Poor calf production and ill thrift in 3–4 mo olds are common limiting problems in raising and maintaining captive muskoxen (Ovibos moschatus). Acute onset of a rapidly progressing enteritis and septicemia in neonatal calves (2–7 days of age) caused by Escherichia coli not normally considered pathogenic in domestic animals is a serious problem in many captive muskoxen facilities. Serum immunoglobulin G (IgG) levels, total protein, albumin, and globulin levels in captive periparturient muskox females and their neonatal calves were compared with levels found in other species in which these parameters have been well described. Results showed that all females in the study had IgG present in their serum (mean = 1,232.14 mg/dl, SD = 178.34 mg/dl, as measured via radial immunodiffusion). Calves were probably born agammaglobulinemic or hypogammaglobulinemic. IgG levels quickly rose in calves after initial colostrum intake to levels similar to those seen in other domestic ruminants. Our results suggest that passive transfer of immunity in muskoxen is similar to what is reported in domestic livestock and that reference ranges from domestic cattle may be used to assist interpretation of serum IgG levels in muskoxen. In addition, the positive relationship between serum protein and globulin levels with serum IgG levels is similar to that reported for Holstein cattle and thus provides a useful indicator of passive transfer in muskoxen.
Muskoxen (Ovibos moschatus) were extirpated from Alaska by the late 1800s and were reintroduced in 1930 by using 34 animals brought from Greenland.2,18 Today, several free-ranging muskox populations exist in northern and western Alaska with a total population of approximately 4,000 animals.15,16,19,30 Muskoxen produce qiviut, a fine underwool shed annually in May that has fiber qualities rivaling cashmere.31 Therefore, although this species has historically been held in captive settings for research or exhibition, there is increasing interest in maintaining muskoxen for fiber production.31 The University of Alaska Fairbanks maintains a herd of muskoxen for research and educational purposes at the Robert G. White Large Animal Research Station (LARS) in Fairbanks, Alaska (64°49′N to 147°52′W). This herd has ranged from the original 16 animals acquired from Nunivak Island in 1980 to a high of 85 animals in the mid-1990s. The herd is currently maintained at approximately 40 animals.
Poor calf production and ill-thrift in 3–4-mo-old calves are common limiting problems in raising and maintaining captive muskoxen.6,7,14,17,34 In general, if captive muskoxen can survive past 6 mo of age in good health and body condition, they are easy to maintain by following good livestock management practices.
Acute onset of a rapidly progressing enteritis and septicemia in neonatal calves (2–7 days of age) caused by Escherichia coli not normally considered pathogenic in domestic animals has been seen at LARS and other facilities. The sudden onset and rapid progression of the disease necessitate aggressive, labor-intensive treatment, often with disappointing results. Free-ranging muskoxen normally calve into a relatively clean, snow-covered environment in late March and April. The calves are extremely well insulated and can easily survive arctic temperatures reaching −40°C and colder. Given the natural calving conditions, it is not surprising that neonates are easily heat stressed at the relatively high ambient temperatures experienced at more southern latitudes and that they may be poorly adapted to accommodate the rapid bacterial colonization of their intestinal tract from the high environmental bacterial counts common in a farm or zoo setting.
In a captive setting, calf morbidity and mortality may be minimized through careful breeding management to ensure calving occurs when ambient temperatures are cooler and a possibility of ground snow still exists. Husbandry practices that ensure calving occurs in an area that is as clean as possible as well as the use of prophylactic antibiotics (e.g., trimethoprim-sulfa or tetracyclines) contribute to control of neonatal disease at LARS.
