Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka.

BACKGROUND: The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed. RESULTS: Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases. CONCLUSIONS: Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

Case-control study on the use of pituitary-derived hormones from sheep as a potential risk factor for the occurrence of atypical scrapie in Great Britain.

A case-control study was conducted in 2013 to investigate the use of pituitary-derived hormones from sheep as a potential risk factor for the presence of atypical scrapie in Great Britain sheep holdings. One hundred and sixty-five holdings were identified as cases. Two equal sets of controls were selected: no case of scrapie and cases of classical scrapie. A total of 495 holdings were selected for the questionnaire survey, 201 responses were received and 190 (38.3 per cent) were suitable for analysis. The variables 'use-of-heat-synchronisation/superovulation' and 'flock size' were significantly associated with the occurrence of atypical scrapie. Farms with atypical cases were less likely (OR 0.25, 95 per cent CI 0.07 to 0.89) to implement heat synchronisation/superovulation in the flock than the control group. Atypical cases were 3.3 times (95 per cent CI 1.38 to 8.13) more likely to occur in large holdings (>879 sheep) than in small flocks (<164 sheep). If the 'use-of-heat-synchronisation/superovulation' is a proxy for the use of pituitary-derived hormones, the significant negative association between having a case of atypical scrapie and the use of these practices rules out the initial hypothesis that using these drugs is a risk factor for the occurrence of atypical scrapie. Flock size was a significant risk factor for atypical scrapie, consistent with a previous generic case-control study.

Goats with aspartic acid or serine at codon 146 of the PRNP gene remain scrapie-negative after lifetime exposure in affected herds in Cyprus.

The results of the study reported here are part of an ongoing integrated research programme aimed at producing additional, robust, evidence on the genetic resistance to classical scrapie in goats, with particular reference to codon 146. The study targeted animals aged ⩾6 years, which were born and raised in infected herds and were being culled for management reasons. A total of 556 animals were tested, and all positive animals (n = 117) were of the susceptible NN genotype. A total of 246 goats heterozygous or homozygous for putatively resistant alleles (S146 and D146) were screened with no positive results. The outcome of this study supports the hypothesis that the D146 and S146 alleles could be used as the basis for a nationwide strategy for breeding for resistance in the Cypriot goat population.

Epidemiological investigations on the potential transmissibility of a rare disease: the case of atypical scrapie in Great Britain.

Multiple cases of atypical scrapie in the same holding and co-existence with classical scrapie have been reported in Great Britain. A two-stage simulation tool was developed by combining a sampling algorithm and a hierarchical Bayesian model to simulate the number of positive cases of atypical scrapie from: (i) random sampling and (ii) using the actual sampled population in Great Britain, being the output probability of detection of flocks with one and more cases. Cluster analysis was conducted to assess the level of geographical over- and under-sampling over the years. The probability of detecting at least two cases of atypical scrapie in the same holding is much lower in simulated random data than in simulated actual data for all scenarios. Sampling bias in the selection of sheep for testing led to multiple sampling from fewer but larger holdings, Scotland, and areas of Wales were under-sampled and the South-West and East of England oversampled. The pattern of atypical scrapie cases observed is unlikely to be explained by a multi-case event epidemiologically linked. The co-existence of classical and atypical scrapie is a rare event with 19 holdings detected in GB and does not suggest an epidemiological link between the two types of disease.

Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds.

Scrapie in goats is a transmissible, fatal prion disease, which is endemic in the British goat population. The recent success in defining caprine PRNP gene variants that provide resistance to experimental and natural classical scrapie has prompted the authors to conduct a survey of PRNP genotypes in 10 goat breeds and 52 herds to find goats with the resistant K222 allele. They report here the frequencies in 1236 tested animals of the resistance-associated K222 and several other alleles by breed and herd. Eight animals were found to be heterozygous QK222 goats (0.64 per cent genotype frequency, 95 per cent CI 0.28 to 1.27 per cent) but no homozygous KK222 goats were detected. The K222 allele was found in Saanen, Toggenburg and Anglo-Nubian goats. The fact that only a few goats with the K222 allele have been identified does not preclude the possibility to design and implement successful breeding programmes at national level.

Cytokine Expression at Different Stages of Influenza A(H1N1)pdm09 Virus Infection in the Porcine Lung, Using Laser Capture Microdissection.

