Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

Seroprevalence of contagious bovine pleuropneumonia (CBPP) in cattle from Karamoja region, North-eastern Uganda.

BACKGROUND: Contagious bovine pleuropneumonia [CBPP] is a transboundary animal disease of cattle caused by Mycoplasma mycoides subsp. mycoides [Mmm]. CBPP causes severe economic losses to livestock producers in sub-Saharan Africa mainly due to high mortality, morbidity, reduction in productivity as well as livestock trade restrictions. This study aimed at determining seroprevalence of Mmm in cattle from Karamoja region, north-eastern Uganda; data that are required to design and implement risk based CBPP control program. METHODS: We randomly collected blood samples from 2,300 cattle spread across Karamoja region. Serum was extracted and screened for antibodies against Mycoplasma mycoides subsp. mycoides [Mmm] using the competitive enzyme linked immunosorbent assay [cELISA]. RESULTS: A quarter [25.4%; 95% CI: 23.7-27.3] of the screened cattle [n = 2,300] were sero-positive for Mmm. Amudat and Kaabong districts recorded the lowest [12.3%] and highest [30.7%] Mmm seroprevalence respectively. Increasing age, overnight stay in cattle kraals and location [certain districts, villages, herds and sub counties] of the cattle herds, the factors that promote animal commingling, were the most significant risk factors of seroconversion with Mmm. CONCLUSION: Results from this study indicated a higher seroprevalence of Mmm in Karamoja region cattle herds. This could be due to the increased frequency of CBPP outbreaks in recent years. To be effective, CBPP vaccination programs should target high risk herds along the international borders and other hotspot areas [e.g., parishes or sub counties] where cattle commingling is high.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

Sero-prevalence of contagious bovine pleuropneumonia in dryland of Borana, southern Oromia, Ethiopia.

Ethiopia is one of the largest African countries where livestock farming represent a relevant resource for the economy and the livelihood of the population. Contagious bovine pleuropneumonia (CBPP) is among the transboundaries animal disease that is hindering cattle farming in Ethiopia. Due to the limited resources of veterinary services, disease control and surveillance is discontinuous and occasional field investigations of target areas contribute to depict disease spreading in the country. The study was conducted to determine the seroprevalence, at herd and animal level, and identify the risk factors involved in CBPP diffusion and persistence in the Borana pastoral zone. A total of 498 serum samples were collected from 120 cattle herds and tested using competitive Enzyme-Linked Immunosorbent Assay (c-ELISA). Of 120 herds sampled, 37 (30.83%; (95% CI = 22.73-39.91%) were tested positive to CBPP antibody. Out of 498 sera samples tested 46 (9.24%; 95% CI = 6.84-12.13%) were positive. The highest prevalence was observed in Teltele (12/95; 12.90%; 95% CI = 6.7-21%) followed by Yabello (12/104; 11.54%; 95% CI = 6.1-19.3%) and Arero (10/91; 10.99%; 95% CI = 95% CI = 5.4-19.3%), whereas the lowest prevalence was observed in Gomole (5/101; 6.42%; 95% CI = 1.6-11.2%) and Dubluk (7/109; 4.95%; 95% CI = 2.6-12.8%) districts and statistically not significant (p > 0.05). Results of multivariate logistic regression analysis revealed that, age, herd movement and herd size of the animals had statistically significant effect on sero-positivity to CBPP (p < 0.05). Sex, season and body condition were not significantly (p > 0.05) associated with the occurrence of CBPP. The study confirms that CBPP is persistent in the territory and remain as a major problem that affects health and productivity of cattle. Therefore, awareness creation to the pastoralists in the study area about the effect of CBPP and designing appropriate control methods has a paramount importance to improve the health and productivity of cattle production in the area.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

Risk factors associated with contagious caprine pleuropneumonia in goats of Amhara region, Ethiopia.

Contagious caprine pleuropneumonia (CCPP) is a serious contagious disease of goats, sheep and wild ruminants caused by Mycoplasma capricolum subspecies capripneumoniae. The disease is known for its high mortality, morbidity and economic losses. A cross-sectional study using multistage cluster sampling technique was conducted in Amhara region from January 2019 to July 2019 to estimate seroprevalence and identify risk factors of CCPP occurrence in the region. A total of 2080 goats from 61 villages and 12 districts of the region were tested for CCPP serostatus using Competitive Enzyme-Linked Immunosorbent Assay (C-ELISA). A multilevel mixed-effect logistic regression model was used to identify risk factors of CCPP seropositivity at animal and flock-level. The serum sample results revealed an overall animal level seroprevalence of 5.1% (95% CI: 3.8-6.6) and flock-level prevalence of 26.0% (95% CI: 19.7-33.4). At individual animal level, presence of other health problems (OR = 45.9 (95% CI: 25.3-83.4)), age (adult age (OR = 6.2 (95% CI:3.4-11.4)) and old age (OR = 13.1 (95% CI: 6.2-27.8))), and breed type (Afar (OR= 32.3 (95% CI: 2.9-366.1)), Central highland (OR=13.7 (95% CI: 1.3-140.6)), and western highland (OR=16.2 (95% CI: 1.4-185.7))) were identified as risk factors for CCPP seropositivity. In contrast, contact with other flocks (OR = 59.9 (95% CI: 6.1-585.6)), presence of trade route (OR = 3.1 (95% CI: 1.0-9.1)) and presence of sheep (OR = 2.6 (95% CI: 1.2-5.7)) were flock-level risk factors for CCPP seropositivity. Generally, CCPP appears to be common among goats of Amhara region. Goat flocks dominated with older age animals; breeds of Afar, central highland, and western highland; raise with sheep; have contact with other flocks; and kept along trade routes are more at risk for CCPP. Hence, awareness creation to the producers, movement control, and regular prophylactic vaccination should be considered to control CCPP in Amhara region.

Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Revealing Genomic Insights of the Unexplored Porcine Pathogen Actinobacillus pleuropneumoniae Using Whole Genome Sequencing.

Actinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems. IMPORTANCE Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI. Moreover, this study also includes 16 APP isolates of Spanish origin with known epidemiological relationships through vertical integrated systems. Genomic comparisons provided a deeper understanding of molecular and epidemiological knowledge between different APP serotypes. Furthermore, determination of resistance and toxin profiles allowed correlation with the presence of mobile genetic elements and specific serotype, respectively.

Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction.

Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level.

IL-5 enhances the resistance of Actinobacillus pleuropneumoniae infection in mice through maintaining appropriate levels of lung M2, PMN-II and highly effective neutrophil extracellular traps.

Interleukin 5 (IL-5) regulates the maturation, activation, proliferation and function of immune cells, and plays an important role in the inflammatory response induced by an allergy. However, its anti-pathogen effect is poorly understood currently, especially on pneumonia. Here, this study was designed to elucidate the immunological role of IL-5 in the infection of mice with Actinobacillus pleuropneumoniae (APP). We established an acute lung infection model of APP in IL-5 knockout mice (IL-5-/-) and wild-type mice (WT) through nasal infusion or intraperitoneal injection, compared the survival rate, clinical symptoms, lung bacterial load, proportion of various immune cells, immune molecular expression, and neutrophil germicidal ability through flow cytometry, RT-qPCR, ELISA and immunofluorescence. Compared to WT mice, the IL-5-/- mice had a lower survival rate, more severe clinical symptoms, significantly increased bacterial load, and inflammatory cell infiltration in the lung after APP infection. In an uninfected state, IL-5 deficiency decreased the number of M1 interstitial macrophages and CD14- monocytes, while after infection, IL-5 deficiency significantly reduced the M2 alveolar macrophages, and increased PMN-II cells in the lung. Furthermore, the expression of IL-10, IL-4, IL-33, TNF-α, iNOS in the lung was lower in IL-5-/- mice under an uninfected condition, and the secretion of IL-18 was significantly increased after infection. In addition, IL-5 deficiency decreased bactericidal ability by inhibiting the formation of neutrophil extracellular traps (NETs). Collectively, these results provide evidence that IL-5 can enhance the resistance of APP infection, and its anti-infection mechanism, implying new targets and ideas for APP or similar respiratory agents' prevention and treatment.

JMM Profile: Actinobacillus pleuropneumoniae: a major cause of lung disease in pigs but difficult to control and eradicate.

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in pigs, its only known natural host. Typical symptoms of peracute disease include fever, apathy and anorexia, and time from infection to death may only be 6 h. Severe lung lesions result from presence of one or two of the ApxI-III toxins. Control is through good husbandry practice, vaccines and antibiotic use. Culture and presence of the species-specific apxIV gene by PCR confirms diagnosis, and identification of serovar, of which 19 are known, informs on appropriate vaccine use and epidemiology.

Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Proposal of a subtype of serovar 4, K4b:O3, of Actinobacillus pleuropneumoniae based on serological and genotypic analysis.

The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14-760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14-760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14-760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14-760, but not with antigens prepared from serovars 1-18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called "K4b:O3″, and we propose isolate 14-760 as the reference strain.

Contagious bovine pleuropneumonia: Seroprevalence and its associated risk factors in selected districts of Afar region, Ethiopia.

