Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

Detection of Bovine Viral Diarrhoea Virus 2 as the Cause of Abortion Outbreaks on Commercial Sheep Flocks

Outbreaks of abortions and lambs born with nervous clinical signs and/or congenital malformations affected different sheep farms in Spain. Initial diagnosis of ‘border disease‐like’ was established, based on clinical signs, serology and/or RNA detection by a pan‐pestivirus RT‐PCR. However, further investigation using immunohistochemical and molecular techniques identified BVDV‐2b as the aetiological agent.     

Evidence for Circulation of Bovine Viral Diarrhoea Virus Type 2c in Ruminants in Southern Italy

Recently, bovine viral diarrhoea virus type 2c (BVDV‐2c) was responsible for a severe outbreak in cattle in northern Europe. Here, we present the results of an epidemiological survey for pestiviruses in ruminants in southern Italy. Pooled serum samples were obtained from 997 bovine, 800 ovine, 431 caprine and eight bubaline farms, and pestiviral RNA was detected by molecular methods in 44 farms consisting of 16 cattle and one buffalo herds and of 21 sheep and six goat flocks. Twenty‐nine and 15 farms were infected by BVDV‐1 and BVDV‐2 strains, respectively. BVDV‐1 strains were recovered mainly from cattle and were heterogeneous, belonging to the subtypes 1b, 1u, 1e, 1g and 1h. In contrast, all BVDV‐2 viruses but two were detected in sheep or goats and were characterized as BVDV‐2c by sequence analysis of 5′UTR. These strains displayed high genetic identity to BVDV‐2c circulating in cattle in northern Europe and were more distantly related to a BVDV‐2c isolate recovered from a cattle herd in southern Italy more than 10 years before. The circulation of a BVDV‐2c in small ruminants suggests the need for a continuous surveillance for the emergence of pestivirus‐induced clinical signs in southern Italian farms.     

Detection of Bovine Leukaemia Virus Antibodies and Proviral DNA in Colostrum Replacers

Great Britain has been bovine leukaemia virus (BLV) disease free since 1999. We recently reported three separate incidents of BLV seropositivity on farms with home‐reared cattle due to the use of colostrum replacer rather than infection with BLV (Emerg. Infect. Dis., 19 , 2013, 1027). These cases were all linked via the use of the same brand of colostrum replacer. Here, we investigate further by examining multiple brands of colostrum replacer for proviral DNA and BLV antibodies. BLV antibodies were detected in 7 of the colostrum replacers tested, with PCR concurring in two cases. Thus, the use of these BLV antibody‐positive colostrum replacers may also lead to false‐positive serological diagnostics.     

Global knowledge gaps in the prevention and control of bovine viral diarrhoea (BVD) virus

The significant economic impacts of bovine viral diarrhoea (BVD) virus have prompted many countries worldwide to embark on regional or national BVD eradication programmes. Unlike other infectious diseases, BVD control is highly feasible in cattle production systems because the pathogenesis is well understood and there are effective tools to break the disease transmission cycle at the farm and industry levels. Coordinated control approaches typically involve directly testing populations for virus or serological screening of cattle herds to identify those with recent exposure to BVD, testing individual animals within affected herds to identify and eliminate persistently infected (PI) cattle, and implementing biosecurity measures such as double‐fencing shared farm boundaries, vaccinating susceptible breeding cattle, improving visitor and equipment hygiene practices, and maintaining closed herds to prevent further disease transmission. As highlighted by the recent DISCONTOOLS review conducted by a panel of internationally recognized experts, knowledge gaps in the control measures are primarily centred around the practical application of existing tools rather than the need for creation of new tools. Further research is required to: (a) determine the most cost effective and socially acceptable means of applying BVD control measures in different cattle production systems; (b) identify the most effective ways to build widespread support for implementing BVD control measures from the bottom‐up through farmer engagement and from the top‐down through national policy; and (c) to develop strategies to prevent the reintroduction of BVD into disease‐free regions by managing the risks associated with the movements of animals, personnel and equipment. Stronger collaboration between epidemiologists, economists and social scientists will be essential for progressing efforts to eradicate BVD from more countries worldwide.     

