Subcutaneous application of hyperimmune serum against Histophilus somni recombinant proteins affects serum antibody reactivity in beef calves.

BACKGROUND: Respiratory tract diseases cause significant economic loss in beef cattle. This study aimed to determine whether the application of hyperimmune serum (HS) containing antibodies against selected antigens of Gram-negative bacteria would improve the health and growth of different breeds of beef calves kept on three farms. Two recombinant protein antigens (Histophilus somni rHsp60 and rOMP40) were used to immunize four cows to produce HS. Eighty seven beef calves (Charolaise n = 36, Limousine n = 34, and crossbreed n = 17) were included into study. One hundred milliliters of serum were administered subcutaneously to 43 beef calves (Charolaise n = 18, Limousine n = 17, and crossbreed n = 8) twice, between 1 and 5 and 21-28 days of life. Calves were examined three times, and blood samples were taken to evaluate immunoglobulin M, G1, and G2, fibrinogen, serum amyloid A, and haptoglobin concentrations and reactivity of these Ig classes of antibodies against H. somni rHsp60 and rOMP40. Average daily weight gain during the first month and until weaning was calculated. RESULTS: HS showed higher (p ≤ 0.05) reactivity in calf sera against H. somni rHsp60 and OMP40 in IgG1 and IgG2. In experimental calves, compared to control calves, the reactivity of IgG1 against rOMP40 in the second sampling was higher in Limousine calves (p ≤ 0.001) and in the other two herds (p ≤ 0.05). Serum IgG2 antibody activity against H. somni rHsp60 in the second sampling was higher in experimental calves than in control calves in charolaise (p ≤ 0.05) and limousine (p ≤ 0.001) herds. The reactivity of IgG2 against rOMP40 in the second sampling of experimental calves was higher in herds with Charolaise and Limousine calves (p ≤ 0.001) and in crossbred calves (p ≤ 0.05). In the third sampling, serum IgG1 antibody reactivity against rOMP40 in Limousine calves was higher (p ≤ 0.05) in the experimental group. Among the other evaluated parameters, only SAA in the second sampling in the herd with Charolaise calves and heart rate in the herd with Limousine calves were significantly higher in the control calves (p ≤ 0.05). CONCLUSION: The application of HS to calves in all herds had an impact on specific reactivity in IgG1 and IgG2 classes against H. somni rOMP40 and rHsp60, antigens which were used for serum production.

Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A.

L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial infections.

Prevalence and antimicrobial resistance of opportunistic pathogens associated with bovine respiratory disease isolated from nasopharyngeal swabs of veal calves in Switzerland.

The composition of the bacterial flora in the calf nasopharynx might influence the risk of bovine respiratory disease (BRD). The aims of the present study were, firstly, to investigate the prevalence of bacteria potentially involved in BRD in the nasopharynx of veal calves and to identify associated risk factors for their presence, and, secondly, to provide data on antimicrobial resistance levels in these bacteria. Deep nasopharyngeal swabs were collected from veal calves on 12 Swiss farms over a period of one year by non-random, but systematic sampling for isolation of Pasteurellaceae and Mycoplasma (M.) bovis and dispar. Associations of potential risk factors with occurrence of these bacteria were tested in multivariable mixed logistic regression analyses, based on information gained from extensive questionnaires completed with the farmers. Antimicrobial susceptibility testing was performed for Pasteurellaceae by broth microdilution method to obtain minimal inhibitory concentrations (MIC). Pasteurellaceae, including Pasteurella (P.) multocida, Mannheimia (M.) haemolytica, Bisgaard Taxon 39 and Histophilus (H.) somni, were almost twice as prevalent as M. bovis and dispar in this study. Continuous stocking was a risk factor for the presence of Pasteurellaceae, especially when calves originated from more than six suppliers. In young calves (≤ 91 days), feeding of California Mastitis Test (CMT) positive milk was an additional risk factor for the presence of Pasteurellaceae whereas transport of calves by farmers and livestock traders (as opposed to transport only by farmers) increased the risk in older calves (> 91 days). Risk factors for the presence of M. bovis/dispar were higher number of calves per drinking nipple in young calves, and no access to an outside pen and feeding of CMT positive milk in older calves, respectively. While further research will have to investigate the observed associations in more detail, this suggests that management can play an important role in the prevalence of nasopharyngeal bacteria with a potential subsequent involvement in BRD. Antimicrobial resistance differed between the three bacterial species tested in this study and was highest to oxytetracycline and spectinomycin in P. multocida, oxytetracycline and penicillin in M. haemolytica, and ampicillin and penicillin in H. somni. Only two European VetCAST breakpoints (for florfenicol in P. multocida and M. haemolytica) have been published to date, matching the MIC distribution of the present isolate populations well, in contrast to certain commonly applied American Clinical and Laboratory Institute interpretive criteria. This highlights the potential for further refinement of clinical breakpoints in veterinary medicine.

