Veterinaria Italiana (Journal)
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Infezione sperimentale di bovini, pecore e capre con il virus emergente EHDV-8
No full textEpizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats. Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats.
Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.Il sierotipo 8 del virus della malattia emorragica epizootica (EHDV-8) è stato identificato per la prima volta in Europa a fine 2022. In questo studio, abbiamo approfondito la cinetica di infezione di EHDV-8 nei vitelli, pecore e capre. In seguito all'infezione sperimentale, quattro vitelli su cinque hanno presentato febbre, mentre in un vitello sono state osservate lesioni ulcerative e crostose sul muso. L'RNAemia ha raggiunto il picco al settimo giorno dopo l'infezione in tutti i vitelli ed è rimasta relativamente stabile fino alla fine dello studio, dopo 78 giorni. Il virus è stato isolato, su colture cellulari, dal campione di sangue di un vitello prelevato 21 giorni dopo l'infezione. Per quanto riguarda pecore e capre, una pecora ha manifestato febbre e due hanno presentato RNAemia costante fino alla fine dello studio. Il virus è stato isolato fino a 7 giorni dopo l'infezione dal campione di sangue di una pecora. Nelle capre non è stata osservata RNAemia. Tutti gli animali infetti hanno mostrato sieroconversione e sono stati evidenziati anticorpi neutralizzanti in tutte e tre le specie utilizzate ma con una risposta più robusta nei vitelli. Questo studio fornisce dati importanti sulla cinetica dell’infezione da EHDV-8 e sulla risposta immunitaria dell’ospite. Inoltre, è stato evidenziato che le pecore potrebbero svolgere un ruolo rilevante nell’epidemiologia e nella trasmissione di EHDV-8. Nel complesso, i dati raccolti in questo studio hanno importanti implicazioni per le strategie di controllo e prevenzione della malattia
O5-2 Improving Rev.1 vaccine safety in pregnant ewes
No full textLack of safety in pregnant animals hinders Rev.1 mass vaccination campaigns, the most cost-effective strategy to control B. melitensis in endemic and low-income countries. In addition to its abortifacient effect, Rev.1 is virulent to humans and streptomycin (Strp)-resistant, an antibiotic of choice for treating human brucellosis. Our goal was to develop a Rev.1 vaccine of improved safety for pregnant sheep. Three Rev.1 mutants were studied: Rev.1ΔwadC, lacking the lipopolysaccharide core lateral branch, that enhances immune system recognition; Rev.1Δery1, unable to catabolize erythritol, involved in Brucella genital tropism; and Rev.1StrpS, carrying the B. melitensis 16M rsmG instead of Rev.1 rsmG (Rev.1ΔrsmG::Tn7BmersmG) and dispaying increased Strp sensitivity. In mice, the mutants protected similarly than Rev.1 against B. melitensis showing the same (Rev.1Δery1) orless (Rev.1ΔwadC and Rev.1StrpS) residual virulence. Two similar but independently conducted safety experiments were carried out in sheep (one for Rev.1ΔwadC and one for Rev.1Δery1 and Rev.1StrpS). Coetaneous 17-month- old Brucella-free ewes were synchronized reproductively and mated. Pregnant ewes were randomly allotted into groups of 12-13 animals. At the middle of pregnancy (73-78 days), each group was vaccinated conjunctively with 1-2 x 109 CFU of the corresponding mu-tant while a control group received the same conjunctival dose of the commercial Rev.1 vaccine (Ocurev®, CZV, Spain). Fever, anorexia, apathy, abortion, perinatal death or lesions were recorded daily. Also, bacterial excretion in vaginal fluids and milk or presence in fetuses was monitored weekly and immediately after abortion/delivery by culturing samples on duplicate plates of both CITA and Farrell selective media. After deliveries, ewes were necropsied and main target organs (spleen, uterus, and mammary, crural, prescapular, iliac and cranial lymph nodes) submitted to bacteriological assessment. The antibody response was assessed weekly by Rose Bengal, Complement Fixation, Agar Gel Immunodiffusion and iELISA tests. Similarly to Rev.1, all mutants induced Brucella antibodies 2 weeks after vaccination. However, whereas 61-75% of Rev.1 controls excreted the vaccine and 46-58% suffered undesirable reproductive symptoms, Rev.1ΔwadC, Rev.1Δery1 and Rev.1StrpS did not induce excretion, abortion or perinatal death. Thus, these three safe Rev.1 mutants are suitable candidates for further efficacy experiments in sheep
