Evaluating automated infrared thermography and vulva exposure tracking as components of an estrus detection platform in a commercial dairy herd.

The primary objective of this study was to develop an automated infrared thermography platform (Estrus BenchMark) capable of measuring skin temperature and tail movements as a means of identifying cows in estrus. The secondary objective was to evaluate the accuracy of Estrus BenchMark to detect estrus compared to in-line milk progesterone (P4) analysis (Herd Navigator System) in a commercial dairy herd managed under a robotic milking system. Data were collected on forty-six cows from 45 to 120 d after calving. Cows were flagged in estrus when milk P4 fell below 5 ng/mL. The Estrus BenchMark true positive estrus alerts (Sensitivity; Se%) were compared to Herd Navigator System estrus alerts at different time-windows (±12 h, ±24 h, ±48 h, and ±72 h) relative to the Estrus BenchMark estrus alerts for all the estrus alerts (AE) and confidence-quality estrus (CQE; >80% quality) alerts identified by Herd Navigator System. The Estrus BenchMark captured skin temperature and tail movements resulting in vulva exposure (left tail movements, LTail; right tail movements, RTail; and pooled tail movements, PTail) for each milking event. Skin temperature tended to increase when the milk P4 concentration (Least-Squares Means ±â€¯SE) dropped for AE (estrus day [d 0]; P4; 3.51 ±â€¯0.05 ng/mL, Skin temperature; 33.31 ±â€¯2.38 °C) compared with d -7 (P4; 20.22 ±â€¯0.73 ng/mL; Skin temperature: 32.05 ±â€¯3.77 °C). The increase in skin temperature, however, was significant in cows with CQE > 80% at d 0 (32.75 ±â€¯0.29 °C) compared to d -7 (31.80 ±â€¯0.28 °C). The prevalence of tail movements to expose vulva was greater (P = 0.01) in AE at d 0 (LTail: 62.50%; PTail; 68.75%; and RTail: 56.25%) compared with d -7 (LTail: 18.75%; PTail: 9.37%: and RTail: 9.37%), and d +4 (LTail: 9.37%; PTail: 9.37%; and RTail: 12.5%). Moreover, the higher prevalence of tail movements at d 0 was observed in cows with CQE > 80% (LTail; 65%, PTail; 80%, and RTail; 70%) compared to those with CQE < 80%. The highest Estrus BenchMark Youden index (YJ; 0.45), diagnostic odds ratio (DOR; 9.04), and Efficiency (0.77) were achieved for AE in a ±48 h window and at ±72 h window for CQE (YJ; 0.66, DOR; 25.29, and Efficiency 0.76) relative to Herd Navigator System estrus alerts. The highest Estrus BenchMark resulted in 58% estrus detection rates for AE and 80% for cows with CQE compared to the Herd Navigator System.

Evaluation of infrared thermography combined with behavioral biometrics for estrus detection in naturally cycling dairy cows.

