Phylogenetic relationship of Pasteurella pneumotropica isolates from laboratory rodents based on 16S rDNA sequence

A 1344 bp fragment of the 16S ribosomal DNA (rDNA) sequence was used to determine the genetic relationship of Pasteurella pneumotropica isolates from laboratory rodents. A total of 30 nucleotide sequences of P. pneumotropica, including 24 wild strains, 3 reference strains, and 3 nucleotide sequences deposited in GenBank, were examined for heterogeneity of their 16S rDNA sequences. Phylogenetic analysis based on 16S rDNA sequence discriminated 5 types of branching lineages. Of these 5 types, 3 types had significant associations with mice or rats, and 2 had significant associations with the beta-hemolytic phenotype. These results suggest that 16S rDNA sequencing of P. pneumotropica isolates demonstrates genetic heterogeneity and phylogenetic discrimination in terms of their hemolytic phenotype and host associations.     

嗜肺巴氏杆菌的临床检测方法的评价

巴氏杆菌具有20个种，嗜肺巴氏杆菌就是其中一个种。由于这个家族地位特殊，巴氏杆菌种目前归属于巴氏杆菌属、放线杆菌属和嗜血流感杆菌属。嗜肺巴氏杆菌具有广泛的宿主，存在于许多啮齿类动物，其它实验动物中，也存在于人中。属中各个种的生化特性比较相似，采用目前的国家标准实验动物微生物检测方法，不能有效区分与嗜肺巴氏杆菌生化特性相类似的其它种。我们通过抽检广东省清洁级以上实验小鼠和大鼠280只．分离到168株巴氏杆菌，通过扩大生化鉴定项目，154株被确认为嗜肺巴氏杆菌，14株被划分为放线杆菌-嗜血流感杆菌-巴氏杆菌复合体。所分离的嗜肺巴氏杆菌表现甘露醇阳性，这与文献报道不符合。因此，在现有的鉴定嗜肺巴氏杆菌的生化标准上，增加了鸟氨酸脱羧酶、甘露醇、ONPG等生化项目，将有利于嗜肺巴氏杆菌的临床检测。     

The genetic classification of the Pasteurella pneumotropica complex

Pasteurella pneumotropica with its biotypes Jawetz and Heyl are the most common bacterial pathogens associated with diseases in rodents. 23 P. pneumotropica biotype Jawetz, biotype Heyl and P. pneumotropica-like rodentia isolates have been investigated phenotypically by characterization of their micromorphology and biochemical fermentation reactions. The taxonomic position within the family Pasteurellaceae has been examined by DNA:DNA hybridisation (optical method). It could be shown that P. pneumotropica biotype Jawetz represents a genus-like cluster containing several species including the V-factor dependent Haemophilus Taxon B and the avian P. pneumotropica-like organism and therefore resembles a new species of the new genus. It is concluded that the biotype Heyl of P. pneumotropica taxonomically remains as a species within the family Pasteurellaceae, however without further relationship to other known genera or genus-like groups.     

Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.     

Subcutaneous abscesses and arthritis caused by a probable bacterial L-form in cats

Three cats in one household developed pyogenic subcutaneous abscesses and arthritis over a period of 9.5 months. Despite vigorous surgical and antibiotic treatment, the infections in each of these cats continued to spread locally and hematogenously to involve other joints and subcutaneous sites. Although the infections did not respond to modern broad-spectrum antibiotics, they were susceptible to tetracycline. In spite of a favorable response to tetracycline, all 3 cats were euthanatized. A causative agent could not be identified by microbiologic culture of tissues obtained prior to death and at necropsy, or with special tissue stains. The infection was transmitted experimentally by sc inoculation with cell-free material from one of the naturally infected cats to a specific-pathogen-free cat. A tissue extract from the experimentally infected cat was, in turn, infectious for another specific-pathogen-free cat. The experimentally induced lesion was a rapidly enlarging necrotizing and pyogenic cellulitis and panniculitis, with no demonstrable causative agent by special tissue stains or microbiologic culture. A probable bacterial L-form was visualized in affected tissues of the experimentally infected cats and propagated in special L-form broth. Like the natural disease, infection in experimentally inoculated cats was progressive in nature, but could be treated successfully with tetracycline.     