Protection from infection by known pathogens as well as environmental organisms is provided to most mammalian neonates by maternal antibodies (primarily immunoglobulin [Ig]G1, but also IgM and IgA), which are passively transferred to the neonate either while in utero (placental transfer) or after birth, mainly through colostrum.5,39 For domestic cattle and sheep, Ig transfer occurs after birth through ingestion of colostrum, where Ig levels are concentrated before parturition to equal that of serum concentrations.24,39 Calves are born with little or no protection (agammaglobulinemic) against disease, and they receive protection only after ingestion of colostrum from the dam.22,33,35,39
IgG is the prominent immunoglobulin in colostrum and has a long serum half-life (approximately 18 days), allowing it to persist in substantial amounts during the perinatal period of high susceptibility to neonatal enteric diseases.4 The primary site of Ig absorption in the neonate is the small intestine, and this absorption declines quickly after birth. In domestic cattle, colostral antibodies are absorbed mainly during the first 12–24 hr of life, and, at 6 hr postparturition, mucosal ability to absorb Igs may already be decreasing.3 Failure of the neonate to receive these protective antibodies usually results in death primarily because of infections by opportunistic organisms.4,22,23,25,36
The susceptibility of neonatal muskoxen to infection by organisms not normally associated with disease in other species has raised questions about calf immune function. It has been speculated that passive transfer of immunity is either less efficient in this species or it is adversely affected by current husbandry practices and the more southerly environment. Little is known about the transfer of protective antibodies in muskoxen, but it is generally assumed to be similar to domestic cattle, based on their placentas sharing characteristics (e.g., synepitheliochorial).13
Measurement of serum Ig levels is a common means for determining success of passive transfer, and radial immunodiffusion is a simple and accurate means for quantification.11,17 The primary goal of this clinical study is to evaluate passive transfer of immunoglobulin in muskoxen and to test for differences in multiple serum parameters (protein, albumin, globulin, and IgG) over time. We compare parameters in captive periparturient muskox females and their neonatal calves to levels found in other species where passive transfer of immunity has been well described.
MATERIALS AND METHODS
Blood was collected from six pregnant adult muskox females, three nonpregnant adult females, and seven calves during March through May 2003 at LARS. All adult females were born at the facility, of known age (6–16 yr), and housed together in a harem. Females were in good body condition at time of calving (body score 4/5 by using a standard bovine body scoring scale),14 and all were multiparous. Cows were vaccinated 3 wk before parturition with a commercially available cattle bacterin following label instructions (ScourGuard 3(K)/C®, Pfizer Animal Health, Exton, Pennsylvania 19341, USA). Within 7 days of parturition, cows were transferred into a clean dedicated calving pen. Reduction of calving interval from 2 wk in previous years to 1 wk minimized contamination of the calving pen and permitted better observations. Cow–calf pairs were transferred out of the calving pen 7 days postpartum. Our typical calf birth protocol was maintained during this clinical study. On the date of birth, calves were caught, examined, and weighed. An oral coronavirus–rotavirus vaccine (CalfGuard®, Pfizer Animal Health) was administered, and navels were wiped with a tincture of iodine. Calves were given five daily treatments of oxytetracycline (5 mg/kg, p.o., Terramycin® Soluble Powder, Pfizer Animal Health) for prophylaxis. All calves had the same sire. Sampling focused on cow–calf pairs except for one calf (216) whose dam (138) was extremely fractious, making sampling impossible for animal and human safety reasons.
Blood samples were collected from cows every 3–4 days for a period of 1 mo leading up to parturition. No samples were collected during the 10– 14-day period immediately before parturition, nor postpartum, to prevent disruption to the cow–calf bond. Sample collection took place in a commercial bison chute (A.E. Thorson and Sons, Corvallis, Montana 59828, USA) adapted for use in muskoxen. Parturition dates were estimated through a combination of remote mounting recorder devices (DDx, Incorporated, Boulder, Colorado 80301, USA) and a progesterone assay (Diagnostic Products Corporation, Los Angeles, California 90045, USA) that was used to confirm the pregnancy after mating.34 Blood samples were collected from calves on the day of birth and every 2–3 days thereafter until they were between 8 and 10 days of age. An effort was made to collect calf serum before its first intake of colostrum; however, cows and calves were not observed continuously. If calves were born during the night (i.e., unobserved times), blood was collected the next morning. Blood was collected into red, tiger-top, serum separator tubes (Vacutainer, BD Biosciences, Franklin Lakes, New Jersey 07417, USA) from the jugular vein. Blood was allowed to clot for 10 min, and then it was centrifuged for 5 min at 4,000 rpm. The separated serum was removed and frozen at −80°C until analysis. A colostrum sample was collected opportunistically during a cesarean section on female 184 included in this study. This sample was frozen at −80°C and later analyzed along with the serum. All procedures were conducted under a protocol approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee.
Standard serum analysis
The protein profile consisting of total protein, albumin, and globulin was performed on a Boehringer Mannheim/Hitachi-912 Chemical Analyzer (Roche Diagnostics, Indianapolis, Indiana 46250, USA). Total protein and albumin were measured calorimetrically. A biuret reagent was used for the total protein procedure, and bromcresol purple reagent was used for albumin. A serum blank process was used to remove background spectral interferences not completely removed by the bichromatic analysis. The globulin level was an automatic instrument calculation and was printed with the panel results.
