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Can Vet J. Mar 2006; 47(3): 229–233.
PMCID: PMC1371050

Language: English | French

Mycoplasma haemolamae infection in a 4-day-old cria: Support for in utero transmission by use of a polymerase chain reaction assay

Abstract

Blood smear examination in a 4-day-old alpaca revealed massive erythrocyte parasitism by Mycoplasma haemolamae. Blood collected from both the nonparasitemic dam and the cria were positive for M. haemolamae by polymerase chain reaction (PCR) analysis. These findings suggest in utero transmission of M. haemolamae in camelids, even when the dam is not parasitemic.

Résumé

Infection à Mycoplasma haemolamae chez un cria âgé de 4 jours : élément de preuve pour une transmission in utero par amplification en chaîne par polymérase. L’examen d’un frottis sanguin chez un alpaga de 4 jours a révélé un parasitisme érythrocytaire massif par Mycoplasma haemolamae. Le sang prélevé chez la mère, qui ne présentait pas de parasitémie, et celui prélevé sur le cria étaient positifs à M. haemolamae lors de l’analyse par amplification en chaîne par polymérase. Ces résultats suggèrent une transmission in utero de M. haemolamae chez les camélidés, même lorsque la mère ne présente pas de parasitémie.

(Traduit par Docteur André Blouin)

A 4-day-old, 14.5 kg female alpaca (Lama pacos) from central Virginia was admitted to the Veterinary Teaching Hospital (VTH) at the Virginia-Maryland Regional College of Veterinary Medicine for evaluation of poor appetite, inability to stand, and possible failure of passive transfer. The cria was born approximately 2 wk prematurely to a 3-year-old primiparous dam. Following uncomplicated parturition, the cria was observed to suckle the dam. Within 48 h, the cria had become inappetent, weak, and no longer able to stand. According to the owner, at 60 and 72 h postpartum, the cria was fed 75 and 45 mL of alpaca colostrum, respectively, since she no longer suckled the dam and remained unable to stand.

Case description

On physical examination at the VTH, the cria was depressed and could not stand, even with assistance. She was estimated to be 8% dehydrated. Capillary refill time was prolonged at 3 s and mucous membranes were tacky. Skin and mucous membranes were hyperemic. The cria was afebrile with a rectal temperature of 39.2°C. Tachycardia (150 beats/min; reference interval, 60 to 100 beats/min) and tachypnea (60 breaths/min; reference interval, 20 to 40 breaths/min) were noted. An abbreviated ophthalmic examination revealed bilateral posterior chamber hyphema. Petechial hemorrhages were evident in both pinnae.

Initial diagnostic tests included a complete blood (cell) count (CBC), serum biochemical analysis, urinalysis, and venous blood gas analysis. In addition, to assess for adequacy of passive transfer of maternal antibodies, serum immunoglobulin (Ig)G concentration was measured by radial immunodiffusion specific for llama and alpaca IgG (Triple J Farms, Bellingham, Washington, USA). A fecal floatation examination for intestinal parasite larvae and ova was negative. Blood was collected from the left jugular vein for aerobic and anaerobic bacterial cultures. Results from the initial laboratory evaluation, including IgG concentration, are shown in Tables 1 and and2.2. While laboratory results were pending, initial treatments included 1 L of lactated Ringer’s solution (100 mL/h), IV; 295 mL plasma, IV (over 1 h), and milk replacer (Grober Nutrition, Cambridge, Ontario), 125 mL, per nasogastric tube, q2h. Due to the potential for failure of passive transfer, and based upon the clinical signs, sepsis was considered likely. Therefore, treatment with ceftiofur sodium (Naxcel; Pfizer Animal Health, New York City, New York, USA), 4.4 mg/kg bodyweight (BW) IM, q24h, was begun. The most significant laboratory test results were severe metabolic acidosis (total CO2, 7 mmol/L; reference interval, 24 to 32 mmol/L), marked hypoglycemia (glucose, 1.28 mmol/L; reference interval, 5.22 to 9.44 mmol/L), marked hyperkalemia (potassium, 9.6 mmol/L; reference interval, 4.6 to 5.9 mmol/L), and severe azotemia (urea nitrogen and creatinine, 119 mmol/L and 831 μmol/L, respectively; reference intervals, 3.57 to 7.50 mmol/L and 97 to 256 μmol/L, respectively) with nearly isosthenuric urine (specific gravity 1.013). To correct the severe metabolic acidosis, 5% sodium bicarbonate was added to the IV fluid treatment regimen (0.5 mL/kg BW/h). The hypoglycemia warranted a change from lactated Ringer’s solution to 5% dextrose in Ringer’s solution, IV (100 mL/h).