P3-05 Regulatory patterns influenced by the Two-Component System BvrR/BvrS in B. abortus
No full textAn essential element required for B. abortus intracellular replication is the two-component system BvrR/BvrS. Intracellularly, the regulatory protein BvrR is activated by phosphorylation in response to cues found in early and late compartments. We generated two-point mutations in the BvrR putative phosphorylated aspartate 58 to understand the BvrR-regulated transcriptional response. Aspartate 58 was substituted witheither an alanine (BvrRD58A-dominant negative) or a glutamic acid (BvrRD58E-dominant positive), and the sequences were inserted in plasmids. We introduced these plasmids expressing bvrR mutants and the wildtype version into a B. abortus bvrR- and analyzed the strains’ phenotypes. The wild type and the BvrRD58E mutant restored the phenotypes affected in B. abortus bvrR- according to polymyxin assays, ex vivo replication, and expression of proteins such as Omp25, VirB8, and VjbR. In contrast to B. abortus bvrR-, we observed that the BvrRD58A mutant is not as attenuated in several phenotypes tested. For example, VjbR expression was recovered in the BvrRD58A mutant compared to B. abortus bvrR- confirming that unphosphorylated BvrR exerts transcriptional control. Interestingly, although BvrRD58A binds to the virb promoter, VirB8 expression was not restored. Based on these results, we propose three regulatory patterns defined by BvrR phosphorylation, (i) unphosphorylated BvrR binds and regulates the expression of genes directly, (ii) phosphorylated BvrR binds and regulates the expression of genes directly, and (iii) unphosphorylated BvrR binds to gene promoters, but additional regulators are needed to promote gene expression. These findings serve as a working model for understanding how the response regulators of two-component systems control gene expression
O6-2 Cross-reaction comparative evaluation of five enzyme-linked immunosorbent assay and Golden Standard methods for porcine Brucellosis diagnosis
No full textPorcine brucellosis is listed as categories D and E disease in the regulation (EU) 2018/1882, Animal Health Law (AHL). The AHL (prescribes measures in the EU member states for trade conditions and surveillance/ notification. Moreover, according to the regulation, the diagnostic methods should be validated in accordance with international standards for this purpose. Rose Bengal and Complement fixation tests are mainly used for Brucella abortus, B. melitensis and B. suis serological diagnosis. Although performed for brucellosis diagnosis in domestic animals, these tests were initially validated only for bovines. Furthermore, in pigs, it seems to have a high level of false positive serological reactions (FPSRs) caused primarily by Yersinia enterocolitica O:9. To date, no method for porcine brucellosis diagnosis has been validated for the intended purpose according to current World Organisation for Animal Health (founded as WOAH) principles. This study aims to compare the FPSRs performances of five ELISA kits for porcine brucellosis diagnosis [1IDScreen Brucella suis, 2ID Screen Brucellosis serum indirect multi-species (Innovative Diagnostics, France), 3Ingezim Brucella Compac, 4Ingezim Brucella Porcina (Ingenasa, Spain), and 5Brucella abortus/melitensis/suis DIVA (VMRD, USA)] comparing to Rose Bengal (RBT) and Complement Fixation test (CFT). A panel of 88brucellosis-free pig sera from artificial insemination centers6 in France, that presented a serological positive reaction in at least one method (RBT, CFT or i-ELISA), were examined for analytical specificity (cross-reaction activity). The false positive reactions were observed in all tests: RBT (80/88), CFT (56/85) and 3 with anti-complementary reaction, Kit7 (6/88), Kit8 (55/88), Kit9 (81/88), Kit10 (44/88), and Kit11 (60/88). The RB and CF methods for brucellosis diagnosis showed a high level of cross-reaction comparing with ELISA kits for porcine brucellosis. ELISA is a robust and simple test to perform, however each ELISA kit presents different performances. Therefore, validation of this method for porcine species is needed for surveillance and trade
P8-08 Seroprevalence of Brucellosis, Isolation and Characterization of Brucella And Identification of the Associated Risk Factors in Small Ruminants with History of Abortion at Two Districts of South Omo Zone, Ethiopia
