Browsing by Author "De Buck, Jeroen M."
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Item Open Access Antimicrobial resistance: Prevalence, genetics and associations with antimicrobial use in food-producing animals(2020-07-27) Borin Nobrega, Diego; Barkema, Herman W.; De Buck, Jeroen M.; Deardon, Rob; Dufour, Simon; Saini, VineetAntimicrobial use (AMU) in livestock has come under growing criticism. There is increasing pressure to optimize AMU in food-producing animals, which will likely entail restrictions and voluntary reductions of their use, as well as implementation of protocols promoting antimicrobial stewardship. In this thesis, 1) methods were compared for obtaining AMU data on dairy farms, 2) factors associated with the prevalence of antimicrobial resistance (AMR) in non-aureus staphylococci (NAS) isolated from intramammary infections were studied, 3) treatment strategies for non-severe clinical mastitis (CM) in dairy cattle were contrasted, and 4) effects of restricted antimicrobial use in food-producing animals towards the prevalence of AMR genes (ARGs) were evaluated. Chapter 2 confirmed that treatment records accurately quantified AMU in well-managed dairy herds. Yet, their widespread adoption into AMU surveillance cannot be recommended, due to an underestimation of AMU in herds with elevated bulk tank somatic cell count. In regard to AMR, Chapter 3 demonstrated that resistance against tetracycline, penicillin and erythromycin in NAS was common in Canadian dairy herds. In Chapter 4, factors associated with AMR were further explored. An association between AMR in NAS and AMU was present when penicillins, 3rd-generation cephalosporins or macrolides were administered systemically, whereas intramammary use of antimicrobials were not associated with AMR. As antimicrobials classified as critically important antimicrobials (CIAs) for humans were associated with AMR, in Chapter 5 a systematic review was done to assess whether CIAs and non-CIAs had comparable efficacy to treat non-severe bovine CM caused by the most prevalent bacteria causing mastitis worldwide. No protocol including the use of CIAs had superior bacteriological cure rates of non-severe CM than protocols relying on non-CIAs. Therefore, no adverse effects in terms of animal health should be expected by ceasing use of CIAs for treating non-severe CM in dairy herds. A second systematic review showed that restricted AMU in food animals was associated with a lower presence of ARGs in bacteria isolated from animals and humans. Reducing use of CIAs to treat non-severe CM in typical dairy herds may reduce load of ARGs without significant impacts on animal health and welfare.Item Open Access Creating a Marked Mycobacterium avium subsp. paratuberculosis Vaccine Strain and Detecting Marker-Specific Immune Responses in Calves(2018-04-17) Luo, Yi Yi; De Buck, Jeroen M.; Dong, Tao G.; Abdul-Careem, Mohamed FaizalMycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a chronic, contagious granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control due to its ability to survive in harsh environmental conditions for long periods of time, its easily transmittable nature through the fecal-oral route of transmission, the difficulty to detect the pathogen, and lack of an effective treatment or cure. The difficulty with detecting the pathogen stems from current diagnostic assays lacking specificity, posing issues with mycobacterial cross-reactivity, and low sensitivity, particularly during subclinical stages of disease. Previously developed vaccines do not prevent infection and are not approved in Canada in part due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were inserted into a field strain of MAP as part of a vaccine capable of differentiating infected from vaccinated animals (DIVA). Specialized transducing mycobacteriophages were used to replace a gene coding for an immunogenic protein (MAP1693c) in the MAP genome with an HA (human influenza hemagglutinin) epitope-tagged immunogenic gene (PepA) via allelic exchange. Once gene replacement was confirmed, these markers were evaluated in a calf infection trial, where 17 Holstein-Friesian dairy calves were inoculated with either 109 CFUs of the marked strain (n = 6), 109 CFUs of a wild-type (WT) field strain (n = 6), or remained uninfected controls (n = 5). Cellular and humoral immune responses were measured using an interferon gamma release assay (IGRA) and enzyme-linked immunosorbent assay (ELISA), respectively. Marker-specific IFN- release was measured by stimulating whole blood with marker peptides and proteins, while antibody response was assessed by incubating serum with the same marker peptides and proteins. Some calves inoculated with the marker strain showed increased cellular immune responses to the HA epitope positive marker. Unexpectedly, a scrambled version of the HA epitope induced a significant IFN- response in marker-infected calves compared to WT-infected (p = 0.016) and uninfected (p = 0.019) groups at 4.5 months post-inoculation. This positive marker thus holds potential as a valuable diagnostic tool as part of a DIVA vaccine for Johne’s disease. We have shown that immune markers, both immunogenic proteins and epitope tags, can be