Association of bovine papillomavirus type-2 and urinary bladder tumours in cattle from Romania

In cattle, bracken fern toxicity is characterized by the presence of haematuria and tumours of the urinary bladder of both epithelial and mesenchymal origin. This syndrome is known as chronic enzootic hematuria (CEH) and is also present in Romania. From January 2006 to April 2007, 90 urinary bladders from slaughtered cows originating from hill-mountain area of Neamt county (Romania), where CEH is endemic, were collected. All samples were histologically examined and Bovine papillomavirus type 2 (BPV-2) DNA was detected by polymerase chain reaction (PCR) analysis in 68% of the analyzed tumours samples. BPV-2 positive urinary bladder tumours were also immunohistochemically analysed for the expression of the major viral oncoprotein E5. We found the expression of E5 intracytoplasmically with a typical juxtanuclear pattern. E5 expression was not observed in normal mucosa, suggesting a causal role for this protein in the neoplastic process. This is the first report of BPV-2 infection in Eastern European country, confirming the role of BPV-2 in naturally occurring bovine urothelial carcinogenesis.     

Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers

Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular forms in the two tumours. Realtime quantitative PCR revealed that equine sarcoids contained higher viral DNA loads compared to bovine bladder cancers. The BPV-1 E5 ORF showed sequence variation but BPV-2 ORF did not. The presence of BPV-1 E5 variations or their absence in the BPV-2 E5 ORF does not appear to have an effect on viral DNA load in either tumour type.     

Multiple glomus tumors of the urinary bladder in a cow associated with bovine papillomavirus type 2 (BPV-2) infection

We report a case of multiple glomus tumors associated with bovine papillomavirus type 2 (BPV-2) infection in the urinary bladder of a 13-year-old cow suffering from severe chronic enzootic hematuria. Macroscopically, multiple submucosal reddish nodules were seen swelling the vesical mucosa. Histologically, neoplastic proliferation was characterized by the presence of numerous blood vessels. These were lined by normal endothelial cells surrounded by round epithelioid cells with central nuclei, prominent nucleoli, acidophilic cytoplasm, and well-defined cytoplasmic borders. Tumor cells were distributed around open vascular lumina and in perivascular spaces. They were immunohistochemically positive for actin and vimentin and negative for cytokeratins, desmin, and factor VIII-related antigen. On the basis of these findings, this tumor was diagnosed as glomus tumor, a neoplasm not previously reported in cattle and exceedingly rare in animals. BPV-2 DNA was amplified from the formalin-fixed, paraffin-processed tissue specimens obtained by laser capture microdissection. This report widens the spectrum of mesenchymal tumors of the bovine urinary bladder. Finally, the microscopic pattern of tumor described here shares striking morphologic and immunohistochemical similarities with the angiomatous form of glomus tumor known to occur in man.     

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.     

牛乳头瘤病毒基因1型广西GX01流行株全基因组克隆及序列分析

为了解牛乳头瘤病毒1型（bovine papillomavirus genotype 1,BPV-1）广西GX01株全基因组序列、结构特征及遗传变异情况,同时了解该毒株引起宿主产生的病理组织学变化情况,本研究选取广西贺州市患病牛皮肤肿瘤样物制作石蜡切片后镜检观察,提取病料DNA,以乳头瘤病毒L1基因的简并引物FAP59/FAP64进行PCR扩增以确定此病毒的基因型,根据GenBank中BPV参考株设计嵌套引物,对GX01株进行全基因组扩增、克隆测序及序列分析。病理组织学检查结果显示,可在病变部位发现表皮细胞增生、肿胀,角质过度及挖空细胞等乳头瘤病毒感染的特征性病变。序列分析结果表明,GX01株为BPV-1,其全基因组长为7 945 bp,包含E1、E2、E4、E5、E6、E7、L1、L2 8个开放阅读框,符合BPV-1型基因组的结构特征;GX01与BPV-1参考株全基因组核苷酸序列同源性为98.6%~99.6%,与BPV-2型参考株（M20219.1）、BPV-13型参考株（JQ798171.1）同源性分别为86.9%和87.2%。GX01株为广西地区首次经检测确认并测定全基因组序列的牛乳头瘤病毒。本研究为广西地区乃至全国的牛乳头状瘤的病原鉴定、流行规律、遗传变异、疫源追溯及科学防控提供了基础数据。     

