Investigation of Mycoplasma haemolamae infection in crias born to infected dams
- S. J. Tornquist, DVM, PhD, DipACVP1,
- L. J. Boeder, BS1,
- S. Lubbers, DVM3 and
- C. K. Cebra, VMD, MS, DipACVIM2
- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4802, USA
- Department of Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4802, USA
- Amazia Veterinary Services, Brush Prairie, WA 98606, USA
- E-mail for correspondence
THE haemotropic mycoplasma Mycoplasma haemolamae adheres to the surface of red blood cells of llamas and alpacas. It is variably associated with anaemia and sometimes with other clinical signs such as depression, fever and weight loss. Many infected camelids do not develop clinical or laboratory abnormalities (Tornquist and others 2002, 2009). Infected camelids that are immunosuppressed, stressed or concurrently infected with other organisms are more likely to show clinical signs and to have organisms visible on blood smears (Hutchison and others 1992, Barrington and others 1997, Lascola and others 2009). It appears that many camelids, once infected, remain chronic carriers regardless of whether they are treated with antibiotics (Tornquist and others 2002, 2009).
The development of a PCR-based assay in 2001 has facilitated the study of M haemolamae and has begun to clarify some aspects of infection and response to treatment, although the organism has still not been successfully cultivated in vitro. The PCR assay is a sensitive and specific method for identifying early infections as well as subclinical infections (Tornquist and others 2009).
The mode of transmission of M haemolamae has not been determined, although biting insect vectors are suspected or confirmed in other haemotropic mycoplasma infections (Messick 2004, Woods and others 2005, Almy and others 2006, Willi and others 2007). In utero transmission has also been suspected, on the basis of the presence of organisms in crias as young as 24 hours of age, although postpartum or colostral transmission were not excluded in these crias (Fisher and Zinkl 1996, Almy and others 2006). Infected crias may be clinically healthy or severely clinically affected at birth (Fisher and Zinkl 1996, Almy and others 2006; S. J. Tornquist, personal communication). In order to investigate the possibility of in utero, parturient or transmammary transmission of M haemolamae in New World camelids, pregnant alpacas known to be positive for the organism were identified and studied.
Blood samples and a sample of colostrum were collected from naturally exposed females and their offspring in three privately owned alpaca herds. Owner consent was obtained for all samples taken. All the dams received regular veterinary care and had tested positive for M haemolamae during herd screening. Blood samples were collected by jugular venepuncture between one hour before to one hour after parturition from five pregnant adult female alpacas ranging from two to four years of age and determined to be positive for M haemolamae at the time they gave birth using the PCR assay, performed as described below. Blood samples were taken from the crias within 30 minutes of birth and before they suckled or had any postpartum contact with another alpaca. A sample of colostrum was taken from one of the dams just after parturition. Postsuckling blood samples were also obtained from each cria within one week of birth. Subsequent blood samples were taken from four of the crias until they were up to six months of age.
The PCR assay for M haemolamae was performed as previously described by Tornquist and others (2009). Briefly, DNA was extracted from 200 µl of the blood using a commercial extraction system (Generation Capture Column; Gentra Systems). To ensure there was no DNA contamination during the extraction process, a negative control consisting of all of the reagents without a DNA sample was included, as well as a positive control that consisted of EDTA-anticoagulated blood from a naturally infected alpaca with large numbers of typical organisms seen on blood smears. The colostrum sample was ultracentrifuged to remove lipids and DNA extraction was performed on the non-lipid fraction.
Amplification was accomplished using primers for a 318 bp sequence that is unique to the hypervariable region of the M haemolamae 16S rRNA region, as identified in GenBank (accession number AF306346) (Messick and others 2002). A negative control consisting of the reaction mixture with no template DNA was included. Confirmed positive blood and distilled water were included in each assay as positive and negative controls, respectively.
All the dams and crias in the study appeared clinically healthy at the time the samples were taken. There was no evidence of concurrent immunosuppression or anaemia. No intercurrent disease problems were known at the farms and there was no history of high mortality. M haemolamae was not detected on blood smears from these alpacas.
Three of the five crias were negative for M haemolamae before suckling as well as after ingesting colostrum. The colostrum sample was also negative. Two of these crias were tested at one- to two-week intervals until they were approximately 15 weeks of age, with negative PCR results for each sample. One of these crias later tested positive when it was two years of age.
The other two crias were positive immediately after birth, both before and after suckling. Blood samples from one of these crias were tested every few months until the animal was 6.5 months of age, and all the samples were positive for M haemolamae. Follow-up samples were not obtained from the other cria that was positive at birth.
One of the positive dams was negative for M haemolamae one year later at the time of parturition and gave birth to a cria that was also negative.
These results indicate that transplacental or parturient transmission of M haemolamae may occur, but it is not inevitable. This study did not provide evidence of transmission in the colostrum but does not preclude the possibility, as the detection limit for the PCR assay in colostrum is not known and a sample size of one animal is clearly insufficient for drawing any conclusion.
Alpacas have diffuse epitheliochorial placentation, which should minimise the intermixing of maternal and fetal blood (Olivera and others 2003, Almy and others 2006). However, there are specialised zones of the maternal-fetal interface in the placenta that may allow exchange of nutrients and hormones, although it is not clear whether an organism could cross at these sites (Olivera and others 2003). It has been suggested that mixing of blood between the dam and cria could occur at the time of parturition, providing an opportunity for transmission of infection. However, experimental transfusions of large amounts of infected blood into negative camelids have shown that M haemolamae is first detected by PCR at four days postinfection at the earliest (Tornquist and others 2009). It seems unlikely that mixing of blood between the dam and cria at parturition could account for a positive PCR test at the time of birth, but the present study does not disprove this possibility. Insect-borne transmission of the infection to these neonatal crias is extremely unlikely.
In summary, dams that are positive for M haemolamae by PCR at the time of parturition may give birth to crias that are either PCR-positive or negative for M haemolamae. This study did not provide evidence that the organism can be transmitted through colostrum. Whether transmission occurs in utero or during parturition is unknown, but other modes of transmission were unlikely in these infected crias.
The authors thank Ron and Stacey Ballensky of Yacolt, Washington, USA.
- British Veterinary Association