Downer cattle would occur due to various causes including traumatic injuries, metabolic disorders, and infections. Although there are no accurate statistics of domestic occurrence, the inability to stand in adult cattle is mainly caused by injuries and metabolic diseases, while infectious diseases are often the causes in calves. Among them, bovine viral diarrhea virus (BVDV) is one of the main pathogens which causes significant economic losses via decreased conception rate, milk production, diarrhea, and suppression of the host immune system. To reduce economic damage, the eradication programs based on the principles of antigenic detection in all cattle and culling of virus-positive animals or of phased control have achieved goals in several countries (7,10).

The BVDV can cross the placenta resulting in fetal death, congenital deformity, and growth retardation when the pregnant cows are infected in the early and middle stages of gestation. Congenital abnormalities of the central nervous system (CNS) such as cerebellar hypoplasia or hydrocephalus are most common in fetal BVDV infection, and the influenced calves have signs of ataxia and characteristic standing postures such as head tilting and wide stance (5).

Growth hormone or somatotropin, one of the main regulatory factors of fetal development, is known to also stimulate and regenerate a variety of animal tissues such as muscle, nerve, and bone by birth (3). Lopez et al. (6) demonstrated nerve regeneration and muscle re-innervation following growth hormone (GH) treatment in a chronic denervation-induced rat model. The rat model, median nerve-injured by surgically, demonstrated axon and myelin regeneration in the affected muscle, and functional recovery.

The aim of the clinical trial of this study was to evaluate the effect of GH treatment on the improvement of standing ability in a BVDV-infected calf showing ataxia in all four limbs. We hypothesized that the symptom would be developed due to the CNS lesion rather than musculoskeletal disorders. Based on the efficacy of GH on neuroregeneration in rodents, the downer calf was treated by GH injection, and the progress was monitored.

A 5-month-old male Holstein downer calf was presented to the hospital due to ataxia. The calf was born normally, but had been reluctant to stand up since around three months after birth. The calf showed a response of the limbs following pain stimulus, and the vital signs were in a normal range. The blood samples were collected at a week after birth and at five-month-old, which is the day of admission to the hospital, and there were no abnomalities in the results of both samples for complete blood cell analysis (Hemavet 950, Drew Scientific, Florida, USA) and blood chemistry (BS-400, Mindray, Shenzhen, China) (Table 1).

Table 1 . Blood analysis results of the patient at the point of one week and 5 months-old.

Parameter	One week old	Five months old
WBC (103/mm3)	3.38	9.88
RBC (106/mm3)	6.48	10.10
Hemoglobin (g/L)	87	137
Hematocrit (%)	21.3	31.3
Total protein (g/L)	63	82
Albumin (g/L)	34	40
Globulin (g/L)	29	42
Total cholesterol (mmol/L)	3.06	2.49
Triglyceride (mmol/L)	0.35	0.11
Glucose (mmol/L)	6.33	6.05
Creatinine (μmol/L)	114.92	61.88
BUN (mmol/L)	6.07	3.93
AST (units/L)	133	118
GGT (units/L)	23	17
Calcium (mmol/L)	2.68	2.25

WBC, white blood cells; RBC, red blood cells; BUN, blood urea nitrogen; AST, aspartate aminotransferase; GGT, γ-glutamyl transpeptidase..

In order to clarify the cause of ataxia, a radiological examination was performed to check skeletal fracture, and myelography was also evaluated by injecting contrast medium (Omnipaque^TM, GE Healthcare, Illinois, USA) into the subarachnoid space through a lumbar puncture. Nevertheless, neither skeletal fracture nor spinal compression was confirmed on the radiograph images (Fig. 1A). RNA was extracted from the feces and cerebrospinal fluid, and a commercial kit (LiliF^TM BVDV RT-PCR Kit, iNtRON, Seongnam, Republic of Korea) was utilized to identify the BVDV infection. The amplified products of BVDV were detected by RT-PCR in the feces as well as the cerebrospinal fluid (Fig. 1C). The blood and feces sample was cultured in blood agar plate (HK, Gunpo, Republic of Korea) and salmonella selection medium (1st; Peptone water, Merck, Darmstadt, Germany, 2nd; Rappaport-Vassiliadis R10 Broth, BD Biosciences, 3rd; XLD Agar, BD Biosciences, 4th; Nutrient Agar, BD Biosciences) to rule out septicemia and salmonellosis respectively, and no pathogen was detected both in blood culture and salmonella selection medium. For indirect diagnosis of white muscle disease, selenium and vitamin B₁₂ emulsion (Selevit, Fatro) was injected subcutaneously for five days (10 mL per day; 1.5 mg of selenium and 700 mg of α-tocopherol), and they did not induce any changes associated with ataxia.

Figure 1. Radiographic images of the patient and molecular detection of BVDV. The lining of the spinal cord was evaluated by injecting contrast medium, and no abnormalities were observed (A). For a few days before death, the patient was suffering esophageal obstruction and megaesophagus followed by ruminal tympany (B). The amplified products of BVDV was detected via RT-PCR in feces and CSF of the patient (C). BVDV, bovine viral diarrheal virus; CSF, cerebrospinal fluid; RT-PCR, reverse transcription polymerase chain reaction; NC, negative control; PC, positive control.

