Antibody responses of calves to Histophilus somni recombinant IbpA subunits

Histophilus somni causes bovine pneumonia and septicemia, but protective immune responses are not well understood and immunodiagnostic methods are not well defined. We previously showed that antibody to a new virulence factor, IbpA, neutralizes cytotoxicity and immunization with a recombinant IbpA domain protects calves against experimental H. somni pneumonia. To further define immune responses to IbpA, we determined isotypic serum antibody responses to three IbpA domains (IbpA3, an N-terminal coiled coil region; IbpA5, a central region of 200 bp repeats and IbpA DR2, a C-terminal cytotoxic domain). ELISA was used to quantitate IgG1 or IgG2 antibodies to each of the IbpA subunits as well as H. somni whole cells (WCs) or culture supernatant (SUP). Calves experimentally infected with H. somni and monitored for up to 10 weeks had the least "0 time" (background) antibody levels to IbpA5, as well as the earliest and highest responses of greatest duration to the IbpA5 subunit. Responses of these calves were high to WC or SUP antigens but with higher "0 time" (background) antibody levels. We concluded that IbpA5 may be a useful immunodiagnostic antigen. Calves immunized with H. somni WC vaccine had antibody responses to WC antigens, but not to IbpA subunits before challenge. After challenge with H. somni, vaccinated calves had slight anamnestic responses to IbpA3 and IbpA5, but not to IbpA DR2. Since IbpA DR2 is a protective antigen, the data suggest the IbpA DR2 would be a useful addition to H. somni vaccines.     

Acquisition of haemoglobin-bound iron by Histophilus somni

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2,336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an approximately 120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.     

First isolation of Histophilus somni from goats

Histophilus somni (former name: Haemophilus somnus) is a Gram-negative, facultative pathogen bacterium that colonises the mucous membranes of cattle and sheep, however it was also described in American bison and bighorn sheep. It can cause local or generalised diseases and asymptomatic carriers can also occur. The presence and the etiological role of this microorganism have not been confirmed in any other domesticated species yet. The purpose of this study was to prove the presence of H. somni in goats by bacterial isolation. Nasal, vaginal or praeputial swab samples were collected from 205 goats in 10 flocks. H. somni strains were isolated from 2 out of 10 flocks; in one flock 10 H. somni strains were isolated from the genital mucosa of 17 goats, while a single H. somni strain was cultured from a vagina of 26 animals in the other flock. Partial amplification and sequencing of the 16S rRNA gene of three H. somni strains verified the identification. The comparative examination of carbon source metabolism using the Biolog Microstation ID System (Biolog, Ca) showed a close relationship of the caprine strains, while they were less related to H. somni type strain CCUG-36157 of bovine origin. H. somni strains were isolated only in the oestrus season from goat flocks with sheep contact. This is the first paper on isolation of H. somni from goats.     

Histophilus somni infection in cows with fertility problems and vaginitis

Histophilus somni associated with vaginitis in cattle; fasciolosis still prevalent in cattle; clostridial metritis in a large goat herd; detachment of the ischial tuberosity in pigs; fowl cholera in commercial and backyard flocks. These are among matters discussed in the Veterinary Laboratories Agency's (VLA's) disease surveillance report for September.     

Genetic diversity and tetracycline resistance genes of Histophilus somni

Nasal swabs were collected at three time points from 2378 calves in four feedlots and cultured for Histophilus somni to assess genetic relatedness and tetracycline resistance. The proportions of animals carrying tetracycline resistant isolates were 0.32% at arrival, 14.82% at interim, and 0.80% at exit. The 606 H. somni isolates recovered were compared by pulsed-field gel electrophoresis (PFGE), screened for the presence of plasmids, and assessed for the tetracycline resistance genes tet(A), tet(B), tet(C), tet(E), tet(G), tet(H), tet(K), tet(L), tet(M) and tet(O) using multiplex polymerase chain reaction. Most of the isolates (98.6%) belonged to one of seven PFGE clusters (A-G) of closely related profiles with 77.7% of the isolates belonging to clusters C and D. Clusters A, B and E were associated with a higher proportion of tetracycline susceptible isolates. Genetic diversity of the isolates was highest at entry in the feedlot and lowest after the period when the animals received in-feed chlortetracycline (interim samples). Clusters A and E were more prominently represented at exit from the feedlot than other clusters. All resistant strains harboured the gene tet(H) while no other tetracycline resistance genes and no plasmids were detected with the methodology employed. It appears that genetic variability in H. somni in Alberta feedlots is low, dissemination likely occurs by clonal expansion, and resistance to tetracyclines is mediated by the tet(H) encoded efflux pump. Pulsotypes associated with tetracycline susceptible strains appear more common at exit suggesting that the in-feed oxytetracycline included throughout the feeding period is not sufficient to exert selective pressure for resistant strains.     

Haemophilus somnus (Histophilus somni) in bighorn sheep.

Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.     

The first report of Histophilus somni pneumonia in Nigerian dairy cattle

Clinical signs of severe bronchopneumonia, including anorexia, coughing, nasal discharge, dyspnoea, diarrhoea, distension of the neck, lethargy, recumbency, lameness preceding collapse, and death were observed among a herd of Holstein–Friesian dairy cattle. The outbreak occurred over a 30-day period, and attack and case-fatality rates were 0.4% and 50%, respectively. At necropsy, extensive consolidation in the cranioventral parts of the lungs was observed. Histologically, a severe acute bronchopneumonia with slight pleuritis was present. Both pathological and bacteriological evaluation of the lungs incriminated Histophilus somni (heavy growth). Supplementary laboratory investigations also isolated Clostridium and Klebsiella species (scanty growth) from the lungs. Histophilosis in cattle was confirmed for the first time in Nigeria.     