Table 1
Hematological data from a 4-day-old alpaca with Mycoplasma haemolamae
Table 2
Biochemical data from a 4-day-old alpaca with Mycoplasma haemolamae

Hematological abnormalities included mild nonregenerative anemia (red blood cell [RBC] count, 9.13 × 1012/L; reference interval, 9.6 to 15.2 × 1012/L; and hemoglobin, 99.0 g/L; reference interval, 101.0 to 149.0 g/L), mild lymphocytosis (6.9 × 109/L; reference interval, 1.7 to 4.7 × 109/L), and hyperfibrinogenemia (14.7 μmol/L; reference interval, 2.9 to 11.7 μmol/L). Actual RBC and hemoglobin values were likely artificially increased, as the cria was moderately dehydrated; hence, the anemia was more severe than the numerical data indicated. More importantly, numerous extracellular parasites, consistent with Mycoplasma haemolamae (formerly Eperythrozoon sp.), were observed both on the erythrocytes and in the plasma space (Figure 1). The organisms were small, basophilic, coccus- and ring-shaped, and affected nearly 100% of the erythrocytes. Many erythrocytes were noted with 20 or more adherent organisms.

Figure 1
Peripheral blood smear from a 4-day-old cria illustrating the light microscopic appearance of Mycoplasma haemolamae and massive erythrocyte parasitemia. Notice the numerous organisms on the surface of the erythrocytes, as well as in the plasma space. ...

Initially, the cria responded favorably to the IV fluid therapy, dextrose, and the plasma transfusion with improvement in hydration status and responsiveness to external stimuli. The cria also began to show some interest in food (milk replacer). She was able to maintain sternal recumbency and could stand unassisted for short periods. However, severe muscle fasciculation affecting all 4 limbs was apparent when standing. Watery diarrhea began approximately 16 h after admission to the VTH. At this time, a CBC was repeated (data not shown) and a fecal sample was collected and submitted for culture for Salmonella spp. Shortly thereafter, the cria became markedly dyspneic, developed generalized seizures, and died approximately 4 h later. A complete necropsy with histologic evaluation was performed.

Gross necropsy findings included a small amount of serosanguinous fluid and a small, free-floating gelatinous fibrin clot in the peritoneal cavity. The wall of the 1st compartment of the stomach was diffusely thickened. The mucosa was dark red in many areas. However, several dull discolored areas of the stomach mucosa were also noted. Blood clots were present in all 3 stomach compartments. The lungs were diffusely and uniformly red, wet, and heavy, and sank in formalin.

The most prominent histologic abnormalities involved the lungs and the 1st compartment of the stomach. There were fibrin and occasional neutrophils in the alveoli. The squamous mucosa of the stomach was ulcerated in some areas, with adherent fibrin. Numerous mixed inflammatory cells infiltrated the muscularis. Bacterial culture of tissue samples from the 1st stomach compartment isolated Staphylococcus spp., Escherichia coli, and Streptococcus uberis. Scattered kidney tubules had necrotic epithelium, but the renal lesions alone were not considered significant enough to account for the marked azotemia and nearly isosthenuric urine. Thus, marked dehydration as a prerenal cause of the azotemia was likely a contributing factor. Additionally, neonates have poor renal concentrating ability, which may explain the low urine specific gravity.

Blood culture for bacteria was negative. The fecal sample submitted for culture was negative for Salmonella spp. Necrotizing gastritis, with overgrowth of bacterial opportunists; pulmonary edema; tubular nephrosis; and marked erythrocyte parasitism was the diagnosis. Failure of passive transfer appeared initially to be the underlying cause of the multiple disease processes, but the concentration of serum IgG was within the reference interval established for neonatal llamas and alpacas (13). Thus, failure of passive transfer as the primary etiology could not be confirmed.