Muskoxen serum samples and the rabbit anti-muskoxen IgG antibody, previously prepared by the Department of Pathology, Microbiology, and Immunology (University of California Davis College of Veterinary Medicine, Davis, California 95616, USA)37 were shipped to the University of Georgia Tifton Veterinary Diagnostic and Investigational Laboratory (Tifton, Georgia 31793, USA). Veterinary Medical Research and Development, Inc. (Pullman, Washington 99163 USA) developed muskoxen-specific single radial immunodiffusion kits from the antibody. The specimens were then analyzed following the kit directions at the University of Georgia. Two Ig concentrations of plates were developed for the kits, one plate with an immunoglobulin concentration of 175–1,400 mg/dl (HLC plate) and one plate with an immunoglobulin concentration of 44–350 mg/dl (LLC plate). When the immunoglobulin reading was less than 175 mg/ dl, the sample was retested using the LLC plate. Bovine standards were used for development of the standard curve by using a protocol derived from the works of Mancini21 and verified with serum electrophoresis data (Miller, unpubl. data). Standards and test samples were added to wells. Wells were read after 24 hr, a standard curve was established, and the diameters of the test samples were compared with the standard curve for determination of the immunoglobulin concentration.
Detectable range of the test kit was from 44 to 1,400 mg/dl; therefore, values outside this range were recorded as 44 mg/dl (lower end) or 1,400 mg/dl (upper end) for statistical analysis. Statistical analyses were performed using the SAS® system (SAS Institute, Cary, North Carolina 27513, USA) and Microsoft Excel (Microsoft, Redmond, Washington 98052, USA). It was assumed all response variables followed a normal distribution. A one-sample t-test was used to test whether average IgG levels of calves were <1,400 mg/dl for each time period. A two-sample t-test was used to test whether average IgG levels of calves differed between calves born to cows <10 yr of age and calves born to cows >10 yr of age. An analysis of variance (ANOVA) was used to test for differences in serum parameters (protein, albumin, globulin, and IgG) over time. Correlations (Pearson correlation coefficient) and regression analyses were run to test for a relationship between average cow IgG and average calf IgG, cow age and rate of change of calf IgG levels, cow parity and rate of change of calf IgG levels, and IgG and serum parameters (protein, albumin, and globulin) in calves and also in cows. A significance level of 0.10 was used for all tests because sample sizes were small. Time period 5 was not used in analyses because data was collected on only one calf.
Serum IgG levels in both pregnant and nonpregnant muskoxen cows were >1,050 mg/dl, which can be considered adequate compared with domestic ruminant standards (foals: 1,000–2,000 mg/dl; calves: >1,000; and lambs: >1,000 at 24–48 hr postbirth).1,10,28 Calf serum Ig levels were low in the first 24 hr (Table 1) with levels <900 mg/dl in five of the seven calves. However, four of these calves had levels of 1,050 mg/dl or above at the next sampling period (days 2–3). One (A, not assigned a number) of the five calves was delivered by caesarian section and had an IgG level below the lowest detectable range (44 mg/dl). Calf A did not nurse, was rejected by its dam (184), and was euthanized. Both colostrum and serum opportunistically collected from cow 184 during the caesarian section delivery had an IgG level of 1,400 mg/dl.
The remaining two calves (216 and 219) had immunoglobulin levels >1,400 mg/dl during the first time sampling period; these values were similar to adult levels. No correlation (r = 0.161; P = 0.76) in average IgG was found between cows and their calves. Calf IgG levels differed among sampling periods (P = 0.06; Fig. 1) and were significantly (P = 0.04) lower than 1,400 mg/dl (cow serum levels) in time periods 1 and 4 but not 2 and 3 (Fig. 1).
No differences were detected (P = 0.41) in IgG levels between calves from cows <10 yr of age (n = 4) and calves from cows >10 yr of age (n = 3). However, rate of change in average calf IgG between time periods 1 and 3 and 1 and 4 was positively related with cow age and parity (r = 0.81– 0.91; P = 0.03–0.09).
Total serum protein, albumin, and globulin levels also were measured. Averaged over all sampling periods, total protein was 7.66 g/dl (SD = 0.63) in the cows and 5.95 g/dl (SD = 0.86) in the calves. Albumin and globulin levels averaged 3.16 (SD = 0.20) and 4.53 (SD = 0.53) in the cows and 2.30 (SD = 0.15) and 3.65 (SD = 0.92) in the calves. IgG positively correlated with protein and globulin in calves and cows, and both protein and globulin were found to be significant predictors of IgG (Figs. 2, 3). No evidence of correlation (r = 0.10–0.65; P = 0.16–0.54) was found between cow and calf protein, albumin and globulin, or average calf protein, albumin, and globulin over time.
