Sero-Prevalence and Risk factors of Contagious Bovine Pleuropneumonia (CBPP) in Afgoye District Lower Shabelle Region, Somalia

Contagious bovine Pleuropneumonia (CBPP) is an infectious and highly contagious respiratory disease of cattle and water buffalo, caused by the Mycoplasma mycoides subspecies mycoides. It induces significant economic losses and leads to a severe livestock production problem, negatively influencing people’s livelihoods of affected countries. In Somalia, there is no updated data on the prevalence and distribution of the disease. Hence, a descriptive cross-sectional study was conducted from September 2022 to June 2023 in different villages under the Afgoye District of lower Shabelle region, Somalia. The main purpose of this study is to assess the sero-prevalence and identify the associated risk factors for the occurrence of the disease. In this study, villages, age, sex, breed, and body condition were considered as risk factors. A total of 90 blood samples were collected and tested in the laboratory using the Anti-CBPP Elisa kit test. Out of 90 serum samples from herd cattle, 32 were positive, resulting in an overall prevalence of 35.5%. In addition, we found a statistically significant variation between the prevalence of the disease and factors such as sex, age, body condition and breeds. In summary, the overall prevalence of Contagious bovine pleuropneumonia in this study area is worth to be considered because there is a low quality of health care and less awareness of the Contagious bovine Pleuropneumonia effects on herds, which warrants the official authorities to act and follow appropriate preventive and control measures to reduce the incidence of the disease and generate appropriate controlling and prevention measures in all regions of Somalia.

The dose regimen formulation of doxycycline hydrochloride and florfenicol injection based on ex vivo pharmacokinetic-pharmacodynamic modeling against the Actinobacillus pleuropneumoniae in pigs

Doxycycline hydrochloride and florfenicol combination(DoxHcl&FF)is an effective treatment for respiratory diseases.In the study,our objective Was to evaluate the activity of DoxHcl&FF against Actinobacillus pleuropneumoniae(APP)in porcine pulmonary epithelial lining fluid(PELF)and the optimal dosage scheme to avoid the development of resistance.The DoxHcl&FF Was administered intramuscularly(IM)at 20mg/kg,and the PELF was collected at differ-ent time points.The minimum inhibitory concentration(MIC)and time-mortality curves were also included in the study.Based on the sigmoid Emax equation and dose equations,the study integrated the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data to obtain the area under concentration time curve(AUCo-24h)MIC values in PELF and achieve bacteriostatic activity,bactericidal activity and the virtual eradication of bacteria.The study showed that the combination of DoxHcl and FF caused no significant changes in PK parameters.The peak concentration(Cmax)of FF in healthy and diseased pigs was 8.87±0.08 and 8.67±0.07μg/mL,the_AUCo-24h were.172.75±2.52 and 18022±3.13 h-μg/mL,the Cmax of DoxHcl was 7.91±0.09 and 7.99±0.05μg/mL,and the AUCo-24h was 129.96±3.70 h-μg/mL and 169.82±4.38 h-μg/mL.DoxHcl&FF showed strong concentra-tion-dependent tendencies.The bacteriostatic,bactericidal,and elimination activity were calculated as 5.61,18.83 and 32.68 h,and the doses were 1.37(bacteriostatic),4.59(bactericidal)and 7.99(elimination)mg/kg.These findings indicated that the calculated recommended dose could assist in achieving more precise administration,increasing the effectiveness of DoxHcl&FF treatment for APP infections.

Identification and Detection of Actinobacillus pleuropneumoniae in Infected and Subclinically Infected Pigs by Multiplex PCR Based on the Genes ApxIVA and OmlA

PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3＇-terminus of the species-specific apxlVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 （26%） of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

An abattoir survey of contagious bovine pleuropneumonia lesions in slaughtered cattle in selected districts in Northern Tanzania

Objective:To establish and estimate incidence of contagious bovine pleuropneumonia(CBPP),using abattoir survey as a diagnostic tool in slaughtered cattle in Northern Tanzania.Methods:A total of 4460 cattle were slaughtered in five abattoirs in 3 northern zone regions(Arusha,Kilimanjaro and Tanga)during the period of January to May 2004.They were examined ante-mortem for‘pneumonia signs',and‘characteristic contagious bovine pleuropneumonia(CBPP)lung lesions'.Results:Forty-one(0.91%)of the slaughtered cattle,the majority of which were Tanzania short horn zebu,had gross lung lesions suggestive of CBPP.The prevalence of lesions was significantly(P<0.05)higher in Karatu abattoir compared to others.No animal was detected to have lesion in Bomang'ombe abattoir.The most observed pneumonic signs included labored breathing(90%),dry cough(57%)and mucopurulent nasal discharge(47%).The gross characteristic CBPP pathological lesion,frequently encountered was left lung lesion(47%),pinkish lung(71%)and pleural adhesion(98%).Epidemiological reports show that the CBPP reported outbreaks increased from 19 in 2002,65 in 2003 and 18 in 2004(January-March).The corresponding number of reported deaths increased from 137 in 2002,269 in 2003 and 77 in 2004(January-March).Conclusions:It's concluded from this study that CBPP is a problem in spite of the extensive awareness and vaccination campaigns.Nevertheless,a continued surveillance programme including routine checks of all cattle carcasses at the abattoir and subsequent epidemiological investigation of suspected cases are recommended.