The epierythrocytic organisms in the peripheral blood were morphologically consistent with Mycoplasma haemolamae, based upon light microscopic examination. To confirm that the organisms were M. haemolamae and to investigate the potential for their vertical transmission from dam to cria, the presence of the 16S rRNA gene of M. haemolamae was tested for by polymerase chain reaction (PCR) and DNA sequencing. Peripheral whole blood was collected from the dam into sterile tubes containing ethylenediaminetetraacetic acid (EDTA) for a CBC, a blood film examination, and PCR. Banked frozen whole blood in EDTA from the cria was tested by PCR.

In contrast to the cria, the dam was not anemic and no hemoparasites were found on blood smear evaluation. Briefly, PCR analysis and DNA testing were performed as follows. By use of a commercially available kit (QIAamp DNA mini kit; Qiagen, Valencia, California, USA), DNA extraction was performed on whole blood in EDTA. A partial 16S ribosomal DNA sequence was amplified from both the cria’s and the dam’s blood by use of a PCR assay, as previously described (4). Routine positive and negative controls were included in the assay procedure. The PCR products from the cria’s and the dam’s blood were ligated into a commercial vector. Plasmids were transformed via heat-shock into Escherichia coli, as described, isolated with a commercial plasmid purification kit according to manufacturer’s instructions, and sequenced. Polymerase chain reaction and gene sequencing analysis of peripheral blood from both the cria and the dam revealed partial 16S rRNA sequences 100% homologous with that of the recently characterized hemotrophic bacterium, M. haemolamae (5).

Discussion

The significance of the marked erythrocyte parasitism with respect to the outcome in this case is uncertain. Neither clinical nor laboratory evidence was definitive for hemolysis, although the cria was mildly anemic; the dehydration may have been masking a more significant anemia. The potential for vasculitis associated with the erythrocyte parasitism was considered a cause for the petechiation, but this was not confirmed on histopathologic examination. A coagulopathy was another possibility, but coagulation testing was not performed for this patient.

On the basis of results of 16S rRNA analysis, organisms belonging to the genera Hemobartonella and Eperythrozoon have been reclassified as hemotrophic Mycoplasma spp. (hemoplasmas) (4,6,7). The hemoplasmas are wall-less bacteria that attach to the surface of erythrocytes by thin fibrils and were originally classified as members of the order Rickettsiales in the family Anaplasmataceae. Different species of these hemotrophic organisms infect many vertebrates, including pigs, sheep, cattle, dogs, and cats (8). In camelids and other nonsplenectomized animals, hemotrophic Mycoplasmas may be an incidental finding on blood smear evaluation, but in ill or immunocompromised animals, they may cause mild to severe anemia (9,10). Several reports of RBC parasitism by hemotrophic bacteria (formerly Eperythrozoon) in juvenile and adult South American camelids exist (1114). However, only one instance of hemoplasmosis (formerly, eperythrozoonosis) by a similar organism has been reported in a neonatal llama (15). Interestingly, the dam of the neonatal llama in that report also was not anemic and no organisms were found on blood smear evaluation.

The typical mode of transmission of M. haemolamae in the llama and alpaca is not known. However, studies in swine, dogs, and cats support blood sucking or biting insects, including lice, fleas, and ticks, as vectors of the hemotrophic Mycoplasma spp. (1619). Oral uptake of blood components and indirect transmission via contact with blood contaminated syringes or surgical instruments following tattooing, tail docking, or castration are additional methods of transmission of Mycoplasma spp. in swine. Another mode of direct transmission in swine may occur during copulation due to contact of the cervix or vagina by boar semen contaminated with infected blood. Although experimental intrauterine transmission of hemoplasmas in swine has been demonstrated (16) and there is an early report of in utero transmission from sows to pigs (20), current data supporting naturally occurring intrauterine transmission in swine is lacking (17). Evidence supporting in utero transmission in other species, including llamas, alpacas, dogs, and cats, has been inconclusive. Neither the cria nor the dam of this report were observed to have lice or tick infestations, although ticks are common in the area. Nonparasitemic animals may harbor the parasite, but due to the very transient nature of the parasitemia (as in feline hemoplasmosis) or low numbers of organisms, blood smear evaluation may not detect affected animals. Thus, some animals are likely infected, but nonparasitemic, carriers.