introduced into the MAP genome using a stable allelic exchange method. The specificity of the scrambled HA epitope as an antigen to test for marker-specific cellular immune responses requires further investigation.Item Open Access Development of a Diagnostic Platform to Detect Protein Biomarkers of Infectious Diseases(2020-04-28) Mukherjee, Sonia; De Buck, Jeroen M.; Schryvers, Anthony Bernard; Gilch, Sabine; Storey, Douglas G.Production limiting diseases are a major cause of economic losses to the dairy industry and often affect animal welfare. The implementation of robust diagnostic tests that can detect diseases at an early stage is essential to decrease the high costs associated with the treatment of chronic cases and lost productivity. In this study, we focus on the development of diagnostic tests for two important production limiting diseases, mastitis and enzootic bovine leukosis (EBL). Somatic cell count is the gold standard test for the diagnosis of subclinical mastitis, but an increase in somatic cell count may not always correlate to infection of the udder as it is influenced by multiple factors. On the other hand, the most common methods to diagnose EBL are agar gel immunodiffusion or ELISA. A major limitation of these tests is the requirement of manual handling of samples and the long processing time, which makes them unsuitable as on-farm diagnostic tests. Therefore, the development of reliable point-of-care technology to detect these diseases in a convenient and cost-effective manner is required. In this regard, biomarkers of infection and disease can be detected with a novel biosensor, based on a novel split trehalase (TreA) enzyme, developed in our lab. Glucose is the output signal for this biosensor platform which can be detected by a colorimetric enzymatic assay or a handheld glucometer. In this thesis, we have applied the existing biosensor platform to detect BLV antibodies in clinical serum by using bacterial surface complementation of fusion proteins, containing split TreA fragments fused to antigens expressed on the outer membrane of E. coli. Our results indicate that using this assay, it is possible to detect anti-BLV antibodies in clinical serum and distinguish between healthy and BLV positive samples. We also applied the split TreA biosensor platform to detect lactoferrin, which is the biomarker of subclinical mastitis in cattle. A phage display library approach was used to identify the peptides with affinity for LF. In conclusion, we expanded the existing split TreA biosensor platform with tests for the detection of production diseases like EBL and mastitis that are compatible with on-farm use.Item Open Access The Development of Immunoreagents to Detect for a Cellular Immune Response(2019-08-20) Ma, Crystal Wai Yin; De Buck, Jeroen M.; Jenne, Craig N.; Santamaria, Pere; van Marle, GuidoCell mediated immunity (CMI) is the adaptive T-cell-mediated defense mechanism against pathogens. Oftentimes, by assessing lymphocyte counts and/or cytokine profiles, the presence of an infection can be detected. While current diagnostic tests (e.g. interferon gamma release assay, delayed type hypersensitivity test) exist, many are not widely adopted due to the requirement of specific equipment for quantification, time restraints and/or false negative results. As such, the discovery of new biomarkers that can accurately detect and quantify a T-cell mediated, antigen specific immune response would be very valuable. We aimed to explore cellular immune responses (CIRs) through the direct detection of antigen specific Major Histocompatibility Complex II - T Cell Receptor (MHC II-TCR) interactions. We anticipated to create bioreagents that could enumerate TCR:MHC interactions, thereby giving possible insights of a previously activated CMI by targeting effector T-cells. To do this, superantigens (SAgs) (here: TSST-1) were split and fused to a reporter protein (YFP), allowing for specific TCR-pMHC interactions on antigen presenting cells (APCs) to be detected through a fluorescent marker. In the presence of a cognate pMHCII:TCR interaction, our split superantigens would be able to bind and reassemble, thereby quantifying the interaction using fluorescence. To further detect for sensitized T-cells, we engineered peptide-recombinant T cell receptor ligands (pRTLs) to be displayed on the surface of fluorescent E. coli using a pAIDA1 expression system. This system is intended to mimic the expression of MHC II on APCs. Interactions between the T-cells and pRTLs on the surface the E. coli can therefore be visualized using microscopy followed by flow cytometry analysis, whereby clustering of GFP-bacteria around effector T-cells would arise as a result of successful peptide display. Our results indicated no binding occurred between the individual C-domain of TSST-1 and T-cells. It was also revealed that our designed pRTL that were expressed on the surface of E.coli were unable to bind to T-cells, possibly due to complications with expressing the pRTL using a bacterial surface expression vector. Here, we describe the methodology of our approach, including construct design, protein production and analysis. We also explore existing technologies,and suggest future approaches and