GtxA is a virulence factor that promotes a Th2-like response during Gallibacterium anatis infection in laying hens.

GtxA, a leukotoxic RTX-toxin, has been proposed a main virulence factor of Gallibacterium anatis. To evaluate the impact of GtxA during infection, we experimentally infected laying hens with a G. anatis wild-type (WT) strain and its isogenic gtxA deletion mutant (ΔgtxA), respectively, and monitored the birds during a 6 day period. Birds inoculated with ΔgtxA had significantly reduced gross lesions and microscopic changes compared to the birds inoculated with the WT strain. To assess the host response further, we quantified the expression of pro-inflammatory cytokines and apoptosis genes by RT-qPCR. In the ovarian tissue, the expression levels of IL-4 and TNF-α were significantly lower in the ΔgtxA group compared to the WT group, while IL-6 and IL-10 levels appeared similar in the two groups. In the spleen tissue of ΔgtxA infected chickens, IL-4 expression was also lower compared to the WT infected chickens. The results indicated that GtxA plays a key role in an acute cytokine-mediated Th2-like response against G. anatis infection in the ovary tissue. The pro-inflammatory response in the ovary tissue of birds inoculated with ΔgtxA mutant was thus significantly lower than the wild-type response. This was, at least partly, supported by the apoptosis gene expression levels, which were significantly higher in the ΔgtxA mutant compared to the wild-type infected chickens. In conclusion, GtxA clearly plays an important role in the pathogenesis of G. anatis infections in laying hens. Further investigations into the specific factors regulating the host response is however needed to provide a more complete understanding of the bacteria-host interaction.

Respiratory and Reproductive Impairment of Commercial Layer Chickens After Experimental Infection with Gallibacterium anatis Biovar haemolytica.

The prevalence of Gallibacterium anatis in poultry production has increased over the last two decades. However, only a few studies have explored the pathogenicity of this bacterium in commercial layer chickens. This trial studied the aspects of the pathogenicity of a Gallibacterium anatis biovar haemolytica local Egyptian isolate (previously registered as strain B14 with GenBank accession no. KJ026147). We used 500 base pairs of a 16S ribosomal RNA gene and the 16S-23S ribosomal RNA intergenic spacer, partial sequence in an experimental infection trial in commercial White Shaver layer chickens aged 19 wk. The hens were divided into three groups of 40 birds each. The hens in Groups 1 and 2 were experimentally infected through the intranasal (IN) and intravenous (IV) routes, respectively, with a dose of 0.2 ml/bird containing 1.2 × 109 colony-forming units/ml. In contrast, Group 3 was kept as a noninfected control group. Both IN and IV infections resulted in a delayed egg laying for 1 wk and a significant (P ≤ 0.05) drop in egg production by 7.81% and 10.28% compared with the control group over 7 wk. Severe lesions in the form of hemorrhagic pneumonia, catarrhal tracheitis, ovarian follicle and oviductal regression, and septicemia were evident on necropsy, demonstrating the pathogenicity of G. anatis as a primary pathogen.

Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves.

