Gamma-Irradiated Fowl Cholera Mucosal Vaccine: Potential Vaccine Candidate for Safe and Effective Immunization of Chicken Against Fowl Cholera

Fowl cholera (FC) caused by Pasteurella multocida is among the serious infectious diseases of poultry. Currently, formalin inactivated FC (FI-FC) vaccine is widely used in Ethiopia. However, reports of the disease complaint remain higher despite the use of the vaccine. The aim of this study was to develop and evaluate gamma-irradiated mucosal FC vaccines that can be used nationally. In a vaccination-challenge experiment, the performance of gamma-irradiated P. multocida (at 1 kGy) formulated with Montanide gel/01 PR adjuvant was evaluated at different dose rates (0.5 and 0.3 ml) and routes (intranasal, intraocular, and oral), in comparison with FI-FC vaccine in chicken. Chickens received three doses of the candidate vaccine at 3-week intervals. Sera, and trachea and crop lavage were collected to assess the antibody levels using indirect and sandwich ELISAs, respectively. Challenge exposure was conducted by inoculation at 3.5×109 CFU/ml of P. multocida biotype A intranasally 2 weeks after the last immunization. Repeated measures ANOVA test and Kaplan Meier curve analysis were used to examine for statistical significance of antibody titers and survival analysis, respectively. Sera IgG and secretory IgA titers were significantly raised after second immunization (p=0.0001). Chicken survival analysis showed that intranasal and intraocular administration of the candidate vaccine at the dose of 0.3 ml resulted in 100% protection as compared to intramuscular injection of FI-FC vaccine, which conferred 85% protection (p=0.002). In conclusion, the results of this study showed that gamma-irradiated FC mucosal vaccine is safe and protective, indicating its potential use for immunization of chicken against FC.

First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera

Background and Aim: Fowl cholera (FC) caused by Pasteurella multocida is a highly contagious bacterial disease of global importance for poultry production. The severity and incidence of FC caused by P. multocida may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment, and the bacterial strain. This study aimed to investigate the genetic diversity of multidrug-resistant P. multocida strains isolated from FC outbreaks in laying hens from commercial farms of Bangladesh. Materials and Methods: We collected 57 samples of suspected FC, including 36 live and 21 dead laying hens. P. multocida isolates were characterized by biochemical and molecular-biological methods. Results: Twenty-two strains of P. multocida were isolated from these samples through phenotypic and genotypic characterization. The strains were grouped into two distinct random amplification of polymorphic DNA (RAPD) biotypes harboring a range of pathogenic genes; exbB, ompH, ptfA, nanB, sodC, and hgbA. In this study, 90.90% and 81.82% P. multocida strains were multidrug-resistant and biofilm formers, respectively. Whole-genome sequencing of the two representative RAPD phylotypes confirmed as P. multocida type B: L2:ST122, harboring a number of virulence factors-associated genes (VFGs), and antimicrobial resistance (AMR) genes (ARGs). In addition, pan-genome analysis revealed 90 unique genes in the genomes of P. multocida predicted to be associated with versatile metabolic functions, pathogenicity, virulence, and AMR. Conclusion: This is first-ever report on the association of P. multocida genotype B: L2:ST122 and related VFGs and ARGs in the pathogenesis of FC in laying hens. This study also provides a genetic context for future researches on the evolutionary diversity of P. multocida strains and their host adaptation.

