The past decade has seen remarkable technical advances in infectious disease diagnosis, and the pace of innovation is likely to continue. Many of these techniques are well suited to pathogen identification directly from pathologic or clinical samples, which is the focus of this review. Polymerase chain reaction (PCR) and gene sequencing are now routinely performed on frozen or fixed tissues for diagnosis of bacterial infections of animals. These assays are most useful for pathogens that are difficult to culture or identify phenotypically, when propagation poses a biosafety hazard, or when suitable fresh tissue is not available. Multiplex PCR assays, DNA microarrays, in situ hybridization, massive parallel DNA sequencing, microbiome profiling, molecular typing of pathogens, identification of antimicrobial resistance genes, and mass spectrometry are additional emerging technologies for the diagnosis of bacterial infections from pathologic and clinical samples in animals. These technical advances come, however, with 2 caveats. First, in the age of molecular diagnosis, quality control has become more important than ever to identify and control for the presence of inhibitors, cross-contamination, inadequate templates from diagnostic specimens, and other causes of erroneous microbial identifications. Second, the attraction of these technologic advances can obscure the reality that medical diagnoses cannot be made on the basis of molecular testing alone but instead through integrated consideration of clinical, pathologic, and laboratory findings. Proper validation of the method is required. It is critical that veterinary diagnosticians understand not only the value but also the limitations of these technical advances for routine diagnosis of infectious disease.

From 2009 to 2011, 163 sheep and 96 goat abortion submissions were received at the Animal Health Laboratory, University of Guelph, Ontario, Canada, for gross and histologic examination, as well as real-time polymerase chain reaction (PCR) testing for Chlamydophila abortus and/or Coxiella burnetii. Additional testing included immunohistochemistry for Toxoplasma gondii and Chlamydophila spp., routine bacterial culture and selective culture for Campylobacter spp., examination of modified acid-fast–stained placenta smears, enzyme-linked immunosorbent assay testing for Chlamydophila spp., and virus isolation. The final diagnosis made for each case by individual pathologists, based on gross and histologic lesions, as well as ancillary testing, was used as a standard to determine the significance of C. abortus and C. burnetii infection. Coxiella burnetii was identified by real-time PCR in 113 of 163 (69.0%) and 72 of 96 (75%) sheep and goat abortion submissions, respectively, but was considered to be significant in causing abortion in only 11 of 113 (10%) sheep and 15 out of 72 (21%) goat submissions that tested positive. Chlamydophila abortus was identified by real-time PCR in 42 of 162 (26%) and 54 of 92 (59%) sheep and goat submissions, respectively, but was considered the cause of the abortion in 16 of 42 (38%) sheep and 34 of 54 (63%) goat submissions that tested positive. Optimal sensitivity and specificity cut points for the real-time PCR copy number for C. abortus and C. burnetii were determined using the final pathology diagnosis as the reference test.

Soybean meal is an excellent and cost-effective protein source; however, its usage is limited in the piglet due to the presence of anti-nutritional factors and the antigens glycinin and β-conglycinin. The objective of the current study was to screen and select for bacteria that can be efficiently adopted to ferment soybean meal in order to solve this problem. Bacteria were isolated from fermented soy foods and the grass carp intestine, and strains selected for high protease, cellulase and amylase activities. The isolated bacteria were characterized as Bacillus cereus, Bacillus subtilis and Bacilus amyloliquefacien, respectively. Fermentation with food-derived Isolate-2 and fish-derived F-9 increased crude protein content by 5.32% and 8.27%, respectively; improved the amino acid profile by increasing certain essential amino acids, broke down larger soy protein to 35 kDa and under, eliminated antigenicity against glycinin and β-conglycinin, and removed raffinose and stachyose in the soybean meal following a 24-h fermentation. Our results suggest these two B. amyloliquefaciens bacteria can efficiently solid state ferment soybean meal and ultimately produce a more utilizable food source for growing healthy piglets.

A real-time polymerase chain reaction assay coupled with high resolution melting curve analysis (PCR-HRM) was developed for identifying and distinguishing Mycoplasma species commonly isolated from ruminant, avian, and canine samples. The real-time PCR used 1 set of universal primers specific for the spacer region between the 16S ribosomal RNA and the 23S ribosomal RNA genes; the melting curve analysis of the PCR product used a high-resolution melt fluorescent dye. The real-time PCR-HRM assay was able to distinguish M. arginini, M. bovigenitalium, M. bovis, M. bovirhinis, M. canadense, M. cynos, M. spumans, M. iowae, M. meleagridis, and M. agalactiae reference strains. The real-time PCR-HRM assay developed was evaluated by testing field isolates of M. bovis, M. arginini, M. bovirhinis, M. bovigenitalium, M. iowae, and M. spumans with results consistent with those of the fluorescent antibody test.

Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities toActinobacillus suisinfection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged withA. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.

Antimicrobial resistance (AMR) in Mycoplasma bovis has been previously associated with topoisomerase and ribosomal gene mutations rather than specific resistance-conferring genes. Using whole genome sequencing (WGS) to identify potential new AMR mechanisms for M. bovis, it was found that a 2019 clinical isolate with high MIC (2019-043682) for fluoroquinolones, macrolides, lincosamides, pleuromutilins and tetracyclines had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% sequence similarity to the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% sequence similarity and low MIC results. Known and potential AMR- associated genetic events were identified through multiple sequence alignment of the three genomes. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Using functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for AMR potential. Seventeen were previously identified from other bacteria as sites of AMR mutation, 38 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.