No full textBrucellosis is one of the contagious neglected bacterial diseases of domestic and wild animals, caused bybacteria of the Genus Brucella, and distributed worldwide including Ethiopia. However, there was scarcityof epidemiological data on its occurrence in pastoral areas. A cross-sectional study was conducted fromSeptember 2018 to June 2019, to estimate the seroprevalence of brucellosis and isolate Brucella from smallruminants in two randomly selected pastoral districts, out of eight districts in South Omo Zone, Ethiopia. Apre-tested questionnaire was used to clarify the purpose. Blood samples were collected from a total of 124small ruminants with history of abortion for serological test. Subsequently, 30 vaginal swabs were inves-tigated from seropositive animals for Brucella isolation. All serum samples collected were screened sero-logically using the modified Rose Bengal Plate Test (mRBPT) and sera positive with mRBPT were confirmedwith Complement Fixation Test (CFT). An overall seroprevalence in small ruminants with history of abortionwas 21% (26/124; 95% CI: 0.14 - 0.28) using combined mRBPT and CFT. A multivariable logistic regressionanalysis revealed that risk factors considered in the study districts: species, history of abortion (OR: 0.28,95% CI: 0.18 - 0.43), and parity numbers (OR: 0.20, 95% CI: 0.059 - 0.72) were significantly associated withBrucella infection. Brucella was isolated from 5(16.7%) of the 30 samples cultured on Brucella SelectiveAgar. All isolates, 5(16.7%) were from vaginal swabs. The isolates were B. melitensis based on biochemical,and bacteriological culture test result, though further test is required at biovariant level. In conclusion, thepresent serological test showed that brucellosis is highly prevalent among aborted small ruminants in thestudy area. Moreover, the isolation of B. melitensis from aborted goats’ vaginal swabs may be consideredone of the confirmatory for the Brucella infection. Therefore, strategic control measures should be imple-mented, such as regular testing of breeding animals to reduce brucellosis is required to reduce its economicimpact and risk of zoonotic infection in the area
O7-2 First analysis of antimicrobial resistance in Brucella strains isolated in Italy
No full textBrucellosis is one of the world’s most widespread zoonosis. The disease affects livestock causing significant economic losses and can be acquired by humans through close contact with infected animal material or consumption of raw dairy products. While the treatment in animal brucellosis is not indicated, human brucellosis is generally treated with doxycycline combined with rifampicin or aminoglycosides. The levelsof antimicrobial resistance (AMR) in Brucella strains circulating in European countries have not been estimated. The aim of this study is to evaluate antimicrobial susceptibility of Brucella strains isolated from livestock and humans in Italy from 2019 to 2022. A total of 165 strains were analyzed: 96 B. abortus, 61 B. melitensis, two B. canis and four B. suis. Additionally, two Ochrobactrum anthropi strains, isolated from acattle and a human, were included in the study. The susceptibility to nine antimicrobials: gentamicin, streptomycin, doxycycline, tetracyciline, chloramphenicol, rifampicin, ciprofloxacin, levofloxacin and trimethoprim/sulfamethoxazole were assessed using broth microdiluition method. Minimum inhibitory concentrations (MIC) values were interpreted according to CLSI guidelines for potential bacterial agents of bioterrorism and for slow-growing bacteria. More than a half of the analyzed Brucella strains (86/163) were susceptible to all tested antibiotics. The other strains were shown to be resistant/non-susceptible to two antimicrobials, rifampicin and doxycycline, only. Four strains of B. melitensis were full resistant to rifampicin (with a MIC of 4ug/ml). Intermediate resistant to rifampicin was detected in 73/163 (44.8%) Brucella strains and six of these, identified as B. abortus, were also non-susceptible to doxycycline (with a MIC of 2 ug/ml). The two O. anthropi isolates were resistant to rifampicin and chloramphenicol and non-susceptible to streptomycin. The O. anthropi strain isolated from cattle was additionally non-susceptible to trimethoprim/sulfamethoxazole. Brucella strains isolated from wild animals show higher susceptibility to some of the tested antibiotics than strains isolated from farmed animals. The data obtained indicate that a considerate part of Brucella strains circulating in Italy show a reduced susceptibility to rifampicin, one of the commonly used antimicrobials for human treatment. This suggest that monitoring of Brucella spp. AMR in animal populations could provide indications for most effective empirical treatment regime for locally-acquired human brucellosis
