An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.

The co-administration of live fowlpox and Newcastle disease vaccines by non-invasive routes to chickens reared by smallholders in Tanzania and Nepal.

The co-administration of commercial live fowlpox (FP) and Newcastle disease (ND) vaccines when given by non-invasive (needle-free) routes was demonstrated to be safe and to elicit immunity in two field studies, one in Tanzania the other in Nepal. Both studies were of a cluster-randomised controlled design in which birds were randomly assigned to one of five treatment groups: (i) administration with FP vaccine alone (feather follicle), (ii) administration with ND vaccine alone (eye-drop), (iii) concurrent administration of FP (feather follicle) and ND (eye-drop) vaccines, (iv) concurrent administration of FP (wing-web) and ND (eye-drop) vaccines, and (v) unvaccinated, acting as environmental sentinels. Data from a total of 1167 birds from seven villages in Hanang District of Tanzania together with 1037 birds from eleven villages in Dhading District of Nepal were collected over a period of 21 and 28 days, respectively. Immune responses to FP vaccination were evaluated by local take reactions, while those to ND vaccination were evaluated serologically by haemagglutination inhibition test. The two studies demonstrated that the concurrent vaccination of free-range, indigenous breeds of chicken with live FP and ND vaccines, both administered by non-invasive routes, was safe and induced immunity against FP and ND that were non-inferior to the administration of FP and ND vaccines alone. These findings are important to appropriately trained small-scale backyard poultry farmers as well as to paraprofessionals and community health workers helping to increase vaccine uptake and the control of both FP and ND in low- to middle-income countries.

Sequence Diversity of Tp1 and Tp2 Antigens and Population Genetic Analysis of Theileria parva in Unvaccinated Cattle in Zambia's Chongwe and Chisamba Districts.

East Coast Fever (ECF), caused by Theileria parva, is a major constraint to improved livestock keeping in east and central Africa, including Zambia. To understand the dynamics and determine the candidates for immunization in Zambia's Chongwe and Chisamba districts, a combination of Tp1 and Tp2 gene sequencing and microsatellite analysis using nine markers was conducted from which an abundance of Muguga, Kiambu, Serengeti and Katete epitopes in the field samples was obtained. Phylogenetic analysis showed six (Tp1) and three (Tp2) clusters with an absence of geographical origin clustering. The majority of haplotypes were related to Muguga, Kiambu, Serengeti and Katete, and only a few were related to Chitongo. Both antigens showed purifying selection with an absence of positive selection sites. Furthermore, low to moderate genetic differentiation was observed among and within the populations, and when vaccine stocks were compared with field samples, Chongwe samples showed more similarity to Katete and less to Chitongo, while Chisamba samples showed similarity to both Katete and Chitongo and not to Muguga, Kiambu or Serengeti. We conclude that the use of Katete stock for immunization trials in both Chongwe and Chisamba districts might produce desirable protection against ECF.

Molecular characterization and population genetics of Theileria parva in Burundi's unvaccinated cattle: Towards the introduction of East Coast fever vaccine.

Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.

Vaccine Quality Is a Key Factor to Determine Thermal Stability of Commercial Newcastle Disease (ND)Vaccines.

Vaccination against Newcastle disease (ND), a devastating viral disease of chickens, is often hampered by thermal inactivation of the live vaccines, in particular in tropical and hot climate conditions. In the past, "thermostable" vaccine strains (I-2) were proposed to overcome this problem but previous comparative studies did not include formulation-specific factors of commercial vaccines. In the current study, we aimed to verify the superior thermal stability of commercially formulated I-2 strains by comparing six commercially available ND vaccines. Subjected to 37 °C as lyophilized preparations, two vaccines containing I-2 strains were more sensitive to inactivation than a third I-2 vaccine or compared to three other vaccines based on different ND strains. However, reconstitution strains proved to have a comparable tenacity. Interestingly, all vaccines still retained a sufficient virus dose for protection (106 EID50) after 1 day at 37 °C. These results suggest that there are specific factors that influence thermal stability beyond the strain-specific characteristics. Exposing ND vaccines to elevated temperatures of 51 and 61 °C demonstrated that inactivation of all dissolved vaccines including I-2 vaccine strains occurred within 2 to 4 h. The results revealed important differences among the vaccines and emphasize the importance of the quality of a certain vaccine preparation rather than the strain it contains. These data highlight that regardless of the ND strain used for vaccine preparation, the appropriate cold chain is mandatory for keeping live ND vaccines efficiency in hot climates.