considerations that can be made to supplement the current research we have performed in this project. In conclusion, our developed biomarkers did not successfully bind to T-cells as we had anticipated, however, we did develop valuable insight regarding the use of SAg and pRTLs are potential bioreagents for the detection of a cellular immune response.Item Open Access Development of Intranasal Bacterial Therapeutics to Mitigate the Bovine Respiratory Pathogen Mannheimia haemolytica(2019-11) Amat, Samat; van der Meer, Frank; Alexander, Trevor W.; Buret, Andre G.; De Buck, Jeroen M.; McAllister, TimThe emergence of multidrug-resistant pathogens associated with bovine respiratory disease (BRD) presents a significant challenge to the beef industry, as antibiotic administration is commonly used to prevent and control BRD in commercial feedlot cattle in North America. Thus, developing antibiotic alternatives such as bacterial therapeutics (BTs) to mitigate BRD is needed. Recent studies suggest that the nasopharyngeal (NP) microbiota, particularly lactic acid-producing bacteria (LAB), are important to bovine respiratory health and may be a source of BTs for the inhibition of BRD pathogens. The research presented in this thesis aimed to develop intranasal BTs to mitigate the BRD pathogen Mannheimia haemolytica and promote NP microbiota stability in feedlot cattle. Results from Study 1 showed that commercial probiotic bacteria were able to inhibit M. haemolytica growth and its adherence to epithelial cells. Study 2 revealed that the NP microbial community structure and relative abundance of LAB families underwent significant changes when cattle transported from the farm to an auction market, then to feedlot. Many of the LAB families were inversely correlated with the BRD-associated Pasteurellaceae family, and isolates from Lactobacillaceae, Streptococcaceae and Enterococcaceae families inhibited growth of M. haemolytica in vitro. This study provided evidence of potential antagonistic competition taking place between LAB and BRD-associated pathogens within the respiratory tract. Following these studies, using a targeted approach based on criteria evaluating M. haemolytica inhibition, adherence to turbinate cells, and immunomodulation, 6 Lactobacillus strains from an initial group of 178 bacterial isolates originating from nasopharynx of cattle were identified as the best BT candidates (Study 3). Intranasal inoculation of these BTs reduced colonization by M. haemolytica and induced modulation of respiratory microbiota in dairy calves experimentally challenged with M. haemolytica (Study 4). Finally, the longitudinal effects of intranasally administered BTs on the NP microbiota and the prevalence of BRD pathogens including Mannheimia were evaluated in post-weaned beef calves (Study 5). A single dose of intranasal BTs induced longitudinal modulation of the NP microbiota while showing no adverse effects on animal health and growth performance. With further characterization of inoculant dose and time of inoculation, the BTs may have potential for application as an antimicrobial alternative for mitigation of M. haemolytica in beef cattle.Item Open Access Disease Control on Dairy Farms with a Focus on Johne's Disease and Veterinary Communication(2018-04-19) Ritter, Caroline Manuela Nancy; Barkema, Herman W.; Adams, Cindy L.; Jansen, Jolanda; De Buck, Jeroen M.; Kelton, David F.; Mason, SteveMotivating dairy farmers to implement disease prevention and control strategies can be challenging. The first objective of this thesis was to assess factors that influence farmers’ management decisions. A literature review was conducted focusing on socio-psychological influences and farmers’ preferred information sources. Additionally, surveys and qualitative interviews with Alberta dairy farmers contributed to the current knowledge by assessing farmers’ attitudes, beliefs, and perceptions in regard to the prevention and control of Johne’s disease, an infectious enteritis that is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is endemic in Canadian dairy cows. Environmental fecal samples were analyzed for MAP to assess whether farm infection status influenced farmers’ decision to enroll in the voluntary Alberta Johne’s Disease Initiative (AJDI). Observed herd prevalence of MAP (i.e., 51%) was similar between AJDI participants and nonparticipants. Results further indicated that farmers have to believe in the importance of the disease and in recommended prevention and control strategies to make changes. In Alberta, the reasons why farmers did not participate in the AJDI or implemented recommended measures included skepticism of the threat and negative effects of Johne’s disease, critique of test sensitivity, required time, and costs. Farmers used a variety of information channels, but herd veterinarians had a major influence on their management. Veterinarians are in an ideal position to communicate and motivate recommended strategies targeted to each farm, and it is well established that effective communication skills can improve adherence with advice and health outcomes. Therefore, the second objective was to assess veterinary communication patterns. First, the suitability of on-farm video recordings for