Low estrus detection rates (>50%) are associated to extended calving intervals, low economic profit and reduced longevity in Holstein dairy cows. The objective of this study was to evaluate the accuracy of infrared thermography and behavioral biometrics combined as potential estrus alerts in naturally (not induced) cycling dairy cows housed in a tie-stall barn. Eighteen first lactation cows were subjected to transrectal ultrasonography to determine spontaneous ovulation. The dominant follicle (DF) disappearance was used retrospectively as an indirect indicator of ovulation, and to establish the estrus period (48-24 h prior the DF disappearance). Raw skin temperature (Raw IR) and residual skin temperature (Res IR) were recorded using an infrared camera at the Vulva area with the tail (Vtail), Vulva area without the tail (Vnotail), and Vulva's external lips (Vlips) at AM and PM milking from Day 14 until two days after ovulation was confirmed. Behavioral biometrics were recorded on the same schedule as infrared scan. Behavioral biometrics included large hip movements (L-hip), small hip movements (S-hip), large tail movements and small tail movements to compare behavioral changes between estrus and nonestrus periods. Significant increases in Raw IR skin temperature were observed two days prior to ovulation (Vtail; 35.93 ±â€¯0.27 °C, Vnotail; 35.59 ±â€¯0.27 °C, and Vlips; 35.35 ±â€¯0.27 °C) compared to d -5 (Proestrus; Vtail; 35.29 ±â€¯0.27 °C, Vnotail; 34.93 ±â€¯0.31 °C, and Vlips; 34.68 ±â€¯0.27 °C). No significant changes were found for behavioral parameters with the exception of S-hip movements, which increased at two days before ovulation (d -2; 11.13 ±â€¯1.44 Events/5min) compared to d -5 (7.30 ±â€¯1.02 Events/5min). To evaluate the accuracy of thermal and behavioral biometrics, receiver operating characteristic curve analysis was performed using Youden index (YJ), diagnostic odds ratio, positive likelihood ratio (LR+), Sensitivity, Specificity and Positive predicted value to score the estrus alerts. The greatest accuracy achieved using thermal parameters was for Res IR Vtail PM (YJ = 0.34) and L-hip PM (YJ = 0.27) for behavioral biometrics. Combining thermal and behavioral parameters did not improve the YJ index score but reduced the false-positive occurrence observed by increasing the diagnostic odds ratio (26.62), LR+ (12.47), Specificity (0.97) and positive predicted value (0.90) in a Res IR Vtail PM, S-hip AM, S-hip PM combination. The combination of thermal and behavioral parameters increased the accuracy of estrus detection compared to either thermal or behavioral biometrics, independently in naturally cycling cows during milking.

Evaluation of infrared thermography compared to rectal temperature to identify illness in early postpartum dairy cows.

This study evaluated and compared infrared thermography (IRT) and rectal temperature (RT) as screening tests to identify sick transition dairy cows. Holstein cows (n = 72; 42 primiparous) had RT and IRT temperatures taken daily from 1 to 12 days in milk (DIM). Health examinations were performed daily to diagnose retained fetal membrane, milk fever and metritis, and blood was analyzed for ß-hydroxybutyrate at 6 and 9 DIM to diagnose ketosis. Plasma concentrations of cortisol, interleukin-6, tumor necrosis factor α and serum amyloid A at 3, 6, 9 and 12 DIM were included as additional indicators of illness. Cows were categorized as true sick if clinically diagnosed with an illness, or if at least 2 blood parameters were above the normal range. Diagnostic test performances for RT and IRT variables were determined for each variable at a test referent value that provided the highest Youden's (J) index. The best performing screening test depended on the definition of true sickness. In general, the J index for RT was 0.15-0.17 whereas the highest J index for the IRT variables was 0.22 for the mean eye temperature and 0.19 for the mean cheek temperature. Infrared thermography was at least comparable to RT and some IRT variables performed better as a screening tests than RT. Future studies into the automation of IRT for surveillance of early postpartum diseases is warranted.

Infrared thermography and behavioral biometrics associated with estrus indicators and ovulation in estrus-synchronized dairy cows housed in tiestalls.

