Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle.

Background: Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain. Methods: Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle. Results: PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia. Conclusion: Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination.

Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis. BCG can also be used to protect livestock against M. bovis; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

Combined Adjuvant Formulations Enhanced an Immune Response of Trivalent Foot and Mouth Disease Vaccine in Cattle.

Introduction: Foot-and-mouth disease is globally one of the most economically important viral diseases of cloven-hoofed animals that can be controlled by different strategies, where vaccination plays an important role. Selection of adjuvant added to vaccine preparation is crucial in ensuring the protective effect of the vaccine. Aluminum hydroxide gel mixed with saponin (AS) is widely used adjuvant, with its suboptimal immune response in FMD vaccine. The present study was undertaken to evaluate different ingredients of adjuvants for inactivated trivalent (A, O and SAT 2) FMD vaccine and to demonstrate the effect of booster dose in cattle. Methods: Cattle were grouped into five; four experimental and one control, with six animals in each group and immunized with trivalent vaccine with various formulations of adjuvants. Immune response was measured using Solid Phase Competitive Enzyme Linked Immune Sorbent Assay (SPCE). Results: The antibody level in cattle immunised with a vaccine formulation containing a mixture of aluminum hydroxide gel and saponin (AS) were significantly lower than AS boosted group for the three serotypes (p<0.05, t-test), which directs the need for booster dose. Whereas the antibody response in the AS + oil group was higher followed by oil alone. The AS preparation with a booster dose has shown better immune response compared to the group without. Conclusion: The findings of this study could suggest that oil based and AS with oil could replace the conventional aluminum hydroxide gel and saponin adjuvants in FMD vaccine preparations. Challenge test was not successful indicating the need for further research on the virus infectivity.

Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia.

Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.

Retrospective study on bovine clinical mastitis and associated milk loss during the month of its peak occurrence at the National Dairy Farm in the Emirate of Abu Dhabi, United Arab Emirates.

Background: Commercial dairy establishments are relatively young in the United Arab Emirates (UAE), and as a result, there is lack of epidemiological data on mastitis in dairy farms. Methods: A retrospective data of seven years (2015-2021) were used to estimate the cumulative average monthly incidence rate of bovine clinical mastitis and evaluate associated milk loss at the National Dairy Farm. Data were extracted from the records of lactating dairy cows (n = 1300-1450) and analyzed using repeated measure and one-way ANOVA, non-parametric Spearman correlation, paired and unpaired t tests. Results: The highest average cumulative monthly incidence rate was 49 cases per 1000 cows-year that was recorded in 2019 while the lowest was 19 cases per 1000 cows-year in 2021. The cumulative average monthly incidence rate of clinical mastitis significantly (p < 0.001) varied among the seven years. The cumulative average monthly incidence rate was associated with average monthly humidity (p < 0.01) and average monthly rainfall (p < 0.05); however, it was not associated with the average monthly temperature (p > 0.05). The average daily milk yield of cows with clinical mastitis (Mean ± SEM; 18.6 ± 0.54 kg) was significantly (p < 0.001) lower than the average daily milk yield of clinical mastitis free cows (40.5 ± 0.29 kg). The largest average monthly milk loss due to clinical mastitis was 5% of the average total monthly milk production in 2019 while the lowest was 2% of the average total monthly milk production in 2021. Conclusion: The result of the study indicated the direct influence of weather conditions such as increased rainfall and humidity, which caused an upsurge in the incidence rate of clinical mastitis, leading to an increased loss in milk and hence the economy of the dairy farm. Proactive preventive measures along with good dairy farm practices that help mitigate the impacts of harsh weather conditions are recommended.

Spoligotype analysis of Mycobacterium bovis isolates from cattle and assessment of zoonotic TB transmission among individuals working in bovine TB-infected dairy farms in Ethiopia.

Bovine tuberculosis (bTB) is a disease with impact on dairy productivity, as well as having the potential for zoonotic transmission. Understanding the genetic diversity of the disease agent Mycobacterium bovis is important for identifying its routes of transmission. Here we investigated the level of genetic diversity of M. bovis isolates and assessed the zoonotic potential in risk groups of people working in bTB-infected dairy farms in central Ethiopia. M. bovis was isolated and spoligotyped from tissue lesions collected from slaughtered cattle as well as from raw milk collected from bTB positive cows in dairy farms from six urban areas of central Ethiopia. From consented dairy farm workers, knowledge and practices related to zoonotic TB transmission, together with demographic and clinical information, was collected through interviews. Sputum or Fine Needle Aspirate (FNA) samples were collected from suspected TB cases. Spoligotyping of 55 M. bovis isolates that originated either from cattle tissues with tuberculous lesion or from raw milk revealed seven spoligotype patterns where SB1176 was the most prevalent type (47.3%). Most isolates (89.1%) were of the M. bovis African 2 clonal complex. All sputum and FNA samples from 41 dairy farm workers with symptoms of TB were culture negative for any mycobacteria. Among the 41 TB suspected farm workers, 61% did not know about bTB in cattle and its zoonotic potential, and over two-third of these workers practiced raw milk consumption. Our spoligotype analysis suggests a wider transmission of a single spoligotype in the study area. The results reported here may be useful in guiding future work to identify the source and direction of bTB transmission and hence design of a control strategy. Isolation of M. bovis from milk, knowledge gap on zoonotic TB and practice of consumption of raw milk in the study population showed potential risk for zoonotic transmission.