BACKGROUND: Bovine coronavirus (BCV) is associated with respiratory infections in cattle of all ages; however, a temporal study to evaluate the effect of BCV immunity on virus shedding and bovine respiratory disease (BRD) incidence in pre-weaned beef calves has not been reported. Thus, we report here a prospective study in three herds of crossbred beef calves (n = 817) with endemic BCV. Serial blood samples for measurement of serum anti-BCV antibody titers and nasal swabs for detection of BCV and other common viral and bacterial BRD pathogens were collected from all calves or subsets of calves at predetermined times from birth through weaning. The calves were monitored for BRD and those that developed signs of respiratory disease were sampled for diagnostic testing. To discover additional risk factors that could have influenced BRD development, sequence analysis of the BCV strain(s) circulating in each herd, and the prevalence of common opportunistic bacterial pathogens in the upper respiratory tract of sick and apparently healthy cattle were also evaluated. RESULTS: Two hundred forty-eight of the 817 study calves (30.4%) were treated for BRD prior to weaning; 246 of those were from a single herd involved in two outbreaks of BRD leading to mass treatment of all calves in that group. Molecular diagnostic testing found BCV and Histophilus somni in nasal swabs taken at the time of BRD treatment. Between herd analyses revealed anti-BCV serum antibody abundance did not associate with the incidence of BRD or BCV shedding, though these measurements may have been hindered by the long periods between sample collections. Analysis of the BCV spike gene hypervariable region revealed four polymorphisms in 15 isolates from the three herds, making strain variation unlikely to account for differences in treatment rates between herds. Persistent or recurrent shedding episodes of BCV occurred in some animals treated for BRD. CONCLUSION: Co-detection of BCV and H. somni at the time of the disease outbreak suggests that these pathogens contributed to disease pathogenesis. Developing appropriate control measures for respiratory BCV infections may help decrease the incidence of pre-weaning BRD. The role of antibodies in protection must still be further defined.

Early migration pattern of Avibacterium paragallinarum in the nasal passage of experimentally infected chicken and Japanese quail by immunohistochemistry.

Infectious coryza (IC) is often a curse for poultry farmers when it occurs concurrently with several pathogens causing swollen head syndrome. The disease is caused by Avibacterium paragallinarum, which inflicts initial damage to the nasal and respiratory epithelium. This facilitates the progression of disease pathology across the nasal cavity, thereby providing a platform for multiplication of opportunistic microbes. In this study, we attempted to investigate the early entrance and migration pattern of A. paragallinarum in chicken and Japanese quail following experimental infection, by employing an in-house developed polyclonal antiserum against this pathogen. Antigenic-specificity of the raised antiserum was subsequently evaluated through immune-dot blot techniques and counter-current immunoelectrophoresis (CIE). The resultant antiserum characterized the antigen localization within formalin-fixed and partially decalcified nasal tissue sections though immunohistochemistry (IHC). Japanese quail showed prominent localization of the bacterial antigen at 12 h post-infection in anterior turbinates. However, the chicken exhibited a higher level of the bacterial pathogen with intense immuno-reactivity at 24 and 48 h post-inoculation. The decline in immunostaining intensity in the nasal tissue of chicken as well as Japanese quail by 72 h post-infection signifies either an attempt to resolve the infection by the resident immune cells across the nasal passage of the host, or its dissipation by certain inherent innate immune factors present across the nasal passage that are still unknown to us. In the present study, we used a moderately virulent pathogen (A. paragallinarum) that inflicted a mild to moderate degree of damage to histo-architecture of the nasal passage and provided a discernible migratory pattern with fewer alterations, along with provision toward unravelling basics of the immuno-pathogenetic mechanism. This knowledge will support efforts towards the development of a future mucosal nasal vaccine in birds affected with IC.

Respiratory pathogens and their association with population performance in Montana and Wyoming bighorn sheep populations.