Evaluation of bovine viral diarrhoea virus control strategies in dairy herds in Hokkaido,Japan, using stochastic modelling

Bovine viral diarrhoea virus (BVDV ) infection in cattle can result in growth retardation, reduced milk production, reproductive disorders and death. Persistently infected animals are the primary source of infection. In Hokkaido, Japan, all cattle entering shared pastures in summer are vaccinated before movement for disease control. Additionally, these cattle may be tested for BVDV and culled if positive. However, the effectiveness of this control strategy aiming to reduce the number of BVDV ‐infected animals has not been assessed. The aim of this study was to evaluate the effectiveness of various test‐and‐cull and/or vaccination strategies on BVDV control in dairy farms in two districts of Hokkaido, Nemuro and Hiyama. A stochastic model was developed to compare the different control strategies over a 10‐year period. The model was individual‐based and simulated disease dynamics both within and between herds. Parameters included in the model were obtained from the literature, the Hokkaido government and the Japanese Ministry of Agriculture, Forestry and Fisheries. Nine different scenarios were compared as follows: no control, test‐and‐cull strategies based on antigen testing of either calves or only cattle entering common pastures, vaccination of all adult cattle or only cattle entering shared pastures and combinations thereof. The results indicate that current strategies for BVDV control in Hokkaido slightly reduced the number of BVDV ‐infected animals; however, alternative strategies such as testing all calves and culling any positives or vaccinating all susceptible adult animals dramatically reduced those. To our knowledge, this is the first report regarding the comparison of the effectiveness between the current strategies in Hokkaido and the alternative strategies for BVDV control measures.     

Genetic diversity and zoonotic potential of rotavirus A strains in the southern Andean highlands,Peru

Interspecies transmission is an important mechanism of evolution and contributes to rotavirus A (RVA) diversity. In order to evaluate the detection frequency, genetic diversity, epidemiological characteristics and zoonotic potential of RVA strains in faecal specimens from humans and animals cohabiting in the same environment in the department of Cusco, Peru, by molecular analysis, 265 faecal specimens were obtained from alpacas, llamas, sheep and shepherd children, and tested for RVA by RT‐PCR. Genotyping was performed by multiplex PCR and sequence analysis. Rotavirus A was detected in 20.3% of alpaca, 47.5% of llama, 100% of sheep and 33.3% of human samples. The most common genetic constellations were G3‐P[40]‐I8‐E3‐H6 in alpacas, G1/G3‐P[8]‐I1‐E1‐H1 in llamas, G1/G3/G35‐P[1]/P[8]‐I1‐E1‐H1 in sheep and G3‐P[40]‐I1/I8‐E3‐H1 in humans. The newly described genotypes P[40] and P[50] were identified in all host species, including humans. Genotyping showed that the majority of samples presented coinfection with two or more RVA strains. These data demonstrate the great genetic diversity of RVA in animals and humans in Cusco, Peru. Phylogenetic analysis suggested that the strains represent zoonotic transmission among the species studied. Due to the characteristics of the human and animal populations in this study (cohabitation of different host species in conditions of poor sanitation and hygiene), the occurrence of zoonoses is a real possibility.     

The Detection and Genetic Characterization Based on the S1 Gene Region of BCoVs from Respiratory and Enteric Infections in Turkey

We investigated bovine coronavirus (BCoV) as an etiological agent in cattle with clinical respiratory and digestive signs using 147 feces and 199 nasal swab samples. A total of 18 test samples (16 feces and 2 nasal swap samples) were detected positive by ELISA and/or RT‐PCR targeting the BCoV N gene. The partial S1 gene regions of BCoVs (An‐4 and An‐11) detected in feces samples from two herd‐mate dairy calves were compared. Virological and serological results indicated that BCoVs are widespread in Turkey and are likely etiological agents in diarrhea cases in calves.     

Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows

The etiology and pathologic findings of bovine respiratory disease (BRD) in adult dairy cows (n = 35) from a commercial dairy herd in Southern Brazil were investigated. Pulmonary samples were examined for histopathologic patterns and specific features within these patterns, while immunohistochemical (IHC) assays were designed to detect the intralesional antigens of viral infectious disease agents and Mycoplasma bovis. Pneumonia was diagnosed in 91.4% (32/35) of these cases; neither pneumonia nor any of the infectious disease pathogens evaluated occurred in three cows. The presence of multiple respiratory pathogens in 75% (24/32) of these cases indicated the complex origin of pneumonia in cattle. Interstitial pneumonia, necrosuppurative bronchopneumonia and suppurative bronchopneumonia were the principal patterns of pulmonary disease identified by histopathology. The most frequent pathogens identified by IHC were bovine viral diarrhea virus (BVDV; n = 18), M. bovis (n = 16) and bovine alphaherpesvirus type 1 (BoHV‐1; n = 14), followed by bovine respiratory syncytial virus (BRSV; n = 11) and bovine parainfluenza virus type 3 (BPIV‐3; n = 5). Obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with M. bovis. Necrohemorrhagic bronchitis with bronchial angiogenesis was associated with BoHV‐1. Necrotizing bronchitis and bronchiolitis were associated with BVDV, BoHV‐1 and BRSV. Ballooning degeneration of the bronchial and bronchiolar epithelia was associated with BRSV and BoHV‐1. This is the first report from Brazil that correlated the histopathologic findings of BRD with the associated infectious disease agents by immunohistochemistry. M. bovis was frequently detected in the tissues of cows with fatal pulmonary disease during this study and may be a possible primary disease pathogen associated with the development of BRD in dairy cows. Additionally, the histopathologic features identified within patterns of pulmonary disease during this investigation may be an efficient diagnostic tool to associate histopathologic findings with specific agents of BRD in dairy cows.     

Detection and Quantification of Bovine Papilloma Virus Type 2 (BPV‐2) by Real‐time PCR in Urine and Urinary Bladder Lesions in Enzootic Bovine Haematuria (EBH)‐Affected Cows

This study was conducted with the objectives of detecting bovine papillomavirus type 2 (BPV‐2) in urine samples and urinary bladder lesions in bovines using polymerase chain reaction (PCR) and real‐time PCR‐based molecular diagnostic tests, and quantifying BPV‐2 in urinary bladder lesions especially in enzootic bovine haematuria (EBH)‐affected animals. BPV‐2 viral DNA was detected in urine samples (50%) and urinary bladder tissue (68.6%). Cloning and sequencing results showed a close homology with other Indian BPV‐2 sequences. Quantitative real‐time PCR (SYBR Green assay) showed that the BPV‐2 load was low and similar irrespective of inflammatory or neoplastic lesions in the bladder. It was concluded that BPV‐2 DNA is frequently present in urine and urinary bladder lesions in cows in an EBH endemic region and virus load was low in urinary bladder lesions.     

Risk Factors Associated with Bovine Tuberculosis and Molecular Characterization of Mycobacterium bovis Strains in Urban Settings in Niger

A retrospective and a longitudinal survey were carried out at the abattoir of Niamey. Results showed a highly significant difference in suspected tuberculosis (TB) gross lesions among different animal species (P < 0.0001). The proportion of carcasses with TB‐like lesions was 0.19% among cattle, 0.11% among camels, 0.001% among sheep and 0.0006% among goats. In cattle, cows are significantly more affected than the other categories (P < 0.001). Also in cattle, TB‐like lesions are mostly localized in the lungs (92.77%) followed by the lymph nodes (50.87%) and the liver (32.40%). The prevalence of gross lesions compatible with bovine TB (BTB) is strongly influenced by the season (P < 0.0001), is closely correlated with the origin of the animals (P < 0.001) and has a negative impact on the weight of affected animals (P < 0.0001). Sixty‐two samples of suspected TB gross lesions were subject to microbiological analysis and molecular typing of strains. Mycobacterium bovis was identified in 18 animals showing five different spoligotypes, belonging to type ‘African 1’ previously identified in Central and West Africa. In addition, a profile (SB1982) not previously reported distinguished by the absence of spacers 3, 4, 9, 16, 22, 30 and 39–43 has been characterized in this study. To assess risk factors for BTB transmission, a questionnaire on animal husbandry practices, food habits, and clinical signs of TB in animals and humans was submitted to the heads of 1131 randomly selected households. The main risk factors identified are consumption of unpasteurized milk (91%) and lack of hygiene within households (32–74%). Clinical signs that could be attributed to TB were also reported both in humans and in animals of the households.     