Pandemic influenza A(H1N1)pdm09 virus has retained its ability to infect swine whilst developing the ability to transmit effectively between humans, thus making the pig a valuable model for studying disease pathogenesis in both species. Lung lesions in pigs caused by infection with influenza A viruses vary in both their severity and distribution with individual lung lobes exhibiting lesions at different stages of infection pathogenic development and disease resolution. Consequently, investigating interactions between the virus and host and their implications for disease pathogenesis can be complicated. Studies were undertaken to investigate the discrete expression of pro- and anti-inflammatory mediators during lung lesion formation in pigs during infection with influenza A(H1N1)pdm09 (A/Hamburg/05/09) virus. Laser capture microdissection was used to identify and select lung lobules containing lesions at different stages of development. Dissected samples were analysed using quantitative RT-PCR to assess pro- and anti-inflammatory cytokine mRNA transcripts. Differential expression of the immune mediators IL-8, IL-10 and IFN-γ was observed depending upon the lesion stage assessed. Upregulation of IFN-γ, IL-8 and IL-10 mRNA was observed in stage 2 lesions, whereas decreased mRNA expression was observed in stage 3 lesions, with IL-8 actively downregulated when compared with controls in both stage 3 and stage 4 lesions. This study highlighted the value of using laser capture microdissection to isolate specific tissue regions and investigate subtle differences in cytokine mRNA expression during lesion development in pigs infected with influenza A(H1N1)pdm09.

Allelic variants at codon 146 in the PRNP gene show significant differences in the risk for natural scrapie in Cypriot goats.

Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

The impact of the National Scrapie Plan on the PRNP genotype distribution of the British national flock, 2002-2012.

In order to determine the PRNP genotype distribution of the sheep population and to assess the impact of the National Scrapie Plan, a total of 9972 brain samples were collected between January 2012 and April 2013 from sheep sampled under the TSE active surveillance programme in Great Britain. Genotype frequencies observed were ARR/ARQ (29.5 per cent), ARR/ARR (28.6 per cent), ARR/AHQ (10.9 per cent), ARQ/ARQ (9.8 per cent) and AHQ/ARQ (7.2 per cent). Of the remaining possible genotypes, none accounted for more than 5 per cent of the sheep sampled. ARR was the most frequent allele (52.3 per cent), followed by the wild-type ARQ (30 per cent), AHQ (11.3 per cent), VRQ (3.4 per cent) and ARH (3 per cent). There has been a positive but limited effect of the breeding for resistance schemes within Great Britain on the genotype distribution in the sheep national flock. The dissemination of the resistant alleles has proven to have been less noticeable than in the breeding flocks. The risk of atypical scrapie remains very similar to that in 2002. Significant differences were observed in the genotype profiles of the abattoir survey and fallen stock survey samples. It would be advisable to promote best practice among farmers to consolidate the level of genetic resistance in their flocks and to set up a national flock genotype monitoring programme in partnership with the industry.

Immunopathology of granulomas produced by Mycobacterium bovis in naturally infected wild boar.

Limited information has been published on the wild boar immune response against bovine tuberculosis (bTB) and the immunopathogenesis of the pathological hallmark (granuloma) in this species. The main objectives of this study were, on the one hand, to characterize the histopathological features (number of acid-fast bacilli (AFB) and multinucleated giant cells (MNGCs) and the immunohistochemical distribution of different cell subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ) in the different developmental stages of granulomas produced by the natural infection of Mycobacterium bovis (M. bovis) in wild boar. On the other hand, the study also aimed to analyze the mechanisms underlying the marked differences in the typical lesional patterns observed in M. bovis infections of wild boar (contained, not generalized) and those previously described in fallow deer (poorly contained, generalized). The majority of granulomas analyzed (95.3%) did not show any AFB with the ZN stain and a low number of MNGCs were identified in the different granuloma stages. The immunohistochemical analysis showed that MAC387 was the only immune marker that produced decreasing positivity by granuloma stage, being statistically significantly lower in stages III and IV when compared to stage I and II. Immune markers for lymphocyte cells (CD3 and CD79a) showed a slight rise in the positivity (which was not statistically significant) in the advanced granuloma stages. In keeping with the presence of large numbers of T cells and macrophages, there was a consistently high level of expression of IFN-γ at all stages of granuloma development without a statistical significant decrease in advanced stages. Also related with the higher presence of macrophages in stage I and II, the expression of iNOS was higher in early stages and sustained until stage III, showing a non statistical significant decrease in stage IV. The macrophage and iNOS activity are more intense and sustained along the granuloma development than those described in fallow deer. Immunohistochemical protocols with a panel of markers for wild boar different cells subsets (CD3+, CD79a+ and MAC387+) and chemical mediators (iNOS and IFN-γ), and their use to further investigate the immune response in this species are provided.

Differential chemokine responses in the murine brain following lyssavirus infection.

