The Infection Formerly Known as Eperythrozoonosis in Camelids:
New information and New Test, Susan J. Tornquist, DVM, PhD, Dip. ACVP
College of Veterinary Medicine, Oregon State University, Corvallis, OR, 97331
An Eperythrozooon species affecting camelids has recently been reclassified as Mycoplasma haemolama based on its 16s rRNA sequence.
1. It is most closely related to M. wenyonii, which affects cattle, then to M. haemosuis (formerly Eperythrozoon suis), which affects pigs and somewhat less to M. haemominutum (formerly one of the Hemobartonella felis isolates) which affects cats. M. haemolama was first described in 1990.
2,3. It is associated with mild to marked anemia and rarely, death, in stressed, immune-suppressed, and debilitated camelids. It has also been identified in low numbers in apparently healthy camelids. Some camelid herds with high rates of M. haemolama based on blood smear examination have experienced acute collapse, chronic weight loss, depression, and lethargy. These clinical signs, and the presence of the hemoparasite on blood smears are sometimes associated with shipping or movement of camelids from one premise to another.
The mode of transmission is not known and little had been reported about prevalence, the effects of treatment on the carrier state, and the pathogenesis of infection and disease with this hemoparasite. This has led to confusion among veterinarians and owners as to whether all infected camelids and their herdmates should be treated and how finding the hemoparasite on blood smears from clinically-healthy camelids should affect pre-purchase exams and shipping of animals.
Grants from the Morris Animal Foundation and the Alpaca Research Foundation allowed us to carry out two projects that were designed to develop a sensitive and specific polymerase chain reaction (PCR)-based assay to detect the presence of M. haemolama even at low levels of parasitemia. We then used the assay to study the kinetics of parasitemia, response to antibiotic treatment, and presence of a carrier state that may recrudesce to a clinical level with stress. The long-term goal of these projects is to study the transmission and risk factors of M. haemolama infection in camelids.
A PCR-based assay to amplify the 16S ribosomal RNA gene of M. haemolama was developed using primers based on GenBank sequence accession AF306346 and blood from a naturally-infected alpaca. Specificity was shown by failure of these primers to amplify related Mycoplasma species. Parasitemia was detected in llamas and alpacas from a variety of geographic location using the assay. The detection limit is estimated to be 1 organism in 3.8 x 10 9 to 7.7 x 10 9 RBCs.
Since the hemotropic mycoplasmas have not been successfully produced in vitro, a splenectomized alpaca infected with M. haemolama provided blood for experimental infection of 8 other alpacas. All developed parasitemia detectable by PCR at least 2 days before organisms were seen on blood smears. Four of the infected alpacas were treated with a commonly-used tetracycline regime and four were not. All animals were monitored for over 6 months by physical examination, body temperature, PCV, total protein, blood smear examination, and PCR for M. haemolama in blood. At the end of 6 months, the animals were given intravenous examethasone at an immunosuppressive dose to simulate the effects of stress or immune suppression and parameters as described above were measured.
4. The results of this study will be published. Overall, the results show that:The PCR assay is now available for $19 through submission of whole, EDTA-anti-coagulated blood to the Veterinary Diagnostic Laboratory at Oregon State University. For questions about the assay, contact Dr. Sue Tornquist at (541) 737-6943 or Susan.Tornquist@oregonstate.edu
- Parasitemia is not cleared by the standard tetracycline regime used in camelids
- Once infected, many camelids may become chronic carriers
- The PCR assay is more sensitive than blood smear exam for diagnosis of M. haemolama
References: 1. Messick JB, Walker PG, Raphael W, Berent L, Shi X. 'Candidatus mycoplasma haemodidelphidis' sp. nov., 'Candidatus mycoplasma haemolamae' sp. nov. and Mycoplasma haemocanic comb. nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phyogenetic and secondary structural relatedness of their 16S rRNA genes to other mycoplasmas. Int J Syst Evol Microbiol 52: 693-698, 2002.
2. McLaughlin BG, Evans CN, McLaughlin PS, Johnson LW, Smith AR, Zachary JF. An Eperythrozoon-like parasite in llamas, J Am Vet Med Assoc 1990 197: 1170-1175, 1990.
3. Reagan WJ, Garry F, Thrall MA, Colgan S, Hutchison J, Weiser MG. The clinicopathologic, light, and scanning electron microscopic features of eperythrozoonosis in four naturally-infected llamas. Vet Pathol, 27 426-431, 1990.
4. Tornquist SJ, Boeder LJ, Parker JE, Cebra CK, Messick J. Use of a polymerase chain reaction assay to study the carrier state in infection with camelid Mycoplasma haemolama, formerly Eperythrozoon spp infection in camelids. (Abs) Vet Path 39: 616, 2002.
"What's an epe -- epe-erythr-- What's an epe?
