Co‐circulation of multiple genotypes of African swine fever viruses among domestic pigs in Zambia (2013–2015)

During 2013–2015, several and severe outbreaks of African swine fever (ASF ) affected domestic pigs in six provinces of Zambia. Genetic characterization of ASF viruses (ASFV s) using standardized genotyping procedures revealed that genotypes I, II and XIV were associated with these outbreaks. Molecular and epidemiological data suggest that genotype II ASFV (Georgia 2007/1‐like) detected in Northern Province of Zambia may have been introduced from neighbouring Tanzania. Also, a genotype II virus detected in Eastern Province of Zambia showed a p54 phylogenetic relationship that was inconsistent with that of p72, underscoring the genetic variability of ASFV s. While it appears genotype II viruses detected in Zambia arose from a domestic pig cycle, genotypes I and XIV possibly emerged from a sylvatic cycle. Overall, this study demonstrates the co‐circulation of multiple genotypes of ASFV s, involvement of both the sylvatic and domestic pig cycle in ASF outbreaks in Zambia and possible trans‐boundary spread of the disease in south‐eastern Africa. Indeed, while there is need for regional or international concerted efforts in the control of ASF , understanding pig marketing practices, pig population dynamics, pig housing and rearing systems and community engagement will be important considerations when designing future prevention and control strategies of this disease in Zambia.     

Detection of African Swine Fever Virus DNA in Blood Samples Stored on FTA Cards from Asymptomatic Pigs in Mbeya Region,Tanzania

The aim of the study was to assess whether blood samples collected onto FTA^® cards could be used in combination with real‐time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource‐poor settings under the assumption that asymptomatically (sub‐clinically) infected pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA^® cards and analysed by real‐time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27–29), and three pigs had a low level of viral DNA (Ct 36–45). Four pigs were positive in one of the duplicate samples only, but clear products of the expected size were obtained when the reactions were analysed by gel electrophoresis. For comparison, blood samples from pigs experimentally infected with either a pathogenic (OURT T88/1) or a non‐pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA^® cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22‐33), whereas infection with non‐pathogenic OURT T88/3 isolate resulted in only low levels of viral DNA (Ct 39) in samples collected at 10–14 days after inoculation.     

Experimental Infection of Domestic Pigs with African Swine Fever Virus Lithuania 2014 Genotype II Field Isolate

An experimental infection was conducted to evaluate horizontal transmission, clinical, virological and humoral response induced in domestic pigs infected with African swine fever (ASF) genotype II virus circulating in 2014 into the European Union (EU). Ten naive pigs were placed in contact with eight pigs experimentally inoculated with the Lithuanian LT14/1490 ASF virus (ASFV) responsible for the first ASF case detected in wild boar in Lithuania in January 2014. Clinical examination and rectal temperature were recorded each day. Blood sampling from every animal was carried out twice weekly. Blood samples were examined for presence of ASF virus‐specific antibodies and for determining the ASFV viral load. From the obtained results, it was concluded that the Lithuanian ASFV induced an acute disease which resulted in 94, 5% mortality. The disease was easily detected by real‐time PCR prior to the onset of clinical signs and 33% of the animals seroconverted. All findings were in accordance with observations previously made in domestic pigs and wild boar when infected with ASF genotype II viruses characterized by a high virulence. One in‐contact pig remained asymptomatic and survived the infection. The role of such animals in virus transmission would need further investigation.     

The evolution of African swine fever virus in Sardinia (1978–2014) as revealed by whole‐genome sequencing and comparative analysis

African swine fever (ASF) is a highly contagious and lethal viral disease of pigs and wild boars, which is enzootic in many African countries and on the Italian island of Sardinia, where it has been present since 1978. Previous genetic analyses of Sardinian ASF virus (ASFV) isolates have revealed that they all belong to p72 genotype I, with only minor sequence variations. However, these studies examined only a few selected genes. To distinguish between these closely related isolates and better investigate ASFV evolution in Sardinia, we sequenced the complete genomes of 12 Sardinian ASFV isolates collected between 1978 and 2012, and compared them with 47/Ss/2008 and 26544/OG10. Most of the observed changes occurred in a time‐dependent manner; however, their biological significance remains unclear. As a whole, our results demonstrate the remarkable genetic stability of these strains, supporting a single‐source introduction of the virus.     

Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans)

Within Eastern Europe, African swine fever virus (ASFV ) has unexpectedly spread to farms with high biosecurity. In an attempt to explain this process, pigs were allowed to ingest flies that had fed on ASFV ‐spiked blood, which had a realistic titre for an infected pig. Some of the pigs became infected with the virus. Thus, ingestion of blood‐sucking flies, having fed on ASFV ‐infected wild boar before entering stables, represents a potential route for disease transmission.     

Symptomatic and asymptomatic cases of African swine fever in Tanzania

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub‐clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease‐endemic regions.     

Genetic characterization and diversity of porcine circovirus type 2 in non‐vaccinated South African swine herds

The porcine circovirus type 2 (PCV2) is a swine infectious viral pathogen of great significance in global swine herds. It was recently detected at another Province of South Africa sequel to the first detection of North American‐like strain (PCV2a) at Gauteng about two decades ago, but there is a dearth of information about the genomic features and diversity of the viral strains in circulation within the country and the entire sub‐Saharan Africa region. To date, only one complete genome of the virus from South Africa is available on global data base. This current effort is therefore geared towards the full‐genome characterization of the circulating PCV2 strains in the pigs of Eastern Cape Province. With the use of conventional polymerase chain reaction method, fifteen complete PCV2 genomes were successfully amplified, sequenced and assembled from field samples obtained from non‐vaccinated pigs in the region. Neighbor Joining and Maximum Likelihood phylogenetic analyses of the ORF2 gene and full genomes unanimously showed that most of the assembled genomes (11) belong to genotype PCV2b. Furthermore, three of the characterized sequences formed clade with other reference mutant PCV2b and PCV2b subtype 1C (i.e. PCV2d) strains from the USA, China and South Korea. The last sequence, however, clustered with other reference strains belonging to PCV2 intermediate clade 2 (PCV2‐IM2), recently identified in a global PCV2 strains phylogenetic analysis. This study reports the first complete genome sequences of PCV2b, PCV2d and PCV2‐IM2 in pigs from South Africa, and it gives a possible insight into the genetic characteristics and variability of the viral strains presently in circulation within the country. It further emphasizes the need for more stringent measures in curtailing the introduction and spread of transboundary swine pathogens in the country and entire Southern African region.     

Preliminary study of rat liver transplantation from non‐heart‐beating donors

Objective : The aim of this study was to investigate the tolerance of warm ischaemia of liver grafts from non‐heart‐beating donors (NHBD). Livers harvested from NHBD would greatly expand the donor pool for transplantation. However, sensitivity of the donor liver to warm ischaemia is a major obstacle to the successful use of livers from NHBD and the limit of non‐heart‐beating time in the donor remains unclear. Materials and Methods : Rat orthotopic liver transplantation was performed in 5 groups (groups 1–5) with non‐heart‐beating time in the donor ranging from zero to 15, 30, 45 and 60 min. The cardiac‐arrest time was counted from clamping the base of the donor’s heart to the beginning of cold flushing the liver. Graft pathological changes, hepatic function and the recipients’ survival rates in each group were compared. Results : With a stepwise increase of the non‐heart‐beating time from zero to 15, 30, 45 and 60 min in donors, the recipient 1‐week survival rates from groups 1–5 were 100%, 75%, 62.5%, 25% and 0%, respectively. The stepwise increase of non‐heart‐beating time significantly reduced recipient survival rates after 30 min. The recipients in groups 1–4 could survive more than 60 days. Conclusions : The liver is less sensitive to warm ischaemia than was formerly believed. Rat liver can be used for transplantation after cardiac arrest for up to 45 min with a chance of survival.      

No evidence for long‐term carrier status of pigs after African swine fever virus infection

This study targeted the assessment of a potential African swine fever virus (ASFV) carrier state of 30 pigs in total which were allowed to recover from infection with ASFV “Netherlands'86” prior exposure to six healthy sentinel pigs for more than 2 months. Throughout the whole trial, blood and swab samples were subjected to routine virological and serological investigations. At the end of the trial, necropsy of all animals was performed and viral persistence and distribution were assessed. Upon infection, a wide range of clinical and pathomorphological signs were observed. After an initial acute phase in all experimentally inoculated pigs, 66.6% recovered completely and seroconverted. However, viral genome was detectable in blood samples for up to 91 days. Lethal outcomes were observed in 33.3% of the pigs with both acute and prolonged courses. No ASFV transmission occurred over the whole in‐contact phase from survivors to sentinels. Similarly, infectious ASFV was not detected in any of the tissue samples from ASFV convalescent and in‐contact pigs. These findings indicate that the suggested role of ASFV survivors is overestimated and has to be reconsidered thoroughly for future risk assessments.     