P6-10 Development and validation of a real-time PCR assay for detection of Brucella spp. in samples of animal origin
No full textBrucellosis is a zoonosis with serious implications for human and animal health. Direct diagnosis in domestic ruminants depends on the isolation of Brucella from abortion material, vaginal secretions, milk or from tissues taken after slaughtering. Even if serological methods are sufficient to determine the sanitary status of a herd, bacteriological isolation is still the gold standard to establish a definitive diagnosis of Brucellosis in a single animal. The culture-based method to detect Brucella spp. can include the use of an enrichment medium that could be incubated for up to 6 weeks. It presents several inconveniences such as: managing laboratory spaces, intensive manipulation of potentially infectious material with increased risk of infection for the laboratory personnel. The aim of this study was to develop and validate a robust and reliable real-time PCR designed to amplify a portion of IS711 genetic element which is unique to Brucella species. It includes a chimeric DNA as internal amplification control that is coamplified with the target sequence to monitor the presence of PCR-inhibitory substances in clinical samples. It allows a higher predictivity in identifying the positive samples, or in case of a negative result, it avoids the long-term culture based-methods. To validate the method a total of 335 samples from domestic ruminants and marine mammals were examined and the results were compared with those obtained using the culture method. The Receiver Operating Characteristic (R.O.C.) curve was calculated from the resulting data which allowed us to identify the optimal threshold value (best cut-off) with results of sensitivity, specificity, accuracy of 91.9%, 94.5%, 94.3%, respectively, which places the diagnostic method in the range of values attributable to highly accurate testing. This developed real-time PCR could represent a valid and timely tool for the diagnosis of brucellosis and, therefore, a support to the eradication program of this zoonosis
P6-11 The Brucellosis conundrum: antibody vs microbial detection.
No full textBrucella is a highly monomorphic genus and consist of gram-negative pathogenic species. The disease is a systemic infection that can affect numerous organs and tissues and is shared between animals in a herd. Small ruminants and cattle are typically infected with Brucella melitensis and Brucella abortus, respectively. The presence o Brucella in livestock contributes to massive economic losses as well as veterinary and public health distress in developing countries. Due to its unspecific signs and symptoms that are similar to those of other febrile diseases, its slow growth rate on culture, and the complexity of its serodiagnosis, brucellosis remains difficult to diagnose. In the present study, samples including blood (serum), liver, kidney, spleen and lymph node tissues were collected from 275 animals (180 cattle, 60 sheep and 35 pigs) from 5 abattoirs in the Eastern Cape province, South Africa. A random sampling method was used for this study.Serum samples were tested using Rose Bengal test (RBT) and confirmation was done using Complement Fixation Test (CFT). Tissue samples were screened using the PCR for the detection of Brucella species and positive samples were subjected for culture. The PCR assay amplified Brucella directly from tissues in 77 of 180 cattle, 28 of 60 sheep and no Brucella was detected from the pigs. The direct culture technique detected B. abortus from 23 cattle and 1 sheep whereas mixed infections of B. abortus and B. melitensis were detected from 4 cattle and 3 sheep using AMOS-PCR assay. RBT revealed a seroprevalence of 2.9% (1 of 35), 6.2% (4 0f 60) and 6.1% (11 of 180) from pigs, sheep and cattle respectively. Only 1 cattle sample was confirmed positive on CFT. The result of this study emphasises the importance of using more than one diagnostic technique for the detection of Brucella species in livestock. The sensitivity and time rate of PCR technique suggest that the assay could be used for Brucella diagnosis in livestock from abattoirs. The study further shows again the low sensitivity and specificity of brucellosis serological tests for diagnosis