Cloning, Expression of apxl Gene of Actinobacillus pleuropneumoniae and Development of ELISA

Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 - 8 326 bp)gene fragment was amplified by PCR from the isolated strain of A. pleuropneumoniae serovar 1. Then, it was cloned into pMD18-T, identified by both restriction endonuclease and sequence analysis, and inserted into pET-28a expression vector to yield the expression plasmid. SDS-PAGE result indicated expression of apxICA in BL21 (DE3), Western blot analysis showed the protein's immunogenicity. Using the expressed protein, ELISA was established to detect serum antibody against ApxI. The feature of ELISA to detect highly virulent A. pleuropneumoniae strains infection was proved by primary clinical application.

Presence of <i>Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae</i>in upper respiratory tract of swine in farms from Aguascalientes, Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms inAguascalientes,Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms);17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2;30% were positive for H. parasuis (93% of farms);23% of the samples to P. multocida (in 79% of farms);and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied;therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.

In Vitro Activity and Postantibiotic Effect of Tulathromycin against Actinobacillus pleuropneumonia

The postantibiotic effect （PAE） is defined as the suppression of bacterial growth persisting after a short exposure of bacterial cultures to an antibiotic. This phenomenon has obvious clinical interest, because antibiotic agents which produce extensive PAEs could be administrated less frequently without reducing efficacy. The minimum inhibitory concentration （MIC） and PAE of tulathromycin and erythromycin on five isolates of Actinobacillus pleuropneumonia were determined. All strains were susceptible to tulathromycin （ MIC： 0.25 - 1.00 μg/ml） and resistant to erythromycin （MIC： 32 -128 μg/ml）. The mean PAEs of 1 xMIC, 2 xMIC, 4 xMIC and 8 xMIC of tulathromycin against the tested isolates were 0.62, 1.53, 2.70 and 4.50, respectively. These results indicated that tulathromycin might be administered at longer time intervals than those already applied. This is in tavorite of the clinical rational use of antimicrobial druqs.

ArcA Gene Expression Level of Actinobacillus pleuropneumoniae in Anaerobic Culture

In the present study,mRNA levels of the ArcA gene of Actinobacillus pleuropneumoniae serotype1 were measured during response to stress by growing under anaerobic conditions.Expression levels were measured by semi-quantitative RT-PCR using the housekeeping gene recF as an internal standard.The expression of ArcA was undetectable until about 3 hours under standard culture conditions,but it was promptly and highly expressed in anaerobic culture.The results are consistent with ArcA being a potential virulence gene of Actinobacillus pleuropneumoniae,and likely playing an important role in pathogenesis caused by this organism.

Study on Formation and Inhibition Mechanism of Biofilm of Pig Actinobacillus pleuropneumoniae

Pig Actinobacillus pleuropneumoniae(App) could induce chronic respiratory tract infection in pigs, which causes major economic losses on pig industry. Bacterial biofilm(BBF) is bacterial community adsorbed on the surface of biomaterials or body cavity, to protect bacteria escape, and recurrent outbreaks of related infectious diseases and chronic infections resulting therefrom are called bacterial biofilm diseases. App BF belongs to polymers with spatial structure in vitro, and its formation is regulated by multiple genes. Among them, gene deletion of the key component TolC of multidrug efflux pumps and type I secretion systems causes that App BF adhesion weakens; gene deletion of catalytic core ClpP of Clp proteolytic complex induces the inhibition of BF formation; outer membrane lipoprotein VacJ of App promotes BF formation; gene deletion of active enzyme LuxS enhances the formation of App BF and decreases bacterial adhesion ability; gene deletion of Adh obviously declines bacterial accumulation, BF formation and adhesion to host cells. In this paper, BF formation or inhibition mechanism in App is elaborated from molecular level, which could provide reference basis for exploring the prevention of its biofilm diseases.

