Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs

Pesquisa Veterinária Brasileira

Klein, Catia S.⁴  Morés, Marcos A.Z.⁴  Plieski, Camila L.A.³  Agnol, Alais M.D.²  Oliveira Filho, João X. de¹  Rebelatto, Raquel⁴  Barcellos, David E.S.N.¹  Morés, Nelson⁴ 

[1] Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;Universidade do Oeste de Santa Catarina, Xanxerê, Brazil;Universidade do Estado de Santa Catarina, Lages, Brazil;Embrapa Suínos e Aves, Concórdia, Brazil

关键词:  Swine pasteurellosis;    necrosuppurative pleuropneumonia;    sepsis.;   

DOI  :  10.1590/S0100-736X2015000800003

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

:In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals.Index Terms: Swine pasteurellosis; necrosuppurative pleuropneumonia; sepsis.Resumo:Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais.Termos de Indexação: Pasteurelose suína; pleuropneumonia necrossupurativa; septicemia.IntroductionRespiratory diseases are responsible for significant economic losses in pig production (Sørensen et al. 2006). Pasteurella multocida is one of the bacterial agents most commonly isolated from pneumonic lesions in pigs (Falk et al. 1991, Høie et al. 1991, Choi et al. 2003). This bacterial species belongs to the family Pasteurellaceae, which includes other species associated with pneumonia and polyserositis in pigs, such as Actinobacillus pleuropneumoniae and Haemophilus parasuis, respectively (Vanalstine 2012). Typically, P. multocida is regarded as a secondary opportunistic agent of enzootic pneumonia caused by Mycoplasma hyopneumoniae (Pijoan & Fuentes 1987, Hansen et al. 2010). In addition to secondary role in bronchopneumonia, P. multocida may cause pleuritis, pericarditis (Pijoan & Fuentes 1987, Ono et al. 2003, Pors et al. 2011a) and necrohemorrhagic pneumonia, similar to that caused by A. pleuropneumoniae ("A. pleuropneumoniae-like") (Cappuccio et al. 2004).The successful experimental reproduction of porcine pneumonia by P. multocida has been achieved only when associated with other agents (Ross 2006). A preliminary study (Kich et al. 2007) verified that a field isolate of P. multocida type A was able to cause marked fibrinous pleuritis and pericarditis and focal necrohemorrhagic pneumonia (A. pleuropneumoniae-like lesions) after intranasal inoculation. Doses of 2.6x107and 2.1x108 colony forming units (CFU)/ml were used in this study (each pig received 1.5 ml/nostril of the inoculum). As a result, the aim of the present study was to establish an experimental model for reproducing pneumonia and systemic lesions characterized by serositis and septicemia following sole inoculation of P. multocida type A strain 11246 in pigs.Materials and MethodsEthics statement. The experiment was conducted at Embrapa Swine and Poultry Research Centre, Concordia/SC, Brazil, in compliance with the Ethics Principles in Animal Experimentation, being approved by the Ethics Committee on Animal Experimentation (CEUA/CNPSA) (Protocol #005/2010).Animal houses. Eight 50 kg Body weigth 100 day-old pigs from a high health status herd raised at the Embrapa Swine and Poultry Research Centre, Concordia-SC, Brazil, facilities were used in this study. The source herd has been negative for the main respiratory pathogens based on six monthly testing of tonsillar swabs for Pasteurella multocida type A and D, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Streptococcus suis by bacterial isolation; Nested-PCR and PCR (Mycoplasma hyopneumoniae and A. pleuropneumoniae, respectively) and ELISA test (M. hyopneumoniae and Porcine Reproductive and Respiratory Syndrome Virus - PRRS). Circulating antibodies against influenza A virus were detected on basal levels by ELISA, however, no viral genetic material was detected by RT-PCR test. Furthermore, respiratory diseases, such as enzootic pneumonia, influenza, polyserositis or Glässer's disease, atrophic rhinitis, pleuropneumonia and pasteurellosis have never been diagnosed since the farm was populated with caesarean derived animals in 2009. The farm respects strict biosecurity guidelines and has health barriers, closed rooms with positive internal pressure, visit restrictions and regular monitoring.The animals were transferred from the farm to the isolation unit facilities (biosafety level 2) at the premises of the Animal Health and Genetics Laboratories at Embrapa Swine and Poultry, Concordia-SC, Brazil. Each group was housed in a different room, two pigs per pen, receiving food and water "ad libitum". Access to animals was restricted to the staff responsible for the experiment.Before inoculation, two swabs were obtained from each pig, one from the palatine tonsils and one from the nasal cavity. A pool of these two swabs was cultured using routine methods for the isolation of bacterial pathogens of the respiratory tract of pigs (A. pleuropneumoniae, H. parasuis, B. bronchiseptica and P. multocida) (Quinn et al. 2011). M. hyopneumoniae testing was undertaken through Nested PCR assay from individual tonsillar swabs, according to Yamaguti et al. (2008).Bacteria culture and inoculum. The P. multocida serotype A, strain #11246 from Embrapa Swine and Poultry Research Centre collection was used. This bacterial strain was isolated from pleura and lung swabs of a finishing pig from a farrow-to-finish farm with 200 sows in the state of Santa Catarina, Brazil, in 2007. The pig had severe respiratory disease and fibrinosuppurative pleuropneumonia. Some animals from the same herd were necropsied and mucopurulent pneumonia affecting large areas of the lung (approximately 60%), diffuse pleurisy and adherence between the parietal and visceral pleura were present. These animals were positive to M. hyopneumoniae and Porcine Circovirus Type 2 (PCV2) by Immunohistochemistry assay (IHC) from lung and lymph nodes