P6-12 Evaluation of a new double-antigen indirect ELISA using a rough-LPS extract from B. suis for the diagnosis of porcine brucellosis
No full textCurrent serological tools (complement fixation test -CFT- or Rose-Bengal test -RBT-) do not offer satisfactory results for swine brucellosis diagnosis. A recently developed indirect ELISA (iELISA) for the detection of anti- B. suis antibodies, based on a combination of two antigens derived from both rough (r-) and smooth (s-)Brucella LPS, was shown to limit false positive serological reactions (FPSR). Based on the same concept, the ID Screen® Brucella suis Indirect is a double- well iELISA using smooth and rough extracts as antigens. Test performance was assessed by testing 3 groups of swine sera (origin: France): i) positive reactors from infected herds (n=286; positive in CFT and RBT) ii) FPRS from free herds (n=202; positive in RBT) iii) negative reactors from free herds (n=720, RBT negative). Results were expressed as S/Pvalues and interpreted as per manufacturer’s instructions. Animals are considered to be infected with B. suis when positive with both the s- and r-LPS antigens. FPRS should only react on the s-LPS, and are considered as negative. A Receiver Operating Characteristic (ROC) curve analysis was performed. Measured sensitivity was 72.7% (IC95% 67.5-77.9%, n=286); specificity was 99.9% (IC95% 99.6-100%, n=720). Out of 202 FPRS tested, 197 were found negative with the new ELISA, drastically reducing the false positive reactors rate: measured specificity on FPRS was 97.5% (IC95% 95.3-99.7%, n=202). Different cut- off values were tested; it is possible to increase the B. suis sensitivity without significantly affecting specificity. Increasing sensitivity could be of interest when testing herds where the presence B. suis has been already confirmed. To conclude, this new iELISA could improve surveillance and control programs of porcine brucellosis by reducing the impact of FPRS in herds, historically negative for Brucella infections
K10 Brucellosis and OneHealth: inherited and future challenges
No full textOneHealth is the collaborative efforts of multiple disciplines to attain optimal health for people, animals and the environment. While few studies have quantified the benefits, it is accepted that OneHealth strategies are necessary to combat brucellosis, a consensus supported by the fact that historically the concept owes much to observations made on brucellosis by Schwabe and Steele, and by experiences in the eradication of cattle and small ruminant brucellosis. Furthermore, the analysis of brucellosis in conflicting scenarios supports the inclusion of political and ethical considerations in the OneHealth concept. In brucellosis, OneHealth actors include Public Health and Veterinary Services (with emphasis on notification systems), microbiologists, medical and veterinary practitioners and breeders, and brucellosis awareness plus a correct use of diagnostic, epidemiological and prophylactic tools are essential. Nevertheless, although the concept is clear, a series of inherited and new challenges pose significant obstacles, some aggravated by global warming and breeding intensification to meet food demands. Studies in endemic scenarios show that disease awareness, stakeholder sensitization/engagement and breeder trust are unresolved issues, all particularly difficult in brucellosis because of the protean characteristics of this zoonosis. Of paramount importance areinfrastructural weaknesses, often accentuated by geography and climate. Capacity building faces misconceptions derived from an uncritical adoption of diagnostic and eradication strategies that were successful in countries with suitable means, and is hampered by technical matters requiring reference laboratories in endemic areas, problems not solved by new tools whose value in the real scenario is unknown. Althoughimproving diagnostics and vaccines is challenging, urgent needs are research in semi-domestic or domestic species other than cattle and small ruminants and in wildlife and to develop a safer small ruminant vaccine. Results of this research, lessening the infrastructure requirements, realistic capacity building, creating reference laboratories in critical areas and a stepwise implementation of measures not directly transposed from the so-called developed countries are prerequisites for OneHealth implementation in the combat against brucellosis