Prevalence of some common bacterial diseases in commercial poultry farm

Bacterial disease of poultry is one of the major constraints to the expansion of poultry industry. The study was undertaken to investigate some common bacterial diseases in commercial poultry farm. A total of 100 sick and dead chickens (67 broilers, 26 layers and 7 sonali) were collected from different poultry farms which were subjected to postmortem examination for tentative diagnosis. After the post-mortem examination, out of 100 collected dead chickens, bacterial diseases were confirmed 58 %. Among them 52 % of the chickens were diagnosed tentatively to be the case of colibacillosis, 4 % salmonellosis, and 2 % of fowl cholera. In post-mortem examination, some pathological lesions like: omphalitis, fibrinopurulent fluid accumulation in peritoneal cavity, air sacculitis, pericarditis and perihepatitis, extreme congestion and septicemia in intestine for colibacillosis infection; unabsorbed yolk mass, bronze discoloration and friable liver, hemorrhages in spleen, misshaped ova for salmonellosis as well as swollen and hardening of comb, congestion of skin, multiple pin point pale color necrotic lesion on liver, pin point hemorrhage on fat muscle of heart were observed for fowl cholera infection. Hence, this study will definitely help to perceive the prevalence of common bacterial diseases like colibacillosis, salmonellosis and fowl cholera infection in commercial poultry farm.

Immunogenicity and Protective Efficacy of Iron-inactivated Pasteurella multocida A:1 Vaccine against Fowl Cholera in Backyard Chicken

Background: Fowl cholera is a highly fatal, contagious bacterial disease that incurs significant economic loss in commercial as well as back-yard poultry. Vaccination is the most effective way in controlling this disease. In this study, we prepared and evaluated the immunogenicity of iron- inactivated Pasteurella multocida A:1 vaccine and its protective efficacy against fowl cholera experimental infection in backyard chicken.Methods: Scaled up Pasteurella multocida A:1 culture with 5 X 108 CFU/ml equivalent to 2.5 mg of antigen per dose was used for preparation of experimental vaccines. Formalin inactivated and mixed with APS adjuvant (FIA), formalin inactivated-Freund adjuvant (FIF), Iron inactivated and adjuvanted with iron (III), Iron inactivated from iron supplemented media and adjuvanted with iron (ISII) and commercial oil emulsion vaccine (CV) were used in the study. A total of 120 Vanaraja birds (n=20/group) of 2 weeks age were immunized with these vaccine and booster were given at 3rd and 6th week with respective vaccine. Specific antibody titers were assessed by iELISA in the serum at weekly intervals. The birds were challenged (n=6 /group) with 5x104 CFU/ml of virulent isolate by intraperitoneal route and morbidity, mortality percentage were observed.Result: Protective antibody titers were induced by iron inactivated vaccine from 4th week of immunization and upon booster doses it induced significantly higher (P less than 0.05) antibody response. The iron inactivated experimental vaccine gave equivalent protection as that of commercial vaccine upon challenge infection.

Immune Responses Induced in Chickens by Capsular Extract of P. Multocida Isolated From Farm Rat

An experiment was conducted to investigate the immune response induced in chickens by capsular extract of Pasteurella multocida isolated from rats wandering in and around the poultry farms. The rat isolate of P. multocida was isolated and identified by cultural, morphological, and biochemical characteristics, followed by capsular extract preparation and experimental vaccine development. The isolated P. multocida was found Gram-negative, non-motile, non-spore forming rod occurring singly or pains and occasionally as chains or filaments in Gram’s-staining method. The isolates consistently produced acid from dextrose, sucrose and mannitol but not fermented maltose or lactose. The Capsular antigen was extracted and confirmed by acriflavine test. Finally, experimental fowl cholera vaccine was prepared. Primary vaccination was performed at the dose rate of 5.6×107 CFU/ml through intramuscular and subcutaneous routes in birds of group A (10 birds) and group B (10 birds) and group C (10 birds) were control birds. Secondary vaccination was similarly performed after 15 days of primary vaccination in groups A and B. The levels of pre-vaccination and post-vaccination sera were determined by passive haemagglutination test. The passive haemagglutination antibody titre was recorded on 15 and 35 days of post vaccination in groups A and B. It was demonstrated that experimental capsular extract fowl cholera vaccine conferred 100% protection (p<0.01) against challenge infection and found to be safe. It could be suggested that after thorough field trial, the experimentally prepared capsular extract FC vaccine using rat isolate of P. multocida may be used side by side with conventional FC vaccine. Res. Agric., Livest. Fish.8(1): 117-124, April 2021

