Transmission and pathogenicity of Gallibacterium anatis and Escherichia coli in embryonated eggs.

In laying hens, Escherichia coli (E. coli) and Gallibacterium anatis (G. anatis) are considered the two major pathogens causing reproductive tract disorders, either as single infections or as co-infections. Vertical transmission has been confirmed for E. coli but remains to be clearly demonstrated for G. anatis. The aim of the present study was to investigate the ability of both G. anatis and E. coli at eggshell transmission using an embryonated egg dipping model, and to investigate the possible interaction between the two organisms in an embryonated egg injection model. Embryonated eggs were dipped into brain heart infusion broth containing 108 CFU/ml either of G. anatis 12656-12 liver, E. coli ST95 or E. coli ST141, respectively. E. coli ST95 and ST141 were re-isolated from the interior egg contents in 60% (12/20) and 85% (17/20) of the eggs, respectively, while G. anatis 12656-12 was only re-isolated from the interior egg contents in 6.7% (3/45) eggs. Eggs were injected with 10-1000 CFU of either G. anatis 12656-12, E. coli ST95 or ST141 into the allantoic cavity. As few as 10 CFU of G. anatis 12656-12 resulted in 100% mortality within 24 h post injection whereas the E. coli injected embryos all died at 48 h post injection. Significant difference in CFU counts were observed for G. anatis when compared G. anatis injection group with either of the two G. anatis - E. coli co-injection groups. Sixteen hours post injection, a significant difference in embryo mortality could be observed when comparing co-injected embryonated eggs (G. anatis and E. coli) and single-injected (G. anatis or E. coli) embryonated eggs. In conclusion, bacterial transmission via the eggshell was demonstrated for both G. anatis and E. coli although at different magnitudes. The embryonated egg injection model revealed that G. anatis in particular was highly pathogenic when exposed directly to the developing embryo.

Transmission dynamics of Mannheimia haemolytica in newly-received beef bulls at fattening operations.

The primary objective of this study was to determine, at the lung level, whether single or multiple clones of Mannheimia haemolytica are present within a pen during a bovine respiratory disease (BRD) episode. A secondary objective was to assess whether M. haemolytica isolates obtained from nasal swabs (NS) are identical to those isolated deeper within the respiratory tract. Sixteen BRD episodes that naturally occurred in 12 pens of eight to 12 bulls (n=112) newly-received at three fattening operations were investigated. One hundred and seventy five M. haemolytica isolates were collected from 239 pairs of trans-tracheal aspirations (TTA) and NS performed during these 16 BRD episodes. M. haemolytica isolates were characterized by pulsed-field gel electrophoresis (PFGE). PFGE types obtained from NS and TTA were then compared. M. haemolytica was isolated during 14 BRD episodes. Two to three different clones of M. haemolytica were recovered during 10 episodes whereas only one clone was recovered in four episodes. A moderate agreement (kappa=0.50) between NS and TTA for M. haemolytica isolation was observed. Identical PFGE types were only observed in 77% of matched NS-TTA pairs. The significant within-pen diversity of M. haemolytica during BRD episodes indicates that the disease is not primarily due to the spread of a single virulent clone among cattle and highlights the importance of predisposing factors that enable the resident flora to overcome the cattle's immune system. The results also demonstrate that isolates recovered from NS are not always representative of the isolates present deeper within the respiratory tract.

Transmission of Mannheimia haemolytica from the tonsils of lambs to the teat of ewes during sucking.

Objective of the work was to study whether Mannheimia haemolytica may be transmitted from the mouth of the lambs into the teat of the dam during sucking. We compared bacterial populations within the teat duct and milk of ewes immediately before and immediately after sucking by the lambs. Tonsils of lambs of the ewes were swabbed. M. haemolytica strain DAG21T recovered from a teat duct of a ewe was compared to strain DAG21R recovered from the tonsils of her lamb by using 16s rRNA sequencing. We used those two isolates and another one of known pathogenicity, for challenging ewes: (i) 2 mm deep into healthy teats, (ii) 2 mm deep into teats with chapping lesions or (iii) into the cistern of healthy mammary glands. Of samples collected before suckling, 20/792 were bacteriologically positive, and of those after, 50/792 were bacteriologically positive (P<0.001); in 37 cases, a negative sample became positive. One M. haemolytica (DAG21T) was recovered after suckling from a teat duct of a ewe. The organism was isolated from 57/90 tonsillar swabs from lambs. Risk of infection of ewe' teats was 0.004 throughout lactation, being greatest (0.021) during the 3rd week of lactation. The 16s rRNA sequences of strains DAG21T and DAG21R were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica. Organism deposition into healthy teats caused subclinical mastitis; deposition into teats with lesions or directly into mammary gland caused clinical mastitis. When results of inoculation of the three strains were compared between them, statistical significance was always P>0.9. Results provide clear evidence that suckling by lambs can lead to transmission of M. haemolytica into the teats of the ewes; the bacteria have the potential to cause mastitis if circumstances are favourable.

A bighorn sheep die-off in southern Colorado involving a Pasteurellaceae strain that may have originated from syntopic cattle.