comprehensive communication analysis using the Roter Interaction Analysis System was demonstrated. Then, veterinary communication during 70 dairy farm visits was assessed. Veterinarians spent most of their talk on farmer education and relationship building. Demographics such as gender and length of the professional veterinarian-farmer relationship affected the use of some communication variables, whereas the effect of previous communication training was minimal. Identification of influences on farmers’ management decisions and of veterinary communication patterns can reveal opportunities to enhance communication, thus improving the uptake of prevention and control measures.Item Open Access Distribution of Treponema species and antimicrobial resistance genes in digital dermatitis lesions(2018-08-04) Beninger, Caroline; De Buck, Jeroen M.; Orsel, Karin; Morck, Douglas W.Digital dermatitis lesions are ulcerative or proliferative masses between the heel bulbs. The most significant clinical outcome of digital dermatitis (DD) is lameness, leading to animal welfare concerns and economic loss for dairy producers worldwide due to premature culling, milk loss, and decreased fertility. While there is insufficient evidence to determine the etiological agent(s) responsible for DD, it is widely accepted that DD is a polymicrobial disease significantly associated with anaerobic bacteria, Treponema. Difficulties in obtaining pure cultures and the nearly exclusive presence of Treponema in diseases as members of a polytreponemal or polymicrobial communities has led to insufficient species identification within lesions and incomparable prevalence estimates based on phylotype. Our primary research objectives were to develop a diagnostic tool to identify Treponema spp. within DD lesions to associate presence and abundance with DD lesion grades. Further, we examined the distribution of antimicrobial resistance genes (AMGs) within sequenced isolates of DD-associated Treponema and designed targeted PCR for Treponema AMGs (AMGsTrep) within lesions. We provide significant evidence that the absolute quantities of Treponema and AMGsTrep are lower in chronic and early, active DD lesions suggesting Treponema spp. within these lesions may be more susceptible to antimicrobials (AMs) compared to species in advanced, active lesions. We have identified potential interactions among Treponema spp. that may facilitate DD progression and enhance pathogenicity and may affect AM susceptibility and lesion chronicity. With the novel diagnostic tool developed here, future research should elucidate interactions among Treponema spp. and in vitro susceptibility profiles and the efficacy of treating early and chronic lesions compared to advanced, active lesions on infection resolution.Item Open Access Effects of transportation to and commingling at an auction market on the bacterial communities of the respiratory tract of beef cattle(2018-07-12) Stroebel, Christina Maria; Timsit, Édouard; Alexander, Trevor W.; De Buck, Jeroen M.; Janzen, Eugene D.Commingling at auction markets is considered a major predisposing factor for bacterial bronchopneumonia (BP) in beef cattle. However, the effects of commingling on the respiratory tract bacterial communities is largely unknown. Therefore, the objective of the thesis was to study the effects of transportation to and commingling at an auction market on the nasopharyngeal and tracheal bacterial communities of recently weaned calves using culture-dependent and independent methods. Two groups of 30 Angus-cross heifers were studied from weaning at the ranches of origin to 28 d after arrival at a feedlot. For each group, half the heifers were either transported directly to a feedlot after weaning (RANC) or transported to and commingled at an auction market for 24 hrs before being placed in a feedlot (AUCT). Heifers from both groups received vaccines against major respiratory viruses and a parenteral injection of a long acting macrolide (tildipirosin) at on-arrival processing (d2). Deep nasal swabs (DNS) and trans-tracheal aspirates (TTA) were collected at weaning (d0) and at on-arrival processing at the feedlot (d2). Deep nasal swab sampling only was then repeated 7 days (d9) and 28 days (d30) after arrival. Bacterial culture and 16S rRNA sequencing did not reveal difference in the nasopharyngeal and tracheal bacterial communities between RANC and AUCT at any sampling days. However, both time after arrival and feedlot where calves were placed affected diversity and composition of the nasopharyngeal bacterial communities. In both groups, there was a reduction in bacterial diversity and a large increase in Mycoplasma after feedlot placement, especially seven days after on-arrival processing. Furthermore, we observed the horizontal transmission of a multi-resistant strain of Pasteurella multocida among calves at d9 and d30 in one of the two groups of heifers. Based on these findings, we concluded that transportation to and commingling at an auction market for 24 hrs did not significantly influence the composition and diversity of the nasopharyngeal and tracheal bacterial communities of recently weaned calves.Item Open Access Exploration of innate immune response during infectious bovine