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Contagious bovine pleuropneumonia (CBPP) is a highly contagious respiratory disease affecting cattle and is widely distributed in the sub-Saharan Africa. The objective of this study was to detect Mycoplasma mycoides subspecies mycoides (Mmm) the causative agent of CBPP from 90 cattle at slaughter using polymerase chain reaction-Restriction fragment length polymorphism. In this study, 450 samples suggestive of CBPP in Maiduguri, Yola and Gombe township abattoirs were processed according to standard protocols. The isolation rate was found to be 3.33% and percentage of identification with PCR-RFLP yielded 1.56%. Subsequently, QIAxcel revealed molecular size of 574 bp for Mycoplasma mycoides subcluster. Further analysis of PCR amplicons with restriction digestion, confirmed the presence of Mmm 16 S rRNA of CAP 21 genomic region with molecular sizes of 180 bp and 380 bp. Thus, the 380 bp fragments delineated Mmm from Mycoplasma mycoides subsp. capri. Three isolates (BL5, BL6 and AL1) were from lungs and four from pleural fluids (APF2, APF8A, APF8B and APF9) were isolated and identified, while a vaccine strain T1/44 was re-detected along with the field isolates. No sample from Gombe had Mmm. In conclusion, the findings of this study have detected the presence of Mmm as causative agent of CBPP. Measures such as surveillance, quarantine and vaccination are hereby recommended for the control of CBPP in Nigeria.

African horse sickness (AHS) is a non-contagious, insect-borne disease of equids caused by a RNA virus (AHSV), which belongs to the genus Orbivirus, family Reoviridae. The disease is endemic in sub-Saharan and western Africa, where prevention strictly depends upon vaccination. The present paper aims at evaluating the serological response and the occurrence of AHS in horses bred under field condition and regularly immunized using the commercially available live attenuated vaccine (LAV) produced by Onderstepoort Biological Products. The study was carried out in a farm located in the district of Windhoek (Namibia), where the disease is endemic. A total of 72 cross-breed horses, out of the 150 housed on the farm, were subdivided in six age groups, from 2 to 7 years-old. Each group consisted of 12 heads which were born during the same breed- ing season and had undergone from four to nine vaccination courses. AHSV specific immune response was evaluated by serum-virus neutralization test. Data about the clinical occurrence of the AHS from 2006 to 2011 were made available. The immune response, in terms of number of seropositive horses and serum neutralizing titers, was quite variable among horses and against different serotypes. Neutralizing antibodies against all serotypes were recorded in all the horses only after eight vaccination courses at 6 years of age onwards. Immune response to AHSV-5 and 9, which are not included in the LAV formulation, were also established. A severe AHS epidemic occurred in Namibia in 2011. On the farm under study, a total of 32 animals were clinically affected, 12 died, 11 of them were 2 year-old or younger. Our data confirm that vaccination with LAV is a useful tool to reduce the severity of the disease in ende- mic areas. However, clinical and sometimes fatal AHS can still affect young vaccinated horses, thus high- lighting the necessity to better understand the immune response to AHSV and to dispose of more effective vaccines. 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.

An immunohistochemical (IHC) technique was optimised using a monoclonal antibody (MAb) to detect Mycoplasma mycoides subsp. mycoides (Mmm), the agent of Contagious Bovine Pleuropneumonia (CBPP), in sections of lung tissue. A panel of MAbs was produced and screened for Mmm speci city and for cross-reactivity against other mycoplasmas belonging and not belonging to the Mycoplasma mycoides cluster, using in parallel indirect ELISA (i-ELISA) and Immunoblotting (IB). Based on i-ELISA and IB characterization data, 1 MAb (clone 3G10E7) was selected and its highest a nity vs Mmm was con rmed by the Quartz Crystal Microbalance (QCM) technology. Afterwards, IHC analyses were conducted to compare MAb 3G10E7 vs rabbit Mmm speci c hyperimmune serum using lung tissue sections of CBPP infected and CBPP negative animals. Results suggest that screening of MAbs using in parallel ELISA, IB, and QCM technology enables to select high a nity target speci c MAbs. Immunohistochemical results demonstrated that MAb 3G10E7 improved IHC performances, showing reduced background staining and no cross-reactivity against Mycoplasma bovis, which is responsible of pneumonia in cattle.

