contagious caprine pleuropneumonia and other pulmonary mycoplasmoses of sheep and goats. Rev Sci Tech 1996;15:1397–1414. 3. Thiaucourt F, Bölske G, Leneguersh B, Smith D, Wesonga H. Diagnosis and control of contagious caprine pleuropneumonia. Rev Sci Tech 1996;15:1415–1429. 4. World Organization for Animal Health. Contagious caprine pleuropneumonia. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (mammals, birds and bees). 5th ed. vol. II Chapter 2.4.6. Paris: Office International Des Epizooties, 2004: 623–634. 5. Friis NF, Mycoplasmas cultivated from the respiratory tract of Danish pigs. Acta Vet Scand 1971;12:69–79. 6. Rose DL, Tully JG, Wittler RG. Taxonomy of some swine mycoplasmas: Mycoplasma suipneumoniae Goodwin et al. 1965, a later,
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