Microcomputed tomography imaging in a rat model of delayed union/non‐union fracture

We aimed to develop a clinically relevant delayed union/non‐union fracture model to evaluate a cell therapy intervention repair strategy. Histology, three‐dimensional (3D) microcomputed tomography (micro‐CT) imaging and mechanical testing were utilized to develop an analytical protocol for qualitative and quantitative assessment of fracture repair. An open femoral diaphyseal osteotomy, combined with periosteal diathermy and endosteal excision, was held in compression by a four pin unilateral external fixator. Three delayed union/non‐union fracture groups established at 6 weeks—(a) a control group, (b) a cell therapy group, and (c) a group receiving phosphate‐buffered saline (PBS) injection alone—were examined subsequently at 8 and 14 weeks. The histological response was combined fibrous and cartilaginous non‐unions in groups A and B with fibrous non‐unions in group C. Mineralized callus volume/total volume percentage showed no statistically significant differences between groups. Endosteal calcified tissue volume/endosteal tissue volume, at the center of the fracture site, displayed statistically significant differences between 8 and 14 weeks for cell and PBS intervention groups but not for the control group. The percentage load to failure was significantly lower in the control and cell treatment groups than in the PBS alone group. High‐resolution micro‐CT imaging provides a powerful tool to augment characterization of repair in delayed union/non‐union fractures together with outcomes such as histology and mechanical strength measurement. Accurate, nondestructive, 3D identification of mineralization progression in repairing fractures is enabled in the presence or absence of intervention strategies. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:729–736, 2008     

Are polymorphisms of molecules involved in bone healing correlated to aseptic femoral and tibial shaft non‐unions?

Fracture healing is a well‐organized process between several molecules and mediators. As known from other diseases, genetic polymorphisms may exhibit different expression patterns in these mediators. Concerning fracture healing, this may lead to an extended healing process or non‐union. We investigated the incidence of polymorphisms in patients with aseptic non‐unions after femoral and tibial shaft fractures as compared to patients with uneventful healing. Exclusion criteria were smoking, diabetes, bilateral fractures, systemic corticoid therapy, and septic non‐unions. Analysis of allele frequencies and genotype distribution of various mediators were carried out following PCR. Clinical parameters such as injury severity and in‐hospital were analyzed. Fifty patients following non‐union (group NU) were enrolled, the control group consisted of 44 patients (group H). A significant association of a PDGF haplotype and non‐unions following fracture could be observed. There was a significantly increased in‐hospital time and amount of surgical procedures in group NU. Polymorphisms within the PDGF gene seem to be a genetic risk factor for the development of non‐unions of the lower extremity following fracture. The early identification of high risk patients could result in an adapted therapeutical strategy and might contribute to a significant decrease of posttraumatic non‐unions. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1724–1731, 2011     

Polymorphisms in BMP4 and FGFR1 genes are associated with fracture non‐union

Fracture healing is a complex process influenced by a multitude of factors and expression of several thousand genes. Polymorphisms in these genes can lead to an extended healing process and explain why certain patients are more susceptible to develop non‐union. A total of 16 SNPs within five genes involved in bone repair pathogenesis (FAM5C, BMP4, FGF3, FGF10, and FGFR1) were investigated in 167 patients with long bone fractures, 101 with uneventful healing, and 66 presenting aseptic non‐unions. Exclusion criteria were patients presenting pathological fractures, osteoporosis, hypertrophic and infected non‐unions, pregnancy, and children. All genetic markers were genotyped using TaqMan real‐time PCR. Chi‐square test was used to compare genotypes, allele frequencies, and haplotype differences between groups. Binary logistic regression analyzed the significance of many covariates and the incidence of non‐union. Statistical analysis revealed open fracture to be a risk factor for non‐union development (p < 0.001, OR 3.6 [1.70–7.67]). A significant association of haplotype GTAA in BMP4 (p = 0.01) and FGFR1 rs13317 (p = 0.005) with NU could be observed. Also, uneventful healing showed association with FAM5C rs1342913 (p = 0.04). Our work supported the role of BMP4 and FGFR1 in NU fracture independently of the presence of previously described risk factors. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1971–1979, 2013     

A suboptimal learning control scheme for non‐linear systems with time‐varying parametric uncertainties

In this paper, learning control is integrated with non‐linear optimal control to enhance control performance of a class of non‐linear systems with time‐varying parametric uncertainties. A suboptimal control strategy based on a control Lyapunov function (CLF) and Sontag's formula provides suboptimal performance as well as stability along the time horizon for the nominal part of the non‐linear dynamic system. The proposed learning mechanism learns the unknown time‐varying parametric uncertainties so as to eliminate uncertain effects. System information both in time horizon and learning repetition horizon are incorporated in a composite energy function (CEF). The proposed control scheme achieves asymptotic convergence along the learning repetition horizon and boundedness and pointwise convergence of the tracking error (perfect tracking performance) along the time horizon. Copyright © 2001 John Wiley & Sons, Ltd.     

