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Gribbles Veterinary Pathology, Clayton, Victoria, Australia (PJM), Department of Primary Industries, Wodonga, Victoria, Australia (JGC), Department of Primary Industries, Leongatha, Victoria, Australia (EMP), and University of Melbourne Veterinary Clinical Centre, Werribee, Victoria, Australia (RFS)
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Abstract |
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Key words: Bovine; chondrodysplasia; deficiency; disproportionate; drought; dwarf; manganese; skeleton.
Sporadic congenital deformities are expected in the bovine population. Outbreaks of higher incidence of congenital disease such as that described here may be associated with considerable losses of animals and consequent financial loss. Across Northeastern Victoria and the Southern Tablelands of New South Wales (NSW), Australia, outbreaks of chondrodysplastic disease have been reported, particularly in years following prolonged drought conditions. Outbreaks occurring in 2 consecutive spring calving seasons (July to November, 2003 and 2004) and involving numerous losses through stillbirth, perinatal loss, and poor growth were investigated to exclude potential infectious disease and to define the gross and histopathologic defects. The cause of the syndrome is still unresolved, although congenital manganese deficiency is suspected; despite marked improvement in the climatic conditions in this area, a lesser number of farms saw deformities among calves in a third consecutive calving season (2005). Investigations are ongoing.
Congenital disease may result from genetic abnormality, infectious disease, or other in utero insults. "Chondrodysplasia" is a general term for abnormalities of cartilage, usually caused by defective nutrition or metabolism of cartilage, which results in disproportionate dwarfism.19
Genetic chondrodysplastic syndromes are reported in Holstein, Dexter, and several other breeds of cattle. Beef breeds selected for short stocky phenotype have an increased prevalence of brachycephalic calves, with longitudinal compression of the vertebrae and shortening of the distal long bones, believed to be hypochondroplasia rather than a dysfunction of endochondral ossification.19
There are many examples of toxic and nutritional causes of musculoskeletal deformity. Hypervitaminosis A causes vertebral abnormalities and may be compounded by protein-energy malnutrition.5 "Crooked calves" are caused by ingestion of certain Lupinus sp. containing toxic concentrations of the quinolizidine alkaloid, anagyrine, by the dam, particularly between 40 and 70 days of gestation.23 This leads to arthrogryposis, spinal deformities, and disordered long bone growth, possibly due to reduced intrauterine motility either through tonic contraction of the uterus or suppressed motility and apparent sedation of the fetus,3 leading to a functional arthrogryposis without identifiable histologic or histochemical lesions in the bone or muscle.1 Many other plant-associated toxins are implicated in skeletal disease, for example teratogenic piperidine alkaloids in Astragalus sp., Oxytropis sp., and Nicotiana glauca, which may act in a similar manner.3 Fungal toxins have been presumptively associated with outbreaks of congenital spinal stenosis and disproportionate dwarfism, leading to myelomalacia and posterior paralysis in 3 outbreaks in Western Canada, where heavy growth of Penicillium sp. and Fusarium sp. in wet straw bales was also causally associated with pruritus and mortality in dams.20 "Acorn calves," described in western regions of North America and also recorded in New Zealand and South Africa, were associated in the original reports with ingestion of acorns.2,19 Affected calves showed joint laxity, varus, and valgus deformities, and disproportionate dwarfism. More recent work has suggested that some of these cases may be consistent with a deficiency in dietary intake of manganese, as supported by small feeding trials in cattle.6,7,10,22
Infectious causes of skeletal deformities seen in cattle in Australia include bovine viral diarrhea virus (BVDV), Akabane and Aino viruses. These tend to cause arthrogrypotic deformity of the skeleton rather than chondrodysplasia.
Consideration and, to a large part, elimination of these differential diagnoses combined with evaluation of the anatomic pathology of several cases led investigators to propose that deficiency of manganese could be responsible for the abnormalities in this outbreak. Manganese deficiency was suspected, although not conclusively proven, in several previous field outbreaks of skeletal disease in calves.24,28
This article presents the gross and histopathologic features of 2 outbreaks of congenital disease in calves, defines the disease, and discusses the potential relationship with manganese deficiency.
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Methods |
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Portions of vertebrae and long bones from calf No. 1, a stillborn calf from a farm in Northern Victoria, were received fixed in 10% neutral buffered formalin (NBF) and examined grossly and histopathologically. Calf No. 2, one live 21-day-old calf from a second farm, and calves Nos. 3 and 4, entire stillborn calves from a third farm, underwent full postmortem and histologic examination using standard methods. Carcasses of calves Nos. 2–4 were weighed, and skeletal components of calves Nos. 3 and 4 were recorded using standard measuring tapes. Radiographic analysis of intact demuscled limb bones and sections through vertebrae were prepared using a band saw, after fixation but before decalcification, on calves Nos. 3 and 4 (not presented).