digital dermatitis and the evaluation of topical therapeutic treatment(2018-09-18) Watts, Kaitlyn; Cobo, Eduardo R.; Barkema, Herman W.; De Buck, Jeroen M.; Jenne, Craig N.Digital dermatitis (DD) is a frequently occurring infectious disease amongst dairy cattle associated with ulcerative and necrotizing lesions. Due to the associated pain and lameness, DD is a recognized animal welfare problem and has economic implications associated with decreased milk production, lower reproduction rates, and premature culling. DD is of polymicrobial etiology, with the main causative agent identified as belonging to the Treponema genus. Current treatments include topical application of antibiotics such as oxytetracycline or foot baths containing caustic chemicals; however clinical cure rates remain highly variable. In this thesis cattle with DD were monitored to explore the skin innate immune response. An exhaustive description of the inflammatory response during disease progression and a novel description of bovine host defence peptides (HDPs) and their contribution to disease resolution are found herein. It was observed that active DD was characterized by necrotic tissue populated with neutrophils and elevated Cxcl-8 and Tlr4 expression. Tracheal antimicrobial peptide (Tap) was vastly increased in active lesions and key for the resolution of DD. An in vitro model utilizing human keratinocytes showed pro-inflammatory cytokines are released in the absence of living treponemes through Tlr2 signaling and that secretory treponeme products induced cathelicidins. The ability to manipulate inflammatory reactions via treatment with vitamin D3 in DD was compared to the commonly-used oxytetracycline. A cohort of cattle with M2 were topically treated with vitamin D3 against powdered oxytetracycline for 5 days. Although vitamin D3 did elevated Tap expression, lesions and inflammatory markers remained unchanged. In contrast, oxytetracycline reduced neutrophil chemoattractant Cxcl-8 while Tlr2 remained elevated. Histologic assessment evidenced scab formation. Taken together, this thesis established the skin innate response and role of host defence peptides (Tap) during DD and supported oxytetracycline as a treatment, providing lesion resolution and aiding in bacterial elimination.Item Open Access Mycobacterium avium subsp. paratuberculosis: herd prevalence and calf-to-calf transmission(2018-05-10) Corbett, Caroline Susan; Barkema, Herman Wildrik; De Buck, Jeroen M.; Orsel, Karin; Kastelic, John Patrick; Buret, Andre G.Johne’s disease results in a progressive chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), leading to economic losses among dairy producers worldwide. In the absence of an effective vaccine to prevent infection and treatment for infected animals, control is primarily based on decreasing the number of new transmissions within a herd. Control programs have been implemented in countries and regions around the world; however, comparisons among prevalence estimates of difference regions and control programs are difficult and unreliable due to different tests used to identify infected animals and herds. Therefore, the first objectives of this thesis were to elucidate the influences of environmental sample characteristics on the outcome status of a herd, and to estimate the prevalence of MAP based on 2 environmental samples (and 3 environmental samples when including young stock) in Canada. Six environmental samples were collected twice, 3.5 years apart, from 148 dairy farms to determine whether difference in prevalence between sampling periods were associated with herd size and sample characteristics. All environmental samples regardless of type, had decreased odds of testing positive in the second sampling, and the largest herds had increased odds of testing positive than smaller herds at both sample periods. Across 4 regions (10 provinces) in Canada, 2 environmental samples, one from the lactating cow area and one the manure storage, were collected from 362 dairy farms, with an additional sample collected from breeding age heifers. Prevalence was lowest among tie-stall herds, in herds ≤ 100 cows, and in Québec; and although breeding age heifer samples did not affect prevalence estimates, they provided additional evidence that young stock are shedding MAP on farm. Therefore, the second objectives of this thesis were to determine the extent to which calf-to-calf transmission occurs among group-housed penmates, and to quantify the amount of fecal shedding that occurs among these infectious calves. An experimental transmission study was conducted, where 32 newborn calves were grouped into 7 experimental groups of 4, consisting of 2 inoculated (IN), and 2 contact exposed (CE) calves, and 1 control pen with 4 non-exposed calves. Calves were group-housed for 3 months, during which fecal, blood and environmental samples were collected frequently. The based reproduction ratio (R0) was estimated as a parameter of transmission of MAP infection using a final size (FS) model with a susceptible-infected-recovered (SIR) model based on ELISA and tissue culture. In addition, transmission rate parameter () was estimated using a GLM with a susceptible-infected-susceptible (SIS) model based on fecal