"It's a mouthful, isn't it? An eperythrozooan is a little bacterium that affects the red blood cells. It actually sits on the red blood cells and the immune system sees that as a problem and figures it has to take out the red blood cells and destroy them. It can lead to severe anemia or mild or moderate anemia particularly in animals that are stressed or immune compromised.
"I don't want to give the impression that this disease is killing alpacas right and left. The organism probably does not kill animals - at least by itself. We see it more often as complicating factor in other diseases, and in that sense it's worth figuring out more about it and how to prevent it."
Dr. Tornquist says that since it was first described in camelids in 1990, we still don't know much more about camelid perythrozoonosis. We still don't know how it's transmitted.
Tetracycline is the treatment of choice, but what is unclear is if the tetracycline actually makes the organism go away, or if it doesn't just suppress the disease to undetectable levels. Then, if an animal carrying low levels of the organism gets stressed by shipping or by some other disease, the eperythrozoons can start multiplying again.
And it is difficult to study a disease if you can't detect it.
"The standard way you would diagnose eperythrozoonosis is take blood, make a blood smear and look for it. But actually it's a little tiny organism and it does fall off the red blood cells if the blood sits on the slide for a while, and it can look like precipitate in the background. So definitely if you have low numbers of it, it can be hard to diagnose with the standard test."
So Dr. Tornquist and her colleagues came up with a better test - a PCR-based assay. What's a PCR?
"PCR stands for preliminary chain reaction. It's a way of amplifying the DNA from a sample. So if you have something that's present in a really low level in a sample, you amplify it greatly, then you can detect it that way."
She explains that for each thing you want to look for, you have to develop a specific test. Dr. Tornquist's team developed a PCR-based test specifically for camelid eperythrozoonosis.
"Then we actually infected eight alpacas, and we've been using the test to monitor how soon after infection do they get the disease. We give tetracycline to half of them and not to the other half. A healthy animal usually fights the eperythrozoonosis off on its own in a couple days.
"Now we're monitoring them. We're going to be doing this for about six months. At the end of that time we're going to see if there's a difference in the two groups as to whether one group still has the organism and the other doesn't, or whether it's wiped out in both, or just exactly how effective is the treatment in absolutely getting rid of the organism.
"The first step was coming up with this more sensitive diagnostic test. "So that's what we're doing with the current project then, hopefully, figure out how it's transmitted."
Is it transmitted from mother to cria?"We haven't shown that for sure but we've diagnosed it in cria as young as a couple days, to me that's pretty good evidence that they can be infected in utero."Was the mother symptomatic?"No. With the better diagnostic test we may pick some more of these that are positive."The other question that comes up a lot is: if a veterinarian does a health test on an alpaca and finds that it has low level eperythrozoonosis but otherwise seems healthy, should you test or should you treat all the other animals in the herd?Usually you don't find eperythrozoonosis in the other animals but it may be there in a low level. And that's where our PCR assay could be helpful in detecting. And then: is it okay to sell an animal that has eperythrozoonosis and say that it's a healthy animal?"The alpacas that you infected for the test, did they fight it off?"We have one - it's been two months now - we continue to pick it up in him."Was he treated or untreated?"The one having the recurrences was treated with antibiotics."Is there any geographical preference for this bug?"It's been described pretty much everywhere."Is it seasonal?"The seasonality question is interesting. Here in Oregon - this is not based on study, just on observation - we tend to see it more in the late summer and fall, not so much in winter. So that sort of fits with maybe there's an insect vector, but that hasn't been proven."Can it spread through mating?"Possible. We can't rule that out. But due to the nature of the organism that it's found in blood and no where else - it makes it a little less likely that that's actually the case."Once the alpaca has recovered on its own once, is it immune?"We don't know that either. But they do make antibodies, which is part of the immune response. We don't know for sure that antibody is protective. And that's another thing that would be good to look at. I don't know if this disease is enough of a problem that we would ever want to develop a vaccine but that's the kind of question that's important when you're developing a vaccine."If we treat the alpacas for it and they still have it, are we making supergerms?"In most of them that are treated, it's never a problem again. Clearly the treatment doesn't work 100% in all cases."What's an owner to do?"At this point as an owner I would say don't worry about it too much. If you have animals that aren't doing right, losing weight, seeming sick, you would naturally probably have a vet check them out."We haven't started doing the new PCR-based test as a regular diagnostic test - which means we don't charge money for it. Obviously, eventually, we'll have to do that because it's not cheap to run, but we're still at that point where we're interested in testing strains from different parts of the country, and so it's actually helpful to get samples from animals in different places. So at this point if someone wanted the PCR assay run, they could contact me and we could set up sending in some blood and testing it. Eventually we're going to have to charge for that, but for now it's still part of making sure we've got a really really good test.
"Having been at both ends - in practice and wishing I had all the answers, then being in research and realizing how long it can take to get anything done - I know it's frustrating for the veterinarians and for the owners. It just takes time, but eventually, if you keep working at it, you get it. Eventually."
- April 2002"
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