Five-millimeter sections were taken from a wide range of axial and appendicular skeletal sites and fixed in 10% NBF for 24–48 hours before decalcification, using a standard aqueous solution of 18% formic acid and 3.5% sodium formate for approximately 1 week at room temperature with continuous slow agitation. Following standard paraffin embedding, 4-µm sections were cut and stained with hematoxylin and eosin (HE). Histochemical stains used to evaluate cartilage and bone included Giemsa and Acid Giemsa, periodic acid–Schiff (PAS), Alcian blue, Alcian blue–PAS, toluidine blue, safranin O/fast green,15 and Verhoeff elastic stain.
For comparison of bone measurements and histopathology, 20 femora of grossly normal young calves randomly collected by staff at an abattoir in an unaffected region were weighed and measured, and histologic sections were prepared from the distal physis of each. Age and sex matching was not possible in this investigation.
Fresh frozen liver was collected from calves Nos. 3 and 4. Glutathione peroxidase was analyzed using an enzymatic method according to the Standing Committee on Agriculture, Commonwealth Scientific and Industrial Research Organ, Australia, 1993. Vitamin B12 was analyzed spectrophotometrically with competitive binding assay, and copper was analyzed by atomic absorption spectrophotometry. Manganese concentration was determined following wet combustion in perchloric/nitric acid solution and dilution in dilute hydrochloric acid to correct volume by flame atomic absorption spectrophotometry at 279.5 nm on an AAnalayst 300 Spectrometer (PerkinElmer, Waltham, MA). The reference ranges available for concentration of each of these elements are based on adult cattle, and no reference range for neonates was available at the start of the study.
Agar gel immunodiffusion was performed on serum samples from calves Nos. 2–4 and several dams and live calves from affected farms to detect BVDV and Akabane virus antigen. Virus neutralization was performed to detect Aino virus antigen. The National Arbovirus Monitoring Program of Animal Health Australia was consulted to assess presence of insect vectors and serologic evidence of arboviruses in the region during the periods of gestation (www.namp.com.au/reports/report_0203.pdf, 2005).
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90%) of cohort groups affected. The spectrum of severity ranged from stillbirths and perinatal deaths (due to inability to stand and respiratory compromise) to calves deformed but able to suckle and follow the dam. Less-affected calves were vigorous and seemed to show some clinical reduction of deformities over time. Total reported losses due to high morbidity and mortality numbered into hundreds of calves; given economic conditions in this region that had suffered severe drought for several years, unreported losses were expected to approximate 1,000 calves. Dams of all age groups produced deformed calves. Affected calves appeared to be clustered in management cohort groups rather than showing a pattern of spread expected with an infectious agent. The groups of dams giving birth to affected calves had predominantly been at pasture on hilly granitic country that had been extremely dry over the majority of each gestation period, although not all dams were in poor body condition. No toxic plants were found on repeated examination of the grazing areas or reported during careful questioning of farmers. Some farms had been blanketed in heavy bushfire smoke during the first gestation period (2003), but there did not appear to be an association between density of smoke and the affected cohort groups.
Common supplemental food, medication, or management procedures, and genetic links were not identified during clinical investigation and epidemiologic study. The calves affected were a range of beef breeds and crosses, including Murray Grey, Hereford, and Angus. Numerous sires were involved, and cows giving birth to deformed calves had previously produced normal calves and, in many cases, went on to produce normal calves in the following season. One mildly affected heifer calf was observed to produce a normal calf at her first calving.
Gross pathology
External examination of the calves revealed similar deformities in all calves presented intact. All had low body weight (Table 1) and short stature, with moderate-to-marked limb-trunk disproportion (Fig. 1). The heads appeared slightly domed in calves Nos. 2–4 (Fig. 1). There was variable brachygnathism, and calves Nos. 3 and 4 showed protrusion of the tongue with oversize relative to the oral cavity.
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The articular cartilages of all cases showed irregular thickness, with some areas of erosion and prominent subchondral vessels. There was variable multifocal lifting and separation of the articular cartilages in calf No. 2, with suppurative polyarthritis and osteomyelitis of the proximal tibia.