Prioritizing smallholder animal health needs in East Africa, West Africa, and South Asia using three approaches: Literature review, expert workshops, and practitioner surveys.

Managing the health needs of livestock contributes to reducing poverty and improving the livelihoods of smallholder and pastoralist livestock keepers globally. Animal health practitioners, producers, policymakers, and researchers all must prioritize how to mobilize limited resources. This study employed three approaches to prioritize animal health needs in East and West Africa and South Asia to identify diseases and syndromes that impact livestock keepers. The approaches were a) systematic literature review, b) a series of expert workshops, and c) a practitioner survey of veterinarians and para-veterinary professionals. The top constraints that emerged from all three approaches include endo/ ectoparasites, foot and mouth disease, brucellosis, peste des petits ruminants, Newcastle disease, and avian influenza. Expert workshops additionally identified contagious caprine pleuropneumonia, contagious bovine pleuropneumonia, mastitis, and reproductive disorders as constraints not emphasized in the literature review. Practitioner survey results additionally identified nutrition as a constraint for smallholder dairy and pastoralist small ruminant production. Experts attending the workshops agreed most constraints can be managed using existing veterinary technologies and best husbandry practices, which supports a shift away from focusing on individual diseases and new technologies towards addressing systemic challenges that limit access to veterinary services and inputs. Few research studies focused on incidence/ prevalence of disease and impact, suggesting better incorporation of socio-economic impact measures in future research would better represent the interests of livestock keepers.

Safety, Immunogenicity and Antibody Persistence of Rift Valley Fever Virus Clone 13 Vaccine in Sheep, Goats and Cattle in Tanzania.

Background: Vaccination is considered to be the best approach to control Rift Valley fever (RVF) in animals and consequently in humans. This study assessed the efficacy and safety of the RVF virus (RVFV) Clone 13 vaccine under field conditions. Methodology: A vaccine trial was conducted in sheep (230), goats (230), and cattle (140) in Ngorongoro district, Tanzania. Half of each of the animal species were vaccinated and the other half received the placebo. Animals were clinically monitored and bled before vaccination and at days 15, 30, 60, 180 and 360 (+/- 10) post-vaccination to measure Immunoglobulin M (IgM) and IgG antibody responses to RVFV. Survival analysis was conducted using cox-proportional hazard regression model to measure the time until an event of interest had occurred and to compare the cumulative proportion of events over time. Results: Of 600 animals included in the study, 120 animals were lost during the study, leaving a total of 480 (243 in the vaccinated group and 237 in the control group) for complete follow-up sampling. There was no adverse reaction reported at the injection site of the vaccine/placebo in all animals. Abortions, deaths, or body temperature variations were not associated with vaccination (p > 0.05). By day 15 post-inoculation, the IgG seroconversion in vaccinated goats, cattle and sheep was 27.0% (n = 115), 20.0% (n = 70) and 10.4% (n = 115), respectively. By day 30 post-inoculation, it was 75.0% (n = 113), 74.1% (n = 112) and 57.1% (n = 70) in vaccinated sheep, goats and cattle, respectively. By day 60 post-inoculation, IgG seroconversion in sheep, goats and cattle was 88.1% (n = 109), 84.3% (n = 108) and 64.60% (n = 65), respectively. By day 180, the IgG seroconversion in sheep, goats and cattle was 88.0% (n = 108), 83.8% (n = 105) and 66.1% (n = 62), respectively. By day 360, the IgG seroconversion in sheep, goats and cattle was 87.2% (n = 94), 85.6% (n = 90) and 66.1% (n = 59), respectively. Only five animals from the vaccinated group were RVFV IgM positive, which included four sheep and a goat. Conclusion: RVFV Clone 13 vaccine was well tolerated by sheep, goats, and cattle. The vaccine induced detectable, but variable levels of IgG responses, and of different duration. The vaccine is considered safe, with high immunogenicity in sheep and goats and moderate in cattle.

