Volume 1, Issue 2 , Pages 143-148, December 2008
Treatment of Mycoplasma wenyonii Infection in Cows with Imidocarb Dipropionate Injection-acupuncture
Article Outline
Abstract
Sixteen shorthorn cows from Xiazhuang farm were admitted to the Veterinary Teaching Hospital at the College of Animal Science and Veterinary Medicine, Shandong Agricultural University for evaluation of poor appetite, listlessness, fever, tachycardia, tachypnea, lethargy, positive jugular venous pulse and anemia. Blood smear examination and polymerase chain reaction analysis in these cows revealed an infection with Mycoplasma wenyonii. The subjects were divided into two groups: control group (three cows) treated with intramuscular injection with imidocarb dipropionate (3 mg/kg/day for 2 days) and the experimental group (13 cows), treated with injection-acupuncture (Imidocarb Dipropionate, 1 mg/kg, once every 3 days for 6 days) at BL17, BL18, BL20, BL25, ST36, SP06 and CV04. At day 15, negative results were found using blood smear examination in all control and experimental groups.
Key Words: acupuncture , cow , imidocarb dipropionate , Mycoplasma
Mycoplasma wenyonii (M. wenyonii, formerly Eperythrozoon wenyonii) is a wall-less hemotrophic prokaryote formerly classified as a rickettsia, but recently reclassified with the genus Mycoplasma on the basis of 16S rRNA analysis [1, 2, 3]. M. wenyonii is an epicellular hemoparasite that attaches to red blood cells (RBC) and causes RBC deformity and damage, but may also be found in plasma unattached eryth-rocytes [4]. Scanning electron microscopic analysis of M. wenyonii infected blood shows deformed eryth-rocytes with invaginations and the presence of either rod or coccoid shaped organisms embedded in the membrane of erythrocytes [3]. The resulting disease, traditionally called bovine eperythrozoonosis, has been reported and is considered a problem in feeder cows, where it manifests as a parasitemia and anemia with infected cattle rarely dying, but on occasions showing acute clinical signs [5]. Swollen teats, edema of the distal portion of the hind limbs, transient fever, prefatorily lymphadenopathy, a rough coat, dramatically decreased milk production and subsequent infertility and weight loss have all been reported [5]. In addition, scrotal and hind limb edema, tachycardia, pyrexia and infertility were reported in a Charolais bull in the United States that was chronically infected with M. wenyonii [6]. Adverse environmental conditions, nutritional mismanagement and co-infections with other microbes have been suggested as important factors predisposing to the development of clinical cow eperythrozoonosis.
In general, doxycycline, terramycin, acromycin, aureomycin, kanamycin and gentamicin were used to treat the infection of Eperythrozoon. Imidocarb dipropionate (N, N′-bis[3-(4,5-dihydro-1 H-imidazol-2-yl)phenylurea]) is a carbanilide derivative that has been used for more than 20 years for the treatment of certain protozoal diseases, including babesiosis and anaplasmosis, in cattle, horses, sheep and dogs. Hung [7] reported that the first injection of imidocarb dipropionate subcutaneously with two doses of 4 mg/kg (24 hours apart) could rapidly reduce the number of Eperythrozoon ovis (E. ovis) parasitemias in non-splenectomized Cheviot and Soay sheep experimentally infected with E. ovis and that E. ovis organisms were not demonstrable at 48 hours after the first treatment.
Acupuncture therapy, laser-acupuncture therapy [8] and moxibustion therapy [9] are well known in veterinary clinical practice. These methods showed excellent therapeutic effects in various animal diseases [10]. Injection-acupuncture has also been studied for treating dairy cattle with reproductive disorders [11], cows with foot rot [12] and horses with lameness and back pain [13]. Physicians could also use injection-acupuncture to inject medications or for vaccination in order to derive the added benefit of acupuncture point stimulation [9, 14, 15]. Injection-acupuncture may also be used for hemo-puncture or injecting autologous blood [16], and for injecting bee venom (apipuncture). This last technique has produced favorable outcomes for canine coxofemoral osteoarthritis [15], decreased milk production in postpartum sows [17] and diarrhea in pre-weaned piglets [18]. Here, we report a favorable therapeutic effect of injection-acupuncture with imidocarb dipropionate in M. wenyonii infection in 16 cows.