Only skeletal elements were examined in calf No. 1; abnormalities of nonskeletal elements were limited to hypoplasia and lateral flattening of the tracheal cartilage rings and extensive primary atelectasis, with only small areas of inflation of the lungs in calves Nos. 3 and 4.
Morphometric analysis
Measurements of skeletal components of calves Nos. 3 and 4 are presented (Table 1) and compared with measurements of the femora of young but not age-matched calves from an abattoir (Table 2). Previously measured skeletal elements of calves are compared (Table 3).
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Histopathologic findings
The cases all showed widespread physeal and metaphyseal abnormalities of varying degrees of severity. At the physis, the zone of cartilage hypertrophy was irregularly reduced, consisting of disorderly columns varying markedly in number and alignment along the growth plate, often with only 3–6 cells (Fig. 7), compared with neat columns of 10–20 cells in control animals (Fig. 8). The zones of resting and proliferating cartilage appeared relatively normal, although small multifocal nodules of increased numbers of proliferative chondrocytes were seen in each case. Multifocal cystic spaces were present both within the zone of hypertrophy and interspersed between the degenerate/calcifying chondrocytes of the zone of mineralization, suggestive of failure of formation of matrix or premature loss of chondrocytes and matrix. These cysts showed basophilic granular-to-fibrillar staining with toluidine blue and PAS, suggesting abnormal cartilage matrix filling the spaces (Fig. 9). A high rate of osteoclastic activity was indicated in these areas by the presence of abundant osteoclasts. Trabeculae within the primary spongiosa were short and irregular; a low proportion showed evidence of fracture close to the physis, with osteoclasts present at the blunt distal ends of trabeculae (Fig. 7).
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Sections of vertebrae examined from each animal showed irregular outlines of the cranial and caudal physes due to variation in depth of both cranial and caudal aspects of the zones of hypertrophy and mineralization (Fig. 13).
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In calf No. 1, the atlas showed a pathologic in utero fracture of a focus of ectopic cartilage, disorderly proliferation of new bone, and cartilage with altered staining characteristics and increased cellularity and proliferation of the periosteum. This caused distortion and compression of the spinal canal and cord.
The proximal tibial physis of calf No. 2 had bacterial osteomyelitis. A necrotic cartilage sequestrum was surrounded by clusters of degenerate neutrophils grouped around colonies of small coccobacilli and associated with dense infiltrates of lymphocytes, plasma cells, and macrophages and a thick rim of fibroblastic connective tissue bridging the physis, thus causing pathologic fracture. In this case, lymph nodes taken from mesenteric and prescapular regions all showed a mild hypertrophy of follicles, consistent with excessive immune stimulation; however, viscera and neurologic tissues showed no significant changes. Microbial cultures were not performed.
Mineral analysis
In calves Nos. 3 and 4, portions of fresh frozen liver were submitted for mineral analysis (Table 4). The manganese concentrations detected in these unsuckled neonates were below the normal reference range; however, since this range was determined for adult cattle, further analysis is required to assess the range for neonatal calves before reliable interpretation can be made.
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Serologic analysis for BVDV, Akabane and Aino viruses performed on these calves was negative as was that from numerous other animals from herds affected during the outbreak (data not presented). The National Arbovirus Monitoring Program did not report any seropositive animal or insect vector detection in the affected regions during the gestation periods.
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Discussion |
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The common causes of chondrodysplasia including genetic defects, most toxic and nutritional causes, and infectious agents were excluded sequentially by epidemiologic and pathologic investigation.
In the affected herds in this outbreak, no familial link could be established, and the range of breeds and crosses affected was felt to preclude any possibility of a genetic etiology.
Vitamin A supplementation was rarely performed; therefore, hypervitaminosis A was excluded. Many farms with diverse management practices were affected; therefore, disease related to management procedures or therapeutic product administration was considered unlikely.
Pastures had been bare for much of the gestation period in these outbreaks. While there was no known access to any toxic plants, under conditions of undernutrition, cattle will graze plants normally rejected as unpalatable. The presence of toxic plants during the lengthy period of development and growth of bone and cartilage (from 40 days onward), and not during investigation several months later, cannot be excluded. The skeletal changes within individual affected calves and the range of severity seen among cohort groups suggest a prolonged period of exposure extending throughout the period of skeletal mineralization and growth, increasing the likelihood of detection of toxic plants. In this region of Australia, lupins are not common, and oak trees are rare; aflatoxicosis was unlikely since there was little rain and silage feeding was uncommon. No epidemiologic association could be made between bushfire smoke intensity and the congenital abnormalities.