Live Attenuated African Swine Fever Viruses as Ideal Tools to Dissect the Mechanisms Involved in Cross-Protection.

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.

Sequence diversity of cytotoxic T cell antigens and satellite marker analysis of Theileria parva informs the immunization against East Coast fever in Rwanda.

BACKGROUND: East Coast fever (ECF) caused by Theileria parva is endemic in Rwanda. In this study, the antigenic and genetic diversity of T. parva coupled with immunization and field challenge were undertaken to provide evidence for the introduction of ECF immunization in Rwanda. METHODS: Blood collected from cattle in the field was screened for T. parva using ELISA and PCR targeting the p104 gene. Tp1 and Tp2 gene sequences were generated from field samples and from Gikongoro and Nyakizu isolates. Furthermore, multilocus genotype data was generated using 5 satellite markers and an immunization challenge trial under field conditions using Muguga cocktail vaccine undertaken. RESULTS: Out of 120 samples, 44 and 20 were positive on ELISA and PCR, respectively. Antigenic diversity of the Tp1 and Tp2 gene sequences revealed an abundance of Muguga, Kiambu and Serengeti epitopes in the samples. A further three clusters were observed on both Tp1 and Tp2 phylogenetic trees; two clusters comprising of field samples and vaccine isolates and the third cluster comprising exclusively of Rwanda samples. Both antigens exhibited purifying selection with no positive selection sites. In addition, satellite marker analysis revealed that field samples possessed both shared alleles with Muguga cocktail on all loci and also a higher proportion of unique alleles. The Muguga cocktail (Muguga, Kiambu and Serengeti) genotype compared to other vaccine isolates, was the most represented in the field samples. Further low genetic sub-structuring (FST = 0.037) coupled with linkage disequilibrium between Muguga cocktail and the field samples was observed. Using the above data to guide a field immunization challenge trial comprising 41 immunized and 40 control animals resulted in 85% seroconversion in the immunized animals and an efficacy of vaccination of 81.7%, implying high protection against ECF. CONCLUSIONS: Antigenic and genetic diversity analysis of T. parva facilitated the use of Muguga cocktail vaccine in field conditions. A protection level of 81.7% was achieved, demonstrating the importance of combining molecular tools with field trials to establish the suitability of implementation of immunization campaigns. Based on the information in this study, Muguga cocktail immunization in Rwanda has a potential to produce desirable results.

Contagious Bovine and Caprine Pleuropneumonia: a research community's recommendations for the development of better vaccines.

Contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP) are major infectious diseases of ruminants caused by mycoplasmas in Africa and Asia. In contrast with the limited pathology in the respiratory tract of humans infected with mycoplasmas, CBPP and CCPP are devastating diseases associated with high morbidity and mortality. Beyond their obvious impact on animal health, CBPP and CCPP negatively impact the livelihood and wellbeing of a substantial proportion of livestock-dependent people affecting their culture, economy, trade and nutrition. The causative agents of CBPP and CCPP are Mycoplasma mycoides subspecies mycoides and Mycoplasma capricolum subspecies capripneumoniae, respectively, which have been eradicated in most of the developed world. The current vaccines used for disease control consist of a live attenuated CBPP vaccine and a bacterin vaccine for CCPP, which were developed in the 1960s and 1980s, respectively. Both of these vaccines have many limitations, so better vaccines are urgently needed to improve disease control. In this article the research community prioritized biomedical research needs related to challenge models, rational vaccine design and protective immune responses. Therefore, we scrutinized the current vaccines as well as the challenge-, pathogenicity- and immunity models. We highlight research gaps and provide recommendations towards developing safer and more efficacious vaccines against CBPP and CCPP.