Pathology of Bovine Tuberculosis in Three Breeds of Dairy Cattle and Spoligotyping of the Causative Mycobacteria in Ethiopia.

Different breeds of cattle were observed to have a variable degree of susceptibility to bovine tuberculosis (bTB). The screening of bTB was conducted on 720 dairy cattle consisting of three breeds using the single intradermal cervical comparative tuberculin (SICCT) test. Besides this, 43 SICCT test-positive cattle were used to compare the severity of the pathology of bTB among the three breeds and to identify the causative mycobacteria using spoligotyping. The overall SICCT test positivity was 17.92% (129/720) by pooling all animals in the three farms. There was a significant difference in SICCT test positivity among the three breeds (χ2 = 71.06; p < 0.001); the highest (25.34%) was recorded in the crossbreed followed by the Boran breed (10.08%), while the least (3.14%) was recorded in the Jersey breed. On other hand, the highest median pathology score (10.0, interquartile range, IQR = 6.0-17.0) was recorded in Boran followed by cross (5.0, IQR = 3.5-7.5), while the least (3.0, IQR = 2.25-3.0) was recorded in Jersey. Thus, the difference in the median pathology scores was significant [Kruskal Wallis χ ( 2 ) 2 = 18.78, p < 0.001] among the three breeds. Furthermore, multivariate analysis using ordinal logistic regression by considering age, sex, breed, reproductive status, and location of the farms also showed a significant [ χ ( 2 ) 2 = 11.97, p < 0.01] difference in pathology scores among the three breeds of cattle. Even at a single-herd level at Holeta, the difference in severity of pathology between the Boran and crossbreeds was significant (U = 33.5; p < 0.01). Culture positivity was 39% in 108 suspicious tissues. Fourteen Mycobacterium bovis (M. bovis) and two Mycobacterium tuberculosis (M. tuberculosis) were isolated from the lesions. All the 14 M. bovis isolates belonged to SB0912, while the two M. tuberculosis belonged to SIT54. In conclusion, although the frequency of the SICCT test positivity was high in the crossbreed, a more severe pathology was observed on the Boran (zebu) breed. In addition M. tuberculosis was isolated from TB lesions of dairy cattle, demonstrating the role of M. tuberculosis in causing TB in cattle.

Cellular and Cytokine Responses in Lymph Node Granulomas of Bacillus Calmette Guérin (BCG)-Vaccinated and Non-vaccinated Cross-Breed Calves Naturally Infected With Mycobacterium bovis.

Local immunological responses at the site of infections, such as at the lymph nodes and lungs, do play a role in containing infection caused by Mycobacterium bovis (M. bovis). This bovine tuberculosis (bTB) study was conducted to evaluate cellular and cytokine responses in the lymph nodes and lungs of BCG-vaccinated and non-vaccinated calves that were naturally infected with M. bovis. Immunohistochemical assays were used for examination of the responses of macrophages, T cells, cytokines and chemical mediators of 40 (22 vaccinated and 18 non-vaccinated) Holstein-Friesian-zebu crossbred calves that were naturally exposed for 1 year to a known bTB positive cattle herd. The incidence rates of bTB visible lesion were 68.2% (15/22) and 89% (16/18) in vaccinated and non-vaccinated calves, respectively. The local responses of CD4+ and CD8+ T cells, and those of IFN-γ and TNF-α within the lesions, were stronger (P < 0.05) in BCG-vaccinated calves than in non-vaccinated calves. However, there was no statistically significant difference between the two groups (P > 0.05) in the response of CD68+ cells. Thus, the findings of this study indicated stronger responses of a set of immunological cells and markers at the local granulomas of BCG-vaccinated calves than in non-vaccinated calves. Furthermore, BCG vaccination may also play a role in reducing the severity of the gross pathology at the primary site of infection.

Evaluation of the Efficacy of BCG in Protecting Against Contact Challenge With Bovine Tuberculosis in Holstein-Friesian and Zebu Crossbred Calves in Ethiopia.

