Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing: a bioprocess review

Mannheimia haemolytica leukotoxin (LKT) is a known cause of bovine respiratory disease (BRD) which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA). Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant engineering problem and further bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites) from a bioprocess engineering perspective, in order to maximize LKT yields.

A fast and simple assay to quantify bacterial leukotoxin activity

Background: Mannheimia haemolytica is the primary bacterial pathogen in causing bovine respiratory disease with tremendous annual losses in the cattle industry. The leukotoxin from M. haemolytica is the predominant virulence factor. Several leukotoxin activity assays are available but not standardized regarding sample preparation and cell line. Furthermore, these assays suffer from a high standard error, a prolonged time consumption and often complex sample pretreatments, which is important from the bioprocess engineering point of view. Results: Within this study, an activity assay based on the continuous cell line BL3.1 combined with a commercial available adenosine triphosphate viability assay kit was established. The leukotoxin activity was found to be strongly dependent on the sample preparation. Furthermore, the interfering effect of lipopolysaccharides in the sample could be successfully suppressed by adding polymyxin B. We reached a maximum relative P95 value of 14%, which is more than seven times lower compared to current available assays as well as a time reduction up to 88%. Conclusion: Ultimately, the established leukotoxin activity assay is simple, fast and has a high reproducibility. Critical parameters regarding the sample preparation were characterized and optimized making complex sample purification superfluous.

Resistencia a antimicrobianos en cepas de Mannheimia haemolytica aisladas de exudado nasal de bovinos productores de leche / Antimicrobial resistance in Mannheimia haemolytica strains isolated from dairy cattle nasal exudate

Two hundred and one strains of M. haemolytica isolated from nasal exudate of dairy cattle were used, 123 strains from clinically healthy (CH) bovines and 78 from clinically ill (CI) bovines affected by pneumonia, obtained from a dairy complex in the Tizayuca region of the state of Hidalgo, Mexico. Strains were previously identified by conventional culture and biochemical tests, and serotyped by indirect haemagglutination. Disk diffusion test was performed to determine antimicrobial resistance to antibiotics, such as: ampicillin, gentamicin, ceftiofiur, penicillin, streptomycin, trimethoprim-sulfamethoxazole, tetracycline and erythromycin. Frequencies of higher antimicrobial resistance were: streptomycin (81.6%) and gentamicin (24.4%), all strains were susceptible to ampicillin and penicillin. Because of the high resistant strain frequency (81.6%) of M. haemolytica to streptomycin, obtained by Kirby-Bauer test, presence of the strA gene, which encodes the enzyme aminoglycoside-3-phosphotransferase that provides resistance to streptomycin, PCR was performed by testing the presence of the strA gene. Of the 201 strains tested, 42.7% showed the gene sfrA, 17.4% of which was serotype A1, 1.4% serotype A6 and 23.8% non-typeable strains. Of the 78 CI strains and 123 CH strains, 80% and 18.7%, had the gene sfrA, respectively.

Se emplearon 201 cepas de Mannheimia haemolytica provenientes de muestras de exudado nasal de bovinos productores de leche, 123 de bovinos clínicamente sanos (CS) y 78 de bovinos enfermos de neumonía (CE), obtenidas de un complejo lechero en la región de Tizayuca, Hidalgo, México, las cuales fueron identificadas previamente mediante pruebas convencionales de cultivo y bioquímicas, y serotipificadas mediante la prueba de hemaglutinación indirecta. Se les realizó la prueba de difusión en placa para determinar la resistencia a diversos antimicrobianos como ampicilina, gentamicina, ceftiofiur, penicilina, estreptomicina, trimetoprim con sulfametoxazol, tetraciclina y eritromicina. Las frecuencias más altas en la resistencia a antimicrobianos se presentaron a la estreptomicina (81.6%) y gentamicina (24.4%), todas las cepas fueron susceptibles a la ampicilina y penicilina. Debido a la alta frecuencia (81.6%) de cepas de M. haemolyfica resistentes a St con la técnica de Kirby-Bauer, se buscó la presencia del gen sfrA. Se realizó la técnica de PCR para comprobar la presencia del gen sfrA que codifica para la enzima aminoglycoside-3-phosphofransferase que proporciona resistencia contra la estreptomicina. Del total de cepas estudiadas (n = 201), 42.7% presentaron el gen sfrA, del cual 17.4% pertenecía al serotipo A1, 1.4% al A6 y 23.8% a cepas no tipificables. De las 78 cepas de CE y las 123 de CS, 80.0% y 18.7% respectivamente, presentaron el gen sfrA.

Ocorrência de pneumonia associada à infecção por Mannheimia haemolytica em ovinos de Minas Gerais / Occurrence of pneumonia associated to infection by Mannheimia haemolytica in sheep of Minas Gerais

O trabalho descreve um surto de pneumonia em ovinos em uma propriedade na região central de Minas Gerais. Clinicamente os animais apresentavam apatia, mostravam dificuldade respiratória durante dois ou três dias ou morriam subitamente. À necropsia as alterações pulmonares eram similares em todos os ovinos. Havia consolidação dos lobos craniais e da parte ventral dos lobos caudais e ao corte fluía exsudato mucopurulento da traquéia e dos brônquios. No parênquima dos lobos craniais havia áreas brancas multifocais a coalescentes com 0,2-0,5cm de diâmetro, levemente proeminentes e intercaladas por áreas vermelho-escuras. Pleurite fibrinosa foi observada nos Ovinos 1, 2 e 3. As lesões de consolidação ocupavam cerca de 70-80 por cento da extensão pulmonar. Microscopicamente, as alterações eram de broncopneumonia fibrinopurulenta com intensa hiperemia, áreas com hemorragia intra-alveolar e espessamento dos septos interlobulares por inúmeros neutrófilos, restos celulares e intensa exsudação de fibrina. Áreas multifocais com necrose de liquefação contendo numerosas colônias bacterianas foram observadas no Ovino 3. Nos lobos craniais dos Ovinos 1, 2 e 3, haviam áreas com neutrófilos degenerados formando aglomerados de células alongadas com formato de "grãos de aveia" associados a colônias bacterianas. As alterações histológicas foram características de pneumonia causada por Mannheimia (M.) haemolytica. Amostras dos lobos craniais de todos os ovinos foram encaminhadas para cultivo bacteriológico e M. haemolytica foi isolada e identificada em todos os animais. Este é o primeiro relato correlacionando os achados patológicos e o isolamento de M. haemolytica como causa de broncopneumonia em ovinos no Brasil.