Sixteen shorthorn cows from Xiazhuang Farm (216 shorthorn cows were raised in this farm) with recurrent pyrexia, poor appetite and listlessness, were referred to the Veterinary Teaching Hospital (VTH) at the College of Animal Science and Veterinary Medicine, Shandong Agricultural University. Before referral to the VTH, the cows were treated intramuscularly at a local veterinary clinic with penicillin (3 mg/kg, Lukang Co., China, once a day for 3 days), cephalosporin (3 mg/kg, Lukang Co., China, once a day for 3 days), ofloxacin (0.15 mL/kg, Yuhan Co., China, once a day for 1 day), terramycin (0.1 mL/kg, Yuhan Co., China, once a day for 2 days) in turn.
On physical examination at the VTH, the cows showed fever (40.5 ± 0.3°C), tachycardia (90 ± 12 beats/min; reference range: 60 to 70 beats/min), tachypnea (45 ± 3 breaths/min; reference range: 10 to 30 breaths/min), lethargy, positive jugular venous pulse and anemia. Initial diagnostic tests included a complete analysis of blood count (CBC) and serum biochemistry. Blood was collected from the left jugular vein for aerobic and anaerobic bacterial cultures but no bacteria were found. The most significant laboratory test results were appreciable metabolic acidosis (total CO2, 21.3 ± 1.2 mmol/L; reference range: 24 to 34 mmol/L), marked hypoglycemia (glucose, 1.28 ± 0.3 mmol/L; reference range: 2.1 to 3.9 mmol/L; Table 2). Hematological abnormalities included mild non-regenerative anemia (RBC count, 3.51 ± 0.93 × 1012/L; reference range: 5.0 to 10.0 × 1012/L; and hemoglobin, 65.0 ± 9.7 g/L; reference range: 80.0 to 150.0 g/L; Table 1). A small drop of anti-coagulated blood was placed at one end of a very clean glass slide and the edge of a second slide was drawn across the drop at an angle so that capillary action spread the drop along the edge. The second slide was then pushed in one smooth motion to the opposite end of the first slide, spreading the drop across the slide to make the smear. After drying in air, this blood smear was scanned under light microscope using 400x magnification. Many extracellular parasites, consistent with M. wenyonii, were observed both on the surface of erythrocytes and in the plasma. The organisms were small, basophilic, coccus- and ring-shaped and affected nearly 90% of the erythrocytes (Figure 1).
Table 2. Serum chemistry values from a cow with M. wenyonii infection
| Items | Result | Reference interval* |
|---|---|---|
| ALT (U/L) | 29 ± 4 | 17–37 |
| AST (U/L) | 128 ± 12 | 48–100 |
| Bilirubin (μmol/L) | 5.10 ± 0.2 | 1.7–5.10 |
| Total protein (g/L) | 66.9 ± 5.6 | 59–77 |
| ALB (g/L) | 26.4 ± 5.7 | 27–43 |
| GLOB (g/L) | 40.5 ± 0.4 | 25–41 |
| GGT (U/L) | 34 ± 12 | 20–48 |
| CK (U/L) | 70 ± 40 | 44–228 |
| Urea nitrogen (mmol/L) | 3.20 ± 1.3 | 3.6–9.3 |
| Creatinine (μmol/L) | 35 ± 13 | 62–97 |
| Potassium (mmol/L) | 4.71 ± 0.4 | 4.0–5.3 |
| Sodium (mmol/L) | 137 ± 15 | 136–144 |
| Chloride (mmol/L) | 104 ± 17 | 94–104 |
| Total CO2 (mmol/L) | 21.3 ± 1.2 | 24–34 |
| Calcium (mmol/L) | 1.87 ± 0.7 | 1.98–2.5 |
| Phosphorus (mmol/L) | 1.87 ± 0.3 | 1.5–2.9 |
| Magnesium (mmol/L) | 1.15 ± 0.1 | 0.7–1.1 |
| Glucose (mmol/L) | 1.28 ± 0.3 | 2.1–3.9 |
Table 1. Hematology values from cows with M. wenyonii infection
| Items | Result | Reference interval* |
|---|---|---|
| RBC count (× 1012/L) | 3.51 ± 0.93 | 5.0–10.0 |
| Hgb (g/L) | 65 ± 13 | 80–150 |
| PCV (L/L) | 0.20 ± 0.07 | 0.24–0.46 |