The time interval between infection and observation of hemotrophic organisms varies among the animal species infected. Low numbers of epierythrocytic organisms have been identified in the peripheral blood of splenectomized pigs as early as 2 d after IV inoculation (21). Intramuscular injection of Eperythrozoon suis in splenectomized pigs resulted in peak parasitemia approximately 7 d postinoculation (21). Nonsplenectomized cats first developed detectable hemoplasmosis in 2 to 17 d (mean of 8.4 d) following IV inoculation (22). The time interval between infection and observable parasitemia in South American camelids is unknown. In contrast to the cria of this report, the 1-day-old llama with hemoplasmosis identified by Fisher and Zinkl (15) was not anemic and had no clinical signs related to the parasitemia; the route of infection was unknown, but transplacental, transmammary (colostrum) or postnatal blood inoculation were all considered possibilities, with the time frame between the birth of the cria and the observation of organisms supporting transplacental infection as the most likely route.

Analysis of the dam’s milk for the presence of M. haemolamae by PCR would have been interesting. While transmammary transmission is theoretically possible, a study in sheep failed to detect infection in lambs born to and suckling Mycoplasma ovis (previously Eperythrozoon ovis) carrier ewes (23). Furthermore, transmammary infection would presume that the organisms are able to survive the harsh gastric environment and pass unaltered into circulation.

Similar to swine, llamas and alpacas have diffuse epitheliochorial placentation that is thought to prevent maternal and fetal blood from intermingling. However, a recent investigation revealed that even though the epitheliochorial alpaca placenta is diffuse, specialized areas of the maternofetal interface impart microregional functions, where nutrition, hormone production, and molecular exchange are prevalent (24). It is unclear if these specialized areas of the alpaca placenta may allow for transplacental infection by larger, single-celled organisms, such as M. haemolamae. Although circumstantial, the short time frame, the significant parasitemia in this cria, and the detection by PCR assay of M. haemolamae in the nonparasitemic dam support transplacental infection as a mode of transmission of hemoplasmosis in neonatal camelids. However, transmammary and postnatal blood inoculation (during the birthing process) as the route of infection in this cria cannot be excluded.

A study of juvenile llamas (25) indicated that immunosuppression does not appear necessary for infection by M. haemolamae, as several of the control animals were parasitemic, but no abnormalities were detected clinically or on hemograms. However, the immunocompromised llamas that were also parasitemic were significantly more anemic than the immunocompromised llamas that were nonparasitemic (25). Although the cria of this report had serum IgG concentrations consistent with successful passive transfer of maternal antibodies, an IgG concentration within the reference interval does not necessarily imply immunocompetence. Other tests, such as a lymphocyte blastogenesis assay, cutaneous delayed type hypersensitivity response, or postvaccination titers, are often better indicators of immunocompetence. The cria in this case died before additional tests of immunocompetency could be performed.

Hypoglycemia has been observed in several cases of hemoplasmosis in llamas (12) as well as in other species. In swine, cattle and sheep, hypoglycemia was directly correlated with maximal parasitemia (2629). The proposed mechanism of hypoglycemia is that glucose utilization by the hemoparasites exceeds gluconeogenesis. It is unclear if the cria in this case was hypoglycemic solely due to massive erythrocyte parasitism or if other etiologies, including anorexia or bacterial sepsis, were contributing. The muscle fasciculation and seizures observed just prior to the cria’s death may have been directly related to the severe hypoglycemia.

Treatment protocols aimed at controlling M. haemolamae infection require vigilant administration of both injectable and oral forms of oxytetracycline for a period of at least 50 d (13,30). However, this treatment may not eliminate the carrier state (5) and the potential for transplacental infection remains in nonparasitemic camelids. Polymerase chain reaction assays can detect nonparasitemic animals, but utilization of this method for routine screening is not currently recommended. CVJ

Footnotes

Dr. Almy’s current address is Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30605 USA.

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