samples, respectively. The isolate was characterized based on phenotypic (Quinn et al. 2011) and molecular features to identified the KMT1 gene (species and Type-specific) and capsular classification by detection of hyaD-hyaC gene (P. multocida capsular Type A) (Townsend et al. 2001). Moreover, it were research the gene Virulence-associated genes encoding haemoglobin binding protein hgbB, filamentous haemagglutinin pfhA, dermonecrotoxin toxA and transferrin-binding protein tbpA were tested by PCR, according to Ewers et al. (2006) and Atashpaz et al. (2009).The recovery of P. multocida type A from the stock was performed by culture on blood agar plates (Blood Agar Base, BD Difco™, 5% sheep's blood) incubated at 37°C for 18-24 hours. A subculture on a Trypticase Soy Agar (TSA) plate (Difco™) was incubated at 37°C for 18-24 hours. The identity of this isolate was confirmed by phenotypic standard (Quinn et al. 2011).Four different concentrations of inocula were prepared. The seed consisted of a suspension of bacterial colonies of the third passage from the TSA medium in 0.9% sterile saline adjusted to 0.7 absorbance (540nm). From this seed, a series of base 10 dilutions were prepared (1:10, 1:100 and 1:1000). Inoculum concentrations were confirmed by counting the CFU.Study design. Four groups (G1, G2, G3 and G4) of two pigs each were challenged with different concentrations of P. multocida type A strain 11246 inoculum as follows: G1: 108 CFU/ml (pigs 131 and 135); G2: 107 CFU/ml (pigs 129 and 130); G3: 106 CFU/ml (pigs 140 and 142) and G4: 105 CFU/ml (pigs 139 and 143). Each pig received 3.0 ml (1.5 ml/nostril) of the inoculum, administered by slow intranasal drip with animals in a sitting position. Another two pigs (177 and 199) housed separately were used as negative controls and inoculated with 3 ml sterile phosphate buffered saline - PBS (1.5 ml/nostril).All pigs were clinically evaluated twice a day (8-9 a.m. and 4-5 p.m.), starting just prior to inoculation (day 0 am) and ending on the 4th day post-inoculation (dpi). Rectal body temperature (with a digital thermometer), dyspnoea (evaluated with pigs lying down) and coughing (5 min. with pigs moving during feeding and cleaning of pens) were evaluated.Necropsy and sampling. Pigs were euthanized by electrocution, bled and necropsied on the 4th dpi. Pigs with clinical signs of severe pneumonia were euthanized immediately due to animal welfare. At necropsy, the type, distribution and severity of lung, pleural and pericardial lesions were recorded. Samples of lung, trachea, mediastinal lymph node, heart, pericardial sac, liver, kidney and spleen were collected for histopathology and bacteriology. One portion of each sample was preserved in 10% buffered formaldehyde for histopathology and immunohistochemistry (IHC) for P. multocida type A. The other fragment was placed in a sterile plastic bag and stored at 2-8 °C for bacteriological examination. Whenever present, fibrinous exudates in the pleura, pericardium, peritoneum and joints were also collected aseptically for bacteriological examination.Histopathology and Immunohistochemistry (IHC). Histopathological slides were prepared by routine procedures and stained with hematoxylin and eosin (Banks 1993). For IHC, a sheep hyperimmune serum against P. multocida was prepared. Briefly, the antigen was produced by growing P. multocida type A (#11246) in TSB, inactivating the bacteria with 0.12% formaldehyde, centrifuging the culture at 12,000g and washing the pellet obtained in phosphate buffered saline (PBS). The pellet was resuspended in PBS with thimerosal (0.2 g/L) and stored at 4-8 °C. This inoculum was used in seven serial inoculations carried out in two sheep (one year of age) by intramuscular route, with the antigen adjusted in a spectrophotometer to a transmittance of 37% (540 nm). The first four doses were administered only with inactivated antigen at two-day intervals. For the last three doses the inactivated antigen was mixed with aluminium hydroxide (AlOH) and administered every seven days.After processing and microtomy, tissue fragments with 3-5 μm thickness were fixed on poly-L-lysine treated slides, dewaxed, hydrated, and then subjected to the following steps: antigen retrieval in tissues by microwave irradiation for 5 min at 700W followed by enzymatic digestion with 0.04% pepsin (pH 7.8) for 10 min at 37°C; blocking of endogenous peroxidase with H2O2; incubation of sections for 2 h at 37°C covered with the anti-P. multocida primary sheep polyclonal antibody in a dilution of 1:500; incubation of sections for 30 min at 37°C with the LSAB® HRP Kit (DakoCytomation®); use of 3-amino-9-ethylcarbazole (AEC) for 5 min at 37°C; and counterstaining with Mayer's haematoxylin for 1 min. PBS (pH 7.4) was employed for washings between each step. All tissues obtained in the experiment were subjected to this technique. A fragment of lung with lesions (in stock) from a previously inoculated pig with the same P. multocida type A isolate was used as a positive control and as a negative control different samples of normal specific pathogen-free (SPF) pig to P. multocida were used.The specificity of the IHC technique was tested using swine tissues samples with typical lesions, experimentally infected with A. pleuropneumoniae, Actinobacillus suis, H. parasuis and M. hyopneumoniae. The Chi-square test (Χ2) was performed to demonstrate the association between the IHC and bacteriology results for the lung, pericardium, trachea, mediastinal lymph nodes, spleen and kidneys samples. Furthermore, the agreement, sensitivity and specificity of the IHC test were calculated adopting the bacteriology results as gold standard method.Microbiology. All tissues, swab samples and exudates collected were plated on blood agar and MacCon

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