Infection with teratogenic viruses was unlikely since serology was negative and the lesions were dissimilar to those expected with infectious deformities. Viral culture and immunoglobulin G analysis was performed in other cases without evidence of viral disease or in utero immune challenge (data not presented). Viral agents known to cause congenital defects (e.g., BVDV, Akabane and Aino viruses) tend to cause neurologic defects and arthrogryposis rather than deformity at the growth plate. No histologic lesions of the nervous system were detected. Nationwide surveillance programs showed that insect vectors responsible for transmission of arboviral disease were excluded from these areas throughout the period of gestation; thus, Akabane and Aino viruses were considered unlikely.
Anecdotal and unpublished data on previous Australian outbreaks similar to this case have suggested a possible association with long periods of drought, deformed calves tending to present following prolonged dry conditions or in the seasons following early onset of more normal rain patterns, particularly in the southeastern regions of NSW. Extensive investigations have always failed to ascertain a definite cause (A. Philbey, T. Ross, personal communication).
Sequential elimination of reported causes of deformities in this investigation led the authors to suspect involvement of manganese deficiency in the pathogenesis. Histopathologic descriptions of experimental manganese deficiency–induced deformities have been sparse to date, although the gross pathology described is similar to that in affected cases presented here. In a field investigation of suspected manganese deficiency, narrowed irregular growth plates with reduced zone of cartilage hypertrophy were described in three 3-month-old calves, but the authors did not describe histologic changes in neonates.24 Growth-plate irregularity with reduced and disorderly proliferating chondrocytes and absence of hypertrophic chondrocytes, with residual necrotic remnants among fibrillar matrix, was described in another field outbreak putatively associated with manganese deficiency.28 Respiratory difficulty has been a common factor in this and in other investigations18,24 and may represent abnormalities of the tracheal cartilage rings leading to reduced luminal diameter, distorted nasal passages, or a combination of these and other factors. Calves not so severely affected as to die in the perinatal period in outbreaks of postulated manganese deficiency have reportedly shown limited-to-good clinical improvement with time, if assistance is given to feed and stand, with reduction in deformation but residual stunting, as noted in this outbreak.28
Evaluation of manganese concentration
Normal reference ranges for manganese concentration are not available in neonates and work to formulate these is still in progress, hampered by the difficulty in obtaining fresh tissues from normal calves in the affected areas. This has limited the interpretation of manganese concentrations in affected calves.
Previous soil and pasture mineral analyses suffer similar difficulty in interpretation of results due to regional variation in soil composition and numerous other compounding factors in manganese absorption (e.g., variable manganese uptake by plants, combination of pasture species, and interference from other elements).
Our review of the literature suggests liver concentration of manganese is most appropriate for analysis in these neonatal calves, although the element can be routinely detected in bone ash, blood, and hair samples.8,9,14 Liver samples are easily obtained by farmers or veterinarians. They are simple to process, and liver concentrations of manganese are relatively high compared with manganese concentrations in other tissues, which may allow more accurate analysis. However, some authors suggest that liver manganese concentration is less well correlated to deficiency states than manganese concentrations in other tissues, including plasma and heart muscle, at least in growing animals.26 Reports of alteration in hair content of manganese with environmental factors unrelated to total body concentration and with hair pigmentation have been identified, so this simple test was not used.9
Manganese in skeletal disease
Manganese is an essential trace element involved as a cofactor in several critical enzymatic pathways of glycosaminoglycan and collagen synthesis and as a component of metalloenzymes, including superoxide dismutase (SOD), among other functions. Deficiency of SOD may be implicated in damage to membranes of mitochondria, endoplasmic reticulum, and golgi bodies and thus lead to loss of integrity, potentially leading to inability to synthesize mucopolysaccharides.14 Manganese is also essential in incorporating carbohydrates into mucopolysaccharides at the epiphyseal plate through manganese-dependant activation of some glycosyltransferases, which transport trisaccharides to link polysaccharide to protein in bone.8,16 Unlike many other metal-activated enzymes, similar metals such as magnesium or iron are less effective than manganese at activation of these particular enzymes.27
The literature on manganese metabolism and deficiency has been thoroughly reviewed.9,14,26,27 Deficiency of manganese has been associated experimentally and clinically with reduced reproductive performance7,22 and skeletal abnormalities7,10,22,24,28 in calves and laboratory animals and with ataxia associated with abnormal development of the otoliths in laboratory rodents but not cattle.13
Manganese bioavailability
Concentrations of manganese in plants vary. Grasses are richer than legumes, and straw and corn silage have particularly low concentrations.9 Uptake of bivalent manganese by plants is more efficient than that of the trivalent or tetravalent forms present in highest concentrations in soil. Low pH soils have a relatively high proportion of bivalent manganese.