| MCV (fl) | 44.1 ± 4.1 | 37–51 |
| MCH (pg) | 14.4 ± 2.1 | 13–18 |
| MCHC (g/L) | 327 ± 25 | 330–370 |
| WBC count (× 109/L) | 13.72 ± 1.6 | 4.0–12.0 |
| Neutrophils (× 109/L) | 0.5 ± 0.1 | 0.6–4.0 |
| Lymphocytes (× 109/L) | 1.3 ± 0.2 | 2.5–7.5 |
| Monocytes (× 109/L) | 1.44 ± 0.2 | 0.03–0.8 |
| Platelets (× 109/L) | 145 ± 24 | 200–730 |
Figure 1.
Light microscopic examination of blood smears from shorthorn cows. Numerous extracellular parasites, consistent with M. wenyonii, were observed both on the surface of erythrocytes and in the plasma space (arrows). The organisms were small, basophilic, coccus-shaped and ring-shaped, and affected nearly 100% of the erythrocytes.
Light microscopy indicated that the epierythrocytic organisms in peripheral blood were morphologically consistent with M. wenyonii. To confirm that the organisms were M. wenyonii, PCR was used to test for the presence of the 16S rRNA gene of M. wenyonii. The PCR method used to amplify the 341 bp fragment of the 16S rRNA gene of M. wenyonii has been described by Zhang et al [19]. The DNA was extracted from 200μL whole blood following the manufacturer's protocol. Standard amplification reactions were carried out in a thermal cycler, using the following primer set: forward: 5′-AGAGTTTGATCCTGGCTCAG3′ and reverse: 5′-TCAGACTTTCGTCCATTGTG3′. The thermal profiles consisted of an initial denaturation step of 5 minutes at 94†C, followed by 36 cycles of 1 minute at 94†C, 45 seconds at 55.4†C and 45 seconds at 72†C, with a final extension for 7 minutes at 72†C, followed by cooling at 4†C until analysis. The predicted 341 bp products were separated by electrophoresis in a 1.2% agarose gel containing 5 μg/mL ethidium bromide. The gels were photographed under ultraviolet light with an Alpha Imager 2200 (Alpha Innotech) imaging system.
Routine positive and negative controls were included in the assay procedure. PCR analysis of peripheral blood from cows was positive (Figure 2).
Figure 2.
PCR based detection of M. wenyonii 16S rRNA in shorthorn cows. The amplified 341 bp product, from the positive sample control (line 1) and positive test samples (lines 2-4) were subjected to electrophoresis in 1.2%-agarose gel and stained with ethidium bromide. Marker 2000 (M) and a negative control free of DNA template (line 5) were run in parallel.
Imidocarb dipropionate (1 mg/kg; Animal Medicine Research Institute of Nongmu University, Jilin, China) was diluted with 6.5 mL physiological saline. Injection-acupuncture was performed in the 13 cows in accordance with the following: after antisepsis cow's skin was pierced to the depth of 1 cm with a number 7 needle according to the injection location of every acupoint. 0.5 mL of the imidocarb dipropionate dilution was slowly injected into each of the following seven acupoints: BL17, BL18, BL20, BL25, ST36, CV04 and SP06 (Table 3), twice a week, for two sessions. Acupoints BL17, BL18, BL20, BL25, ST36 and SP06 were treated bilaterally (total 12 points). At session 2, the appetite of the cows was much better than at session 1 and the client expressed contentment with the improvement of the clinical signs. To demonstrate the efficacy of injection-acupuncture with imidocarb dipropionate, 3 cows infected M. wenyonii as the control group were treated intramuscularly with imidocarb dipropi-onate (3 mg/kg). The cows were injected again after 24 hours by using the same dose.
