The recent detection of Highly Pathogenic Avian Influenza (HPAI) A(H5N1) in dairy cattle in the United States raises significant concerns regarding potential human exposure to the virus. This development follows several months of documented circulation of HPAI A(H5N1) in avian populations, poultry farms, and various mammalian species, including cattle. The extensive presence of the virus increases the likelihood of human contact, emphasizing the potential severity and zoonotic risk associated with certain HPAI variants. This situation highlights the urgent need for enhanced vigilance and stringent biosecurity measures to prevent the spread of the virus among animals and to safeguard human health. [8, 10, 11, 13]. One of the effective strategies for managing Avian Influenza Virus (AIV) in Birds involves vaccination programs, which are pivotal for its control. In Egypt, both monovalent and combined inactivated vaccines against AIV are currently employed to prevent infection [16, 20].
This study aimed to evaluate the efficacy of various doses of an inactivated H5 AI vaccine in cattle, as well as the antibody transmission in milk, against a recent bovine isolate of Highly Pathogenic Avian Influenza (HPAI) A(H5N1, clade 2.3.4.4b). The evaluation was conducted using the Hemagglutination Inhibition (HI) test and ELISA.
Calves from Groups 1 to 4 were subcutaneously inoculated with various doses of the AI vaccine, while the lactating cows in Group 5 were inoculated four weeks post-inoculation of the initial four groups to determine the appropriate dose. In our safety evaluation, the localized reactions observed in Groups 3 (mild to moderate) and 4 (moderate) for 4 ml (8x) and 6 ml (12x), respectively, were consistent with hypersensitivity type III responses, primarily affecting the injection site. Importantly, the administration of the vaccine did not impact milk production in Group 5 cattle (2 ml (4x)), and the rectal temperatures remained within normal ranges in all vacinated Groups and were comparable to the control values.indicating its safety concerning this critical aspect of cattle farming. In summary, our evaluation supports the overall safety of the inactivated H5 AI vaccine in cattle for doses of 2 ml (4x) and 3 ml (6x) for Groups 1 and 2, respectively, highlighting localized inflammatory responses as the primary concern at higher doses in Groups 3 and 4. These results provide valuable insights for optimizing vaccine formulations and administration protocols in cattle husbandry. Interestingly, another study in cattle observed a slight increase in body temperatures and local reactions at the inoculation site a few days post-vaccination with their inactivated Lumpy Skin Disease Virus vaccine, which persisted for several days[21]. Another study evaluated the safety of a Newcastle Disease-Avian Influenza (ND-AI) bivalent vaccine in chickens using Marcol white mineral oil adjuvant and Montanide ISA70 adjuvant. The results indicated that both vaccine candidates were free from foreign contaminants and safe for vaccinating chickens, as no detectable signs of infection were observed. Histopathological examinations revealed inflammatory lesions at the injection site. Tissue samples taken on days 3, 7, and 56 post-vaccination showed the presence of local inflammatory lesions, indicating a localized reaction to the vaccine [22].
In our study assessing the humoral immune response in calves immunized with various doses of an inactivated H5 AI vaccine, hemagglutination inhibition (HI) titers against the AIV clade 2.3.3.4b were measured. The HI titers at one week post-vaccination were 4, 5, 5, and 6 log2 for Groups 1, 2, 3, and 4, respectively. These results indicate a robust immune response to the inactivated H5 AI vaccine across all groups, even at the lower dose administered to Group 1 (2 ml 4x). By the fourth week post-vaccination, the HI titers peaked at 8, 9, and 9 log2 for Groups 2, 3, and 4, respectively, and remained elevated through the end of the study at six weeks post-vaccination. In contrast, Group 1 exhibited a stable HI titer of 6 log2 from the second week through the sixth week post-vaccination. In a similar study conducted on ducks and chicken using inactivated AI vaccines, it was observed that the ducks developed antibodies by the 3rd week post-vaccination against the H5 virus. The mean HI antibody titers against the H5-Re13 and H5-Re14 viruses ranged from 6.7 log2 to 7.2 log2. Additionally, the mean HI antibody titers against the DK/FJ/S1424/20 (H5N6) and WS/SX/4 − 1/20 (H5N8) viruses were 6.5 log2 and 6.2 log2, respectively, on the other hand, the mean HI antibody titers at 3 weeks post-vaccination in chickens against the vaccine strains H5-Re13 and H5-Re14 viruses ranged from 7.5 log2 to 8.1 log2. Furthermore, the mean HI antibody titers in chickens against the DK/FJ/S1424/20 (H5N6), WS/SX/4 − 1/20 (H5N8), DK/GX/S30428/21 (H5N6), and DK/GD/S4525/21 (H5N1) viruses were 7.3 log2, 7.5 log2, 6.9 log2, and 6.8 log2, respectively [23].
The statistical analysis revealed that the Friedman test statistic was computed as χ2 = 15, with a corresponding p-value of 0.0018. This finding indicates statistically significant differences in HI values among the vaccinated groups (Groups 1 to 4), as detailed in Table 1. The use of Python with SciPy facilitated a robust statistical examination of the HI values, offering valuable insights into the comparative immune responses triggered by various doses of the H5 AI vaccine in cattle. This analysis underscores the importance of dosage in eliciting distinct immune reactions, contributing to the understanding and optimization of vaccine strategies for bovine populations.