Most Canadian dairy herds operate in tiestall housing (61%), where average estrus detection rates may be lower than 54%. The objective of this study was to evaluate infrared thermography and behavioral biometrics as indicators of estrus in dairy cows. Eighteen cyclic multiparous cows (Synch) were subjected to an estrus synchronization protocol, and 18 pregnant cows (control) received a sham protocol on the same schedule and frequency as the cyclic cow treatment. A decline in plasma concentrations of progesterone and the appearance of a dominant follicle using transrectal ultrasonography were used as indirect indicators of estrus, and the disappearance of a dominant follicle was used to confirm ovulation. All cows were monitored via visual cameras to determine the frequency of treading, drinking, neighbor interaction, tail movement, lying, and shifting behaviors. Infrared thermograms were recorded at the eye, muzzle, cheek, neck, front right foot, front left foot, rump, flank, vulva area, tail head, and withers. To evaluate the accuracy of behavioral and thermal parameters, a predefined minimum acceptable value (i.e., threshold) for estrus alerts (>0.30 Youden J index and >0.60 area under the curve) was used. Ovulation was confirmed in 14 (77.7%) out of 18 Synch cows. Eye, cheek, neck, rump, flank, vulva area, and wither thermograms exhibited higher temperatures at 48 h [cycle threshold (Δt) = +0.30 to 1.20°C] and 24 h before ovulation compared with 4 d prior to ovulation (Δt = 0.06 to 0.11°C) and during ovulation day (Δt = 0.03 to 0.32°C) in the Synch group. In addition, control cows exhibited greater treading activity per day compared with Synch cows (20.84 ± 0.39 vs. 16.35 events/5 min ± 0.34), and tail movement frequency was greater in Synch cows compared with control cows (14.84 ± 2.7 vs. 10.11 ± 4.7 events/5 min). However, within Synch cows, tail movement was the only behavior that significantly increased in frequency 2 d before ovulation (11.81 ± 1.71 events/5 min) followed by a decrease in frequency 1 d before ovulation (4.67 ± 1.05 events/5 min) compared with ovulation day (0 d; 6.10 ± 1.25 events/5 min) and during luteolysis (3 d before ovulation; 6.01 ± 1.25 events/5 min). Upon evaluation of all variables (thermograms and behavior frequencies) as estrus indicators at 48 and 24 h before ovulation, treading and tail movements before milking and 9 thermal locations satisfied the predefined minimum acceptable value for estrus alerts. This study demonstrates that fluctuations in radiated temperature measured at specific anatomical locations and the frequency of tail movements and treading behaviors can be used as a noninvasive estrus alerts in multiparous cows housed in a tiestall system.

Energy utilization in cattle with steady state and non-steady state methods: the importance of thermal neutrality.

The efficiency by which animals utilize dietary energy is fundamental to the cost of production for protein of animal origin and to the carbon footprint an animal industry has. Hence, the development of cost effective methodology for determining these measurements of efficiency is important. The objective of the present study was to investigate the use of infrared thermography in a rapid, non-steady state method for measuring energy loss in cattle. Data from 241 yearling bulls and steers as well as heifers and mature cows are presented. Infrared images were collected following a 24h feed withdrawal period. The infrared thermal response in these animals was significantly ranked (P < 0.03) with conventional measurements of feed efficiency using residual feed intake values for animals demonstrated to be within a thermal neutral zone. When animals were not within a thermal neutral zone there was no significant ranking. The data suggests that the use of a non-steady state approach using infrared thermography for identifying metabolic efficiency in animals may be a more rapid and less expensive method for identifying differences in energy utilization. The data also demonstrates the importance of maintaining thermal neutrality when measuring metabolic efficiency irrespective of the methodology.

The automated analysis of clustering behaviour of piglets from thermal images in response to immune challenge by vaccination.

An automated method of estimating the spatial distribution of piglets within a pen was used to assess huddling behaviour under normal conditions and during a febrile response to vaccination. The automated method was compared with a manual assessment of clustering activity. Huddling behaviour was partly related to environmental conditions and clock time such that more huddling occurred during the night and at lower ambient air temperatures. There were no positive relationships between maximum pig temperatures and environmental conditions, suggesting that the narrow range of air temperatures in this study was not a significant factor for pig temperature. Spatial distribution affected radiated pig temperature measurements by IR thermography. Higher temperatures were recorded in groups of animals displaying huddling behaviour. Huddling behaviour was affected by febrile responses to vaccination with increased huddling occurring 3 to 8 h post-vaccination. The automated method of assessing spatial distribution from an IR image successfully identified periods of huddling associated with a febrile response, and to changing environmental temperatures. Infrared imaging could be used to quantify temperature and behaviour from the same images.

Infrared thermography detects febrile and behavioural responses to vaccination of weaned piglets.