Table 3. The acupoint, location and clinical use in the present study
| Code | Name | Acupoint location | Clinical uses |
|---|---|---|---|
| BL17 | Geshu (Diaphragm Shu) | Lateral to the caudal border of the spinous process of the seventh thoracic vertebra, along the longitudinal line of the costal tubercles | Chronic hemorrhagic diseases, blood dyscrasia |
| BL18 | Ganshu (Liver Shu) | Lateral to the caudal border of the spinous process of the tenth thoracic vertebra, along the longitudinal line of the costal tubercles | Liver and gallbladder problems |
| BL20 | Pishu (Spleen Shu) | Lateral to the caudal border of the spinous, process of the twelfth thoracic vertebra, along the longitudinal line of the costal tubercles | Pancreatic disorders, pancreatitis, vomiting, anemia, digestive disorders |
| BL25 | Dachangshu (Large Intestine Shu) | Lateral to the caudal border of the spinous process of the fifth lumbar vertebra, along the longitudinal line of the thoracic costal tubercles | Gastrointestinal disorders: constipation, diarrhea, chronic colitis |
| ST36 | Zusanli (Foot Three Li) | About one finger-width lateral to the tibial crest, in the lateral part of the cranial tibial muscle | Gastrointestinal disorders, general tonification point for any weak condition and endocrine and metabolic diseases |
| SP06 | Sanyinjiao (Three Yins Crossing) | On the medial aspect of the hindlimb, caudal to the tibial bone, three sixteenths the distance from the medial malleolus of the tibia to the stifle joint, or 3 cun proximal to the medial malleolus | Gastrointestinal disorders, general tonification especially for geriatrics, fatigue, weakness; important point for allergic and immune-mediated disorders, endocrine disorders; also in liver, kidney, and pancreatic disorders |
| CV04 | Guanyuan (Gate of Origin— Uterus) | On the ventral midline of the abdomen, midway between the umbilicus and the pubic tubercle | Urogenital disorders, urine retention, incontinence, enuresis |
After 8 days, 10 cows were discharged as clinically normal; 15 days after treatment, all cows treated by injection-acupuncture with imidocarb dipropionate had been discharged as clinically normal and blood smear examination gave negative results. The efficacy of injection-acupuncture was nearly the same as the control group.
M. wenyonii, first reported by Adler in 1934 [20], is a member of a newly defined group of mycoplasmas that parasitize the RBCs of animals and humans. However, until now, there are few reports on the treatment of this disease in cattle.
Here, we report that cows diagnosed with M. wenyonii infection responded favorably to injection-acupuncture with imidocarb dipropionate. Injection-acupuncture produces better therapeutic effects on various diseases than those of conventional treatment methods, such as subcutaneous or intramuscular injection with the same drugs [11, 21, 22, 23, 24, 25].
Injection-acupuncture is the injection of saline or distilled water into acupoints. The goal is to provide a prolonged mechanical stimulus in the acupoint. Other substances may also be used for this purpose like glucose and vitamins [26]. Autologous blood (hemopuncture) and bee venom (apipuncture) are injected mainly for anti-inflammatory purposes [11, 21, 23, 24, 25]. Recently, pharmacopuncture, i.e., injection of subclinical doses of drugs in acupoints, has been adopted with successful results. Chinese authors claimed that this technique enhances the mechanical stimulus period and produces similar effects as conventional doses [27, 28, 29]. Pharmacopuncture has been a useful tool in veterinary practice. In large animals, it reduces undesirable drug side effects, residues in animal consumption products and treatment costs [30]. A subclinical dose of somatotropin (50 mg) injected in the Bai Hui (GV20) acupoint in adult cows was equally effective in increasing the weight gain of their lactating calves aged 30–40 days as the conventional dose (500 mg) injected intramuscularly [30]. Injection-acupuncture stimulates the acupoints before the injected drugs are absorbed and the absorbed drugs can play a pharmacological role [31]. Kim et al [31] reported that injection-acupuncture with ketamine at GV05 and the lumbosacral space prolonged the duration of anesthesia when compared with intramuscular injection of ketamine alone.