Experimental atrophic rhinitis in 2 and 4 month old pigs infected sequentially with Bordetella bronchiseptica and toxigenic type D Pasteurella multocida.

Experimental infections with Bordetella bronchiseptica and/or toxigenic type D Pasteurella multocida were studied in 2- and 4-month-old primary specific-pathogen-free pigs. None of the 2-month-old pigs inoculated with B. bronchiseptica or P. multocida alone developed turbinate atrophy. All the pigs inoculated with B. bronchiseptica (10(7) CFU/head) and P. multocida (10(9) CFU/head for 5 consecutive days) together, however, developed clinical and post-mortem signs of atrophic rhinitis (AR) similar to the naturally occurring disease. Slight to severe turbinate atrophy was observed in the 4-month-old pigs inoculated with B. bronchiseptica and P. multocida (at the same concentration as above) at necropsy.     

Comparative study of experimental inclusion body hepatitis of chickens caused by two serotypes of avian adenovirus.

Twenty 1-day-old specific-pathogen-free chickens each were given an intraabdominal inoculation of either a type-8 avian adenovirus, [AMG 5 (2a], or a type-5 avian adenovirus, inclusion body hepatitis virus (IBHV). The diseases produced were similar. High (60-100%) mortality and statistically significant depression of body weights occurred in both infections. There were necrotizing hepatitis and pancreatitis, lymphoid depletion in the spleen, bursa of Fabricius and thymus, hydropericardium, nephritis and enteritis. Intranuclear inclusions occurred in affected organs. Fluorescent-antibody staining, the Feulgen reaction for deoxyribonucleic acid and electron microscopic studies, as well as studies from the literature, indicated that basophilic inclusions consisted of assembled adenovirions.     

PCR assay for the cell-free copro-DNA detection of Angiostrongylus cantonensis in rat faeces

To facilitate improved detection of the first stage larvae (L1) of Angiostrongylus cantonensis from rat faeces, a TaqMan(?) probe real-time PCR method for the detection in situ was developed targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. cantonensis. The assay was capable of detecting a single L1 in a grain of fresh faeces (weight 320 ± 125 mg) from the experimental infected Sprague-Dawley rats, and the method can also detect cell-free copro-DNA from positive faeces placed for up to 12 months at ambient environment. The present study exhibited a high level of specificity for A. cantonensis, with no fluorescence signals were observed in reference control consisting of four parasite species commonly found in the intestine of rat. This approach can overcome the limitations of DNA-based identification that faecal materials should be stored in 70% ethanol or kept as frozen samples for further tests, and thus it might be suitable and feasible for the detection of target DNA in faecal materials preserved at ambient temperature, but the detecting efficiency will depend on the amount of DNA in the samples and the time placed for the samples due to DNA degradation.     

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.     

Correlation of results of pulmonary computed tomography and pathologic findings in mice with Pasteurella-induced pneumonia

OBJECTIVE: To determine correlation between results of computed tomography (CT) versus pathologic examination for determining the volume percentage of affected lung in mice experimentally infected with Pasteurella pneumotropica. ANIMALS: 30 adult mice. PROCEDURE: After helical CT scans on day 0, mice were inoculated intranasally with P. pneumotropica. Repeat CT scans were performed on days 1, 2, 3, 4, 6, 8, 10, and 13. Regions of interest (affected areas) were manually drawn on the CT images, and percentage volume of normal lung was calculated by use of 3 methods: first-day volume, largest volume, and last-day volume. Three mice were euthanatized for pathologic evaluation after each scan day. The lungs were examined with a dissection microscope, and lesion scores were assigned on the basis of percentage volume of pneumonia. Correlation coefficients comparing results of the 3 CT methods with results of gross examination were calculated. RESULTS: Lung abnormalities were detected via dissection microscopy by postinfection day 2 and via CT by days 2 or 3. Correlation coefficients for the 3 CT methods of analysis, compared with pathologic findings, were 0.7 via first-day lung volume, 0.8 via largest lung volume, and 0.8 via last-day lung volume. CONCLUSIONS AND CLINICAL RELEVANCE: Results of CT correlated well with results of dissection microscopy for estimating percentage volume of lung affected by pneumonia in mice experimentally infected with P. pneumotropica. This method may be useful for longitudinal studies of pneumonia in mice.     