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.     

Pathogenicity of different strains of Histophilus somni in the experimental induction of ovine epididymitis

The purpose of this study was to determine any differences in pathogenicity when sheep are experimentally infected with different Histophilus somni isolates: a) 2336 bovine origin strain; b) an isolate from ram orchitis and epididymitis; c) an isolate from the brain of a sheep with neurological signs; d) an isolate from the vagina of a clinically healthy ewe. A total of 20 rams divided in groups of 5 animals each were inoculated in the epididymis with 1 × 10⁷ CFU/mL of H. somni; a negative control group of 5 rams was used. All groups inoculated with H. somni showed some epididymitis, but the most pathology was caused by the epididymitis isolate, followed by the vaginal isolate. It was demonstrated that there is a difference in experimental infection capacity among isolates from different origins, as epididymitis occurred and the bacteria was recovered only from groups inoculated with isolates originating from epididymitis and vaginal exudate.     

Antimicrobial susceptibility of Haemophilus parasuis and Histophilus somni from pigs and cattle in Denmark

A total of 52 Haemophilus parasuis and 80 Histophilus somni isolates were tested for antimicrobial susceptibility by MIC-determinations. None of the isolates were resistant to ampicillin, ceftiofur, ciprofloxacin, erythromycin, florphenicol, penicillin, spectinomycin, tetracycline, tiamulin, or tilmicosin. Two H. parasuis isolates were resistant to trimethoprim + sulfamethoxazole. Six H. parasuis isolates had reduced susceptibility (0.06-0.5 microg/ml) to ciprofloxacin and 10 reduced susceptibility to TMP + sulfamethoxazole (1-2 microg/ml). This study showed that Danish isolates of H. parasuis and H. somni in general are fully susceptible to antimicrobial agents currently used for treatment of infections with these pathogens.     

The role of lipooligosaccharide phosphorylcholine in colonization and pathogenesis of Histophilus somni in cattle

Histophilus somni is a Gram-negative bacterium and member of the Pasteurellaceae that is responsible for respiratory disease and other systemic infections in cattle. One of the bacterium’s virulence factors is antigenic phase variation of its lipooligosaccharide (LOS). LOS antigenic variation may occur through variation in composition or structure of glycoses or their substitutions, such as phosphorylcholine (ChoP). However, the role of ChoP in the pathogenesis of H. somni disease has not been established. In Haemophilus influenzae ChoP on the LOS binds to platelet activating factor on epithelial cells, promoting bacterial colonization of the host upper respiratory tract. However, ChoP is not expressed in the blood as it also binds C-reactive protein, resulting in complement activation and killing of the bacteria. In order to simulate the susceptibility of calves with suppressed immunity due to stress or previous infection, calves were challenged with bovine herpes virus-1 or dexamethazone 3 days prior to challenge with H. somni. Following challenge, expression of ChoP on the LOS of 2 different H. somni strains was associated with colonization of the upper respiratory tract. In contrast, lack of ChoP expression was associated with bacteria recovered from systemic sites. Histopathology of cardiac tissue from myocarditis revealed lesions containing bacterial clusters that appeared similar to a biofilm. Furthermore, some respiratory cultures contained substantial numbers of Pasteurella multocida, which were not present on preculture screens. Subsequent biofilm experiments have shown that H. somni and P. multocida grow equally well together in a biofilm, suggesting a commensal relationship may exist between the two species. Our results also showed that ChoP contributed to, but was not required for, adhesion to respiratory epithelial cells. In conclusion, expression of ChoP on H. somni LOS contributed to colonization of the bacteria to the host upper respiratory tract, but phase variable loss of ChoP expression may help the bacteria survive systemically.     

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.     

Single nucleotide polymorphisms in the bovine Histophilus somni genome; a comparison of new and old isolates

Histophilus somni, a causative agent of the bovine respiratory disease complex, can also cause a variety of systemic disorders, including bronchopneumonia, myocarditis, pericarditis, arthritis, pleuritis, and infectious thrombotic meningoencephalitis. The purpose of this study was to determine if currently circulating strains differ from those of the 1980s by identifying genomic changes. Single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites were examined by whole-genome sequencing in 12 samples, 6 old and 6 new. The 31 028 SNP/INDELs recorded were compared against the reference genome sequence of the pathogenic H. somni strain 2336. The distribution of about 75% of these SNPs within a specified gene differed between old and new isolates and did not follow any particular pattern. The other 25% clustered into 2 groups containing the same SNPs in various genes: group I included 5 old isolates and 1 new isolate; group II included 5 new isolates and 1 old isolate. For putative virulence genes there were more SNPs in group I compared with strain 2336, itself an older isolate, than in group II. Although only 25% of all the SNPs formed 2 clusters, the results suggest some genetic difference in various genes between old and new strains.     

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays.     

Optimization of Salmonella enteritidis recombinant heat shock protein 60 production

The aim of the study was to optimize conditions for producing Salmonella Enteritidis recombinant heat shock protein 60 (rHsp60). Seven Escherichia coli host strains (Rosetta, Turner, C41, C43, Origami, BL21pLys, Rosetta pLys) were transformed by a recombinant plasmid containing Hsp60 gene from Salmonella Enteritidis, and then cultured and induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The highest S. Enteritidis rHsp60 yield was obtained using E. coli strain C41. Induction of this strain using IPTG allowed the yield 400 microg of S. Enteritidis Hsp60 protein/2L of culture, but by autoinduction the yield exceeded 800 microg/2L.