culture during group housing. Throughout group housing, all IN and CE calves had MAP-positive fecal samples, and although there was a difference between frequency of shedding, there was no difference between the quantities of MAP shed in feces. All IN calves had positive MAP-tissue samples, and 7 (50%) of CE calves had positive tissue samples. Based on fecal shedding, the basic reproduction ratio R0 for CE calves (R0CE) was 3.24 (95% CI: 1.14, 7.41). R0I (based on interferon- results from blood samples) was 0.90 (95% CI: 0.24, 2.59), and R0T (based on tissue) was 1.36 (95% CI: 0.45, 3.94). Additionally, the effects of freezing on the ability to identify MAP in tissue samples were found to be minor; however, there may be a greater effect for CE calves that should be considered when freezing tissue samples. In conclusion, environmental samples characteristics did not influence the infection status of a herd, and collecting 2 environmental samples could be used to estimate prevalence and compare differences among regions. Shedding calves transmit infection to fellow penmates; therefore, future control programs should consider monitoring and testing of young stock to further decrease new transmissions on farm.Item Open Access Novel split trehalase-based biosensors for the detection of biomarkers of infectious diseases(2019-05-15) Drikic, Marija; De Buck, Jeroen M.; Gilleard, John Stuart; Dong, Tao G.; Turner, Raymond Joseph; Savchenko, Alexei; Campbell, Robert D.Infectious diseases remain a serious public health challenge in the field of human and veterinary medicine. For instance, infectious diseases were among the leading causes of mortality worldwide in 2016 according to the WHO. In veterinary medicine, infectious diseases have a significant negative impact on animal productivity and welfare leading to decreased profits and increased health-associated costs. Furthermore, the WHO estimates that 61% of diseases affecting the human population are zoonotic. Therefore, programs to control and eradicate these diseases are of crucial importance. Correct diagnosis, supported by robust diagnostic tests, is an essential first step in achieving these goals. An ideal diagnostic test needs to be accurate and robust while being easily accessible and widely-available. Although different studies over the years have aimed to develop such a diagnostic test, there have not been many that have managed to enter into clinical practice. The most successful class of biosensors currently on the market is the glucometer used to monitor blood glucose. Glucometer's sensitivity and specificity, as well as its performance in clinical samples, have been mainly perfected. Measuring devices have been miniaturized and their production costs optimized. In this thesis, we developed and characterized a novel biosensor able to detect antibodies and other infectious disease markers. The biosensor is based on the protein complementation principle and uses an E. coli glycolytic enzyme, trehalase (TreA), as a reporter. Trehalase converts trehalose to glucose, which can then be detected by a glucometer. TreA was split into two non-functional fragments, and each fragment was fused to the sensing element that was specific for the targeted analyte. In the presence of the analyte, the sensing elements bind to it and induce the dimerization of the TreA which then becomes active. In the second chapter, we demonstrated that conditional complementation of the trehalase fragments leading to trehalose hydrolysis and glucose production could be used to detect antibodies, bacterial cells, small molecule, protein-protein interaction, and protein aggregation. We also demonstrated the retention of activity of split TreA in undiluted clinical samples like blood or milk. In the third chapter, we attempted to increase the sensitivity and shorten the time for signal detection of the TreA biosensor by introducing split inteins. We placed the inteins within the previously developed biosensor, but we were not successful in achieving analyte-mediated dimerization of the two biosensor components. In the fourth chapter, we applied this TreA biosensor to develop and validate a new diagnostic tool for the quantification of the total amount of immunoglobulins in bovine colostrum and serum (named STIGA). We demonstrated that STIGA performs more efficiently in quantifying total immunoglobulins in colostrum and calf serum than other diagnostic tools (Colostrometer and Brix) that are used on a farm. Therefore, it is a suitable detection assay to establish colostrum quality and calf immune status in the field. We also proposed a modified format of STIGA for this on-farm application and demonstrated that its performance remained high. In the fifth chapter, we explored the applicability of a modified STIGA with the anticipation to detect immunoglobulins in different animal species. We analyzed 29 animal species and proved that the same detection protocol could be applied with success to the majority of animal species. In conclusion, we engineered a trehalase-based biosensor that requires minimal sample preparation and can be integrated with existing glucometers or sensors, which offers a versatile and convenient method for point-of-care applications.