This paper describes an outbreak of pneumonia in a sheep herd in the central region of Minas Gerais, Brazil. Clinically, the animals presented apathy, exhibited respiratory difficulty during 2 to 3 days or sudden death. The animals were not medicated and found dead. Grossly, the pulmonary findings were similar in all sheep. The pulmonary cranial lobes and the ventral portion of caudal lobes were consolidated and purulent exsudate streamed out of the airways. In the parenchyma of the cranial lobes there were white slightly prominent multifocal to coalescent areas with 0.2 to 0.5cm in diameter intercalated with dark red areas. Consolidated lesions occupied 70 to 80 percent of the lungs. Fibrinous pleuritis was observed in sheep 1, 2 and 3. Microscopically, the findings were fibrinopurulent bronchopneumonia with intense hyperemia, areas with intra-alveolar hemorrhage and thickening of interlobular septa with numerous neutrophils, cellular rests and scattering fibrin. Multifocal areas with liquefaction necrosis containing numerous bacterial colonies were observed in sheep 1, 2 and 3. In the cranial lobes of these sheep, there were areas with degenerated neutrophils forming clusters of basophilic cells with alongated nuclei ("oat cells") associated with bacterial colonies. The histological findings were characteristic of pneumonia caused by Mannheimia (M.) haemolytica. Samples of the cranial lobes were sent for bacterial culture, and M. haemolytica was isolated and identified in all animals. This is the first report correlating pathological findings and the isolation of M. haemolytica as cause of bronchopneumonia in sheep in the country.

Mannheimiose pulmonar experimental em bezerros: swab nasal e nasofaringeano como auxílio diagnóstico: [revisão] / Experimental pneumonic mannheimiosis in calves: nasal and nasopharingeal swabs for diagnostic: [revision]

Um modelo experimental de mannheimiosepneumônica bovina (MPB) foi utilizado com o objetivo de avaliar as espécies bacterianas das cavidades nasais e nasofaringeanas em diferentes momentos do curso da doença, bem como verificar a eficiência diagnóstica do exame microbiológico dos swabs nasais (SN) e nasofaringeanos (SNF). Um total de 28 bezerros foi distribuído aleatoriamente em quatro grupos experimentais (G1 a G4). SN e SNF foram colhidos sete dias antes e 12 (G1), 24 (G2), 48 (G3) e 72 (G4) horas após a inoculação intrabronquial de Mannheimia haemolytica. Após a indução da MPB, a bactéria M. haemolytica biotipo A foi predominante nos SN e SNF, sendo isolada em todos os momentos avaliados, com exceção de um SN colhido 24 horas após a indução da infecção. Não houve diferença significativa nas taxas de isolamento de Pasteurella multocida nos SN ou SNF, colhidos antes e após a indução da MPB. Contudo, esta bactéria passou a ser isolada mais freqüentemente após a indução da MPB, principalmente no SNF. Portanto, pode-se concluir que o exame microbiológico de SN e SNF é um teste auxiliar no diagnóstico da MPB.

An experimental model of bovine pneumonic mannheimiosis (BPM) was used to evaluate the nasal and nasopharynx bacterial species of calves during the course of the disease and for checking the diagnostic efficiency of nasal swab (NS) and nasopharingeal swab (NPS) microbiological exams. A total of 28 calves were randomized into four experimental groups (G1-G4). NS and NPS were obtained 7 days before and 12 (G1), 24 (G2), 48 (G3) e 72 (G4) hours after intrabronchial inoculation of Mannheimia haemolytica. After the induction of BPM, M. haemolytica biotype A was the predominant isolated bacterium in NS and NPS in all evaluated sampling times, except for one NS (harvested 24 hours). There were no significant statistical differences for the rates of Pasteurella multocida isolation in NS and NPS, harvested before and after the induction of BPM. However, this bacterium was isolated more frequently after the induction of BPM, mainly in NPS. Therefore, the microbiological NS and NPS exams were important auxiliary tests for diagnosing BPM.

Ocorrência de Mannheimia haemolytica e de Pasteurella multocida em ovinos sadios e com enfermidade respiratória / Mannheimia haemolytica and Pasteurella multocida occurrence in healthy and affected lambs

Frequency of Mannheimia haemolytica and Pasteurella multocida in the respiratory tract of lambs in the region of Botucatu, SP, Brazil, was studied. Nasopharingeal and oropharingeal swabs were obtained from 262 animals: 180 from healthy and 82 from animals with respiratory diseases. M. haemolytica was the most prevalent (47 percent), followed by the association of M. haemolytica and P. multocida (27 percent), and P. multocida (11 percent). Animals with respiratory disease presented higher occurrence of P. multocida in the nasopharynx as compared to healthy animals (P<0.05). No significant difference in isolation rate of M. haemolytica, P. multocida, and association of these microorganisms in the oropharynx of healthy and affected animals was observed