Peste des petits ruminants (PPR) virus belongs to the family Paramyxoviridae and represents a major threat to small livestock industry. In recent years, outbreaks of PPR have occurred in Turkey and North Africa. In endemic areas, disease prevention is accomplished using live‑attenuated vaccines. However, the use of live vaccines in non‑endemic regions, such as Europe, is not approved by Veterinary Authorities. In these regions inactivated vaccines are then the only viable alternative. In this study an inactivated vaccine (iPPRVac) was formulated with either a water‑in‑oil emulsion (ISA 71 VG) or with delta inulin adjuvant, alone (AFSA1) or combined with a TLR9 agonist oligonucleotide (AFSA2). These formulations were then tested for immunogenicity on rats. The iPPRV formulation with AFSA2 adjuvant induced 100% seroconversion in rats after 2 injections and was subsequently evaluated in goats. Five goats were immunised twice subcutaneously, 36 days apart with iPPRVac + AFSA2. The immunised goats all seroconverted to PPR by day 9 and remained seropositive until the end of the experimental period (133 days). These data indicate that the rat model is useful in predicting vaccine responses in goats and that inactivated vaccine, when formulated with a delta inulin adjuvant, represents a promising alternative to live attenuated vaccines for PPR vaccination campaigns in non‑endemic areas.

Contagious ecthyma is caused by the orf virus, a member of the family Poxviridae, genus Parapoxvirus. Morbidity in affected sheep flocks is approximately 100%, while mortality varies between 1% and 10%. A live attenuated vaccine was produced by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale'. Quality control was performed in accordance with the European Pharmacopoeia. A wild virus strain was attenuated through serial passages on primary chicken embryo fibroblast tissue cultures. The virus suspension was treated according to standard procedures and freeze dried. The immunising dose was 1 ml containing 10(4.5)TCID(50), administered intramuscularly. The safety of the vaccine was successfully tested by intramuscular inoculation of 20 susceptible sheep and 20 lambs with the routine dose, 10 times the immunising dose and two normal doses administered at seven-day intervals. The efficacy of the vaccine was tested using three groups of susceptible animals. The first group included 10 lambs and the second 10 adult sheep; the animals were immunised intramuscularly with 1 ml of the reconstituted vaccine. The third group, used as controls, included five sheep and five lambs. Serological reactivity was monitored by indirect enzyme-linked immunosorbent assay (ELISA). The animals were challenged 30 days later with a pathogenic strain administered intradermally along the labial area. Vaccinated animals did not show any clinical signs of disease, whereas all the controls developed typical signs of contagious ecthyma. To confirm the efficacy of the vaccine, a field trial was conducted in four flocks affected by the disease. The trial showed that the vaccine was able to block the normal course of the disease and induce rapid recovery.

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10 6.3 TCID 50 of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3–9.3 log 2 (ED50%/50 μl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.

African horse sickness (AHS) is a vector‑borne viral disease of equids, endemic in Sub‑Saharan Africa. This article reports the clinic‑pathological and laboratory findings observed in the framework of passive surveillance during the AHS outbreaks which occurred in Namibia between 2006 and 2013. This study was conducted in the framework of the collaboration among the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (Teramo, Italy), the Namibian Ministry of Agriculture Water and Forestry, and the Namibian National Veterinary Association. A total of 92 horses were investigated, showing different clinical form of AHS: peracute/acute (n = 43), sub‑acute (n = 21) and mild AHS fever (n = 19). Clinical data were not available for 9 horses, because they were found dead. Pathological findings have been recorded for 35 horses. At necropsy, pulmonary and subcutaneous oedema, haemorrhages and enlargement of lymph nodes were mainly observed. Diagnosis was confirmed by laboratory testing, AHS virus (AHSV) was isolated from 50 horses and the identified serotypes were: 1, 2, 4, 6, 7, 8, and 9. The phylogenetic analysis of the S10 genome sequences segregated the Namibian AHSV strains in the same clusters of those circulating in South Africa in recent years. The description of AHS clinical, pathological, and laboratory features of AHS provided in this article is of value for differential diagnosis and control of AHS, especially in areas currently free from this disease.

