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Can Vet J. 2010 August; 51(8): 877–880.
PMCID: PMC2905008

Language: English | French

Upper alimentary tract papillomas in calves related to papillomavirus infection

Abstract

This study reports 3 cases of spontaneous papillomavirus infection in 1-week-old calves. Thickening of the omasum and abomasum wall, with acute inflammation, necrosis, ulceration, and neoplastic changes were seen in 1 calf. In the other 2, small papillomas were observed in the omasal mucosa, exhibiting proliferation of the parakeratinized epithelium. Papillomavirus antigens were detected by immunohistochemistry and virus-like particles were seen through electron microscopy.

Résumé

Papillomes du tractus alimentaire supérieur chez des veaux liés à une infection à papillomavirus. Cette étude signale 3 cas d’infection spontanée à papillomavirus chez des veaux âgés de 1 semaine. L’épaississement de la paroi de l’omasum et de l’abomasum, accompagné d’une inflammation aiguë, d’une nécrose, d’une ulcération et de changements néoplasiques ont été observés chez 1 veau. Chez les 2 autres, de petits papillomes ont été observés dans la muqueuse omasale, avec des signes de prolifération de l’épithélium parakératosique. Des antigènes du papillomavirus ont été détectés par immunohistochimie et des particules d’aspect viral ont été observées en microscopie électronique.

(Traduit par Isabelle Vallières)

Introduction

Papillomaviruses (PV) belong to the Papillomaviridae family; they have a double-stranded DNA genome in an unenveloped, 50- to 60-nm icosahedral capsid (1,2). These viruses are oncogenic, infecting cutaneous and mucous epithelia in various animal species and humans. They induce hyperproliferative benign tumors known as papillomas or warts, which occasionally undergo malignant transformation ending in squamous cell carcinoma (1,2). The most commonly observed neoplasms include ano-genital and upper respiratory tract carcinomas as well as skin cancer in humans and rabbits (13). In bovines, fibropapilloma of the skin and epithelial papillomas of the upper alimentary tract and bladder have also been reported (1,2,4). In this species, malignant transformation is related to bracken fern ingestion (5,6). This infection, affecting the upper alimentary tract, has been reported as early as 15 mo of age (7), but, to our knowledge, there are no reports of neonatal papillomavirus infection in bovines.

Material and methods

One 7-day-old Holstein calf (calf 1) was submitted for postmortem examination to the Pathobiology Department, at the National Institute of Agricultural Technology (INTA) in Castelar, Buenos Aires province, Argentina. The calf belonged to a dairy farm in Venado Tuerto, Santa Fé province. It was in poor body condition, unable to stand, eat, or drink. Diarrhea had been noticed by the clinician in several weaned calves from the same farm, affecting almost 10% of them, including the 3 calves in this study. Both rotavirus antigens and Escherichia coli had been detected in stools from some of these calves by enzyme-linked immunosorbent assay (ELISA) (8,9) and bacterial culture, respectively. All animals were fed a balanced ration and milk. Water was provided ad libitum. All affected calves were treated for neonatal diarrhea by the veterinarian; treatment included antibiotic therapy. Although mild recovery was noticed in most calves, the diarrhea would start again after 2 wk post treatment, resulting, in some cases, in the death of the affected calf. Two other calves (calves 2 and 3) from the same herd, exhibiting similar signs were euthanized together with calf 1 and postmortem examinations were performed on all 3.

Tissue specimens from the omasum, abomasum, small and large intestine, liver, spleen, kidney, lung, heart and brain were obtained for histological examination. The tissues were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 4 μm and stained with hematoxylin and eosin (H&E).

Immunohistochemistry (IHC) was performed on tissue sections from papillomas in the omasum and abomasums of all 3 calves, displaying either malignant-like or non-malignant changes. A rabbit polyclonal antibody to detect PV antigens (Dako Cytomation, Carpinteria, California, USA) was used. This antibody, however, was not type-specific, cross-reacting with any human or bovine papillomavirus.