An automated, non-invasive system for monitoring of thermoregulation has the potential to mitigate swine diseases through earlier detection. Measurement of radiated temperature of groups of animals by infrared thermography (IRT) is an essential component of such a system. This study reports on the feasibility of monitoring the radiated temperature of groups of animals as a biomarker of immune response using vaccination as a model for febrile disease. In Study A, weaned pigs were either treated with an intramuscular vaccine (FarrowSure Gold), a sham injection of 0.9% saline or left as untreated controls. An infrared thermal camera (FLIR A320) was fixed to the ceiling directly above the pen of animals, and recorded infrared images of the treatment groups at 5 min intervals. The effect on temperature of the spatial distribution of pigs within the pen was significant, with higher temperatures recorded when pigs were grouped together into a single cluster. A higher frequency of clustering behaviour was observed in vaccinated animals compared with controls during a period of the afternoon ~4 to 7 h post-vaccination. The daily mean of the maximum image temperature was significantly higher in vaccinated animals compared with control and sham-treated animals. In the vaccination treated group, the 24 h mean of the maximum temperature was significantly higher during the post-vaccination period compared with the 24 h period before vaccination. Increased temperature in the vaccinated animals occurred from ~3 h, peaked at ~10 h, and remained elevated for up to 20 h post-vaccination. In Study B, the effect of prevalence was tested in terms of the difference in maximum temperature between control and vaccination days. A thermal response to vaccination was detected in a pen of 24 to 26 animals when <10% of the animals were vaccinated. The results support the concept of radiated temperature measurements of groups of animals by IRT as a screening tool for febrile diseases in pig barns.

Prophylactic nitric oxide treatment reduces incidence of bovine respiratory disease complex in beef cattle arriving at a feedlot.

Bovine respiratory disease complex (BRDc), is a challenging multi-factorial health issue caused by viral/bacterial pathogens and stressors linked with the transport and mixing of cattle, negatively impacting the cattle feedlot industry. Nitric oxide (NO) is a naturally occurring molecule with antimicrobial attributes. This study tests whether NO can prevent the symptoms associated with BRDc. Eighty-five, crossbred, multiple-sourced, commingled commercial weaned beef calves were monitored and scored for temperature, white blood count, clinical score, hematology, cortisol levels and neutrophil/lymphocyte ratio. NO treatment or placebo were given once on arrival to the stockyard. After one week 87.5% of sick animals were from the control while 12.5% from treatment groups and after two weeks 72% and 28% respectively. Treatment was shown to be safe, causing neither distress nor adverse effects on the animals. These data show that NO treatment on arrival to the feedlot significantly decreased the incidence of BRDc in this study.

The non-invasive and automated detection of bovine respiratory disease onset in receiver calves using infrared thermography.

Bovine respiratory disease complex (BRD) causes considerable economic loss and biosecurity cost to the beef industry globally and also results in significant degradation to the welfare of affected animals. The successful treatment of this disease depends on the early, timely and cost effective identification of affected animals. The objective of the present study was to investigate the use of an automated, RFID driven, noninvasive infrared thermography technology to determine BRD in cattle. Sixty-five calves averaging 220 kg were exposed to standard industry practices of transport and auction. The animals were monitored for BRD using conventional biometric signs for clinical scores, core temperatures, haematology, serum cortisol and infrared thermal values over 3 weeks. The data collected demonstrated that true positive animals for BRD based on a gold standard including core temperature, clinical score, white blood cell number and neutrophil/lymphocyte ratio displayed higher peak infrared thermal values of 35.7±0.35 °C compared to true negative animals 34.9±0.22 °C (P<0.01). The study also demonstrated that such biometric data can be non-invasively and automatically collected based on a system developed around the animal's water station. It is concluded that the deployment of such systems in the cattle industry would aid animal managers and practitioners in the identification and management of BRD in cattle populations.

Spectroscopic (multi-energy) CT distinguishes iodine and barium contrast material in MICE.