We investigated a pasteurellosis epizootic in free-ranging bighorn sheep (Ovis canadensis) wherein a Pasteurellaceae strain carried by syntopic cattle (Bos taurus) under severe winter conditions appeared to contribute to pneumonia in affected bighorns. Twenty-one moribund or dead bighorn sheep were found on the "Fossil Ridge" herd's winter range, Colorado, USA, between 13 December 2007 and 29 February 2008. Eight carcasses examined showed gross or microscopic evidence of acute to subacute fibrinous bronchopneumonia. All eight carcasses yielded at least one ß-hemolytic Mannheimia haemolytica biogroup 1(±(G)) strain, and seven also yielded a ß-hemolytic Bibersteinia trehalosi biogroup 4 (CDS) strain; evidence of Pasteurella multocida, Mycoplasma ovipneumoniae, and parainfluenza 3 and bovine respiratory syncytial viruses was also detected. Isolates of ß-hemolytic Manneimia haemolytica biogroup 1(G) from a bighorn carcass and a syntopic cow showed 99.5% similarity in genetic fingerprints; B. trehalosi biogroup 4(CDS) isolates were ≥94.9% similar to an isolate from a nearby bighorn herd. Field and laboratory observations suggested that pneumonia in affected bighorns may have been caused by a combination of pathogens including two pathogenic Pasteurellaceae strains--one likely of cattle origin and one likely of bighorn origin--with infections in some cases perhaps exacerbated by other respiratory pathogens and severe weather conditions. Our and others' findings suggest that intimate interactions between wild sheep and cattle should be discouraged as part of a comprehensive approach to health management and conservation of North American wild sheep species.

Aerosol infection of calves with Histophilus somni.

The purpose of this study was to develop and evaluate an aerosol infection method with Histophilus somni that closely resembles the natural way of infection of calves. Another aim was to compare the virulence of two H. somni strains by collecting clinical and postmortem data of experimentally infected and control animals. Seventeen conventionally reared 3-month-old calves were divided into three groups. Two groups of six animals each were exposed to suspensions containing H. somni on three consecutive days using a vaporiser mask. The third group of five animals was used as control. The data of individual clinical examination were recorded daily. All animals were exterminated, and gross pathology of all lungs was evaluated on the 15th day after the first infection. Both H. somni strains caused an increase of rectal temperature, respiratory signs, decrease of weight gain, and severe catarrhal bronchopneumonia in both infected groups. Although some chronic lesions were detected in the lungs of the control animals as well, the histopathological findings in the infected and control groups were different. H. somni was recultured from all lungs in the challenged groups but it could not be reisolated or detected by PCR examination in the control group. This is the first paper on aerosol challenge of calves with H. somni using repeated infection and verified by detailed pathological, bacteriological and histopathological examination. The infection method proved to be successful. There was no difference in the virulence of the two H. somni strains used in the trial.

Prevalence and transmission of haemolytic Gallibacterium species in chicken production systems with different biosecurity levels.

A stratified cross-sectional study consisting of four strata of biosecurity based on production system type, including organic/free-range layer, battery-cage layer, layer parent, broiler parent and broiler grandparent flocks, was performed to estimate the prevalence of haemolytic Gallibacterium spp. Thirty birds were sampled by tracheal and cloacal swabs in each flock. A flock was considered infected when just one bird tested positive. A total of 27 flocks was included in the study. All chickens from the broiler grandparent flocks sampled negative, whereas 28% of the broiler parents, 40% of the layer parents, 67% of the battery-cage layers and 96% of the organic/free-range chickens sampled positive. A total of 95.9% (standard deviation +/- 7.6%) of birds from infected flocks was colonized by haemolytic Gallibacterium species. A significantly higher number of tracheal swabs was positive compared with cloacal swabs. The probability of vertical transfer was investigated by sampling offspring from an infected as well as a non-infected parent flock. None of the samples were found positive. In conclusion, we showed that haemolytic Gallibacterium spp. were widely distributed within the Danish commercial chicken production systems. However, prevalence proportions were highly influenced by the production system and found to be significantly associated with the biosecurity level observed in the flocks. In general, flock infections resembled an 'all or none' type of colonization as practically all of the chickens in infected flocks sampled positive. There was no evidence of vertical transmission of Gallibacterium.

Development and evaluation of PCR test for detection of Taylorella equigenitalis.

A PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis, was developed and evaluated. A genus-specific primer-probe set was derived from the 16S ribosomal DNA sequences. The PCR was specific and amplified a 585-bp product from all 64 available T. equigenitalis isolates. This PCR product hybridized with a specific probe in a dot spot assay. A variety of microorganisms from the genital tracts of horses or with a close phylogenetic relationship to T. equigenitalis did not yield a visible PCR product and were all negative in the dot spot hybridization assay. The results of the PCR assay were compared with those of culture by using 191 genital swabs from horses of several breeds. We demonstrate that the sensitivity of the PCR assay is superior to that of culture. The assay is most sensitive when DNA from culture plates incubated for at least 2 days is used. Of the tested samples, 1.5% were positive in the culture assay, whereas 35% were positive in the culture PCR assay. PCR-positive samples were obtained from all breeds tested. This means that many T. equigenitalis-carrying horses go unidentified by the current culturing technique. This affects current views about the spread and control of T. equigenitalis.