Serologic study of Pasteurella aerogenes sp n.

The antigenic attributes of Pasteurella aerogenes sp n were serologically compared with species of the genera Actinobacillus and Pasteurella. Examination included the tube-agglutination and double-immunodiffusion techniques. The results indicated the possibility of serologically different strains of P aerogenes. Antisera prepared from strains of P aerogenes also reacted well with antigens prepared from Yersinia pseudotuberculosis (P pseudotuberculosis) and P pneumotropica.     

An outbreak of avian urolithiasis on a large commercial egg farm

A significant outbreak of avian urolithiasis was observed on a large commercial egg farm. From the initial outbreak site (a single laying house), the incidence of urolithiasis slowly spread in the ensuing months to numerous other laying houses. Increasing mortality associated with urolithiasis commenced during late growout to early lay and then leveled off when egg production peaked. At the height of the outbreak, mortality was typically 0.5% per week; 75% of this mortality was due to urolithiasis. The clinical and pathologic features of this condition are described. Both infectious bronchitis virus (IBV) and fowl adenoviruses were isolated from organ homogenates of sampled birds. A clone of the IBV strain was found to induce nephritis in specific-pathogen-free white leghorns.     

Equine neural angiostrongylosis

Verminous encephalomyelitis due to Angiostrongylus cantonensis larvae was diagnosed in 2 foals at necropsy. The principal clinical feature was tetraparesis, although history and neurological examination revealed progressive and multifocal neurological disease. At presentation, a tentative diagnosis of parasitic larval migration involving the central nervous system (CNS), presumably due to Strongylus vulgaris, was proposed. Dissection of the spinal cord in one case resulted in recovery of intact larvae of both sexes of A. cantonensis. In both foals, histopathology of the brain and spinal cord revealed nematode sections which were consistent with A. cantonensis larvae.     

Observations on Alcaligenes faecalis infection in turkeys

Experiments were initiated to study the pathogenicity of 5 Alcaligenes faecalis isolates in specific-pathogen-free poults. The isolates were recovered from commercial flocks suffering from a respiratory disease. There were no differences between cultural or biochemical characteristics of the isolates, but differences in antibiotic sensitivity were detected. All 5 isolates were capable of initiating a respiratory disease in poults similar to that seen in the early stages of turkey coryza. The infection, clinical signs, and lesions were limited to the upper part of the respiratory tract, but there were substantial differences in the severity of disease initiated by different isolates. There were also differences in the persistence of infection in the host. Secondary infections in the tracheas and sinuses were higher in poults infected with A. faecalis. The disease observed in the experimentally infected birds was milder than in 4 naturally infected flocks that also had complicating Escherichia coli infections. There was no evidence of infection with infectious bursal disease virus in 4 naturally occurring outbreaks in Ohio. It is proposed that the term turkey coryza be used to describe the disease initiated by A. faecalis.     

Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage.

Modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, both tissue-culture-origin (TCO) and chicken-embryo-origin (CEO), were passaged 20 times in specific-pathogen-free chickens. After serial bird-to-bird passage, increased virulence was observed for CEO virus but not TCO virus. Increased mortality and increased severity and duration of respiratory disease were observed in chickens inoculated with chicken-passaged CEO viruses; only mild respiratory disease (no mortality) occurred in chickens inoculated with chicken-passaged TCO viruses. These findings suggest that ML ILT vaccine viruses may increase in virulence after bird-to-bird passage.     