Expression of Inflammatory Cytokines (TNF‐αα, IL‐1, IL‐6 and IL‐8) in Colon of Pigs Naturally Infected with Salmonella typhimurium and S. choleraesuis

The expression of mRNA encoding tumour necrosis factor‐α (TNF‐α), interleukin‐1 (IL‐1), IL‐6 and IL‐8 was studied, by in situ hybridization with a non‐radioactive digoxigenin‐labelled probe, in formalin‐fixed, paraffin wax‐embedded colonic tissue from pigs naturally infected with Salmonella typhimurium and S. choleraesuis. By in situ hybridization, a distinct positive signal for TNF‐α, IL‐1, IL‐6 and IL‐8 was detected in colon from all 12 infected pigs. Hybridization signals for all four inflammatory cytokines were detected primarily inflammatory cells infiltrating the lamina propria and submucosa. In comparison, expression of all four inflammatory cytokines was minimal in non‐lesional colon of infected pigs and in normal colon from control pigs. The results suggest that these cytokines play an important role in the pathophysiological processes in porcine salmonellosis.     

Immunization of African Indigenous Pigs with Attenuated Genotype I African Swine Fever Virus OURT88/3 Induces Protection Against Challenge with Virulent Strains of Genotype I

The attenuated African swine fever virus genotype I strain OURT88/3 has previously been shown to induce protection of European breeds of domestic pigs against challenge with virulent isolates. To determine whether protective immune responses could also be induced in indigenous breeds of pigs from the Kinshassa region in Democratic Republic of Congo, we immunized a group of eight pigs with OURT88/3 strain and challenged the pigs 3 weeks later with virulent genotype I strain OURT88/1. Four of the pigs were protected against challenge. Three of the eight pigs died from African swine fever virus and a fourth from an unknown cause. The remaining four pigs all survived challenge with a recent virulent genotype I strain from the Democratic Republic of Congo, DRC 085/10. Control groups of non‐immune pigs challenged with OURT88/1 or DRC 085/10 developed signs of acute ASFV as expected and had high levels of virus genome in blood.     

Oral 5‐aminolevulinic acid‐mediated photodynamic diagnosis using fluorescence cystoscopy for non‐muscle‐invasive bladder cancer: A multicenter phase III study

Objective

To confirm the reproducibility of the effectiveness and safety in photodynamic diagnosis of non‐muscle‐invasive bladder cancer using 5‐aminolevulinic acid in a prospective multicenter non‐randomized phase III trial.

Methods

A total of 61 patients with primary or recurrent non‐muscle‐invasive bladder cancer were prospectively enrolled from five hospitals between May 2015 and March 2016. 5‐Aminolevulinic acid (20 mg/kg) was orally administered 3 h before transurethral resection of bladder tumors using white light or fluorescent light. Of 60 evaluable patients, 511 specimens were obtained from tumor‐suspicious lesions and normal‐looking mucosa. The primary end‐point was sensitivity. The secondary end‐points were specificity, positive and negative predictive values, and safety.

Results

The sensitivity of the fluorescent light source (79.6%) was significantly higher (P < 0.001) than that of the white light source (54.1%). In total, 25.4% (46/181) of tumor specimens were diagnosed as positive with only the fluorescent light source. In nine (15%) of 60 patients, the risk classification and recommended treatment after transurethral resection of bladder tumors were changed depending on the additional types of tumor diagnosed by the fluorescent light source. The specificity of the fluorescent light versus white light source was 80.6% versus 95.5%. No grade 4–5 adverse event was noted. Hypotension and urticaria were severe adverse events whose relationship to oral 5‐aminolevulinic acid could not be excluded.

Conclusions

These findings confirm the diagnostic efficacy and safety of photodynamic diagnosis with 20 mg/kg of oral 5‐aminolevulinic acid, and show that transurethral resection of bladder tumors with a fluorescent light source using oral 5‐aminolevulinic acid is well tolerated.     