Bovine viral diarrhea virus (BVDV) antigens were detected in the small intestine by IHC (10). For both IHC tests (PV and BVDV), the binding of the antigens to the antibodies was identified by a streptavidin-peroxidase complex (LABS 2 System HRP, Dako Cytomatation) according to the manufacturer’s specifications. Tissues from PV- and BVDV-noninfected and infected cattle were used as negative and positive controls, respectively.

A 1-mm square section of abomasum was obtained from calf 2 and fixed in 2% buffered glutaraldehyde for 24 h, then post-fixed in 1% osmium tetraoxide for 2 h. Ultra-thin sections were cut with a diamond knife, mounted on copper grids, stained with uraniyl acetate and lead citrate, and examined using a transmission electron microscope (Jeol 1200 EX II, Jeol, Tokyo, Japan).

Results

In calf 1, the predominant gross changes were thickening of the edematous walls of the omasum and abomasums. The mucosa was dark red in both organs, with hemorrhages in the serosa and mucosa and ulcers in the abomasum. Small and large intestines exhibited petechiae in the mucosa and a strong dark color in Peyer’s patches.

In calves 2 and 3, the only gross changes observed were several papillomas, 1-mm in diameter in the omasal mucosa and a reddish color in the small intestinal mucosa. All 3 calves had diarrhea, though in calf 1, the feces had a dark discoloration.

In calf 1, the abomasum displayed areas of erosion and ulceration. Degenerative and anaplastic changes with frequent mitotic figures were observed in the epithelial cell lining in combination with necrosis of the mucosa and leukocyte infiltration. Thickening of the sub-mucosa due to a fibrino-suppurative infiltration was observed in the region adjacent to the omasum. In this region, neovascular formations, composed of strongly basophilic anaplastic angioblasts, exhibiting no specific orientation (Figure 1A) and many cells with high nucleus to cytoplasm ratio were also seen. These neovascular formations were often surrounded by areas of diffuse hemorrhage.

Figure 1
Tissue section from the omasum of calf 1.

The omasum exhibited focal locally extensive suppurative inflammation, erosion, and necrosis of the mucosa. The omasal wall was thickened due to hyperplasia of the parakeratinized epithelium which exhibited numerous anaplastic cells displaying increased nucleus to cytoplasm ratio, vacuolation and hyaline inclusions in the cytoplasm and ballooning cell degeneration, resembling koilocytes (Figure 1B). In this area, fibrino-suppurative hemorrhagic exudates were present, extending into the lumen of the gut. Exfoliated leukocytes, together with cellular debris, were abundant in the lumen of the omasum. In the small intestine, diffuse fibrino-supurative enteritis with necrosis of Peyer’s patches was the most relevant change observed. Mesenteric lymph nodes exhibited edema in the sinusoids with mononuclear and polymorph infiltration.

The liver displayed mild centro-acinar vacuolation and mono-nuclear proliferation in the sinusoids. Fibrinous thrombi in arterioles and capillaries were also observed. Glomerulonephritis with mononuclear and polymorphonuclear cell infiltration was seen in the kidneys, together with cellular debris and protein in the lumen of the tubules. Vacuolar change was also present in the tubular cells.

In calves 2 and 3 the papillomas observed in the omasum exhibited thickening of the parakeratinized epithelium supported by a core of highly vascularized connective tissue (Figure 2A), interspersed with areas of erosion. Vacuolar change of the squamous cells was abundant (Figure 2B). Both jejunum and ileum displayed shortening of the villi with erosion, edema, and hemorrhages. Polymorphonuclear cell infiltration of the mucosa and lamina propria was seen. Mesenteric lymph nodes were atrophic, exhibiting hyperplasia of the monocytic-phagocytic system together with mild polymorphonuclear cell infiltration and edema. Fibrinoid change in small blood vessels was observed in the small intestinal sub-mucosa, kidney, spleen, lymph nodes, and brain. Mild mononuclear cell infiltration of blood vessels was also present in the kidneys.