Genetic Evolution Analysis of Actinobacillus pleuropneumoniae

In order to explore the genetic evolution of Actinobacillus pleuropneumoniae(App) in different countries and clarify the relationships among different App in each region, the 16 S rRNA gene of App in the NCBI nucleotide database was analyzed and compared by the bioinformatics method. The phylogenetic tree was constructed after tailoring alignment. The results showed that a stable genetic phenomenon was indicated in the evolutionary process of App. The isolates derived from China were clustered and showed a high degree of conservation. They had a certain genetic relationship with the British and American strains, but had far relationship with the strains from Japan which was a neighboring country of China. The isolates from different countries in the Eurasian continent shared high homology. The isolates of the two regions originated from common ancestors.

Cloning and Expression of Actinobacillus pleuropneumoniae Gene Coding for TbpA and Development of an Indirect TbpA-ELISA

This study presents the cloning and expression of gene encoding transferrin-binding protein A from Actinobacillus pleuropneurnoniae in Escherichia coli expression system and the development of an indirect TbpA-ELISA. The gene coding TbpA was amplified from the A. pleuropneumoniae serotype 2 genome using polymerase chain reaction and cloned to pET-28b expression vector under the control of strong, inducible T7 promoter. The recombinant plasmid was expressed in E. coli BL21 （DE3）. The expressed fusion protein was analyzed using SDS-PAGE and Western blotting. The diagnostic potential of recombinant TbpA （rTbpA） was evaluated through an antibody-detection indirect ELISA based on the purified rTbpA. The TbpA antibodies were detectable in mice on day 7 after vaccination with purified rTbpA protein or infection with A. pleuropneumoniae serotype 10 with the TbpA-based ELISA. In addition, the TbpA-ELISA was able to detect 12 serotyping rabbit antisera postinoculation （PI） with A. pleuropneumoniae 12 serotypes experimentally. The comparable result was obtained by detecting the 117 clinical serum samples using, respectively, the TbpA-ELISA and indirect hemagglutination test （IHA） based on multiplex antigen. The result indicates that the TbpA-ELISA was the more sensitive method compared with the Mix-IHA method because of its consistent presence in A. pleuropneumoniae serotypes. In conclusion, the conserved TbpA of A. pleuropneumoniae can be used for the development of a cross-serotype diagnostic method for the detection of antibodies against A. pleuropneurnoniae.

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

A Serological Survey of Contagious Caprine Pleuropneumonia in Taizhou City,Jiangsu Province

[ Objective] This study aimed to investigate the prevalence of contagious caprine pleuropneumonia in Taizhou City, Jiangsu Province. [ Method] By using indirect hemagglutination assay kits for detection of antibodies against Mycoplasma capricolum subsp, capripneumoniae （ Mecp）, Mycoplasma mycoides subsp. capriclonum （Mmc） and Mycopalsam ovipneumonia （ Movi）, 1 157 goat sera from Taizhou City of Jiangsu Province were detected. [ Result ] The average positive detection rate of Mccp and Mmc in infected goats was 51.85% and 39.81%, respectively; the average positive detection rate of Mccp and Mmc in non-infected goats was 10.52% and 5.84%, respectively, and the total positive detection rate of Mycoplasma infections was 16.37%. [ Conclusion ] Mycoplasma infections oc- cur commonly in Taizhou City. The prevention and control of Mycoplasma infections should be strengthened.

Diagnosis and Treatment on Contagious Pleuropneumonia and Escherichia coli Mixed Infection in Sheep

[ Objective] In order to provide a reference for prevention and control of contagious pleuropneumonia and E. coli mixed infection. [ Meth- od] A comprehensive introduction of the two diseases was given on the following aspects： morbidity, clinical symptoms, pathological changes, la- boratory diagnosis, treatment, prevention, and prevention and control measures. [ Results] Sound development of sheep production was seriously influenced by the two diseases, effective prevention and control measures must be adopted to inhibit the occurrence and prevalence of sheep conta- gious pleuropneumonia and colibacillosis. [ Conclusion] The two epidemic diseases were controllable.

Development of Contagious Caprine Pleuropneumonia Inactivated Vaccine( M1601 Strain)

Three batches of contagious caprine pleuropneumonia inactivated vaccine( M1601 strain) developed by the laboratory were studied from the aspects of safety,minimum immune dose,immunity duration and storage life. The results showed that the vaccine was safe to goats under different physiological conditions.Regardless of lambs or adult goats,the minimum immune dose was 3 m L,and the immunity duration and the storage life were 6 and 12 months,respectively.