Discussion with nutritionists in this investigation led to a suggestion that rumen pH may have a similar effect on bioavailability of manganese. At the time of writing, investigations were being undertaken to review the current knowledge of this interaction and determine whether manipulation of rumen pH rather than direct supplementation of manganese may aid in improving manganese absorption (T. Parker, personal communication).
Overall bioavailability from ingested plant matter is low, with <1% intestinal absorption in cattle. It appears from some studies that high dietary levels of iron, copper, zinc, sulphur, and, to a lesser extent, calcium and phosphorus lead to further reduction in manganese bioavailability.8,9 Although the soil in the affected area is high in granite, which usually has a high level of manganese, the bioavailability of the manganese may be reduced by high dietary intake of other elements in soil such as copper, cobalt, calcium, phosphorus, and iron, which show significant interference with manganese.26 Under severe drought conditions, cows may ingest a high volume of soil and dust, leading to altered dietary mineral balance.
Manganese has been shown in rats and sheep to be preferentially sequestered by the placenta in conditions of manganese deficiency of the dam, but this sequestration is insufficient to return the fetal manganese concentration to normal.11 Unlike neonates, fetuses are not able to preferentially concentrate manganese in the liver.14 It is believed that manganese concentrations in the liver of neonatal presuckled calves represent the manganese status of the dam.26
Retention of absorbed manganese varies with age; calves may apparently alter the percentage of dietary manganese retained after intestinal absorption according to the concentration of the element in the food and in response to deficiency.12 Manganese concentration in tissues has been shown to change rapidly after birth and onset of suckling in calves if there is high manganese concentration in the diet, as indicated by radioactive isotope studies of manganese metabolism.4 Young calves are able to rapidly incorporate supplemental manganese from oral and intravenous administration into tissues, and the percentage retained varies according to the dietary and whole body concentrations of the element at the time of administration of manganese.4 Once suckling commences, it is probable that the manganese concentration in liver rises rapidly and more markedly than in other tissues. This is attributed to the liver's role in manganese homeostasis through storage in mitochondria and excretion in bile. Higher manganese concentrations were found more often in suckled affected calves of a few days old (data not presented) than in neonatal unsuckled affected calves in this outbreak. Results suggested that within a few days, the liver concentration of manganese in affected calves approached normal levels despite the low concentration of manganese found in milk.
The percentage of dietary manganese absorbed in adults is relatively constant. Manganese is largely removed from first-pass portal circulation by the liver, and peripheral blood has a very low concentration of manganese bound to 
-2-macroglobulin.
The apparent difference in manganese absorption may reflect the incorporation of the majority of dietary manganese into ruminal micro-organisms in adults, as evidenced by more efficient cellulose digestion by micro-organisms in the presence of supplemental manganese.17 The reason adult cows are unable to enhance retention of manganese if deficient is unclear.
Feeding of silage alone through winter in northern latitudes has been identified as a risk factor for a condition known as congenital joint laxity and dwarfism, which has some similarities to experimental manganese deficiency.21 It is postulated by some authors that this may relate in part to manganese deficiency indirectly through reduced bioavailability of manganese present in silage.10
In this outbreak, drought conditions may have led to rumen content with a high percentage of dry matter and thus a relatively high pH. Under these conditions, it is postulated that manganese is present within the rumen mostly as poorly bioavailable forms and thus absorption may be reduced. One potential avenue of investigation at present is analysis of the effects of rumen pH on manganese absorption; alterations of rumen pH may be more readily achieved through the use of supplemental grain feeding or dietary cation-anion–deficit rations without the need to supplement manganese directly.
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Manganese deficiency remains the main postulated cause at the time of writing. Confirmation of manganese deficiency in calves is difficult and time-consuming. Further studies are ongoing, and veterinary nutritional specialists are assessing advice to be given to farmers in the area. Ideally, supplementation will be planned to provide a controlled experiment of the effect of dietary manganese on the birth of deformed calves in this region, provided sufficient resources are available to gather the epidemiologic data and for manganese concentration analysis and other ongoing exclusions. One review has recommended the addition of 0.5 g/day per head of manganese as sulphate, the most soluble form, or as oxide, carbonate, or methionine salts to cattle.8 Avoidance of management procedures that increase the pH of soil such as liming may also aid in disease prevention.26
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