OBJECTIVE: Spectral CT differs from dual-energy CT by using a conventional X-ray tube and a photon-counting detector. We wished to produce 3D spectroscopic images of mice that distinguished calcium, iodine and barium. METHODS: We developed a desktop spectral CT, dubbed MARS, based around the Medipix2 photon-counting energy-discriminating detector. The single conventional X-ray tube operated at constant voltage (75 kVp) and constant current (150 microA). We anaesthetised with ketamine six black mice (C57BL/6). We introduced iodinated contrast material and barium sulphate into the vascular system, alimentary tract and respiratory tract as we euthanised them. The mice were preserved in resin and imaged at four detector energy levels from 12 keV to 42 keV to include the K-edges of iodine (33.0 keV) and barium (37.4 keV). Principal component analysis was applied to reconstructed images to identify components with independent energy response, then displayed in 2D and 3D. RESULTS: Iodinated and barium contrast material was spectrally distinct from soft tissue and bone in all six mice. Calcium, iodine and barium were displayed as separate channels on 3D colour images at <55 microm isotropic voxels. CONCLUSION: Spectral CT distinguishes contrast agents with K-edges only 4 keV apart. Multi-contrast imaging and molecular CT are potential future applications.

Comparisons among serum, egg albumin and yolk concentrations of corticosterone as biomarkers of basal and stimulated adrenocortical activity of laying hens.

1. Serial blood samples from individual birds were analysed for corticosterone concentrations under basal and stimulated conditions, and matched to eggs from the same birds for comparison to albumin and yolk concentrations of corticosterone. 2. Serum corticosterone exhibited increases in response to stimulation by ACTH and Handling stress. There were no significant increases in egg albumin or yolk concentrations of corticosterone following stimulation. 3. Several significant correlations were observed between the mean and area under the curve (AUC) measurements of serum corticosterone concentrations with albumin and yolk corticosterone concentrations in eggs laid from 1 to 2 d later. 4. The results demonstrated a relationship between endogenous concentrations of serum corticosterone that reflected daily adrenocortical output with albumin and yolk corticosterone concentrations in eggs laid the following day. 5. The results do not support the concept of albumin and yolk concentrations of corticosterone as biomarkers of acute adrenocortical responses to stimulation.

Spectroscopic biomedical imaging with the Medipix2 detector.

This study confirms that the Medipix2 x-ray detector enables spectroscopic bio-medical plain radiography. We show that the detector has the potential to provide new, useful information beyond the limited spectroscopic information of modem dual-energy computed tomography (CT) scanners. Full spectroscopic 3D-imaging is likely to be the next major technological advance in computed tomography, moving the modality towards molecular imaging applications. This paper focuses on the enabling technology which allows spectroscopic data collection and why this information is useful. In this preliminary study we acquired the first spectroscopic images of human tissue and other biological samples obtained using the Medipix2 detector. The images presented here include the clear resolution of the 1.4mm long distal phalanx of a 20-week-old miscarried foetus, showing clear energy-dependent variations. The opportunities for further research using the forthcoming Medipix3 detector are discussed and a prototype spectroscopic CT scanner (MARS, Medipix All Resolution System) is briefly described.

The use of infrared thermography as an early indicator of bovine respiratory disease complex in calves.

Bovine respiratory disease (BRD) complex causes considerable distress to domestic livestock and economic hardship to the beef industry. Furthermore, the resulting extensive use of antimicrobial treatments is a growing concern from the perspective of facilitating antibiotic resistant microbes. The earlier detection of BRD would enable an earlier, more targeted treatment regime and earlier isolation of infected individuals. The objective of the present study was to investigate the use of non-invasive infrared thermography in the early detection of BRD in cattle. Studies were conducted on 133 head of weaned calves. Data demonstrated that infrared thermography was able to identify animals at early stages of illness, often several days to over one week before clinical signs were manifest. Data indicated that 4-6 days prior to the onset of clinical symptoms of BRD, greater positive and negative predictive values and test efficiency for infrared thermography (80%, 65% and 71%, respectively) compared to the industry standard practice of clinical scoring (70%, 45% and 55%, respectively).

