Mycoplasma haemolamae and intestinal parasite relationships with erythrocyte variables in clinically healthy alpacas and llamas

Abstract Background Mycoplasma haemolamae (Mhl) and gastrointestinal nematodes can cause anemia in camelids. Control programs aim to suppress parasitism without promoting anthelminthic resistance, but few evidence‐based guidelines define acceptable parasite loads in camelids. Hypothesis/Objectives In clinically healthy nonanemic camelids, compare erythrocyte variables to Mhl real‐time PCR status and to fecal egg count (FEC). Determine the FEC threshold above which erythrocyte variables are consistently below reference interval medians. Animals One hundred fourteen client‐owned adult alpacas and llamas. Methods In a cross‐sectional study, whole blood in ethylenediaminetetraacetic acid (EDTA) was assessed for packed cell volume (PCV) by centrifugation, erythrocyte count (RBC), and hemoglobin concentration (HGB) using an ADVIA120 analyzer, and Mhl using real‐time PCR. Trichostrongyle eggs per gram (epg) were counted by modified McMaster test on freshly collected feces. Significant differences in erythrocyte variables based on Mhl status and FEC thresholds were assessed by independent t test and one‐way ANOVA, respectively. Results Packed cell volume, RBC, and HGB were not significantly different between Mhl‐positive and Mhl‐negative animals, but were significantly lower in animals with FEC >1000 epg compared to those with <500 epg. All animals with FEC >600 epg had RBC and HGB below the reference interval median. All animals with FEC >750 epg had PCV below the reference interval median. Conclusions and Clinical Importance In healthy nonanemic camelids, positive Mhl PCR is not associated with lower erythrocyte variables and such animals may not warrant treatment. Fecal egg count >600‐750 epg has a negative effect on erythrocyte variables, and may be a guide for deworming protocols.


| INTRODUCTION
Mycoplasma haemolamae (Mhl) is a hemotropic bacterium that can cause clinically apparent anemia in alpacas and llamas. Clinically relevant anemia is reported most often in infected animals that are immunosuppressed, stressed, debilitated, or suffering from a concurrent illness, but organisms still can be observed on peripheral blood smears from some clinically healthy animals. 1 Haemonchus contortus is a trichostrongylid nematode that resides in the third gastric compartment (C3). It is the most common and clinically important endoparasite of alpacas and llamas, and is a common cause of clinically relevant anemia. 2,3 The gold standard test for diagnosis of Mhl is a real-time PCR assay that is specific for detection of the organism's 16S rRNA gene. 4,5 However, bacteremia in infected animals is transient and cyclical, and it therefore can be difficult to identify Mhl as an etiologic agent in animals with anemia. 6 Additionally, most infections are subclinical, resulting in a carrier state that persists despite antibiotic treatment of infected animals. 1 Previous studies have reported that infection with Mhl (based on positive real-time PCR results) is not significantly associated with low PCV. 5,7 The gold standard for diagnosis and quantification of intestinal nematodes is adult worm counts at necropsy. Antemortem, trichostrongylid infections can be diagnosed and semi-quantified using the fecal egg count test (FEC), which measures the number of trichostrongyle eggs per gram (epg) of feces. However, this test cannot differentiate H. contortus eggs from other trichostrongyle eggs (eg, Teladorsagia circumcincta, Trichostrongylus axei). 3 In addition, no established thresholds define clinically relevant FECs in camelids. The FEC has been shown to be negatively correlated with PCV and other erythrocyte variables (erythrocyte count [RBC], hemoglobin concentration [HGB]) in clinically ill alpacas and llamas. 3,8,9 However, no documentation has been published of any correlation between FEC and erythrocyte variables in clinically healthy, nonanemic camelids. Tools used to identify animals in need of deworming treatment, such as the Faffa Malan Chart system (FAMACHA©), 10 only identify animals that are already anemic, potentially missing nonanemic animals with high nematode burdens.
Conversely, deworming all animals in a herd regardless of their nematode burden promotes parasite resistance to anthelminthic medications.
Because PCV is only 1 index of erythrocyte mass, a goal of our study was to perform a more comprehensive assessment of the relationship between parasitism and erythrocyte variables, including PCV, RBC, and HGB. We hypothesized that clinically healthy, nonanemic animals that were PCR-positive for Mhl would not have significantly lower results for erythrocyte variables compared to those with negative PCR results, but that erythrocyte variables would be lower in animals with high FEC compared to those with low FEC. Additionally, we hypothesized that there would be an FEC threshold above which erythrocyte variables in clinically healthy, nonanemic alpacas and llamas would be consistently below the reference interval medians, suggesting that although the animals were not clinically anemic, they may still have adverse hematologic effects and would benefit from parasite control strategies.