Identification and virulence characterization of fowl adenoviruses in Korea

Since 2007, 55 adenovirus strains have been isolated from commercial chicken flocks in Korea and have been identified and the pathogenicity of these isolates was confirmed in specific-pathogen-free chickens of different age. Based on sequencing analysis of the hexon gene, 55 FAdV isolates were genetically related to the IBH-2A strain of FAdV3 (4 isolates, 99.2% to 100%), the KR5 strain of FAdV4 (22 isolates, 97.9% to 99.2%), the 764 strain of FAdV9 (11 isolates, 99.1% to 99.3%), and the 1047 strain of FAdV11 (18 isolates, > 99%). Experimental infections with four serotypes of FAdV resulted in high mortality of 18-day-old chicken embryos and 1-day-old chicks with marked liver necrosis similar to those observed in the natural outbreaks. Notably, specific hydropericardium was observed in chicks challenged with the K531 strain (serotype 4). However, 3-wk-old chickens challenged with FAdVs, regardless of serotype, did not show any clinical signs or mortality except histologic lesions of focal hepatocytic necrosis with mild lymphocytic infiltration. The results indicate that four FAdV serotypes (3, 4, 9, and 11) are the dominant serotypes of FAdVs in the Korea and are pathogenic enough to cause clinical disease in young chicks. The present investigation provides important information on the epidemiology and pathogenesis of FAdVs and highlights the importance of control strategies against FAdV infection in Korea.     

Antemortem diagnosis of canine neural angiostrongylosis using ELISA

A 5-month-old female Kelpie developed paraparesis, hind limb ataxia and spinal hyperaesthesia 4 days after ovariohysterectomy. Neurological examination demonstrated upper motor neuron signs in the pelvic limbs with lower motor neuron signs in the tail. Cerebrospinal fluid analysis demonstrated an increased protein concentration and marked eosinophilic pleocytosis. The dog was known to have eaten rats, snails and slugs. A tentative diagnosis of neural angiostrongylosis was made and later confirmed using an ELISA based on soluble antigens obtained from larval 4 Angiostrongylus cantonensis. Antibody titres from the patient's serum and CSF were 800 and 6400, respectively. The dog was treated successfully with prednisolone. ELISA testing of serum may provide a non-invasive means for diagnosing neural angiostrongylosis in dogs.     

Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.

A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains.     

鸡传染性支气管炎病毒CK/CH/LHLJ/04V的分离鉴定及致弱的初步研究

从黑龙江省某鸡场发病鸡群的肾脏中分离到一株病毒,通过病毒致SPF鸡胚病变特征、对鸡外周血红细胞凝集特性、病毒粒子形态学特征以及RT-PCR鉴定等方面的研究,表明该病毒为鸡传染性支气管炎病毒,并命名为CK/CH/LHLJ/04V。通过对该病毒基因型分析以及对SPF鸡致病性试验发现该病毒为我国近年来流行的一类重要IBV的代表。将该毒株在SPF鸡胚连续传代110代(P110),取不同代次毒进行动物实验。结果显示,该毒株对SPF雏鸡的致病力随鸡胚传代次数的增加而逐渐降低。P110代毒以105.0 EID50/0.1 mL的剂量通过点眼滴鼻接种15日龄SPF雏鸡,鸡群发病率和死亡率均为0。不同代次毒接种SPF鸡对同源毒株P3代强毒的攻击均具有100%保护性。实验表明,IBV毒株CK/CH/LHLJ/04V P110对SPF雏鸡已无致病性,但仍具有良好的免疫原性,可作为研制IB弱毒疫苗的候选毒株。     

Induced atrophic rhinitis in rats

Infections with Bordetella bronchiseptica and Pasteurella multocida were inducted in newborn specific-pathogen-free rats. Turbinate atrophy was quantified by measuring the length of the osseous core of the ventral turbinates. Bordetella bronchiseptica readily colonized the nasal cavity. Inoculated rats developed serum agglutinating antibodies to B bronchiseptica. Turbinate atrophy, correlating with a severe inflammatory reaction, was observed after dual inoculation with B bronchiseptica or after a single inoculation when the inoculum contained relatively large numbers of B bronchiseptica. Pasteurella multocida only rarely colonized the nasal cavity, even after prior instillation of weak acetic acid solution or B bronchiseptica.