BACKGROUND: In pastoral and lowland areas of the country particularly in Afar region, studies suggested higher prevalence of contagious bovine pleuropneumonia (CBPP) than mid and highland agro-ecologies. Though CBPP is a prime constraint to cattle productivity in the region, research outputs pertaining to CBPP are unavailable compared to highland areas. Thus, the objectives of the current study were to determine seroprevalence of CBPP and assess risk factors in selected districts of Afar region. METHODS: A cross-sectional study was conducted on cattle aged 6 months and above from February 2018 to January 2019 in selected districts of the region. A total of 420 blood samples were collected and sera were separated for further serologic analysis. Using competitive enzyme linked immunosorbent assay (c-ELISA), antibodies against Mycoplasma mycoides subspecies mycoides small colony (MmmSc) were detected at National Veterinary Institute, Ethiopia. Data were analysed using Stata version 14.0. RESULT: Of 420 samples tested by c-ELISA, 158 samples were found to be positive for CBPP providing an overall seroprevalence of 37.6%. Among the three risk factors considered (age, sex and district) assessed, only two (age and district) were found to be associated significantly with the disease (p < 0.05) at 95% CI and p-value less than 5% applying logistic regression. CONCLUSION: The study has revealed a higher prevalence of CBPP over the study areas urging a coordinated act to be set in place.

Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the "typical" apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Acute fibrinous pleuropneumonia and septicaemia caused by Bibersteinia trehalosi in neonatal calves in New Zealand.

Case history: In July and August 2019, 15/40, ≤48-hour-old calves became acutely ill. The calves were all born on-farm, transferred to pens soon after birth, and fed with "gold" colostrum. The hygiene, biosecurity and ventilation in the pens were poor. Of the 15 calves, 11 died or were euthanised and four calves, ≤48-hour-old, that became acutely ill later in the outbreak were treated with cefquinome, a fourth-generation cephalosporin, and recovered. Clinical findings: The affected calves presented with acute recumbency, lethargy, tachypnoea, tachycardia, increased lung sounds, inability to stand or feed, and dehydration without pyrexia. Pathological findings: Gross findings in a calf that died naturally included fibrinous pleuropneumonia, marked oedematous expansion of the interlobular septa, especially in the ventral lung lobes, fibrinous polyserositis and fibrinous polyarthritis. A second calf that was euthanised had strikingly similar lung lesions. Histologically, the pulmonary interlobular septa of both calves were prominently expanded by oedema, dilated lymphatics and the infiltration of numerous neutrophils and macrophages interspersed with small Gram-negative rod bacteria. Likewise, the visceral pleura showed fibrinopurulent inflammation with numerous small Gram-negative rods. Microbiological findings: Microbial culture and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry identified Bibersteinia trehalosi in the lung, stifle joint and peritoneal cavity of the first calf and lung of the second. Diagnosis: B. trehalosi acute fibrinous pleuropneumonia and septicaemia. Clinical relevance: This is the first report of the clinical findings and histological lesions of B. trehalosi pleuropneumonia and septicaemia in calves in New Zealand. The pathogen is isolated with increasing frequency from cases of bovine respiratory disease in dairy cows, feedlot cattle and calves in the United Kingdom and North America. The importance of microbial culture in cases such as this with unusual lung lesions in calves <48 hours of age, cannot be over emphasised. Cefquinome was administered to all remaining heifer calves and four calves that became ill later in the outbreak recovered after cefquinome treatment.

The first isolation and molecular characterization of Mycoplasma capricolum subsp. capripneumoniae Pakistan strain: A causative agent of contagious caprine pleuropneumonia.

PURPOSE: Mycoplasma capricolum subsp. capripneumoniae (Mccp) causes a severe, usually fatal disease in goats known as Contagious Caprine Pleuropneumonia (CCPP). CCPP is listed by OIE as a notifiable animal diseases, causing economic losses in terms of high morbidity and mortality. Thus far, very limited information is available on the molecular characterization of the unique Mccp strains prevalent in Pakistan. The study was aimed to isolate Mccp local strain for the development of diagnostics and vaccines. METHODS: Samples were collected during November 2017-December 2018 at Northern areas of Pakistan from 10 goat flocks each in Gilgit-Baltistan, Chitral, Swat, Buner, and Hazara. 900 samples were collected; nasal swabs (n = 400), tracheal swabs (n = 150) from naturally infected goats showing clinical signs of CCPP, and lungs tissue (n = 200), pleural fluid (n = 150) from goats at necropsy. RESULTS: The clinical signs recorded were mucopurulent nasal discharges, cough, abdominal respiration and hyperthermia. The post-mortem revealed, pulmonary consolidation, fibrinous pleuropneumonia, and accumulation pleural fluid. The fried egg like growth was observed on agar in 16 (4%), 11 (7.3%), 38 (19%), and 24 (16%) nasal swab, tracheal swabs, lungs and pleural fluid samples, respectively. PCR targeting 16S rRNA gene revealed isolates, belongs to Mycoplasma mycoides cluster, in 72 (8%) samples. Forty one (4.5%) isolates were Mccp by specie specific PCR generating an amplicon of 316 bp. CONCLUSIONS: We successfully isolated local strain of Mccp for the first time in Pakistan. This Mccp strain could be further utilized for the development of diagnostics and control measures against Mccp infection in goats.