Novel multi-strain probiotics reduces Pasteurella multocida induced fowl cholera mortality in broilers

Pasteurella multocida causes fowl cholera, a highly contagious poultry disease of global concern, causing significant ecological and economic challenges to the poultry industry each year. This study evaluated the effects of novel multi-strain probiotics consisting of Lactobacillus plantarum, L. fermentum, Pediococcus acidilactici, Enterococcus faecium and Saccharomyces cerevisiae on growth performance, intestinal microbiota, haemato-biochemical parameters and anti-inflammatory properties on broilers experimentally challenged with P. multocida. A total of 120 birds were fed with a basal diet supplemented with probiotics (108 CFU/kg) and then orally challenged with 108 CFU/mL of P. multocida. Probiotics supplementation significantly (P < 0.05) improved growth performance and feed efficiency as well as reducing (P < 0.05) the population of intestinal P. multocida, enterobacteria, and mortality. Haemato-biochemical parameters including total cholesterol, white blood cells (WBC), proteins, glucose, packed cell volume (PCV) and lymphocytes improved (P < 0.05) among probiotic fed birds when compared with the controls. Transcriptional profiles of anti-inflammatory genes including hypoxia inducible factor 1 alpha (HIF1A), tumor necrosis factor- (TNF) stimulated gene-6 (TSG-6) and prostaglandin E receptor 2 (PTGER2) in the intestinal mucosa were upregulated (P < 0.05) in probiotics fed birds. The dietary inclusion of the novel multi-strain probiotics improves growth performance, feed efficiency and intestinal health while attenuating inflammatory reaction, clinical signs and mortality associated with P. multocida infection in broilers.

Genomic diversity and molecular epidemiology of Pasteurella multocida

Pasteurella multocida is a bacterial pathogen with the ability to infect a multitude of hosts including humans, companion animals, livestock, and wildlife. This study used bioinformatic approaches to explore the genomic diversity of 656 P. multocida isolates and epidemiological associations between host factors and specific genotypes. Isolates included in this study originated from a variety of hosts, including poultry, cattle, swine, rabbits, rodents, and humans, from five different continents. Multi-locus sequence typing identified 69 different sequence types. In-silico methodology for determining capsular serogroup was developed, validated, and applied to all genome sequences, whereby capsular serogroups A, B, D, and F were found. Whole genome phylogeny was constructed from 237,670 core single nucleotide variants (SNVs) and demonstrated an overall lack of host or capsular serogroup specificity, with the exception of isolates from bovine sources. Specific SNVs within the srlB gene were identified in P. multocida subsp. septica genomes, representing specific mutations that may be useful for differentiating one of the three known subspecies. Significant associations were identified between capsular serogroup and virulence factors, including capsular serogroup A and OmpH1, OmpH3, PlpE, and PfhB1; capsular serogroup B and HgbA and PtfA; and capsular serogroup F and PtfA and PlpP. Various mobile genetic elements were identified including those similar to ICEPmu1, ICEhin1056, and IncQ1 plasmids, all of which harbored multiple antimicrobial resistance-encoding genes. Additional analyses were performed on a subset of 99 isolates obtained from turkeys during fowl cholera outbreaks from a single company which revealed that multiple strains of P. multocida were circulating during the outbreak, instead of a single, highly virulent clone. This study further demonstrates the extensive genomic diversity of P. multocida, provides epidemiological context to the various genotyping schemes that have traditionally been used for differentiating isolates, and introduces additional tools for P. multocida molecular typing.