Evaluation of new generation macrolides for the treatment and metaphylaxis of contagious bovine pleuropneumonia (CBPP) in cattle experimentally infected with Mycoplasma mycoides subspecies mycoides.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subspecies mycoides (Mmm) is an important disease of cattle that causes serious economic losses. With the known effectiveness of new generation macrolides, tulathromycin and gamithromycin were assessed in comparison with oxytetracycline as a positive control and saline as a negative control for effectiveness in inhibiting lung lesion development, promoting resolution, preventing spread and bacteriological clearance in susceptible local cattle breeds in two separate studies in Kenya and Zambia. Animals were monitored for clinical signs, sero-conversion as well as detailed post-mortem examination for CBPP lesions. RESULTS: Using the Hudson and Turner score for lesion type and size, tulathromycin protected 90%, gamithromycin 80%, and oxytetracycline 88% of treated animals in Kenya. In Zambia, all animals (100%) treated with macrolides were free of lung lesions, while oxytetracycline protected 77.5%. Using the mean adapted Hudson and Turner score, which includes clinical signs, post-mortem findings and serology, tulathromycin protected 82%, gamithromycin 56% and oxytetracycline 80% of the animals in Kenya whereas in Zambia, tulathromycin protected 98%, gamithromycin 94% and oxytetracycline 80%. The saline-treated groups had 93 and 92% lesions in Kenya and Zambia respectively, with Mmm recovered from 5/14 in Kenya and 10/13 animals in Zambia. Whereas the groups treated with macrolides were free from lesions in Zambia, in Kenya 5/15 tulathromycin-treated animals and 6/15 gamithromycin-treated animals showed lesions. Oxytetracycline-treated animals showed similarities with 3/14 and 4/15 showing lesions in Zambia and Kenya respectively and Mmm recovery from one animal in Kenya and six in Zambia. In both studies, lesion scores of saline-treated groups were significantly higher than those of the antibiotic treated groups (p < 0.001). In sentinel animals, CBPP lesions were detected and Mmm recovered from one and two animals mixed with the saline-treated groups in Kenya and Zambia respectively. CONCLUSIONS: This study demonstrated that tulathromycin, a mycoplasmacidal, can achieve metaphylactic protection of up to 80%, while non-recovery of Mmm from sentinels suggests macrolides effectiveness in preventing spread of Mmm. It is recommended that further studies are conducted to evaluate strategies comparing vaccination alone or combining vaccination and antibiotics to control or eradicate CBPP.

Reproduction of contagious caprine pleuropneumonia reveals the ability of convalescent sera to reduce hydrogen peroxide production in vitro.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae is a severe disease widespread in Africa and Asia. Limited knowledge is available on the pathogenesis of this organism, mainly due to the lack of a robust in vivo challenge model and the means to do site-directed mutagenesis. This work describes the establishment of a novel caprine challenge model for CCPP that resulted in 100% morbidity using a combination of repeated intranasal spray infection followed by a single transtracheal infection employing the recent Kenyan outbreak strain ILRI181. Diseased animals displayed CCPP-related pathology and the bacteria could subsequently be isolated from pleural exudates and lung tissues in concentrations of up to 109 bacteria per mL as well as in the trachea using immunohistochemistry. Reannotation of the genome sequence of ILRI181 and F38T revealed the existence of genes encoding the complete glycerol uptake and metabolic pathways involved in hydrogen peroxide (H2O2) production in the phylogenetically related pathogen M. mycoides subsp. mycoides. Furthermore, the expression of L-α-glycerophosphate oxidase (GlpO) in vivo was confirmed. In addition, the function of the glycerol metabolism was verified by measurement of production of H2O2 in medium containing physiological serum concentrations of glycerol. Peroxide production could be inhibited with serum from convalescent animals. These results will pave the way for a better understanding of host-pathogen interactions during CCPP and subsequent vaccine development.