Figure 2
Tissue section from the omasum of calf 2.

Immunohistochemistry was positive for PV antigens in the tissue sections (Figure 3) from both omasum and abomasum from the 3 calves and the positive control, but not from the negative control. Antigens from BVDV were only detected in the ileum of calf 2. Transmission electron microscopy of the omasum sample from calf 2 revealed several hexagonal virus-like particles, scattered in the cytoplasm of some epithelial cells (Figure 4). These cells displayed nuclear, organelle, and cytoskeleton disintegration. The virus-like particles were composed of an electron-dense core (50 to 65 nm in diameter) surrounded by a capsid of lower electron density, and an electron-lucent space between both.

Figure 3
Omasum from calf 1, showing positive immunostaining for PV antigens. Bar = 100 μm.
Figure 4
Transmission electron microscopy of keratinocytes from the omasum of calf 2, showing hexagonal virus-like particles (arrows). Bar = 50 nm. × 160 000.

Discussion

The gross and histological changes observed in the present study were predominantly in the upper gastrointestinal tract, consisting of inflammation and neoplastic changes in the mucosa of the abomasum and omasum. In calf 1, the omasal and abomasal mucosa exhibited acute infiltration, necrosis, and ulceration in combination with malignant neoplastic changes of the epithelial cell lining. These changes may have altered the mucosal permeability, allowing bacteria and/or toxins to flow into the bloodstream, inducing changes in other organs. Lesions in calves 2 and 3 appeared more benign and proliferation of the parakeratinized epithelium was the most relevant change observed. Both of these features have been previously reported in BPV type-4 infections in cattle (2,4,5).

Papillomaviruses have star-like capsomer projections, which reveal a 2-nm space between core and capsid (1). The virus-like particles observed by electron microscopy in this study were hexagonal, suggesting an icosahedral form with a small gap between core and capsid. This is compatible with papillomavirus particles. The affected cells were keratinocytes from the spinous to the squamous layers. Papillomavirus antigens were also demonstrated through IHC in these cells. Unfortunately, the virus type was not determined, due to the lack of virus-type specificity of the polyclonal antibody used.

Although malignant transformation has been previously reported in BPV infections, it is known to occur as a result of ptaquiloside, the major carcinogen of bracken fern (5). This substance was detected in milk from cows experimentally fed on bracken fern and whose milk has been shown to cause cancer in rats (5). Although no fern was found in the farm of the present study, all calves received milk from cows which had been fed on hay, brought from an area in Argentina were bracken fern is present. Therefore, it is likely that bracken fern leaves might have been mixed in the hay, thus contaminating the milk. Unfortunately, neither hay nor milk was available at the time of this study, thus leaving this hypothesis open.

Tsirimonaki et al (7) reported neoplastic change in a 15-month-old steer, naturally infected by BPV-4, grazing on a pasture with no evidence of bracken fern. These authors noted that the animal may have been infected with BVD-virus, and therefore immunosuppressed, enhancing the BPV virulence. Similarly, the calves of this study had a history of neonatal diarrhea, BVDV, and E. coli infection. It is likely that these calves may have also been immunosuppressed, due to the diarrhea, thus enhancing the pathology of the disease. The changes observed in the intestine of the 3 calves were related to either BVDV or E. coli infection rather than to PV infection.

Human papillomavirus infection is known to occur in the neonatal period and children born to HPV positive parents are at higher risk (3). In cattle, BPV infection of the upper gastrointestinal tract has been reported in 15-month-old animals or even older (7), but, to our knowledge, this is the first report of both benign and malignant changes of the upper gastrointestinal tract in young calves, related to papillomavirus infection. CVJ

Footnotes

Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (gro.vmca-amvc@nothguorbh) for additional copies or permission to use this material elsewhere.

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Articles from The Canadian Veterinary Journal are provided here courtesy of Canadian Veterinary Medical Association