Characterisation and pathogenicity of Pasteurella multocida capsular serogroup A isolates from Awassi sheep in Syria

The aim of the present study was to evaluate the prevalence of serogroup A of Pasteurella multocida in Syrian Awassi sheep. Of 1630 samples collected from nasal swabs of healthy and pneumonic sheep (125 herds) and pneumonic sheep lungs, a total of 228 (13.9%) strains were isolated and identified as P. multocida subsp. multocida by phenotypic and biochemical characterisation. However, of them only 117 (51.3%) were identified as serogroup A of P. multocida when PCR assay with specific primers for serogroup A strains was applied. The highest rate of serogroup A isolation was from apparently healthy sheep (49.6%) with consideration that all lung isolates (23 isolates) belonged to serogroup A. Geographical and seasonal distribution showed that about 60% of positively isolated bacteria originated from Syrian desert (29 isolates) and central parts of semi-arid step zone (41 isolates). A significant increase (P≤0.05) in the rate of positive isolates was observed in winter as compared to spring. Pathogenicity tests of 10 isolates with 50 or 10 LD50 values showed that 5 isolates were able to induce symptoms of fowl cholera in challenge-exposed chickens indicating that migratory Awassi sheep might serve as a carrier for serogroup A of P. multocida and that ovine isolates may be virulent for local breed of chickens.

THE EFFICACY OF VACCINATION OF LAYER CHICKENS WITH INACTIVATED FOWL CHOLERA BACTERIN PREPARED FROM LOCAL EGYPTIAN STRAINS OF Pasteurella multocida

This study was carried out to evaluate the efficacy of vaccination of layer chickens with inactivated FC bacterin prepared from local Egyptian strains of Pasteurella multocida (P. multocida). A total of 200 layer chickens were divided into 5 equal groups, 40 for each. At the age of 6 weeks, chickens in groups (A) and (B) were vaccinated with P. multocida serotypes A:1 and A:3, respectively, booster doses were given after 3 weeks (9 weeks old) and challenge was done with virulent serotypes A:1 and A:3 at 2 weeks later (11 weeks old). Chickens in groups (C) and (D) were not vaccinated, only challenged with P. multocida serotype A:1 and A:3, respectively. Birds in group (E) were kept as non-vaccinated and non-challenged. Blood samples were collected weekly from all groups for humoral immune response. All the birds were kept under observation for signs, mortalities, lesions and re-isolation of challenging organism and for histopathological examination. Results of the mean Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that the highest level was at 5 weeks post vaccination as the titers reached to 3970 in group (A) and 3905 in group (B). The clinical signs, mortality rate and lesions were mild in the vaccinated birds while severe lesions were in non-vaccinated and challenged birds. The protection rates were 85 % and 80 % in groups (A) and (B); respectively, while 10 % and 20 % in groups (C) and (D); respectively. The re-isolation rates of P. multocida after challenge were 95 % and 90 % in non-vaccinated-challenged birds with P. multocida serotypes A:1 and A:3; respectively, while they were 25 % and 15 % in vaccinated-challenged groups with P. multocida serotypes A:1 and A:3; respectively. Histopathological examination of P. multocida vaccinated-challenged birds revealed mild to no microscopic lesions when compared with non-vaccinated challenged chickens. In conclusion, the prepared FC inactivated bacterin from the local Egyptian predominant P. multocida serovars proved efficacy and protection of layer chickens. Key words: Pasteurella multocida; chickens; immunization; protection; Egypt UČINKOVITOST CEPLJENJA KOKOŠI NESNIC Z INAKTIVIRANO BAKTERIJO KOLERE PERJADI, PRIPRAVLJENE IZ LOKALNIH EGIPTOVSKIH SEVOV BAKTERIJE Pasteurella multocida Povzetek: Raziskava je bila izvedena z namenom ocenitve učinkovitosti cepljenja kokoši nesnic z inaktivirano bakterijo FC, pripravljeno iz lokalnih egiptovskih sevov bakterije Pasteurella multocida (P. multocida). Skupno 200 kokoši nesnic je bilo razdeljenih v 5 enakih skupin. V vsaki skupini je bilo 40 kokoši. Pri 6 tednih smo kokoši v skupinah A in B cepili s serotipoma P. multocida A:1 in A:3, po 3 tednih, ko so bile živali stare 9 tednov, so dobile poživitvene doze cepiva. Po dveh tednih (v starosti 11 tednov) so bile kokoši okužene z virulentnima serotipoma A:1 in A:3. Piščanci v skupinah C in D niso bili cepljeni temveč samo okuženi s serotipoma A:1 in A:3. Kokoši v skupini E niso bile niti cepljene, niti okužene. Vzorci krvi so bili odvzeti pri vseh skupinah tedensko za preverjanje humoralnega imunskega odziva. Vse kokoši smo stalno opazovali in beležili prisotnost bolezenskih znakov, različnih ran in umiranje kokoši. Pri poginulih kokoših smo osamili bakterije ter opravili histopatološki pregled. Rezultati encimsko-imunskega testa (ELISA) so pokazali da je bila najvišja stopnja zaščite dosežena 5 tednov po cepljenju, saj so titri dosegli 3970 v skupini A in 3905 v skupini B. Klinični znaki, stopnja umrljivosti in rane so bili pri cepljenih kokoših blagi, hude rane pa so bile vidne pri necepljenih in okuženih kokoših. Stopnja zaščite je bila v skupinah A in B 85- oziroma 80-odstotna, v skupinah C in D pa 10- oziroma 20-odstotna. Stopnje ponovne izolacije P. multocida po okužbi so bile 90 in 95 odstotkov pri kokoših, ki niso bile cepljene, medtem, ko so bile v skupinah, ki so bile okužene s P. multocida serotipa A:1 in A:3 15- in 25-odstotkov. Histopatološki pregled cepljenih in okuženih kokoši je pokazal popolno odsotnost ali prisotnost blagih mikroskopskih poškodb, medtem ko so imele necepljene okužene kokoši bolj obsežne histopatološke poškodbe. Pripravljena inaktivirana bakterija FC iz lokalnih egiptovskih prevladujočih serovarov P. multocide se je izkazala za učinkovito zaščito kokoši nesnic.Ključne besede: Pasteurella multocida; kokoši; imunizacija; zaščita; Egipt