The hallmark of lyssavirus infection is lethal encephalomyelitis. Previous studies have reported distinct lyssavirus isolate-related differences in severity of cellular recruitment into the encephalon in a murine model of infection following peripheral inoculation with rabies virus (RABV) and European bat lyssavirus (EBLV)-1 and -2. In order to understand the role of chemokines in this process, comparative studies of the chemokine pattern, distribution and production in response to infection with these lyssaviruses were undertaken. Expression of CCL2, CCL5 and CXCL10 was observed throughout the murine brain with a distinct caudal bias in distribution, similar to both inflammatory changes and virus antigen distribution. CCL2 immunolabelling was localized to neuronal and astroglial populations. CCL5 immunolabelling was only detected in the astroglia, while CXCL10 labelling, although present in the astroglia, was more prominent in neurons. Isolate-dependent differences in the amount of chemokine immunolabelling in specific brain regions and chemokine production by neurons in vitro were observed, with a greater expression of CCL5 in vivo and CXCL10 production in vitro after EBLV infection. Additionally, strong positive associations between chemokine immunolabelling and perivascular cuffing and, to a lesser extent, virus antigen score were also observed. These differences in chemokine expression may explain the variation in severity of encephalitic changes observed in animals infected with different lyssavirus isolates.

Comparative pathology of the natural infections by Mycobacterium bovis and by Mycobacterium caprae in wild boar (Sus scrofa).

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB-like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south-western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid-fast bacilli (AFBs) were quantified in the Ziehl-Neelsen-stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high-risk source of infection for other animals.

Upregulation of IL-17A, CXCL9 and CXCL10 in early-stage granulomas induced by Mycobacterium bovis in cattle.

To gain further insight into the immunopathogenesis of bovine tuberculosis (bTB), the cytokine and chemokine expression of cattle experimentally infected with Mycobacterium bovis was analysed in TB granulomas, using immunohistochemistry (IHC) and laser capture microdissection (LCM) followed by qPCR. Immunohistochemistry was conducted for cell types using labelling for CD68, CD3, CD4, CD8, WC1 and CD79a and for the cytokines IFN-γ, TNF-α and TGF-ß as well as inducible form of nitric oxide synthase (iNOS). qPCR was conducted for mRNA expression of IFN-γ, TNF-α, TGF-ß, IL-17A, IL-22, IL-2, granzyme A and the chemokines CXCL9 and CXCL10. Early stages of granuloma were primarily comprised of epithelioid MΦs expressing high levels of IFN-γ and iNOS, with significantly upregulated expression of CXCL9 and CXCL10 when compared with control tissue. These chemokines displayed a trend of decreasing mRNA expression as lesion progressed, suggesting a higher level of importance during the early stages of the immune response to mycobacterial infection. IL-22 levels showed a strong trend of decrease through granuloma development, and IL-17A was shown to be upregulated, supporting its investigation as a potential biomarker of bTB. The use of LCM and qPCR may prove especially useful for the study of IL-17A as previous attempts to analyse its expression using IHC and in situ hybridization proved unsuccessful.

The risk of introducing scrapie from restocking goats in Great Britain.

The goat population in Great Britain (GB), which is mostly oriented to milk production, is small compared to that in other European Union (EU) countries and contributes a very small fraction of the total livestock production. The recent confirmation and cull of scrapie-affected goat herds has raised the concern that the risk of re-introducing scrapie by mass restocking after the cull of a scrapie-affected herd, may not have been fully considered at the time of implementing statutory eradication measures. A conditional probability model has been developed to estimate the probability of introducing at least one animal infected with classical scrapie into a British goat herd under two scenarios: restocking over one year under normal operating conditions (Scenario 1); and restocking post a whole herd cull as part of the compulsory eradication measures (Scenario 2). Several of the parameters were based on expert opinion, as there is a paucity of data regarding goat industry norms for all sectors. Considering all herds, of which 99% have less than 100 animals, the probability of introduction is approximately 2 times higher for Scenario (2) than for Scenario (1). The risk of subsequently re-introducing the disease through the introduction of replacement stock is not insignificant, although it can be considered very low for the vast majority of herds (>99%). In the case of very large herds (>1000 heads), mass restocking would almost certainly reintroduce the disease since it would require purchases from a very large number of herds.

Histological and immunohistochemical characterisation of Mycobacterium bovis induced granulomas in naturally infected fallow deer (Dama dama).

Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role in the epidemiology of bovine tuberculosis (bTB), together with other deer species. There is little knowledge of the pathogenesis of bTB in fallow deer. The aim of this study was to perform a histopathological characterisation of the granulomas induced by M. bovis in this species and the immunohistochemical distribution of different cell subsets (CD3+, CD79+, macrophages) and chemical mediators (iNOS, TNF-α, IFN-γ) in the different developmental stages of granulomas. Stage I/II granulomas showed a marked presence of macrophages (MAC387+) expressing high iNOS levels while stage III/IV granulomas showed a decrease in the number of these cells forming a rim surrounding the necrotic foci. This was correlated with the presence of IFN-γ expressing cell counts, much higher in stage I/II than in stage III/IV. The number of B cells increased alongside the developmental stage of the granuloma, and interestingly the expression of TNF-α was very low in all the stages. This characterisation of the lesions and the local immune response may be helpful as basic knowledge in the attempts to increase the vaccine efficacy as well as for disease severity evaluation and for the development of improved diagnostic tools. Immunohistochemical methods using several commercial antibodies in fallow deer tissues are described.

