Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin

One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.     

Within-population diversity of koala Chlamydophila pecorum at ompA VD1-VD3 and the ORF663 hypothetical gene

Infection of koalas by Chlamydophila pecorum is very common and causes significant morbidity, infertility and mortality. Fundamental to management of the disease is an understanding of the importance of multi-serotype infection or pathogen virulence in pathogenesis; these may need consideration in plans involving koala movement, vaccination, or disease risk assessment. Here we describe diversity of ompA VD1-3, and ORF663 hypothetical gene tandem repeat regions, in a single population of koalas with diverse disease outcomes. We PCR amplified and sequenced 72 partial ompA segments and amplified 25 tandem repeat segments (ORF663 hypothetical gene) from C. pecorum obtained from 62 koalas. Although several ompA genotypes were identified nationally, only one ompA genotype existed within the population studied, indicating that severe chlamydial disease occurs commonly in free-ranging koalas in the absence of infection by multiple MOMP serotypes of C. pecorum. In contrast, variation in tandem repeats within the ORF663 hypothetical gene was very high, approaching the entire range reported for pathogenic and non-pathogenic C. pecorum of European ruminants; providing an impetus for further investigation of this as a potential virulence trait.     

Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis

The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA.     

Epizootiology of Chlamydia infections in two free-range koala populations

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.     

Biological properties and genetic analysis of the ompA locus in chlamydiae isolated from swine.

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and by the morphologic features and ultrastructure of intracellular inclusions. Amplified chlamydial ompA DNA fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (RB) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet RB were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 RB, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the RB as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.     

Pasteurella multocida and bovine respiratory disease

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, bovine pathogens and common isolates from bovine respiratory disease (BRD), both enzootic calf pneumonia of young dairy calves and shipping fever of weaned, stressed beef cattle. P. multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping. Development of P. multocida-induced pneumonia is associated with environmental and stress factors such as shipping, co-mingling, and overcrowding as well as concurrent or predisposing viral or bacterial infections. Lung lesions consist of an acute to subacute bronchopneumonia that may or may not have an associated pleuritis. Numerous virulence or potential virulence factors have been described for bovine respiratory isolates including adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide, polysaccharide capsule and a variety of OMPs. Immunity of cattle against respiratory pasteurellosis is poorly understood; however, high serum antibodies to OMPs appear to be important for enhancing resistance to the bacterium. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature.     

Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.     

山羊流产衣原体ompA和CPAF基因的序列分析及原核表达

为了解山羊流产衣原体陕西分离株ompA和CPAF基因的遗传变异情况及制备检测抗原,根据GenBank收录的ompA和CPAF基因序列设计合成特异性引物,分别进行ompA基因和CPAF基因的基因克隆、序列分析及原核表达.结果表明,成功克隆出大小为1 053 bp的ompA基因和大小为1 056 bp的CPAF基因;核酸序...     

鸡源鸭疫里默杆菌的分离鉴定及ompA基因遗传进化分析

从病死鸡肝脏中分离到8株革兰阴性多形杆菌,对8株分离株进行形态学和16SrDNA基因鉴定并分析ompA基因及其推导的蛋白序列遗传进化特征.16S rDNA进化分析显示,8株鸡源分离株与22株鸭疫里默杆菌(Rieme-rellaanatipestifer,RA)参考株形成一个大的进化分支,与RA模式菌株ATCC11845...     

The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome

Clostridium perfringens has been implicated in a broad array of enteric infections including the fatal haemorrhagic enteritis/enterotoxaemia syndrome in cattle. The beta2 toxin (CPB2), encoded by cpb2, is suspected to be implicated in this syndrome. However, among C. perfringens isolates from cattle suspected of clostridial disease, an atypical allele was recently found to predominate at the cpb2 locus and atypical corresponding CPB2 proteins were shown to be poorly expressed, thus arguing against a biologically significant role of the beta2 toxin in clostridial diseases in cattle. This study compared genotype and phenotype of the beta2 toxin between C. perfringens isolates from a group of healthy calves (n=14, 87 isolates) and from a group of enterotoxaemic calves (n=8, 41 isolates). PCR results revealed the exclusive presence of the typical "consensus"cpb2 in the enterotoxaemic group. Western blot analysis demonstrated that the typical variant of CPB2 was often expressed in isolates from enterotoxaemic calves (43.9%) and infrequently in isolates from healthy cattle (6.9%). These data suggest that the typical variant of the CPB2 toxin may play a role in the pathogenesis of cattle enterotoxaemia.     