African horse sickness (AHS), a non-contagious infectious disease caused by a RNA virus in the Orbivirus genus within the Reoviridae family affecting all equids, is endemic in sub-Saharan Africa. The virus is transmitted by some species of biting midges in the genus Culicoides (Diptera: Ceratopogonidae). In April 2011, 8 Culicoides collections were performed in 6 districts of 4 regions of the Republic of Namibia (Africa), all within a 400 km radious from the capital Windhoek. Six farms - Khomas (Windhoek and Steinhausen), Erongo (Karibib and Omaruru), Otjozondjupa (Okahandja), and Omaheke (Gobabis) involved in the AHS outbreaks, were sampled. Overall 194,211 Culicoides were collected and identified. Culicoides imicola was largely the most abundant species at all farms (99.4%). A total of 18,687 parous and gravid Culicoides females were assayed for AHS virus (AHSV) by real time RT-PCR. Of the 248 assayed pools, 227 consisted of C. imicola, 13 of Culicoides pycnostictus and 5 of Schultzei complex. Only 1 pool each of Culicoides nivosus, Culicoides leucostictus, and Culicoides tropicalis was assayed. Of the 248 pools examined by real time RT-PCR, 81 tested positive for AHSV, all consisting of C. imicola collected at Omaruru, resulting in a field vector infection rate of 0.91%. No viable AHSV could be isolated from 88 of the tested pools (n = 1,463). However, bluetongue virus (BTV) serotype-1 and 10 were isolated from 3 of these pools, each consisting of 100 C. imicola collected at Windhoek. The present study confirms the relative low infection prevalence in field collected Culicoides and the strict relationship between the high abundance of C. imicola and outbreaks of AHSV.

Mycoplasma mycoides subsp mycoides (Mmm) is a major pathogen of cattle, causing contagious bovine pleuropneumonia (CBPP), a severe–frequently fatal-disease that is widespread in sub-tropical countries. Despite recognition as a pathogen in the 19th century and advances made through genome sequencing and synthetic biology, many of the molecular characteristics of Mmm remain poorly characterized. Using a proteomics approach, this study aimed to define Mmm major protein determinants, including surface membrane proteins and those involved in host colonization and pathogenicity. For this purpose, both whole cells and a Triton X-114 enriched membrane fraction of a highly pathogenic African field strain (N6) and a vaccine strain (KH3J) were analysed and compared using gelbased methodologies i.e. 2-DGE and SDS-PAGE in combination with MALDI-MS/MS and LC-ESI-MS/MS. A total of 318 proteins, (around 31% of the predicted proteome) were identified, comprising lipoproteins, prolipoproteins and hypothetical proteins with predicted membrane locations and possible roles in pathogenicity. One hundred and forty-five of these proteins, with different predicted sub-cellular locations and functions but also including uncharacterized proteins, have not previously been reported as expressed by Mmm. Many more proteins were detected by LC-ESI-MS/MS (n=315) than by 2-DGE and MALDI-MS (n=52), however 3 proteins were detected only by the latter approach. Most proteins were observed to be common to both strains although a substantial number of proteins were detected exclusively in only one strain with possible implications for fitness and pathogenicity. To our knowledge, this study represents the most extensive proteomics characterization of Mmm to date and has identified characteristics on which to base future studies of the physiology and pathogenicity of this prokaryote.

Background Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. Results Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. Conclusions Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

The Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZS A&M) has been engaged for many years in research and studies designed to increase knowledge and expertise when dealing with ‘exotic diseases’ namely, those diseases which are not present in a country. To achieve these objectives, it is important to create cooperation networks with laboratories and research organisations at national and international levels. The relationship between the IZS A&M with Namibia in particular and, more recently with Botswana, are proving to be very valuable to mutual technical/scientific growth. In 2005, the National Reference Centre for the Study of Exotic Diseases set up its own Virology Laboratory at the Windhoek Central Veterinary Laboratory where the Namibian and IZS A&M personnel, working towards common goals, share diagnostic responsibilities and scientific research. The authors describe the activities involved in this joint project.

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK21c13 or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained. © 2008 Elsevier B.V. All rights reserved.