The humoral immune response observed was consistent with the ID Screen ELISA Influenza A Antibody Competition Multi-Species results. Initially, Group 1 showed negative results in the first week post-vaccination, whereas Groups 2, 3, and 4 displayed positive results. From the 2nd week through the 6th week post-vaccination, ELISA results indicated persistent and robust antibody presence in Groups 1, 2, 3, and 4, with Group 4 consistently exhibiting the strongest positivity. Although lower vaccine doses provided significant protection, the highest dose was most effective. Non-vaccinated cattle did not develop measurable antibodies against the virus, underscoring the necessity of vaccination. Interestingly, In a similar study, all chickens vaccinated with the inactivated AIV H9N2 vaccine seroconverted within 14 days post-vaccination as confirmed by competitive ELISA (cELISA). Conversely, all non-vaccinated birds remained seronegative. These findings were consistent with the results obtained from the HI test [24].
In lactating cows (Group 5) inoculated with 2 ml (4x) of inactivated H5 AI vaccine, Milk Antibody Transfer was observed. Initially, ID Screen ELISA Influenza A Antibody Competition Multi-Species results indicated negative antibody results in serum samples at the 1st week post-vaccination, while milk samples showed doubtful results. By the 2nd week post-vaccination, serum samples from Group 5 tested positive, whereas milk samples exhibited a strong positive response in ID ELISA testing. Our findings clearly demonstrated that the ID ELISA test had higher sensitivity for detecting antibodies against AIV in milk samples compared to serum samples. This discrepancy may be attributed to the larger volume of milk samples used in ID ELISA, as recommended by guidelines provided in Addendum FLUACA ver 220424 EN(related information), potentially reducing troubleshooting and interference issues observed with serum samples. In a 2014 study, a high level of agreement (94.4%) and concordance (kappa = 0.865) was reported between 125 paired serum and individual milk samples. The study suggested that individual milk could serve as a viable alternative to blood collection and serum testing[25]. Regarding AIV in cattle, no studies have yet established the duration for which antibodies can be detected in milk after natural infection or post-vaccination. Consequently, the results reported in this study should be considered in light of these limitations.
The limitations of this study can be addressed by noting that the humoral immune response and Milk Antibody Transfer were assessed using an AIV vaccine designed for birds. Various doses of the AI vaccine were administered based on the recommended chicken doses, and the number of animals in each group was sufficient for statistical analysis. a larger sample size would provide more reliable data. Nonetheless, the study proceeded with a carefully planned duration and well-managed sampling intervals. This step is crucial to establish the feasibility of the immune response of cattle to the current AI vaccine. To enhance the precision of the study, future investigations should delve deeper into factors influencing the formulation of AI vaccines for cattle, such as adjuvant and antigen content. Understanding and addressing these variables will contribute to a more accurate assessment of bovine immune response and immune receptors. Additionally, extending the monitoring period post-vaccination, increasing the sample size, and exploring different vaccine formulations could provide more comprehensive insights into the efficacy and longevity of the immune response in cattle. These efforts will help optimize vaccine strategies for bovine populations, ensuring better protection against AIV. By addressing these limitations and refining the study parameters, we can improve our understanding of the immune response in cattle to AI vaccines and develop more effective vaccination protocols tailored to bovine needs.
The study revealed a clear dose-dependent pattern in the cattle's immune response to the H5 avian influenza vaccine, with higher doses resulting in stronger and more sustained antibody responses. Even the lower doses provided significant antibody protection, achieving titers within the protective range of 5 to 7 log2, as established in chickens. A minimum hemagglutination inhibition (HI) titer of ≥ 5 log2 has been suggested as a serological potency standard indicative of protection, safeguarding birds against mortality. Furthermore, HI titers of ≥ 7 log2 have been associated with the prevention of virus shedding from the oropharynx in vaccinated birds [19, 26]. This response was corroborated by the ID Screen ELISA results. Notably, the humoral immune response in all vaccinated groups elicited a rapid and high antibody response within the early weeks post-vaccination. This response stabilized quickly and persisted consistently throughout the experimental period.
However, the cattle's immune response to the H5 AI vaccine differed markedly from their responses to other vaccines, such as those for Foot and Mouth Disease Virus (FMDV) [27, 28, 29] and Lumpy Skin Disease Virus (LSDV) [30]. Several factors could explain these differences. The adjuvant and antigen composition of the H5 AI vaccine may be particularly effective in inducing a strong and stable immune response in cattle, unlike the formulations used for FMDV and LSDV vaccines. Additionally, the H5 AI vaccine may possess higher immunogenicity in cattle compared to the FMDV and LSDV vaccines, leading to a more robust production of antibodies. The nature of the H5 avian influenza virus itself may trigger a different immune mechanism in cattle[31], resulting in a distinct and more effective antibody response compared to other viruses. Furthermore, the observation of a dose-dependent response underscores the importance of optimizing vaccine dosage, as higher doses of the H5 AI vaccine achieved better immune responses, a factor that might need consideration for other vaccines. Finally, the rapid stabilization and persistence of the antibody response to the H5 AI vaccine suggest that the immune system of cattle responds more favorably to this particular vaccine's stimulation.