Respiratory disease caused by Mycoplasma ovipneumoniae and Pasteurellaceae poses a formidable challenge for bighorn sheep (Ovis canadensis) conservation. All-age epizootics can cause 10-90% mortality and are typically followed by multiple years of enzootic disease in lambs that hinders post-epizootic recovery of populations. The relative frequencies at which these epizootics are caused by the introduction of novel pathogens or expression of historic pathogens that have become resident in the populations is unknown. Our primary objectives were to determine how commonly the pathogens associated with respiratory disease are hosted by bighorn sheep populations and assess demographic characteristics of populations with respect to the presence of different pathogens. We sampled 22 bighorn sheep populations across Montana and Wyoming, USA for Mycoplasma ovipneumoniae and Pasteurellaceae and used data from management agencies to characterize the disease history and demographics of these populations. We tested for associations between lamb:ewe ratios and the presence of different respiratory pathogen species. All study populations hosted Pasteurellaceae and 17 (77%) hosted Mycoplasma ovipneumoniae. Average lamb:ewe ratios for individual populations where both Mycoplasma ovipneumoniae and Pasteurellaceae were detected ranged from 0.14 to 0.40. However, average lamb:ewe ratios were higher in populations where Mycoplasma ovipneumoniae was not detected (0.37, 95% CI: 0.27-0.51) than in populations where it was detected (0.25, 95% CI: 0.21-0.30). These findings suggest that respiratory pathogens are commonly hosted by bighorn sheep populations and often reduce recruitment rates; however ecological factors may interact with the pathogens to determine population-level effects. Elucidation of such factors could provide insights for management approaches that alleviate the effects of respiratory pathogens in bighorn sheep. Nevertheless, minimizing the introduction of novel pathogens from domestic sheep and goats remains imperative to bighorn sheep conservation.

Fecal microbiome signatures are different in food-allergic children compared to siblings and healthy children.

BACKGROUND: Intestinal microbes have been shown to influence predisposition to atopic disease, including food allergy. The intestinal microbiome of food-allergic children may differ in significant ways from genetically similar non-allergic children and age-matched controls. The aim was to characterize fecal microbiomes to identify taxa that may influence the expression of food allergy. METHODS: Stool samples were collected from children with IgE-mediated food allergies, siblings without food allergy, and non-allergic controls. Stool microbiome characterization was performed via next-generation sequencing (Illumina) of the V1V3 and V4 variable regions of the 16S rRNA gene. Bacterial diversity, evenness, richness, and relative abundance of the operational taxonomic units (OTUs) were evaluated using QIIME. ANOVA and Welch's t test were utilized to compare groups. RESULTS: Sixty-eight children were included: food-allergic (n = 22), non-food-allergic siblings (n = 25), and controls (n = 21). When comparing fecal microbial communities across groups, differences were noted in Rikenellaceae (P = .035), Actinomycetaceae (P = .043), and Pasteurellaceae (P = .018), and nine other distinct OTUs. Food-allergic subjects had enrichment for specific microbes within the Clostridia class and Firmicutes phylum (Oscillobacter valericigenes, Lachnoclostridium bolteae, Faecalibacterium sp.) compared to siblings and controls. Identification of Clostridium sp. OTUs revealed differences in specific Clostridia drive the separation of the allergic from the siblings and controls. Alistipes sp. were enriched in non-allergic siblings. CONCLUSIONS: Comparisons in the fecal microbiome of food-allergic children, siblings, and healthy children point to key differences in microbiome signatures, suggesting the role of both genetic and environmental contributors in the manifestation of food-allergic disease.

Presence of Avibacterium paragallinarum and Histopathologic Lesions Corresponds with Clinical Signs in a Co-infection Model with Gallibacterium anatis.

Recently we demonstrated that co-infection with Avibacterium paragallinarum and Gallibacterium anatis leads to increased severity of clinical signs of infectious coryza in birds. The present study examined the interaction of these two pathogens in chickens by evaluation of histologic lesions in sinus infraorbitalis and nasal turbinates, applying a defined scoring scheme ranging from 0 to 3. Furthermore, for the first time, an in situ hybridization (ISH) technique was applied to detect A. paragallinarum in tissues. The samples were received from vaccinated and nonvaccinated birds that were infected with A. paragallinarum and/or G. anatis. Vaccinated birds were mostly devoid of any histopathologic lesions except a few birds with lesion score 1 at 7 and 14 days postinfection (dpi). Likewise, nonvaccinated birds infected with G. anatis only did not present microscopic changes in the sinus infraorbitalis, except in a single bird at 7 dpi. Interestingly, median lesion scores caused by G. anatis infection were significantly higher in the nasal turbinates of infected birds than in negative control at 7 and 14 dpi. The most prominent histologic changes were recorded from sinus infraorbitalis and nasal turbinates of nonvaccinated birds that were infected either with A. paragallinarum only or together with G. anatis. ISH demonstrated positive signals for A. paragallinarum in exudates present in the lumen or attached to the epithelial layer of investigated tissues. Such signals were mainly detected in tissues from birds with the highest histopathologic lesion scores.