Field-derived estimates of costs for Peste des Petits Ruminants vaccination in Ethiopia.

In 2015, the OIE and FAO launched a global eradication programme for Peste des Petits Ruminants (PPR). Vaccination is a major component of this strategy yet the costs of implementing a campaign are unknown or based on assumptions without field-based verification necessary for effective economic planning. This study used experiences of attending four PPR vaccination campaigns in Ethiopia to estimate various cost components in pastoral and mixed-crop livestock systems. These components included: cost of vaccine; vaccine transport from the producer to the local storage facility; storage of vaccine at the local facility; delivery and administration of vaccine in the field; opportunity cost of farmer's time to attend the vaccination; co-ordination of vaccination campaign; publicity and mobilisation costs; vaccine wastage from missed shots and vaccine discard. The overall cost of vaccination was approximately 6 Ethiopian birr (ETB) or US$0.2 per animal in the mixed-crop livestock system compared to approximately 3ETB or US$0.1 in pastoral areas. The relative importance of cost components varied in the two systems with farmer time being the largest contributor in the mixed-crop livestock system while field delivery was the main cost in pastoral areas. Notable vaccine wastage was observed particularly through missed shots that were typically between 0 and 10% but as high as 33%. At the national level, the output of the stochastic model showed the cost of vaccination to be highly variable particularly in the mixed-crop livestock system. These results highlight the importance of doing economic assessments of vaccination campaigns and issues that may be compromising efficiency of delivery and vaccine coverage. It is recommended that the framework be used for further economic evaluations of vaccination for PPR and other livestock diseases particularly when limited public or donor funds are being used, and that the approach be expanded to other countries and regions.

Efficacy of a Parapoxvirus ovis-based immunomodulator against equine herpesvirus type 1 and Streptococcus equi equi infections in horses.

The efficacy of Zylexis®, an immunomodulator in horses based on inactivated Parapoxvirus ovis (iPPVO), was assessed using an equine herpesvirus type 1 (EHV-1) challenge model in the presence of a natural infection with Streptococcus equi equi (S. equi). Eleven horses were treated with iPPVO and twelve were kept as controls. Six horses were challenged with EHV-1 and commingled with the horses on study. Animals were dosed on Days -2, 0 (just before commingling) and Day 7. On Day 11 significantly less nasal discharge, enlarged lymph nodes, EHV-1 shedding and lower rectal temperatures were observed in the iPPVO-treated group. In addition, iPPVO-treated horses showed significantly fewer enlarged lymph nodes on Days 17 and 19, significantly less lower jaw swelling on Day 3 and significantly lower rectal temperatures on Days 12 and 13. Dyspnoea, depression and anorexia were only recorded for the control group. Following challenge seven out of 11 horses in the iPPVO treated group shed EHV-1 but on Days 11, 12, 13, 14, 15 and 16 quantitative virus detection in this group was significantly lower as compared to the controls. All animals shed S. equi but the percentage of animals with positive bacterial detection was lower in the iPPVO group than in the control group from Day 14 through Day 28. This difference was significant on Day 24. No injection site reactions or adverse events were observed. In conclusion, Zylexis administration is safe and reduced clinical signs and shedding related to both EHV-1 and S. equi infections.

Protection of horses from West Nile virus Lineage 2 challenge following immunization with a whole, inactivated WNV lineage 1 vaccine.