To alleviate the symptoms of astasia, 125 mg of recombinant bovine somatotropin (rBST; Bustin-s, LG Chem., Seoul, Korea) was experimentally administered as much amount of one quarter of adult cattle subcutaneously once every two days for one week. The recommended dosage for a cow to increase milk production is about 500 mg every two weeks, but an indication for a patient with neurogenic disorders was not suggested. We set the dosage of the medication according to the body weight of the calf, about 150 kg, and shortened the dosing interval to evaluated the therapeutic effect. The second trial with double dosage was continued right after the first for a week, and the third trial was conducted after one month with the same dosage and period with the second. In addition, the calf went under rehabilitation for 30 min a day by using an adjustable sling (Fig. 2).

Figure 2. Rehabilitation of the patient with standing frame. Standing practice was started two weeks after hospitalization, and the patient still hung on the rehabilitation sling on day 46 (A) and day 54 (B) after visit. The patient could maintain the standing position for a while when recombinant bovine somatotropin administration was combined (C, day 84), but the symptoms were not improved significantly (D, day 113).

After rBST administration and rehabilitation, the calf could gradually maintain the standing position without support for a while, but the posture was still similar to the typical clinical signs of cerebellar lesion representing the tilted head and wide-based stance. Even though continuous treatment, the symptoms were not improved anymore, and the calf died three weeks after stopping rehabilitation at nine months old. The necropsy showed severely enlarged esophagus (Fig. 1B, 3), congested and pus-infiltrated lung, and abomasal ulcer. The hemisphere of the cerebrum and cerebellum was intact without shrinkage or cavitation, but the cerebellum was observed to be softened (Fig. 3).

Figure 3. Post-mortem examination of the calf. Congestion of mesenteric blood vessels (A) and lungs (B), abomasal ulcer (C), and normal sized but softened cerebellum (D) were observed in clinical necropsy. The esophagus was enlarged (E) and fulfilled with undigested hay (F).

Gait ability and standing posture can be affected by numerous pathologic conditions such as musculoskeletal disorders, metabolic disorders, cardiovascular diseases, toxic plants, nutritional conditions, or lesion of the CNS. In this case, the molecular detection of BVDV supported that the cerebellar disorder induced by viral infection would be the suspected cause of the symptoms. In most BVDV cases when a fetus is infected at 100 to 180 days in gestation, congenital cerebellar hypoplasia or dysplasia can be presented at birth, and the affected calves die shortly after birth (1). If a pregnant cow was vaccinated adequately, the fetus and the new-born calf can be protected by maternal antibodies until the first few months after birth (4). In the present study, however, the calf was born from a non-vaccinated cow, and the symptom of standing inability was shown gradually since the calf was three months old. It is assumed that the BVDV infection occurred postnatally, but the possibility of intrauterine transmission cannot be ruled out. There have been several reports of cerebellar abiotrophy which are genetically-induced disorders in various cattle breeds (2,11,12), and the affected animals have clinical signs and variable microscopic findings including loss of Purkinje’s cells in a focal area of the cerebellum, axon swelling, and gliosis. However, morphologic change of cerebellar abiotrophy was observed in only some ataxic animals, and they suggested that electrophysiological disturbance is likely to be responsible for cerebellar ataxia (12). In the present patient, a presumable cause of the cerebellar ataxia, while uncertain, is BVDV infection. The symptoms had been represented gradually over a few months after birth, and it is assumed that the cerebellar disorders following BVDV infection were developed by degrees, and thus the host was able to live for nine months not showing additional and severe symptoms. To prevent the transmission of BVDV, early detection and culling of PI are recommended, but since the patient in this case was the one whose genetic superiority had to be verified, experimental treatment was attempted in an isolated space.

In mice with congenital neurogenic disorders (13) or zika virus infection (14), growth hormone treatment preserved normal locomortor functions and improved growth delay. Growth hormone activate neuroendocrinal pathways and promote progenitor cells (3), and in the present case, clinical trials of rBST were conducted together with rehabilitation from 20 days after hospitalization for the recovery of standing ability. Since the dosage for a calf was not provided, the first rBST administration was initiated as much amount of one quarter of adult cattle. At the first trial of rBST, the status of the calf was not improved, but during the second trial with double-dose rBST, there was a support of the voluntary muscle to maintain the standing posture immediately after rehabilitation. Furthermore, at the third trial after one month, the calf could take two or three steps without a support sling. Two days after every administration, however, the strength of the muscle supporting the posture weakened again without the continuous effect of the rBST. Although GH appears to be directly involved in fetal CNS development during the prenatal period, it has a variety of beneficial effects on postnatal and adult CNS functions including reversal of nerve cell damage, stimulation of neurogenesis, and prevention of apoptosis (8). In this case, it is not clear whether the administration of rBST worked in the cerebellum level or it only stimulated the peripheral nerves and muscles, however, there was a slight improvement of standing posture maintenance while the symptoms including astasia and head tilt were not corrected.

The calf was suffering pressure sore and bloating accompanied by anorexia for about four months in the hospital, and died at nine months of age. The necropsy revealed that the cause of death was the bloat due to esophageal obstruction and severe pneumonia. Swallowing and relaxation of the esophagus are modulated by multiple nervous pathways, and the cerebellum contributes to mediate motor control of deglutition (9). Esophageal obstruction would be induced by incoordination of muscle movements, followed by regurgitation and aspiration pneumonia.

The present case showed the gradually developed symptoms of cerebellar disorders which are induced by BVDV infection, and the typical symptoms were not improved completely in spite of the rBST administration for functional recovery. However, rBST showed potential efficacy representing the ability of balance and movement also in the calf.

We are thankful to LG Chem, Ltd. which greatly assisted the research and provided the recombinant bovine somatotropin (Bustin^®-s).

The authors have no conflicting interests.