Modelling the variation in skin‐test tuberculin reactions,post‐mortem lesion counts and case pathology in tuberculosis‐exposed cattle: Effects of animal characteristics,histories and co‐infection

Correctly identifying bovine tuberculosis (bTB ) in cattle remains a significant problem in endemic countries. We hypothesized that animal characteristics (sex, age, breed), histories (herd effects, testing, movement) and potential exposure to other pathogens (co‐infection; BVDV , liver fluke and Mycobacterium avium reactors) could significantly impact the immune responsiveness detected at skin testing and the variation in post‐mortem pathology (confirmation) in bTB ‐exposed cattle. Three model suites were developed using a retrospective observational data set of 5,698 cattle culled during herd breakdowns in Northern Ireland. A linear regression model suggested that antemortem tuberculin reaction size (difference in purified protein derivative avium [PPD a ] and bovine [PPD b ] reactions) was significantly positively associated with post‐mortem maximum lesion size and the number of lesions found. This indicated that reaction size could be considered a predictor of both the extent (number of lesions/tissues) and the pathological progression of infection (maximum lesion size). Tuberculin reaction size was related to age class, and younger animals (<2.85 years) displayed larger reaction sizes than older animals. Tuberculin reaction size was also associated with breed and animal movement and increased with the time between the penultimate and disclosing tests. A negative binomial random‐effects model indicated a significant increase in lesion counts for animals with M. avium reactions (PPD b− PPD a <  0) relative to non‐reactors (PPD b− PPD a =  0). Lesion counts were significantly increased in animals with previous positive severe interpretation skin‐test results. Animals with increased movement histories, young animals and non‐dairy breed animals also had significantly increased lesion counts. Animals from herds that had BVDV ‐positive cattle had significantly lower lesion counts than animals from herds without evidence of BVDV infection. Restricting the data set to only animals with a bTB visible lesion at slaughter (n  = 2471), an ordinal regression model indicated that liver fluke‐ infected animals disclosed smaller lesions, relative to liver fluke‐negative animals, and larger lesions were disclosed in animals with increased movement histories.     

The PB2 and M genes are critical for the superiority of genotype S H9N2 virus to genotype H in optimizing viral fitness of H5Nx and H7N9 avian influenza viruses in mice

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1‐like M and PB2 genes for the earlier F/98‐like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1‐like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98‐like counterpart, while the G1‐like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1‐like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.     

Detection and Genetic Characterization of Lineage IV Peste Des Petits Ruminant Virus in Kazakhstan

Peste des petits ruminant (PPR) is endemic in many Asian countries with expansion of the range in recent years including across China during 2013–2014 (OIE, 2014). Till the end of 2014, no cases of PPR virus (PPRV) were officially reported to the Office Internationale des Epizooties (OIE) from Kazakhstan. This study describes for the first time clinicopathological, epidemiological and genetic characterization of PPRV in 3 farm level outbreaks reported for the first time in Zhambyl region (oblast), southern Kazakhstan. Phylogenetic analysis based on partial N gene sequence data confirms the lineage IV PPRV circulation, similar to the virus that recently circulated in China. The isolated viruses are 99.5–99.7% identical to the PPRV isolated in 2014 from Heilongjiang Province in China and therefore providing evidence of transboundary spread of PPRV. There is a risk of further maintenance of virus in young stock despite vaccination of adult sheep and goats, along livestock trade and pastoral routes, threatening both small livestock and endangered susceptible wildlife populations throughout Kazakhstan.     