Case-control study of cases of bovine spongiform encephalopathy born after July 31, 1996 (BARB cases) in Great Britain.

This paper reports the results of a case-control study of the bovine spongiform encephalopathy (BSE) cases born in Great Britain after the statutory reinforcement of the ban (BARB) on the feeding of mammalian-derived meat and bone meal on 31 July 1996. A total of 499 suspect clinical cases of BSE, born after 31 July 1996, and reported negative by July 31, 1996 and were compared with the set of 164 confirmed Great BARB cases in Great Britain detected by both passive and active surveillance. Animal-level risk factors (age and type of feed offered) and herd-level risk factors (herd size and type, number of prereinforced feed ban BSE cases born on the holding, the presence of other domestic species and waste management) were obtained for the analysis. BARB cases were 2.56 times (95 per cent CI 1.29 to 5.07) more likely to be exposed to homemix or a combination of homemix and proprietary feeds were 0.59 times (95 per cent CI 0.50 to 0.69) as less likely to be exposed to the unit increases in the number of prereinforced feed ban BSE cases diagnosed on the natal holding. A supplementary spatial analysis of these cases revealed three areas of excess BARB density: Northwest and Southwest of Wales and Northeast of Scotland.

Herd contact structure based on shared use of water points and grazing points in the Highlands of Ethiopia.

The use of shared common water points (WPs) and grazing points (GPs) at two different levels of administrative aggregation (village and kebelle) in a region of the Highlands of Ethiopia was explored by means of a questionnaire survey and social network analysis. Despite GPs being more abundant than WPs (208 and 154, respectively), individual GPs provide more contact opportunities for animals. There was great variability in the contact structure of the selected villages within kebelles for both networks, with this variability being higher in the GP networks for each kebelle. Contrary to the commonly held view that WPs are critical for the potential transmission of infectious diseases, intervention at GPs in the Ethiopian Highlands may have greater impact on contacts and thereby opportunities for transmission of infectious diseases between flocks. Some villages appear naturally at much lower risk of introducing disease. These findings could help the design of surveillance and control activities for directly transmitted infectious diseases.

Calf mortality as a welfare indicator on British cattle farms.

The objective of this study was to test calf mortality as an indicator of on-farm welfare and its use for welfare targeted surveillance. Calf mortality data were retrieved for three UK counties to estimate calf mortality rates at holding and county level. A selection criterion based on upper quartiles of calf mortality for the county of concern was defined. Its predictive ability was tested in a field study. The death risk of calves less than 6 months of age in 2002 was 1.76% in Inverness, 5.83% in Cheshire and 4.8% in Norfolk. Fifty-two welfare inspections matched by parish were conducted between October 2004 and January 2005. The positive predictive value was 26.92% and the negative predictive value was 65.38%. The addition of herd type, county and membership to an assurance scheme improved the predictive value. This study shows that calf mortality can be the starting point to design targeted welfare inspections in countries with centralized animal data recording systems.

Use of social network analysis to characterize the pattern of animal movements in the initial phases of the 2001 foot and mouth disease (FMD) epidemic in the UK.

Aggregated movement data do not take into account the relative position of the units within a higher-level structure. Social network analysis (SNA) and graph theory provide a tool to organise and analyse relational data overcoming the limitations of standard methods where the position of individuals/observations does not affect the result of the analysis. Some recorded movements of cattle and sheep during the initial phase of the 2001 foot and mouth disease (FMD) outbreak in the UK, before the ban on animal movements was imposed, are analysed descriptively using SNA. With the data available, a directed dichotomized network with 653 nodes and 797 arches was analysed. Most of the 10 nodes with the highest betweenness (3 farms, 4 markets and 3 dealers) were identified as key players in the initial spread of the infection. Three groups of nodes with distinctive proportion of k < or = 2 neighbours would result in three different theoretical outbreak dimensions assuming that the infection is only disseminated by the movements included in the network: no spread, spread up to 7% and around 25%. There are three hierarchical clusters with 308, 215 and 130 nodes, respectively. Farms in cluster 1 appear to be more similar in their movement patterns to non-farm holdings than to farms in clusters 2 and 3. Relative betweenness, k-neighbours and structural equivalence using hierarchical clustering were able to identify key actors in the evolution of the initial phases of the FMD outbreak such as markets, dealers and farms with atypical movement patterns. Holdings with high betweenness, large number of k < or = 2 neighbours and with movement pattern as in cluster 1 should be targeted in disease control activities once primary actors like markets, dealers and slaughter houses have been contained.