Study on Antibody Dynamics against Porcine Contagious Pleuropneumonia in Piglets

In order to provide basis for establishing a scientific and reasonable immune procedure against porcine contagious pleuropneumonia in pig farms, antibody dynamics in piglets inoculated with two vaccines against porcine contagious pleuropneumonia （ APP-1,2, 7 ） were detected. The result showed that average antibody blocking rate and antibody positive rate in piglets inoculated with two vaccines against porcine contagious pleuropneumonia exhibited similar variation trend. At 21 day after primary vaccination, average antibody blocking rate and antibody positive rate against porcine contagious pleuropneumonia in piglets reached the peak and then declined gradually. After secondary vaccination, average antibody blocking rate and antibody positive rate against porcine contagious pleuropneumonia in piglets increased gradually, which reached the second peak at 30 day after secondary vaccination （60 day after primary vaccination） ; subsequently, average antibody blocking rate declined gradually but still remained at a high level; antibody positive rate had little change. Average antibody blocking rate against APP-1 was slightly lower than the critical level of antibody protection at 44 day after primary vaccination ; antibody positive rate was slightly higher than the qualification rate at 37 day after primary vaccination; average antibody blocking rate against APP-7 was close to the critical level of antibody protection at 30 day after primary vaccina- tion, suggesting that piglets should be inoculated twice with polyvaccine against porcine contagious pleuropneumonia during the entire growth period, and the secondary vaccination should be performed at 30 -37 day after primary vaccination.

The QseB/QseC two-component system contributes to virulence of Actinobacillus pleuropneumoniae by downregulating apf gene cluster transcription

Actinobacillus pleuropneumoniae(APP)is the major pathogen of porcine contagious pleuropneumoniae(PCP).The QseB/QseC two-component system(TCS)consists of the regulator QseB and the kinase QseC,which relates to quorum sensing(QS)and virulence in some bacteria.Here,we investigated the role of QseB/QseC in apf gene cluster(apfABCD)expression of APP.Our results have showed that QseB/QseC TCS can potentially regulate the expression of apf gene cluster.The△qseBC,△apfA,△apfB,△apfC and△apfD strains are more sensitive to acidic and osmotic stressful conditions,and exhibite lower biofilm formation ability than wild-type(WT)strain,whereas the complemented strains show similar phenotype to the wr strain.In additon,the mutants have defective antiphagocytosis,adhesion and invasion when they come into contact with the host cells.In experimental animal models of infection,mice infected with△qseBC,△apfA,△apfB,△apfC and △apfD strains showed lower mortality and bacterial loads in the lung and the blood than those infected with wr strain.In conclusion,our results suggest that QseB/QseC TCS contributes to stress resistance,biofilm formation,phagocytosis,adhesion,invasion and virulence by downregulating expression of apf gene cluster in A.pleuropneumoniae.

Effect Found in the Combination of Kitasamycin with Other Antibiotics Used against <i>Actinobacillus pleuropneumoniae</i>

Background: Combinations of antibiotics are used to increase the therapeutic options via the simultaneous activities of two compounds. The goals of combination therapy are to resolve the bacterial infection while reducing the treatment cost. The use of kitasamycin is used frequently and in addition to other antibiotics, the desired combinations were not found. Methods: Fields strains: we use serotypes 1, 3, 5 y 7. Microbial culturing: The serotypes 1, 3, 5 y 7 was identified. Antibiotics and Reagents: The antibiotics used were kitasamycin;enrofloxacin and oxolinic acid. The following combinations were tested: a) kitasamycin with enrofloxacin;b) kitasamycin with norfloxacin;and c) kitasamycin with oxolinic acid. Preparation of bacterial suspensions: The cultures were adjusted to a concentration of 5 colony-forming units per ml (CFU/ml) using the same sterile culture medium as the diluent. Determination of the fractional inhibitory concentrations (FICs): To test each combination and their interactions and to calculate the fractional inhibitory concentration (FIC) of each antibiotic. Calculation of the index of the fractional minimum inhibitory concentration (FMIC): The FMIC index was calculated by applying the following formula: FIC index = FIC of antibiotic A/FIC of antibiotic B. Results: What was found showed that: A combination of kitasamycin and norfloxacin has been shown to have an indifferent effect. A combination of kitasamycin and enrofloxacin had antagonistic effects against all of the Actinobacillus pleuropneumoniae serotypes studied. Finally, a combination of kitasamycin and oxolinic acid had antagonist effects against serotypes 1, 3 and 5 and a synergistic effect against serotype 7. Conclusion: The “in vitro” combination Kitasamycin-Norfloxacin showed an indifferent effect, and combination Kitasamycin and Enrofloxacin demonstrated an antagonistic interaction between these two antibiotics. A combination of Kitasamycin and Oxolinic Acid had synergistic effect against serotype 7.