Over the last years West Nile virus (WNV) lineage 2 has spread from the African to the European continent. This study was conducted to demonstrate efficacy of an inactivated, lineage 1-based, WNV vaccine (Equip WNV) against intrathecal challenge of horses with a recent isolate of lineage 2 WNV. Twenty horses, sero-negative for WNV, were enrolled and were randomly allocated to one of two treatment groups: an unvaccinated control group (T01, n=10) and a group administered with Equip WNV (T02, n=10). Horses were vaccinated at Day 0 and 21 and were challenged at day 42 with WNV lineage 2, Nea Santa/Greece/2010. Personnel performing clinical observations were blinded to treatment allocation. Sixty percent of the controls had to be euthanized after challenge compared to none of the vaccinates. A significantly lower percentage of the vaccinated animals showed clinical disease (two different clinical observations present on the same day) on six different days of study and the percentage of days with clinical disease was significantly lower in the vaccinated group. A total of 80% of the non-vaccinated horses showed viremia while only one vaccinated animal was positive by virus isolation on a single occasion. Vaccinated animals started to develop antibodies against WNV lineage 2 from day 14 (2 weeks after the first vaccination) and at day 42 (the time of onset of immunity) they had all developed a strong antibody response. Histopathology scores for all unvaccinated animals ranged from mild to very severe in each of the tissues examined (cervical spinal cord, medulla and pons), whereas in vaccinated horses 8 of 10 animals had no lesions and 2 had minimal lesions in one tissue. In conclusion, Equip WNV significantly reduced the number of viremic horses, the duration and severity of clinical signs of disease and mortality following challenge with lineage 2 WNV.

Vaccination of dogs with canine parvovirus type 2b (CPV-2b) induces neutralising antibody responses to CPV-2a and CPV-2c.

Since the identification of canine parvovirus type 2, three variants have subsequently been observed differing from the historical CPV-2 and each other by 1-2 amino acids only. As a result there has been considerable research into differential diagnostics, with some researchers indicating there is a need for new vaccines containing different strains of CPV-2. In this study we investigated whether vaccination with a CPV-2b containing vaccine would induce cross-reactive antibody responses to the other CPV-2 variants. Two studies where dogs were vaccinated with a multivalent vaccine, subsequently challenged with CPV-2b and sera samples analysed are presented. Six week old pups with defined serological status were vaccinated twice, three weeks apart and challenged either 5 weeks (MDA override study) or one year after vaccination (duration of immunity study). Sera samples were collected before each vaccination and at periods throughout each study. In each study the antibody profiles were very similar; serological responses against CPV-2a, CPV-2b and CPV-2c were higher than those for CPV-2. Nevertheless, responses against CPV-2 were well above levels considered clinically protective. In each study dogs also showed a rapid increase in antibody titres following vaccination, reached a plateau following second vaccination with a slight decline to challenge after which rapid anamnestic responses were seen. Evaluation of the serological responses suggests vaccination with CPV-2b would cross-protect against CPV-2a and CPV-2c, as well as against CPV-2 which is now extinct in the field. In conclusion we have demonstrated that vaccination of minimum aged dogs with a multivalent vaccine containing the CPV-2b variant strain will induce serological responses which are cross-reactive against all currently circulating field strains, CPV-2a and CPV-2c, and the now extinct field strain CPV-2.

Duration of immunity of a multivalent (DHPPi/L4R) canine vaccine against four Leptospira serovars.