Genetic and antigenic analysis of Chlamydia pecorum strains isolated from calves with diarrhea

Chlamydia pecorum (designated 22–58) was isolated in 2010 in HmLu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in Yamaguchi Prefecture, Japan. Immunohistochemical staining identified C. pecorum positive reactions in the jejunal villi. C. pecorum, designated 24–100, was isolated from the feces of a calf with diarrhea in another farm in Yamaguchi Prefecture in 2012. A significant increase in neutralizing antibody titers against C. pecorum was confirmed in paired sera. Nucleotide sequence identities of omp1 genes of the 2 isolates were 100%. The isolates were genetically and antigenically more closely related to C. pecorum Bo/Yokohama strain isolated from cattle with enteritis in Japan than to the other prototype strains, Bo/Maeda isolated from cattle with pneumonia and Ov/IPA isolated from sheep with polyarthritis. These results indicate that C. pecorum strains similar to 22–58 and 24–100 might be endemic in Yamaguchi Prefecture and cause enteric disease in cattle.     

Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum

Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.     

Chlamydiaceae in cattle: commensals, trigger organisms, or pathogens?

Epidemiological data indicate that infection of cattle with chlamydiae such as Chlamydophila (C.) pecorum, C. abortus, C. psittaci and Chlamydia suis, is ubiquitous with mixed infections occurring frequently. The apparent lack of association between infection and clinical disease has resulted in debate as to the pathogenic significance of these organisms, and their tendency to sub-clinical and/or persistent infection presents a challenge to the study of their potential effects. However, recent evidence indicates that chlamydial infections have a substantial and quantifiable impact on livestock productivity with chronic, recurrent infections associated with pulmonary disease in calves and with infertility and sub-clinical mastitis in dairy cows. Data also suggest these infections manifest clinically when they coincide with a number of epidemiological risk factors. Future research should: (1) use relevant animal models to clarify the pathogenesis of bovine chlamydioses; (2) quantify the impact of chlamydial infection at a herd level and identify strategies for its control, including sub-unit vaccine development; and (3) evaluate the zoonotic risk of bovine chlamydial infections which will require the development of species-specific serodiagnostics.     

A clinically silent respiratory infection with Chlamydophila spp. in calves is associated with airway obstruction and pulmonary inflammation

This study was aimed at evaluating functional and inflammatory consequences of persistent chlamydial infections on the respiratory system in clinically inconspicuous calves aged 2-7 months. Thirteen calves persistently infected with Chlamydophila (C.) abortus and/or C. pecorum (Chl+) were compared to 12 calves without chlamydial infections (Chl-). In order to evaluate lung function, 36 non-invasive impulse oscillometry tests were performed per animal within 6 months. The group of chronically infected animals was distinguished by significantly higher peripheral airway resistance (indicating peripheral airway obstruction), significantly higher respiratory rates, and significantly higher minute volumes of ventilation. At the age of seven months, all calves were necropsied, broncho-alveolar lavage fluid (BALF) was obtained ex vivo, and lungs were examined histologically. Significantly higher concentrations of total protein and 8-iso-prostane (8-IP), as well as higher activities of matrix metalloprotease 2 were measured in BALF samples of Chl+ calves. Histologically, markedly activated bronchus-associated lymphoid tissue (BALT) causing partial obstruction of bronchiolar lumina was found in the apical pulmonary lobes of Chl+ calves. Chlamydial DNA was detected in the lung tissue of 7 out of 13 Chl+ calves by real-time PCR. In conclusion, respiratory chlamydial infection appeared to be associated with chronic inflammation of the lungs and airways. Despite the lack of clinical symptoms, pulmonary dysfunctions persisted in calves until the age of seven months. Data obtained in this study provide new insight illustrating the impact of nearly ubiquitous subclinical infections on the respiratory system.     

Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus

Assuming a synergistic or additive effect of Chlamydiaceae in coexistence with other enteropathogenic agents, the viral/bacterial interaction between a cell culture adapted porcine epidemic diarrhea virus (ca-PEDV) and different Chlamydiaceae strains was studied in vitro. Vero cells were dually infected with ca-PEDV and one of the three chlamydial strains Chlamydia trachomatis S45, Chlamydophila abortus S26/3 or Chlamydophila pecorum 1710S. Three experimental protocols were designed varying the inoculation sequence. Cell layers were first inoculated with Chlamydiaceae and 20 h later with ca-PEDV in protocol one. In protocol two, both agents were administered concurrently, whereas in protocol three, ca-PEDV was applied 20 h in advance of the Chlamydiaceae. Immunofluorescence techniques, immunohistochemical (IH) staining and electron microscopy were subsequently employed to investigate the cell layers. Using indirect immunofluorescence (IF) labeling, all mixed infections revealed dually infected cells, however, only incidentally and in low numbers. Characteristically, ca-PEDV syncytia with one or more chlamydial inclusions were detected but dually infected single cells were absent. Some syncytial cells contained enlarged C. abortus or C. pecorum inclusions with abnormally large developmental forms. In comparison with simultaneously conducted monoinfections, larger chlamydial inclusions were observed in dually infected cell layers. Experiments with C. trachomatis showed significantly increased numbers of chlamydial inclusions in dually infected cell layers compared to monoinfected ones. These findings indicate an influence of ca-PEDV on the chlamydial developmental cycle and in the case of C. trachomatis, a positive effect on chlamydial colonization in mixed infections.     

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.     

Detection and molecular characterization of calf diarrhoea bovine coronaviruses circulating in South Korea during 2004-2005

Although the widespread occurrence of calf diarrhoea (CD) bovine coronavirus (BCoV) infections have been reported in most cattle producing countries, only the genetic differences in the BCoVs from American and Canadian isolates and/or strains have been identified and compared. Hence, it is unclear if the BCoVs circulating in the other countries have distinct genetic characteristics. The aim of this study was to determine the prevalence and genetic diversity of CD BCoVs based on the deduced amino acid (aa) sequences of the spike (S) and haemagglutinin/esterase (HE) proteins in South Korea. RT-PCR and nested PCR using the primer pairs specific to the nucleocapsid gene, BCoVs detected the BCoVs in 56 (15.6%) of 359 diarrhoeic faecal samples. Phylogenetic analysis of the entire S gene indicated that 10 Korean CD BCoV strains clustered with other Korean BCoV strains with different clinical forms but were different from the American and Canadian BCoV strains. Moreover, the phylogenetic data of the aa sequences of the HE gene revealed all the Korean CD strains to be distinct from the other Korean BCoV strains with different clinical forms. These results suggest that the Korean BCoVs cause endemic infections in diarrhoeic calves in Jeonnam province and have taken a different evolutionary pathway from the BCoVs in other countries. Moreover, the different BCoV strains are circulating in the different clinical forms in South Korea. These results also suggest that vaccines against the BCoVs can be developed with each Korean BCoV in different clinical forms.     

Seroprevalence of chlamydial infection in cattle in Ireland

Although few studies have investigated the prevalence of chlamydial infections in cattle, reported prevalence rates vary hugely. In order to assess the prevalence of this infection in cattle in Ireland, serum samples (100 herds, 20 samples/herd) collected for statutory screening for brucellosis were examined by soluble chlamydial antigen indirect ELISA. The assay detects antibodies to the two most common Chlamydiaceae spp. affecting cattle, namely Chlamydia abortus and Chlamydia pecorum. A total of 95 samples from 57 herds were seropositive, representing an observed prevalence rate of 4.75%. The parametric bootstrap estimate of the mean disease prevalence in the population was 6.04% (95%, CI 4.70-7.50). The results suggest the prevalence of chlamydial infection is low in cattle in Ireland.     

A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds.     

Diversity of avian Pasteurella multocida strains based on capsular PCR typing and variation of the OmpA and OmpH outer membrane proteins

One hundred avian Pasteurella multocida isolates recovered from cases of fowl cholera and related infections in England and Wales over a 13-year period were characterised by capsular PCR typing and analysis of outer membrane protein (OMP) profiles. Sixty-eight percent of the strains were of capsular type A, 14% were type F, 5% were type D, 4% were type B and 9% were untypable. Nineteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Fifty-six percent of the isolates were represented by 15 OMP-types, whereas 44% of the isolates were associated with four OMP-types. The extensive molecular mass heterogeneity of the OmpA and OmpH proteins supports previous findings that avian P. multocida strains are very diverse. Furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. The high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of P. multocida are opportunistic pathogens of relatively low virulence. Strains of capsular types B, D and F, as well as the untypable isolates, were associated exclusively with specific OMP-types and represent distinct and widely disseminated clonal groups. These observations support the view that avian strains of P. multocida have a clonal population structure. Based on previous studies, the molecular mass heterogeneity of the OmpA and OmpH proteins might provide a selective advantage to P. multocida by generating antigenic variation.