In spring 2003, muskox cows and their calves housed at LARS had IgG levels similar to those seen in periparturient domestic ruminants.1,10,28 Levels observed are interpreted as being sufficient for muskox calves, because no morbidity and mortality occurred in any animal during the clinical study period or afterward. This was attributed primarily to improved reproductive management focused on early breeding the previous August resulting in earlier calving in April, vaccination of cows, dedicated calving pens, reduced calving interval, and strict calf birth protocols. All cows remained healthy throughout the study and calved normally without assistance, except for one small cow that required a caesarian section.
Increasing concentration of IgG into the mammary gland begins several weeks before parturition and ceases near or at parturition in cattle.8 In bovids, evidence suggests that there are two distinct populations of mammary receptors for IgG present: the first with a weak affinity for IgG present throughout lactation and the second with a strong affinity occurring during colostrum formation.32 The increase in colostrum-forming mammary receptors (and thus IgG concentration) is accompanied by a drop in maternal blood IgG concentration in cattle.9 A drop in the blood concentration of IgG was not noted in adult muskoxen (pregnant or nonpregnant) at any time during the study period; however, this finding is most likely a factor of sampling given that no samples were collected during the last 10–14 days of gestation, nor postpartum (Table 1). The drop in the blood concentration of IgG would have been expected during this time period.
The immunoglobulin levels in the calves are consistent with that found in domestic ruminants.1,10,28,38 We suggest that muskox calves are born agammaglobulinemic, like domestic cattle, sheep, and goats, and that they obtain immunoglobulins by passive transfer from dams via colostrum.24,29 Average calf IgG levels followed a trend of time period 1< time period 4 < time period 3 < time period 2, suggesting that IgG levels start low, rise high with colostrum uptake, decrease slightly, and then likely stabilize (Fig. 1). Further investigations are needed to test whether these trends follow the IgG levels of colostrum versus milk.
The calves with Ig levels at or near the cow serum levels were those whose serum samples were collected after they had already suckled colostrum. Calf A of cow 184 was rejected after caesarian section delivery. This calf had not yet suckled at the time of serum collection on time period 1 based on the colostral IgG levels in this cow, serum IgG levels in the calf (<44 mg/dl), and observation before euthanasia. The reason for the drop to inadequate levels on day 7 in calf 222 is unknown. No clinical illness was observed in this calf, and it was found to be clinically normal at the time of sampling. It experienced no major health problems in the subsequent months, although these findings do not rule out the presence of a pathogen, nor do they rule out an abnormality in intake or processing of milk during the days before this measurement.
Although the statistical correlation of age and parity are similar to those found in domestic cattle, our results may be misleading because these correlations are dependent on the levels measured at time period 1.12,26 When reviewing the time period 1 IgG levels, we found that two of the calves in the younger cow group had most likely suckled before blood collection (i.e., calves born late evening and not seen until the next morning). Thus, time period 1 levels in these two calves were probably reflective of colostral IgG.
Our results suggest that passive transfer of immunity in muskoxen is similar to what is reported in domestic livestock (foals: 1,000–2,000 mg/dl; calves: >1,000; lambs: >1,000 at 24–48 hr postbirth)1,10,20,28 and that reference ranges from domestic cattle can be used to assist interpretation of serum IgG levels in muskoxen (Table 1). The positive relationships between serum protein and globulin levels and serum IgG levels are similar to those reported for Holstein cattle; therefore, they provide useful indicators of passive immunity in muskoxen.27 Further clinical assessment is necessary to determine what IgG levels might be associated with calf morbidity. Other factors, such as the relationship of heat stress in muskox calves to nursing behavior and to intestinal absorption, also warrant investigation because muskox calves are well equipped to maximally conserve body temperature.
We thank Sandy Garbowski, Bill Hauer, Peter Reynoldson, Chris Terzi, and the staff of Robert G. White Large Animal Research Station and Veterinary Services at the University of Alaska Fairbanks for assistance with this project and Dr. Sandy Baldwin for support of the laboratory testing. We thank Veterinary Medical Research and Development, Inc., specifically Travis McGuire, for help with IgG analysis.