The use of digital video recorders (DVRs) for capturing digital video files for use in both The Observer and Ethovision.

Before switching a laboratory from analog to digital, for the recording of video files for use in Noldus software such as Ethovision and The Observer, researchers need to proceed with caution. There are obvious advantages in moving to digital recording for behavioral work, including increased storage capacity; no requirement to purchase video tapes; immediate search by date, time, or event; digital images are of higher quality; ability to view study sites remotely by Internet connection; and "smart" features, such as motion detection. But before you throw away your time-lapse video recorders, time code generators, and video multiplexors, there are some important cautions to take account of. Some research groups have bought digital surveillance systems on the assumption that they work with Ethovision and The Observer, only to be disappointed. The vast majority of systems depend on proprietary compression software that must then be converted to work properly in Ethovision or The Observer.

Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII.

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.

Adrenocorticotropic hormone increases hydrolysis of B-6 vitamers in swine adrenal glands.

Shipping stress is an economic problem because of its effect on meat quality. Because shipping increases plasma cortisol and pyridoxal 5'-phosphate interacts with steroid hormones, we examined the interaction between adrenocorticotropic hormone (ACTH) and vitamin B-6 metabolism in pigs. Six crossbred pigs with ear vein catheters received 50 IU of porcine ACTH intravenously at 3-h intervals from 0800 to 2100 h on d 1-3 and 100 IU intramuscularly at 0800, 1400 and 2000 h on d 6 and 7. Controls received saline. ACTH had no effect on pyridoxal 5'-phosphate in adrenal tissue but decreased pyridoxamine 5'-phosphate from 6.1 +/- 0.7 to 4.7 +/- 1.0 nmol/g (P < 0.05). Adrenal pyridoxal and pyridoxamine concentrations were 0.4 +/- 0.1 nmol/g in controls and 1.1 +/- 0.3 and 1.3 +/- 0.5 nmol/g, respectively, in ACTH-treated pigs (P < 0.01). Pyridoxal 5'-phosphate phosphatase activity [median (25-75 percentile value)] at pH 7.4 in adrenal tissue was 66.6 (47.8-75.5) nmol/(g. min) in the controls and 764 (626-771) in the ACTH-treated pigs (P < 0.01). There was no significant difference in pyridoxal kinase activity. However, kinase activity in the adrenals was about twice as high as in other tissues. These data suggest an active turnover of vitamin B-6 in adrenal tissue.

Paraspinal abscess following facet joint injection.

Injection to the zygapophysial joint is a procedure which is performed frequently for diagnostic or therapeutic reasons in the management of back pain. It is generally considered to be free of significant complications. We report a patient who developed a paraspinal abscess following a lumbar facet joint injection.

Systematic assessment of World Wide Web materials for medical education: online, cooperative peer review.

OBJECTIVE: : To develop a generic methodology for the online assessment of medical education materials available on the World Wide Web and to implement it for pilot subject areas. DESIGN: An online questionnaire was developed, based on an existing scheme for computer-based learning material. It was extended to involve five stages, covering general suitability, local suitability, the user interface, educational style, and a general review. It is available on the Web, so expert reviewers may be recruited from outside the home institution. The methodology was piloted in three subjects areas--clinical chemistry, radiology, and medical physics--concentrating initially on undergraduate teaching. MEASUREMENTS: The contents of completed questionnaires were stored in an offline database. Selected fields, likely to be of use to students and educators searching for material, were input into an online database. RESULTS: The online assessment was used successfully in clinical chemistry and medical physics but less well in radiology. Fewer resources were found to fit local needs than expected. CONCLUSION: The methodology was found to work well for topics where teaching is highly structured and formal and is potentially applicable in other such disciplines. The approach produces more structured and applicable lists of resources than can be obtained from search engines.

Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.

Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.