Bovine tuberculosis (bTB) is prevalent in intensive dairy farms in Ethiopia. Vaccination could be an alternative control approach given the socio-economic challenges of a test-and-slaughter control strategy. The efficacy of the BCG was evaluated on 40 Holstein-Friesian (HF) and zebu crossbred calves recruited from single intradermal cervical comparative tuberculin (SICCT) test negative herds and randomly allocated into two groups. Twenty-two calves were vaccinated within 2 weeks of age, and 18 were kept as a control. Six weeks post-vaccination, the two groups were exposed and kept mixed with known SICCT test positive cows for 1 year. Immune responses were monitored by interferon gamma (IFN-γ) release assay (IGRA), SICCT test, and antibody assay. Vaccinated calves developed strong responses to the SICCT test at the sixth week post-vaccination, but did not respond to ESAT-6/CFP-10 peptide antigen-based IGRA. During the exposure, IFN-γ response to the specific peptide cocktail [F (2.44, 92.67) = 26.96; p < 0.001] and skin reaction to the specific proteins cocktail [F (1.7, 64.3); p < 0.001] increased progressively in both groups while their antibody responses were low. The prevalence of bTB was 88.9% (95% CI: 65.3-98.6) and 63.6% (95% CI: 40.7-83.8) in the control and vaccinated calves, respectively, based on Mycobacterium bovis isolation, giving a direct protective efficacy estimate of 28.4% (95% CI: -2.7 to 50.1). The proportion of vaccinated calves with lesion was 7.0% (34/484) against 11.4% (45/396) in control calves, representing a 38% (95% CI: 5.8-59.4) reduction of lesion prevalence. Besides, the severity of pathology was significantly lower (Mann-Whitney U-test, p < 0.05) in vaccinated (median score = 2.0, IQR = 0-4.75) than in control (median score = 5, IQR = 3.0-6.25) calves. Moreover, survival from M. bovis infection in vaccinated calves was significantly (log-rank test: χ2 = 6.749, p < 0.01) higher than that of the control calves. In conclusion, the efficacy of BCG was low, but the reduced frequency and severity of lesion in vaccinated calves could suggest its potential role in containing onward transmission.

Evaluation of the safety, immunogenicity and efficacy of three capripoxvirus vaccine strains against lumpy skin disease virus.

The safety, immunogenicity and efficacy of three commercially available vaccines against lumpy skin disease (LSD) in cattle have been evaluated using a combination of vaccine challenge experiments and the monitoring of immune responses in vaccinated animals in the field. The three vaccines evaluated in the study included two locally produced (Ethiopian) vaccines (lumpy skin disease virus (LSDV) Neethling and Kenyan sheep and goat pox (KSGP) O-180 strain vaccines) and a Gorgan goat pox (GTP) vaccine manufactured by Jordan Bio-Industries Centre (JOVAC). The latter vaccine was evaluated for the first time in cattle against LSDV. The Ethiopian Neethling and KSGPO-180 vaccines failed to provide protection in cattle against LSDV, whereas the Gorgan GTP vaccine protected all the vaccinated calves from clinical signs of LSD. There was no significant difference in protective efficacy detected between two dosage levels (P=0.2, P=0.25, and P=0.1 for KSGP, Neethling and Gorgan vaccines, respectively). Additionally, the Gorgan GTP vaccinated cattle showed stronger levels of cellular immune responses measured using Delayed-Type Hypersensitivity (DTH) reactions at the vaccination site indicating higher levels of immunogenicity produced by the GTPV vaccine in cattle, as opposed to the other two vaccines. This study indicated, for the first time, that the Gorgan GTP vaccine can effectively protect cattle against LSDV and that the Neethling and KSGP O-180 vaccine were not protective. The results emphasise the need for molecular characterization of the Neethling and KSGP O-180 vaccine seed viruses used for vaccine production in Ethiopia. In addition, the potency and efficacy testing process of the Ethiopian LSD Neethling and KSGP O-180 vaccines should be re-evaluated.

Study on seroprevalence, risk factors, and economic impact of foot-and-mouth disease in Borena pastoral and agro-pastoral system, southern Ethiopia.

Cross-sectional serological study and questionnaire survey were conducted in Borana pastoral and agro-pastoral area to determine seroprevalence and risk factors associated with foot-and-mouth disease (FMD) infection and to assess community perceptions as to importance of the disease. A multistage random sampling was carried out to select cattle for seroprevalence and households for interviews. Totally, 768 sera were collected from 111 herds. The overall individual level seroprevalence of 23.0% (n = 177) and herd level seroprevalence of 58.6% (n = 65) were recorded using 3ABC ELISA test. The variation of individual level seroprevalence in districts were statistically significant (P < 0.05) which was 29.9% in Arero, 24.0% in Yabello, and 15.7% in Teltele. From multivariate logistic regression analysis, herd size and age were seen to be significantly (P < 0.05) associated with FMD seroprevalence. The result of the questionnaire survey based on 120 respondents indicated that, the daily milk yield of cows infected with FMD during outbreaks is reduced to an average of 0.5 L for 25.5 days while cows developing heat-intolerance syndrome after acute infection gave an average 0.67 L for 3.8 months and their calving interval prolonged about 12 months. The questionnaire survey in agro-pastoral area of Borena also indicated that FMD-infected oxen remained off-plough for one season when outbreaks occur in cropping time, whereas heat-intolerant oxen were no longer used for traction. These findings of the present study indicated that FMD is a highly prevalent and economically important disease in the Borana pastoral and agro-pastoral production systems which need effective control strategy for the disease.