Contagious agalactia due to Mycoplasma spp. in small dairy ruminants: epidemiology and prospects for diagnosis and control.

Contagious agalactia (CA) is a serious disease of small dairy ruminants that has a substantial economic impact on the goat and sheep milk industries. The main aetiological agent of the disease is Mycoplasma agalactiae, although other species, such as Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens, are pathogenic in goats. There are two clinical-epidemiological states of CA in sheep and goats; herds and flocks may exhibit outbreaks of CA or may be chronically infected, the latter with a high incidence of subclinical mastitis and only occasional clinical cases. The complex epidemiology of CA is related to the genetic characteristics and mechanisms of molecular variation of the Mycoplasma spp. involved, along with presence of CA-mycoplasmas in wild ruminant species. In goats, the situation is particularly complex and asymptomatic carriers have been detected in chronically infected herds. The coexistence of other non-pathogenic mycoplasmas in the herd further complicates the diagnosis of CA and the design of efficient strategies to control the disease. Routes of infection, such as the venereal route, may be involved in the establishment of chronic infection in herds. Current challenges include the need for improved diagnostic methods for detection of chronic and subclinical infections and for the design of more efficient vaccines.

Dynamics of an infectious keratoconjunctivitis outbreak by Mycoplasma conjunctivae on Pyrenean Chamois Rupicapra p. pyrenaica.

Between 2006 and 2008, an outbreak of Infectious Keratoconjunctivitis (IKC) affected Pyrenean chamois Rupicapra p. pyrenaica, an endemic subspecies of mountain ungulate that lives in the Pyrenees. The study focused on 14 mountain massifs (180,000 ha) where the species' population is stable. Cases of IKC were detected in ten of the massifs and, in five of them, mortality was substantial. The outbreak spread quickly from the first location detected, with two peaks in mortality that affected one (2007) and three (2008) massifs. In the latter, the peak was seasonal (spring to autumn) and, in the former, the outbreak persisted through winter. To identify the outbreak's aetiology, we examined 105 Pyrenean chamois clinically affected with IKC. TaqMan rt-PCR identified Mycoplasma conjunctivae in 93 (88.5%) of the chamois. Another rt-PCR detected Chlamydophila spp. in 14 of chamois, and 12 of those had mixed infections with mycoplasmas. In the period 2000-2007, the chamois population increased slightly (λ 1.026) but decreased significantly during the IKC outbreak (λ 0.8, 2007-2008; λ 0.85, 2008-2009) before increasing significantly after the outbreak (λ 1.1, 2009-2010). Sex-biased mortality shifted the adult sex ratio toward males (from 0.6 to 0.7 males per female) and reduced productivity slightly. Hunting was practically banned in the massifs where chamois experienced significant mortality and allowed again after the outbreak ended. Long-term monitoring of wild populations provides a basis for understanding the impacts of disease outbreaks and improves management decisions, particularly when species are subject to extractive exploitation.

Survival of Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri in heat treated goat colostrum.

The viability of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) was assessed in goat colostrum treated at different temperatures. Samples of colostrum were inoculated with reference strains of M. agalactiae (PG2) and Mmc (PG3) and heated at 56°C or 60°C for 0, 30, 60, 90 or 120 min. Viable colonies of M. agalactiae were recovered after all treatments and there was a significant reduction in the concentration of viable M. agalactiae after 30 min at 56°C and 60°C. No viable colonies of Mmc were observed after 60 min at 60°C.

Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain.

BACKGROUND: The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes. RESULTS: Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas. CONCLUSIONS: The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers.

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.

Viability of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in goat milk samples stored under different conditions.

Control programs for contagious agalactia (CA) involve monitoring milk samples to detect this disease. This study was designed to establish the effects of the preservatives generally used in dairy laboratories and storage temperature on the viability of Mycoplasma (M.) agalactiae (Ma) and M. mycoides subsp. capri (Mmc) in goat milk samples. In total, 1440 determinations were conducted for each mycoplasma species in milk samples subjected to different storage temperatures (refrigeration at 4°C or freezing at -20°C), preservation strategies (no preservative, NP; azidiol, AZ; or bronopol, BR) and storage times at each temperature (0, 2, 4, 6, 8, 10 and 24h at 4°C and 48h, 1 week, 2 weeks and 4 weeks at -20°C). Our findings reveal the similar viability of Mmc in milk samples stored at 4°C for 24h under the three preservation conditions examined. In contrast, the isolation of Ma in refrigerated milk samples was compromised by the presence of BR, and in smaller measure by the treatments AZ and NP. Freezing milk samples considerably reduced the viability of both mycoplasmas. Given the different sensitivity of the two mycoplasma species to BR, refrigerated milk samples treated with AZ could be used to detect infections caused by both species through culture-based methods.

Latent infection of male goats with Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri at an artificial insemination centre.

Contagious agalactia affects goats and is caused by several species of mycoplasma including Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc). Male goats, latently infected with M. agalactiae and Mmc, were identified at a dairy goat breeding artificial insemination centre. In three samplings, conducted over 1 year, ear swabs were assessed for both of the above organisms using culture and PCR techniques. Serological examination for antibodies against these organisms was performed at each time-point and conjunctival, nasal, rectal and preputial swabs were taken from a sub-sample of animals. Mycoplasma mycoides subsp. capri and M. agalactiae were detected in 80 and four ear swabs, respectively and serology confirmed the presence of both agents. A point prevalence of 0.06 goats infected with Mmc at the first sampling point increased to 0.97 at the last sampling, suggesting spread of infection. Both organisms were also detected in preputial and conjunctival swabs suggesting the shedding of these pathogens by other routes. These findings should inform World Organisation for Animal Health (OIE) guidelines on avoiding the introduction of such pathogens into artificial insemination centres and suggest the need to review current recommendations.

Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in ear swabs taken from goats.

This study was designed to evaluate the validity of PCR for the direct detection of Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two species most frequently causing contagious agalactia (CA) in goats. The PCR method was compared with the traditional culture technique to determine which method was most efficient at identifying all auricular carriers present in herds. The samples analyzed were 307 ear swabs taken from goats reared in a CA endemic area. We assessed the validity of each technique to detect each species and agreement between both methods. For each species, the result was taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246 were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117 showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR-positive samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative culture results and 4 culture-positive samples tested negative using PCR (2 for each species). Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae) and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M. agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and sensitive method for the detection of mycoplasmas in the external ear of asymptomatic carriers. Tools such as this are needed to adopt efficient control measures against CA.