Pathological study of non-neoplastic urinary bladder lesions in cattle and buffaloes: a preliminary report

A total of 236 urinary bladders (94 cattle and 142 buffaloes) collected from Bareilly, Uttar Pradesh, India, were studied for spontaneous lesions. These adult animals belonged to Institute’s organized dairy farm and rural areas in the Rohilkhand region of Uttar Pradesh. Grossly, congestion, hemorrhages, and cystoliths in urinary bladders were diagnosed. Histopathologically, the major conditions diagnosed were acute cystitis, 44 (18.64%), including, congestion, hemorrhages, sub-acute cystitis; chronic cystitis, 74 (31.35%), including chronic cystitis un-complicated type, lymphocytic cystitis, plasmolymphocytic cystitis, follicular cystitis, hyperplasia, nodular/acinar hyperplasia, and cystolithiasis; and nothing unusual diagnosed, 118 (50.00%). Similar types of pathological conditions were diagnosed in both species of animals with exception of follicular cystitis and nodular/acinar hyperplasia which was diagnosed respectively only in buffaloes and cystoliths in cows. In addition, a good number of 17/25 (68%) urinary bladder samples tested were found positive for presence of bovine papillomavirus type-2 (BPV-2) by polymerase chain reaction. These included eight cases of acute cystitis, an equal number of cases of chronic cystitis, and one normal bladder. BPV-2 is known as potential source of enzootic bovine hematuria along with other co-factors in enzootic areas. Lesions of zoonotic significance, like tuberculosis, etc., were not diagnosed. None of the observed lesions represented conditions, which, by themselves, would warrant carcass condemnation in buffaloes.     

Lack of Correlation Between Papillomaviral DNA in Surgical Margins and Recurrence of Equine Sarcoids

Bovine papillomavirus (BPV) types 1 (BPV-1) and 2 (BPV-2) are causally associated with the development of equine sarcoid tumors. Recurrence rates after surgical excision of sarcoids are estimated to be 30%–40%. We hypothesized that the presence of BPV DNA in histologically tumor-free surgical margins of sarcoids is associated with risk of recurrence, and increased quantity of BPV DNA is associated with increased risk of recurrence. Formalin-fixed sarcoids classified as “completely excised” histologically were obtained from two institutions. A total of 25 tumors were included, eight of which recurred within 1 year of excision. Qualitative and quantitative polymerase chain reaction (PCR) tests for detection of BPV-1 and BPV-2 were performed on neoplastic tissue and tumor-free surgical margins in formalin-fixed paraffin-embedded biopsy specimens following DNA extraction. Bovine papillomavirus-1 was found in all tumor samples and in histologically “clean” margins of 21 samples, whereas BPV-2 was found in only two tumor samples. Although quantitative PCR was more sensitive than qualitative PCR in detecting BPV DNA in surgical margins, there was no significant difference in the presence of BPV-1 or BPV-2 DNA in margins of tumors that recurred versus those that did not recur for either test. Although this study is limited by sample size, our results suggest that PCR analysis of surgical margins for BPV DNA is not a reliable method to predict equine sarcoid recurrence after resection.     