Retrospective study on livestock vaccine coverage and trends in Digelu-tijo district, Arsi zone

Ethiopia has huge numbers of livestock hampered with high prevalence of infectious disease due to poor disease prevention and control. A five-year retrospective study was conducted to collect data on commonly diagnosed bacterial and viral disease and associated vaccine available at district government veterinary clinic in 2019. The commonly encountered bacterial and viral cattle diseases recorded in the case record book were LSD, CBPP Black leg, Anthrax, Bovine pasteurellosis, and Mastitis. Sheep and goat pox disease, Ovine Pasteurellosis, PPR, CCPP, and anthrax were the common diseases of sheep and goat in the study district. Regarding diseases of poultry, New castle, Infectious Bursal Diseases (Gumboro), Infectious coriza, chicken pox, Coccidiosis, Fowl Typhoid, Fowl Cholera, and Marex are the major once. The commonly available vaccines in the study district were LSD, Black leg, Anthrax, Bovine pasteurellosis, Ovine pasteurellosis, Sheep and Goat Pox, PPR, and vaccine for chicken disease like New castle disease vaccine, Gumboro, Fowl Pox, Fowl typhoid and Fowl cholera were used. Vaccination coverage for Lumpy Skin Disease, Sheep and Goat pox and Newcastle viral diseases were good compared with other diseases. The majority 36% of the population of cattle were vaccinated for LSD in 2019 and the lowest 16% in 2015.The lower portion of shoat 8% and half of the population 50% were vaccinated in 2017 and 2019 respectively. Maximum proportion of Newcastle and Gumboro vaccination coverage were 53% and 42%. The vaccination trends for most of the vaccines were fluctuating from year to year whereas the vaccination trend for LSD was increasing from year to year.