Also, since 2000, our VTH has used imidocarb dipropionate to treat many swine infected with Mycoplasma suis with a cure rate of circa 85%. Compared with other medicines such as doxycycline, terramycin, acheomycin, aureomycin and diminazene, imidocarb dipropionate has many advantages. These include reliable curative effect, lower recurrence rate, easy absorption, mild local response and low toxicity and side effects in treating swine infected with M. suis. But due to high costs, imidocarb dipropionate is seldom used to treat M. wenyonii infection in cows. The dose of imidocarb dipropionate usually recommended for treating M. wenyonii-infected cows is 3–5 mg/kg. However, using injection-acupuncture, a dose of only 1 mg/kg produced a favorable therapeutic effect in our cases.
The acupoints we selected in our present study have a number of significant functions. BL17 is the influential point of blood, so it can be used to treat various blood disorders such as chronic hemor-rhagic diseases, blood dyscrasia and anemia. BL18 is used to treat lassitude due to dysfunction of the stomach, liver and spleen, suggesting that acupuncture at this point may strengthen the function of the liver in cows. BL20 is one of the places where the spleen Qi transfers and can be used to reinforce the spleen Yang, benefit Ying-nutrient blood, support the resource of Qi and blood production, remove water and dampness, and assist transportation and transformation [32], so it can be used to treat pancreatic disorders, pancreatitis, vomiting and anemia. BL25, ST36 and SP06 are the points for the treatment of gastrointestinal disorders such as constipation, diarrhea, chronic colitis and nausea and vomiting, suggesting that acupuncture at these three points may stimulate functional gastrointestinal motility in cows. Acupuncture at ST36 stimulates the parasympathetic pathway and accelerates colonic transit in rats [33]. Because clinical signs of cows infected with M. wenyonii are fever, lethargy, anorexia, gastrointestinal atony, anemia, icterus, tachy cardia, tachypnea, weight loss, decreased milk production, all the above acu-points could be used to treat them.
In conclusion, 16 shorthorn cows from Xiazhuang Farm were found to be infected with M. wenyonii using blood smear examination and PCR analysis. Injection-acupuncture with imidocarb dipropionate (1 mg/kg, once every 3 days for 6 days) was used at BL17, BL18, BL20, BL25, ST36, SP06 and CV04 to treat 13 cows. At day 15, all cows treated by injection-acupuncture with imidocarb dipropionate had been discharged as clinically normal and blood smear examination gave negative results.
References
- . Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis, and Eperythrozoon suis . J Clin Microbiol . 1997;35:823–829
- . The cell wall-less rickettsia Eperythrozoon wenyonii is a Mycoplasma . FEMS Microbiol Lett . 1997;156:287–291
- . Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus Mycoplasma haemofelis’, ‘Candidatus Mycoplasma haemomuris’, ‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyonii’ . Int J Syst Evol Microbiol . 2001;51:891–899
- . Multiple infection in bovines from the tropics: observation of blood parasites by scanning and transmission electron microscope . Parassitologia . 2002;44:173–178
- . Eperythrozoon wenyonii infection in dairy cattle . J Am Vet Med Assoc . 1990;196:1244–1250
- . Infertility associated with Eperythrozoon wenyonii infection in a bull . J Am Vet Med Assoc . 1994;204:261–263
- . Chemotherapeutic efficacy of imidocarb dipropionate on experimental Eperythrozoon ovis infection in sheep . Trop Anim Health Prod . 1986;18:97–102