Surveillance and control of African Swine Fever in free‐ranging pigs in Sardinia

African swine fever (ASF) is a notifiable infectious disease, caused by the ASF virus (ASFV), which is a DNA virus belonging to the family Asfarviridae, genus Asfivirus. This disease has gained importance in the last decade after its spread in several countries in Eastern and Central Europe, and more recently, in China. Despite the efforts made to eradicate it, ASF is still present on the Mediterranean island of Sardinia (Italy) and has been since 1978. ASF risk factors on the island have been analysed in previous studies; the role of free‐ranging pigs in virus persistence has been suggested, but has not been fully elucidated. The most recent eradication plan provides more stringent measures to combat free‐ranging pigs and any kind of illegality in the pig sector. From December 2017 to June 2018, a total of 29 depopulation actions were performed in 13 municipalities in central Sardinia, during which 2,281 free‐ranging pigs were culled and more than 50% of them were tested for ASFV and antibody presence (1,218 and 1,416, respectively). A total of 651 pigs were seropositive, with a mean seroprevalence of 53.4% (CI 95% = 50.6–56.3), and 38 were ASFV positive (virus prevalence = 2.6%; CI 95% = 2.1–3.0). To the best of our knowledge, the present study is the first to provide a complete evaluation of this millennial system of pig farming and ASFV prevalence in free‐ranging pigs. Furthermore, it has emphasised the necessity of combining the maintenance of an epidemiological surveillance program with continuous education of farmers and other people involved in pig husbandry, based on cultural and economic aspects.     

First Detection of Antibodies Against African Swine Fever Virus in Faeces Samples

African swine fever (ASF) is a viral, highly lethal haemorrhagic disease of swine with no available vaccine or effective treatment. Introduction of ASF into a country triggers immediate restriction measures that cause significant economic losses and threatens spread to neighbouring countries. Wild boar populations have been recently assigned an essential role in the spread of African swine fever virus (ASFV) to European countries. Therefore, effective surveillance and monitoring of wild boar populations is required, but sampling wild boar is logistically challenging and expensive. This study assessed the feasibility of detecting antibodies against ASFV in faeces for later implementation in surveillance and control programmes. Two groups of pigs were experimentally infected with an attenuated ASFV isolate Ken05, and blood, oral fluid and faecal samples were tested for the presence of viral DNA using quantitative real‐time polymerase chain reaction (qPCR) to monitor infection progress. Faecal samples were analysed using two indirect enzyme‐linked immunosorbent assays (ELISAs) based on semipurified viral protein (vp) 72 or purified recombinant vp30 expressed in mammalian cells. Faecal samples from 9 of 10 pigs with non‐haemorrhagic diarrhoea tested positive for antibodies against ASFV using the two ELISA tests that showed a positive correlation. The serum sample results from the two indirect ELISAs were compared against results from the reference ELISA technique and the immunoperoxidase test. Our findings indicate the feasibility of faecal sampling for detecting anti‐ASFV antibodies, which may provide a practical non‐invasive alternative for sampling wild boar populations. In conclusion, the application of these ELISA tests to faecal field samples could be particularly useful to screen for the presence of ASF in field conditions.     

Cancer‐specific and non‐cancer‐related mortality rates in European patients with T1a and T1b renal cell carcinoma

OBJECTIVE

To examine cancer‐specific and non‐cancer‐related mortality rates in 451 patients with T1a–bN0M0 renal cell carcinoma (RCC) treated with either radical or partial nephrectomy (RN or PN) in Europe.

PATIENTS AND METHODS

Between 1987 and 2007, 451 patients with T1a–bN0M0 RCC were treated for histologically confirmed RCC with RN or PN at one of seven participating European institutions. The preoperative American Society of Anesthesiology (ASA) score was available for all patients and was used to control for baseline comorbidities. The preoperative glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease study group equation. We used univariate and multivariate competing‐risks regression analyses to test the effect of the ASA score, GFR, T stage (T1a vs T1b) and nephrectomy type (RN or PN) on RCC‐specific mortality and non‐RCC‐related mortality.

RESULTS

In patients with T1a–b RCC cancer‐ specific mortality was unaffected by stage, nephrectomy type or GFR. Conversely, non‐RCC‐related mortality was strongly affected by the ASA score and GFR. Unlike in a previous report, nephrectomy type did not affect non‐RCC‐related mortality. This lack of significance relative to RN may stem from the relatively high rate of PN use in the present series.

CONCLUSION

PN or RN virtually eliminate the risk of cancer‐specific mortality in patients with T1a–b RCC. Poor preoperative ASA score and impaired renal function appear to represent relative contra‐indications to surgical management of T1a–b lesions.