Detection and Quantification of Soluble Intercellular Adhesion Molecule-1 (slCAM-1) in the Serum and Urine of Patients with Bladder Cancer

Background : A possible role for intercellular adhesion molecules in tumor progression and metastasis has been strongly suggested. To investigate the effect of soluble intercellular adhesion molecule-1 (slCAM-1) on bladder cancer, slCAM-1 serum and urinary concentrations were measured in patients with superficial or invasive bladder cancer and in patients with prostatic hypertrophy.
Methods : Serum and urine samples were obtained from 26 patients with transitional cell carcinoma of the bladder (mean age, 66.8 years) and 14 patients with benign prostatic hypertrophy (BPH; mean age, 70.5 years). Fifteen healthy volunteers served as control patients. Samples were collected before surgery and 5 days after surgery. The serum and urinary slCAM-1 levels were measured by an ELISA.
Results : The preoperative serum concentration of slCAM-1 was significantly higher in patients with invasive bladder cancer (351.8 ±158.0 ng/mL)than in the healthy controls (233.1 ±96.1 ng/mL; P<0.05) or BPH patients (224.7 ± 80.5 ng/mL; P< 0.05). In addition, serum slCAM-1 levels were significantly higher in patients with tumors greater than 3 cm in size (412.7 ± 147.6 ng/mL) than in patients with smaller tumors (246.6 ± 101.2 ng/mL; P<0.05). Urinary slCAM-1 levels in patients with invasive bladder cancer were also significantly higher than in the patients with superficial cancer prior to surgery.
Conclusion : Our results suggested that slCAM-1 may play an important role in the progression of bladder cancer, and that elevated serum slCAM-1 levels may be related to tumor size.     

改良法构建Ⅱ型重组腺相关病毒介导人TIMP1基因及鉴定

目的改良法构建携带金属蛋白酶抑制剂1（TIMP1）基因的Ⅱ型重组腺相关病毒（rAAV2）。方法采用PCR法从pDNR-LIB质粒中扩增人全长TIMP1基因，利用基因重组的方法将TIMP1全长cDNA插入通用型AAV2载体质粒pSNAV的多克隆位点，构建成pSNAV-TIMP1；用脂质体转染的方法将重组质粒转入BHK-21细胞中，G418细胞筛选得到转入重组质粒并能表达目的基因的细胞系BHK21／rAAV2-TIMP1；用具有rAAV2包装功能的重组Ⅰ型单纯疱疹病毒（rHSV1-rc／△UL2）感染BHK-21／rAAV2-TIMP1，纯化后得到rAAV2-TIMP1。结果用改良法成功构建rAAV2-TIMP1，病毒滴度达到1&#215;10^12v.g．／ml。结论成功构建rAAV2-TIMP1，为其进一步应用于基因治疗的研究提供实验基础。     

Phylogenetic Characterization Genome Segment 2 of Bluetongue Virus Strains Belonging to Serotypes 5, 7 and 24 Isolated for the First Time in China During 2012 to 2014

Bluetongue is endemic in China, and Bluetongue virus (BTV) strains belonging to eight different serotypes (BTV‐1, BTV‐2, BTV‐3, BTV‐4, BTV‐9, BTV‐12, BTV‐15 and BTV‐16) had been isolated between 1996 and 1997. However, there has been a long pause in investigating the epidemiology of BTV infection since then. During 2012–2014, eight BTV strains belonging to serotypes 5, 7 and 24 were isolated for the first time in Yunnan and Guangdong provinces from the blood of sentinel animals. Phylogenetic analyses of genome segment 2 of these Chinese BTV strains grouped them into nucleotypes E, F and A, respectively, along with the reference strains of the same serotype. For each serotype, Chinese strains cluster together closely to form a China's sublineage. In addition, these strains were most closely related to strains from Africa, indicating that they may share a recent common ancestry with African strains. To our knowledge, this is the first time that BTV‐5, BTV‐7 and BTV‐24 strains have been isolated in South‐East Asia. These data will be beneficial for understanding the BTV epidemiology and improving diagnostic assays and control measures against bluetongue in China and its neighbouring countries in the Asia–Pacific region.