- . Laser acupuncture theory and light dosage selection . Acta Laser Biology Sinica . 2006;3:35–36
- . The anti-emetic effect of needling acupuncture, aquapuncture, and moxibustion at BL-21 and CV-12 in xylazine induced vomiting of dogs . J Vet Clin . 2005;22:202–205
- . The effect of laserpuncture and aquapuncture with methionine on the recovery in artificially induced hepatic damaged rats . J Vet Clin . 2002;19:125–131
- . Aquapuncture therapy of repeat breeding in dairy cattle . Am J Chin Med . 2002;30:397–404
- . The therapeutic effect of aquapuncture on bovine foot rot . J Vet Clin . 2002;19:115–120
- . Acupuncture in the treatment of back and hindleg pain in sport horses . AAEP Proc . 1997;43:325–326
- . Immunomodulatory effect of aquapuncture with canine parvovirus vaccine . J Vet Clin . 2001;18:368–373
- . Bee-venom acupuncture treatment of hip osteoarthritis in a dog . J Vet Clin . 2006;23:190–193
- . Autologous blood injection for refractory lateral epicondylitis . J Hand Surg [Am] . 2003;28:272–278
- . Effect of bee venom treatment in sows with oligogalactic syndrome post-partum . Am J Chin Med . 2003;31:149–155
- . Effect of apitherapy in piglets with preweaning diarrhea . Am J Chin Med . 2003;31:321–326
- . Sequencing and phylogenetic analysis of partial 16S rRNA gene of Eperythrozoon wenyonii . Chin J Preve Vet Med . 2006;28:152–155
- . Eperythrozoon prevalence and its statistical appraisal . Auist Vet J . 1989;66:122–123
- . Use of acupuncture for the treatment of chronic back pain in horses: stimulation of acupuncture points with saline solution injections . J Am Vet Med Assoc . 1987;190:1177–1180
- . Analysis of the curative effect of acupuncture and point-injection of huangqi injection on viral myocarditis . Shanghai J Acupunct Moxibust . 1998;17:13–14
- . An overview of bee venom acupuncture in the treatment of arthritis . Evid Based Complement Alternat Med . 2005;2:79–84
- . Antinociceptive effect and the mechanism of bee venom acupuncture (Apipuncture) on inflammatory pain in the rat model of collagen-induced arthritis: mediation by alpha2-Adrenoceptors . Brain Res . 2006;1073–1074:305–310
- . Acupuncture plus point-injection for 32 cases of obstinate urticaria . J Tradit Chin Med . 2006;26:22–23
- . The effect of Eperythrozoon ovis infection on the glucose level and some acid-base factors in the venous blood of sheep . Aust Vet J . 1977;53:478–481
- . Analysis on therapeutic effect of acupoint injection on chronic hepatitis B (Chinese) . Zhongguo Zhen Jiu . 2005;25:25–26
- . On effects of acupoints and drugs in acu-point injection treatment (Chinese) . Zhongguo Zhen Jiu . 2005;25:46–48
- . Effects of acupoint-injection of Kaixilai on TG, TC, MDA, SOD, AST and ALT in the patient of non-alcoholic fatty liver (Chinese) . Zhongguo Zhen Jiu . 2006;26:100–102
- . Global acupuncture research: previously untranslated studies. Studies from Brazil . In: Schoen AM editors. Veterinary Acupuncture: Ancient Art to Modern Medicine . St Louis: Mosby; 2001;p. 53–57
- . Electroacupuncture analgesia for surgery in cattle . Am J Chin Med . 2004;32:131–140
- . Treatment of 28 cases of Tourette's Syndrome by puncturing Back-Shu points . J Acupunct Tuina Sci . 2008;6:54–55
- . Electroacupuncture at ST-36 stimulates colonic motility and transit in freely moving conscious rats . Am J Physiol Gastrointest Liver Physiol . 2006;290:G285–G292
- . In: Veterinary Laboratory Medicine Interpretation and Diagnosis . 3rd edition. Elsevier Inc.; 2004;p. 310–311
PII: S2005-2901(09)60035-2
doi:10.1016/S2005-2901(09)60035-2
© 2008 Korean Pharmacopuncture Institute. Published by Elsevier Inc. All rights reserved.
Volume 1, Issue 2 , Pages 143-148, December 2008