Despite effective vaccines against common Leptospira serovars, the development of new products with long duration of immunity is still important to protect dogs against leptospirosis. The results from four challenge studies performed one year after vaccination of dogs with a multivalent vaccine containing four Leptospira antigens are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 367 days later. Clinical observations were recorded, while blood (culture, biochemistry and haematology), urine (culture) and liver and kidney (culture) samples were collected throughout the study or at necropsy. All control dogs remained seronegative until challenge, when they seroconverted. Antibody titres to Leptospira antigens were seen in vaccinated dogs 21 days after first vaccination and peaked three to six weeks after the second vaccination. Titres decreased in all studies over the following 12 months, until challenge when anamnestic responses were observed. In all studies control dogs demonstrated various abnormal clinical signs, while no vaccinated dogs were affected; differences between groups were only significant following L. bratislava challenge. Analysis of blood cultures showed all control and five of the 24 vaccinated dogs were Leptospira positive after challenge; all studies showed significant differences between treatment groups in mean number of days with positive cultures. Significant differences between vaccinated and control groups in mean number of days with positive urine cultures were also observed, with all non-vaccinated and one vaccinated dog Leptospira positive. The urine culture positive vaccinated dog also gave positive culture from kidney and liver samples. All except one control dog also showed positive Leptospira isolation from kidney or liver, with significant differences between vaccinated and control groups observed. The results demonstrate that administration of a new vaccine to six week old puppies induces immunity which is still effective up to one year later as demonstrated by challenge.

A new multivalent (DHPPi/L4R) canine combination vaccine prevents infection, shedding and clinical signs following experimental challenge with four Leptospira serovars.

Although effective vaccines have been developed against the common Leptospira serovars, they are still reported in clinical cases, while others are increasingly prevalent. The results from four challenge studies following vaccination of dogs with a new combination vaccine (DHPPi/L4R) containing inactivated L. serovars, L. canicola, L. icterohaemorrhagiae, L. bratislava and L. grippotyphosa conducted to satisfy the requirements of the European Pharmacopoeia monograph (01/2008:0447), are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 25 days later with different isolates of the L. serovars. Clinical observations were recorded, and blood, urine and tissue samples were collected for analysis. Following challenge, non-vaccinated dogs demonstrated various clinical signs, while no vaccinated dogs were affected; significant differences in mean clinical scores were observed. Measurable antibody titres to each Leptospira antigen were seen in vaccinated dogs 21 days following the first vaccination, with further increases in antibody titres observed following challenge with the respective Leptospira strain. Non-vaccinated dogs remained seronegative until challenge. Leptospira were re-isolated from the blood, urine, kidney and liver of all non-vaccinated dogs following challenge. In contrast no vaccinated dogs had Leptospira re-isolated from the same tissues. Significant differences were seen in number of days with positive isolation (blood and urine) and in number of dogs with positive samples (kidney and liver). In conclusion, vaccination of dogs with the new vaccine induces protective immunity 25 days after second vaccination with protection against infection, renal infection and clinical signs following challenge.

Vaccination of piglets up to 1 week of age with a single-dose Mycoplasma hyopneumoniae vaccine induces protective immunity within 2 weeks against virulent challenge in the presence of maternally derived antibodies.

Enzootic pneumonia, resulting from infection with Mycoplasma hyopneumoniae, is of considerable economic importance to the pig industry and normally is controlled through active vaccination of piglets. We have demonstrated that administration of an inactivated Mycoplasma hyopneumoniae vaccine to piglets less than 1 week old is efficacious under field conditions and reduces the level of lung lesions observed in comparison to that in control pigs. Here, the results of two separate studies, one in piglets with and the second one in piglets without maternal antibodies, conducted to satisfy the requirements of the European Pharmacopoeia (monograph no. 07/2009:2448), are reported. Piglets received either minimal titer Suvaxyn MH-One or saline at less than 1 week of age and were challenged with Mycoplasma hyopneumoniae 2 weeks later. The number of lung lesions was recorded 4 weeks after challenge, and bronchial swab and lung tissue specimens were analyzed for quantification of Mycoplasma hyopneumoniae DNA. In the presence and absence of maternal antibodies, vaccination of piglets at less than 1 week of age was efficacious, with vaccinated piglets having significantly lower percentages of lung with lesions and lower Mycoplasma hyopneumoniae counts detected in bronchial swab and lung tissue specimens at necropsy. In conclusion, the vaccination of piglets at 1 week of age with Suvaxyn MH-One is efficacious in the presence of high levels of maternal antibodies.