Bovine papillomavirus E5 oncoprotein upregulates parkin-dependent mitophagy in urothelial cells of cattle with spontaneous papillomavirus infection: A mechanistic study

This study aimed to provide mechanistic insights into mitophagy pathway associated with papillomavirus infection in urothelial cells of cattle. The elimination of mitochondria via autophagy, termed mitophagy, is an evolutionarily conserved mechanism for mitochondrial quality control and homeostasis. PINK1/parkin-mediated mitophagy, a ubiquitin-dependent selective autophagy of dysfunctional mitochondria, has been described here, for the first time, in urothelial cells from 25 bladder cancers in cattle infected by bovine papillomavirus (BPV). The expression of BPV-2 and BPV-13 E5 oncoprotein was detected by RT-PCR. Abnormal mitochondria delimited by expanding phagophores, were peculiar ultrastructural features of neoplastic urothelial cells. High levels of mitochondrial phosphorylated PINK1/parkin were observed in neoplastic urothelial cells infected by BPVs. Phosphoparkin interacted with mitofusin 2 (Mfn2) and ubiquitin (Ub), which confirmed that Mfn2 is a parkin receptor at the mitochondrial level, where parkin interacted also with Ub. Furthermore, parkin established a complex that was comprised of optineurin, p62, LC3, laforin, and embryonic stem cell-expressed Ras (ERAS), that interacted with BPV E5 oncoprotein, and Bag3, which, in turn, regulated the formation of a complex composed of Hpc70/Hsp70, CHIP, an HSC70-interacting E3 ubiquitin ligase. It is conceivable that ERAS is involved in mitophagosome maturation via phosphatidylinositol 3-kinase (PI3K) pathway. Bag3, in association with Hsc70/Hsp70, may contribute to the transport and degradation of CHIP-ubiquitinated cargo as this complex recognises ubiquitinated cargos and transports them to aggresomes to be degraded. Furthermore, Bag3 may be involved in mitophagosome formation as it interacted with synaptopodin 2, which is known to play a role in mitophagosome biogenesis.     

Evaluation of ovine herpesvirus type 2 infections, as detected by competitive inhibition ELISA and polymerase chain reaction assay, in dairy cattle without clinical signs of malignant catarrhal fever

OBJECTIVE: To monitor ovine herpesvirus type 2 (OvHV-2) infection status and the association between OvHV-2 infection and development of clinical signs of malignant catarrhal fever (MCF) in cattle. DESIGN: Longitudinal study. ANIMALS: 30 mature adult cows and 18 cattle submitted for necropsy. PROCEDURE: Blood and milk samples were collected at monthly intervals from 30 adult cows for 20 consecutive months. Nasal and ocular swab specimens were also collected during months 9 through 20. Polymerase chain reaction (PCR) assay for detection of OvHV-2 was performed on blood, milk, nasal swab, and ocular swab specimens. Competitive inhibition ELISA (CI-ELISA) for detection of antibodies against MCF viruses was performed on serum samples obtained prior to study initiation and monthly during the last 12 months. Tissues obtained from herdmates without clinical signs of MCF that were submitted for necropsy were analyzed for OvHV-2 DNA via PCR assay for possible sites of latency. RESULTS: Initially, 8 of 30 cows had positive CI-ELISA results. Seroconversion was detected in 4 cows. Ovine herpesvirus type 2 DNA was intermittently detected in blood, milk, nasal secretions, or ocular secretions from 17 of 30 cows. Twenty-one cows had positive CI-ELISA or PCR assay results. No cattle in the study developed clinical signs of MCF. Results of PCR assays performed on tissue samples from 2 of 18 animals submitted for necropsy were positive for OvHV-2. CONCLUSIONS AND CLINICAL RELEVANCE; OvHV-2 infection can occur in cattle without concurrent development of clinical MCF. Ovine herpesvirus type 2 DNA was detected intermittently, suggesting fluctuating viral DNA loads or reinfection in subclinical cattle. A definitive site of latency was not identified from tissues obtained during necropsy.     

Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2).     