| Patient population and sample collection
Venous whole blood was collected from clinically healthy adult (≥ 1-yearold) alpacas and llamas from 11 farms in eastern Tennessee between July and October 2015. Blood also was collected from 2 blood donor alpacas housed by the University of Tennessee during the same time period. Health was defined as a lack of clinically relevant abnormalities on physical examination as determined by an experienced veterinarian, a FAMACHA score ≤ 3/5, 10 a body condition score (BCS) ≥ 2.5 /5, 11 and no history of illness or injury within the previous 3 months. All examinations and sample collections took place on the farms, and informed client consent was obtained for all procedures (approved Institutional Animal Care and Use protocol 2298-0914).
Samples were collected from no more than 12 and no fewer than A CBC was performed on each sample using the ADVIA120 hematology instrument 12 (Siemens Healthcare Diagnostics, Tarrytown, NY), and samples were excluded if the PCV, HGB, or RBC were below the UTVMC reference intervals for camelids.
Manual PCVs were determined after 1 minute of centrifugation in a rapid fixed angle head microhematocrit centrifuge (HemataStat II, EKF Diagnostics, Boerne, TX). The centrifuge's read function was used to determine PCV; this function is calibrated for the microhematocrit's centrifuge speed. Proper microhematocrit centrifuge function was verified using an electronic tachometer before beginning the study, and a series of test centrifugations of camelid blood (data not shown) confirmed that there was no clinically relevant difference in PCV with centrifugation times >1 minute. Determinations of PCV were made manually and using the ADVIA120 analyzer by a licensed medical technologist trained in instrument use and following laboratory standard operating procedures. The ADVIA120 underwent daily quality control using 3-level quality control material (OPTIpoint, Siemens Healthcare Diagnostics, Tarrytown, NY).
A minimum of 1 blood smear was prepared from each sample, and reviewed by a medical technologist. Blood smear review included a leukocyte differential count, erythrocyte morphology review, and manual platelet estimate. Each blood smear also was reviewed for Mhl parasites by a board-certified veterinary clinical pathologist.
A fresh fecal sample was collected digitally from the rectum of each animal by trained personnel, placed in an individual, clean plastic bag, and transported in a cooler to the UTVMC parasitology laboratory.

| M. haemolamae and intestinal parasite testing
After performing the CBC, the remaining EDTA-anticoagulated whole blood from each animal was stored at −20 C for up to 30 days.
Extraction of DNA and real-time PCR for Mhl were performed using previously described methods 5   Significance was based on a P value <.05. The sample population also was empirically stratified into groups with low (<500 epg), moderate (500-1000 epg), and high (>1000 epg) FEC. Erythrocyte variables were compared among the different FEC strata using a 1-way ANOVA or Kruskal-Wallis test, and significance was based on a P value <.05. Comparison of erythrocyte variables also was performed between Mhl-positive and Mhl-negative animals in the different FEC strata.
In concordance with the observation of decreasing erythrocyte variables with increasing FECs, scatterplots of FEC versus PCV, RBC, and HGB were visually inspected to determine if there was an FEC threshold above which all patient erythrocyte variables were below the median of the reference interval, suggesting that an FEC above that threshold may have a subclinical effect on erythrocyte variables. These thresholds were apparent at >750 epg for PCV and at >600 epg for RBC and HGB (Figure 3). One limitation, however, is that FEC testing using the modified McMaster's test is relatively imprecise. Counts can vary substantially among observers, with different portions of the same fecal sample, and with variations in the concentration of the flotation solution, sample dilution, and volume of the counting chamber. 16 Additionally, the correlation between trichostrongylid egg numbers in feces and adult worm burden of a particular species (ie, Haemonchus spp.) in the GI tract is uncertain. Our study only allowed for assessment of the association between FEC and erythrocyte variables at a single point in time. It therefore would be useful to perform a prospective cohort study in the future, to determine whether an FEC >600 epg or >750 epg is truly predictive for the development of anemia.
Additionally, the exclusion of alpacas and llamas <1 year old from our study precluded evaluation of the potential effects of Given that cases of clinical anemia have been reported in young, potentially immunocompromised camelids, 6,17 the possibility that negative erythrocyte effects may be more likely to be observed in association with infection in animals <1 year old should be considered for future study. The inclusion of young camelids (<1 year old) in any future studies also would be helpful to assess the effects of GI nematodes on erythrocyte variables in this age group. Similarly, inclusion of both clinically healthy and clinically ill animals (with high FAMACHA, low BCS, or both) would allow for comparison of M. haemolamae prevalence between these groups, and for more definitive assessment of the subclinical nature of M. haemolamae infection.
Additional limitations of our study included inability to exclude the effects of other factors on the measured erythrocyte variables. These variables reflect an animal's overall erythrocyte mass, which is affected by multiple factors including age, hydration status, nutrient balance, and renal and bone marrow function. Despite the presence of known causes of anemia (Mycoplasma spp., GI nematodes), it is difficult to attribute changes in erythrocyte variables (or lack thereof) solely to the presence or absence of these organisms. For example, decreased erythrocyte variables may reflect iron deficiency or the effects of inflammation on erythropoiesis, rather than blood loss from endoparasites.
In conclusion, our results confirm the findings of previous studies that demonstrated a lack of association between M. haemolamae infection and PCV in alpacas and llamas, and expand these findings to also include a lack of association with RBC or HGB. Overall, these findings suggest that treatment of clinically healthy M. haemolamae PCR-positive animals is not warranted, although M. haemolamae infection cannot be ruled out as a contributing cause to the future development of clinically relevant anemia in these camelids. Finally, with additional research and method validation, the use of a FEC threshold of >600 epg or > 750 epg in clinically healthy alpacas and llamas may provide a component for guiding deworming strategies.