Use of a polymerase chain reaction assay to study response to oytetracycline treatment in experimental Candidatus Mycoplasma haemolamae infection in alpacas

Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
American Journal of Veterinary Research (Impact Factor: 1.34). 10/2009; 70(9):1102-7. DOI: 10.2460/ajvr.70.9.1102
Source: PubMed


To develop a PCR assay for Candidatus Mycoplasma haemolamae (CMhl) infection in alpacas and use it to study the efficacy of oxytetracycline treatment and development of a subclinical carrier state.
8 healthy adult alpacas.
Alpacas initially had negative results for CMhl in blood samples via PCR assay and were experimentally infected with CMhl; 4 were treated with oxytetracycline, and 4 were not treated. All were monitored regularly via PCR assay, blood smear examination, PCV, rectal temperature, and physical examination. At 6 months after treatment, all alpacas were immunosuppressed by administration of dexamethasone and tested for CMhl.
7 of 8 alpacas had positive PCR assay results 4 to 6 days after experimental infection. When organisms were detectable on a blood smear, they were seen 2 to 6 days after positive results of PCR assay. Infection was often associated with mild anemia that was usually transient. No alpacas became hypoglycemic. Oxytetracycline treatment was not associated with faster clearance of organisms or resolution of anemia, and 4 of 4 treated alpacas still had positive results of PCR assay when immunosuppressed 6 months later; 0 of 3 nontreated alpacas had positive results of PCR assay following immunosuppression. Transient fever was detected in 3 alpacas during immunosuppression.
The PCR assay was more sensitive than blood smear examination for detection of infection. Clinical signs, anemia, and fever were not necessarily associated with infection. Oxytetracyline administration did not consistently clear CMhl infection. Although treated with oxytetracycline, infected alpacas remained chronic carriers.

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    • "This suggests a component of the dam's immune system is capable of reducing the number of parasites to below the threshold for detection, while producing antibodies specific to this organism. This is also compatible with prior reports (Tornquist et al., 2009, 2011). It is therefore possible that prior infection may provide the immunologic mechanisms necessary to clear infection and protect the newborn that acquires the organism early in life while colostral antibodies are still in circulation. "
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    ABSTRACT: Mycoplasma haemolamae from dam to cria, whether colostral transmission of M. haemolamae occurs and provide preliminary data on colostral M. haemolamae specific antibody from pregnant alpacas on a farm with known prevalence of infection. M. haemolamae specific PCR was performed on blood and colostrum from pregnant alpacas and their cria (n = 52 pairs). Indirect fluorescent antibody testing was performed on a subset (n = 43) of the colostrum samples. Total immunoglobulin concentrations of colostrum and cria sera and M. haemolamae specific IgG (prior to and after ingesting colostrum) were determined by turbidometric immunoassay and indirect fluorescence antibody testing respectively. Sixteen of 52 dams (30.7%) pre-partum and one of 52 cria post-partum (1.9%; prior to ingesting colostrum) were PCR positive for M. haemolamae, while 36/52 dams (69%) and 51/52 cria (98%) tested negative for M. haemolamae by PCR. All 43 colostrum samples and 52 of 52 post-colostrum cria blood samples (100%) were negative by PCR. The dam giving birth to the M. haemolamae PCR positive cria was PCR negative. Statistically, it was no more likely for a PCR positive dam to give birth to a M. haemolamae, PCR positive cria (prior to colostrum ingestion) than a PCR negative dam (p = 0.3077). M. haemolamae specific IgG was present in 22 of 43 (51%) of colostrum samples at a 1:10 dilution and 14 of 22 (64%) at a 1:100 dilution. There was no relationship between the PCR status of the dam and whether or not M. haemolamae specific antibodies were present in colostrum. Among the animals tested, in utero transmission of M. haemolamae was rare (1/52 pre-colostral alpaca cria), and all colostrum samples were negative for M. haemolamae by PCR. These data indicate that colostrum from positive dams is unlikely to harbor this parasite and therefore does not serve as a source of infection to newborn cria. Colostrum derived from both PCR positive and negative dams contained M. haemolamae specific antibodies. Our findings suggest that M. haemolamae specific antibodies may play a role in immunity to this hemoparasite; however, challenge studies are necessary to fully evaluate the role of M. haemolamae specific antibodies. Furthermore, antibody prevalence and detectable titers may provide different estimates than those available from current PCR based prevalence studies. Our findings also suggest that M. haemolamae isolates from geographically distinct regions do not differ significantly from each other.
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    • "Most recently, a real-time PCR method based on the SYBR Green principle has been published that permits initial screening for suspected hemoplasma infection in a broad range of mammalian species (Willi et al., 2009). The assay described herein allows quantification of bacterial burden with a sensitivity of one copy per 5 ml of blood, which is more sensitive than what had been published for a conventional CMhl PCR assay (Tornquist et al., 2009). The analytical specificity of the CMhl real-time TaqMan 1 PCR assay predicted in silico was confirmed by testing DNA from different hemotropic and non-hemotropic mycoplasma species. "
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