国内部分省市肉兔及蜱虫携带土拉杆菌的流行病学调查

本调查旨在探明土拉杆菌在国内部分省份的流行情况。利用本实验室建立的土拉杆菌通用及亚种PCR检测研究方法，对国内肉兔饲养密度比较大的省份和牛羊存栏的部分省市开展了肉兔和蜱虫中土拉杆菌的检测，并对阳性样本进行了土拉杆菌亚种的鉴定和基因测序验证。研究结果表明，肉兔肝脏组织和牛羊携带蜱虫中土拉杆菌DNA检测阳性，其中218份肉兔样本中12份阳性（阳性率5.5%），490份蜱虫样本中15份阳性（阳性率3.1%）；从地区分布来看，山东、河南、四川肉兔病料检测阳性，云南、山东蜱虫阳性率高；PCR检测显示其亚种为F.h，为土拉杆菌毒力较强的亚种。本次调查显示土拉杆菌在肉兔、蜱虫中存在，山东、云南地区存在土拉杆菌病公共卫生安全风险，应引起有关科研院校、医疗单位和政府部门的高度重视。     

Epidemiological Investigation of Francisella tularensis Transmitted by Rabbits and Ticks in Some Provinces of China

The aim of this investigation was to find out the prevalence of Francesella tularensis in some provinces of China. Using the general and subspecies PCR detection method for Francesella tularensis established in our laboratory, PCR detection of Francesella tularensis in rabbits and ticks was carried out in provinces with high-density rabbits feeding and some provinces and cities with sheep and cattle stock. The results showed that DNA detection of Francesella tularensis from rabbit tissues and ticks carried by cattle and sheep was positive. 12 out of 218 rabbit samples were positive (5.5% positive rate), and 15 tick samples were positive (3.1%) of 490 tick samples. In terms of geographical distribution, most of the positive rabbit samples were from Shandong, Henan and Sichuan province, however, ticks collected from Yunnan and Shandong province showed a higher positive rate. PCR detection showed that Francesella tularensis subspecies in this investigation was F.h, which was a subspecies with strong toxicity. This investigation revealed that Francesella tularensis presented in rabbits and ticks, public health and safety risks of Francesella tularensis existed in Shandong and Yunnan province, which should be paid more attention by the relevant scientific research institutions, medical institutions and government departments.     

Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29-31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.     

Identification of unreported putative new bovine papillomavirus types in Brazilian cattle herds

The amplification by degenerate primers FAP59/FAP64 and sequencing allowed the detection of 15 putative new BPV types in cutaneous warts as well as in healthy skin. Four of these isolates were recently recognized as new BPV types (BPV-7, -8, -9, and -10) after determination of their complete genome sequences. In Brazil, investigations involving the definition of BPV types present in skin warts are still rare. The aim of the current study was to identify the BPV types associated with cutaneous papillomatosis observed in Brazilian cattle herds. Twenty-two cutaneous papilloma specimens were submitted to PCR assay employing the FAP primer pair. All PCR products with approximately 480 bp were submitted to direct sequencing. Cloning was performed for the amplicons which prior analysis revealed as putative new BPV types. From 16 cutaneous lesions, BPV-1, -2, and -6 were identified in two, six, and eight papilloma specimens, respectively. In addition, four putative new BPV types were identified in other six skin warts, and then designated as BPV/BR-UEL2 to -5. The detection of the BPV-1, -2, and -6 types in skin wart specimens supports the existence of these BPV types throughout the Brazilian cattle herd. In addition, the identification of four putative new BPV types is the first report of the presence of different BPV types in the American continent.     

Detection and characterisation of papillomavirus in skin lesions of giraffe and sable antelope in South Africa

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.     

Clotting profile in cattle showing chronic enzootic haematuria (CEH) and bladder neoplasms

Primary haemostasis (bleeding and blood clotting time), activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin III (ATIII), protein C, protein S, fibrinogen and D-dimer were determined in 13 cattle affected by chronic enzootic haematuria (CEH) and bladder neoplasms and 10 healthy cattle (control group). Increases in antithrombin III and protein S activities (P<0.01) and protein C and fibrinogen plasma levels (P<0.05) were observed in sick animals, while activated partial thromboplastin time, prothrombin time, and D-dimer did not show significant differences when compared to healthy animals. The clotting profile observed does not seem responsible for the chronic bleeding typical of CEH. The observed modification of some coagulation markers may derive from multiple interactions among cancer, inflammation and viral infection status typical of this syndrome.