Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples.

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Comparison of real-time PCR and ELISA for the detection of crustacean shellfish allergens.

Food allergies are a significant public health concern, and crustacean shellfish represent one of the major FDA regulated food allergens. Allergic individuals must avoid foods containing crustaceans, and this necessitates highly sensitive and accurate detection methods. Two of the major methods used are protein-based ELISA and DNA-based real-time PCR. In order to properly compare these very different methodologies, we used identical split samples for a side-by-side comparison and analysed them using four different real-time PCR methods and two different commercial ELISA kits. Three real-time PCR assays targeting the mitochondrial 12S genes of shrimp, crab, and lobster were compared to a commercial ELISA assay for total crustacean protein. A fourth real-time PCR assay targeting the tropomyosin gene of shrimp was compared to an ELISA assay for shrimp tropomyosin. All comparisons were carried out in two different food matrices: Manhattan clam chowder and fish sauce. PCR assays had a more broad dynamic range (0.1-106 mg/kg) as compared to ELISA (200-4000 mg/kg) and did not show matrix interference like ELISA. In cases where the ELISA assays did not have matrix interference, there was good qualitative agreement between PCR and ELISA.

Homeostasis model assessment, serum insulin and their relation to body fat in cats.

BACKGROUND: Obesity is associated with insulin resistance (IR) and considered a risk factor for diabetes mellitus (DM) in cats. It has been proposed that homeostasis model assessment (HOMA-IR), which is the product of fasting serum insulin (mU/L) and glucose (mmol/L) divided by 22.5, can be used to indicate IR. The objectives of this study were threefold: (i) to evaluate associations between body fat, fasting insulin, and HOMA-IR, (ii) to determine population-based reference interval of HOMA-IR in healthy lean cats, and (iii) to evaluate biological variation of HOMA-IR and fasting insulin in cats. RESULTS: 150 cats were grouped as lean or overweight based on body condition score and in 68 of the cats body fat percentage (BF%) was estimated by computed tomography. Fasting serum insulin and glucose concentrations were analysed. Statistical differences in HOMA-IR and insulin between overweight or lean cats were evaluated using Wilcoxon rank-sum test. Robust method with Box-Cox transformation was used for calculating HOMA-IR reference interval in healthy lean cats. Relations between BF% and HOMA-IR and insulin were evaluated by regression analysis. Restricted maximum likelihood ratio was used to calculate indices of biological variation of HOMA-IR and insulin in seven cats. There were significant differences between groups with overweight cats (n = 77) having higher HOMA-IR (p < 0.0001) and insulin (p = 0.0002) than lean cats (n = 73). Reference interval for HOMA-IR in lean cats was 0.1-3.0. HOMA-IR and fasting insulin concentrations showed similar significant positive association with BF% (p = 0.0010 and p = 0.0017, respectively). Within-animal coefficient of variation of HOMA-IR and insulin was 51% and 49%, respectively. CONCLUSIONS: HOMA-IR and fasting insulin higher in overweight than lean cats and correlate to BF%. The established population-based reference interval for HOMA-IR as well as the indices of biological variation for HOMA-IR and fasting insulin may be used when interpreting HOMA-IR and fasting insulin in cats. Further studies are needed to evaluate if HOMA-IR or fasting insulin is useful for identifying cats at risk of developing DM.

Evaluation of variation within the barcode region of Cytochrome c Oxidase I (COI) for the detection of commercial Callinectes sapidus Rathbun, 1896 (blue crab) products of non-US origin.

Callinectes sapidus Rathbun, 1896 is a western Atlantic species with a disjointed natural geographic range from Massachusetts, USA to Venezuela (distribution area 1) and from Alagoas, Brazil to northern Argentina (distribution area 2). It is the only species of portunid crab commercially harvested in the continental United States but is also imported into the US from several Latin American countries, Venezuela and Mexico in particular. In the United States, crab products labeled as "blue crab" and "Product of the USA" may not legally contain other species of crab or C. sapidus not harvested in the United States. The present study documents nucleotide variation within the barcode region of cytochrome c oxidase I (COI) in 417 reference specimens of C. sapidus collected from throughout its natural range. The goal of this study is to determine if this variation can be utilized to detect mislabeled C. sapidus products sold in interstate commerce by comparing genetic signatures in reference specimens to those observed in commercial crabmeat labeled as "Product of the USA" and "Product of Venezuela." In reference specimens, we observed high levels of genetic variation in the barcode region. However, three lineages were consistently observed with significant pairwise F st values between the lineages. Lineage 1 was observed throughout the natural geographic range but predominated in the continental US and was the only lineage observed in the major crabmeat-producing states (MD, LA, VA, NC). Lineage 2 primarily occurred in the Caribbean region of distribution area 1 but was also infrequently encountered in the South Atlantic Bight region of the US coast. Finally, Lineage 3 was only observed in Brazilian waters and had the lowest haplotype and nucleotide diversity values. Lineages 1 and 2 were separated by a mean pairwise distance (p-distance) of 3.15%, whereas Lineage 3 had a mean p-distance of 2.55% and 1.35% to Lineages 1 and 2, respectively. Within lineage mean p-distances were 0.45%, 0.19%, and 0.07% for Lineages 1, 2, and 3, respectively. Among all vouchered reference specimens collected from the continental United States, Mexico, Puerto Rico, and Venezuela, we identified 22 phylogenetically informative sites that drive observed lineage divergences. Haplotypes identified from barcode COI sequences from commercial C. sapidus products labeled as originating from the US all aligned with haplotypes from Lineage 1 reference specimens and haplotypes from commercial products labeled as originating from Venezuela all aligned with Lineage 2, suggesting that these lineages may be useful for indicating whether products originate from the continental US or are imported when package labeling is in question.

Evaluation and comparison of pain questionnaires for clinical screening of osteoarthritis in cats.

BACKGROUND: Feline osteoarthritis (OA) is a common cause of long-standing pain and physical dysfunction. Performing a physical examination of a cat is often challenging. There is a need for disease-specific questionnaires or the so-called clinical metrology instruments (CMIs) to facilitate diagnosis and evaluation of treatment of feline OA. The CMI provides the owners an assessment of the cat's behavioural and lifestyle changes in the home environment. The purpose of the study was to evaluate readability, internal consistency, reliability and discriminatory ability of four CMIs. METHODS: This is a prospective, cross-sectional study with 142 client-owned cats. Feline OA was diagnosed based on medical history, orthopaedic examination and radiography. RESULTS: The results indicate that all four instruments have sound readability, internal consistency, are reliable over time and have good discriminatory ability. Preliminary cut-off values with optimal sensitivity and specificity were suggested for each instrument. The osteoarthritic cats showed significant changes in behavioural response to pain during orthopaedic examination, compared with sound cats. CONCLUSION: The results indicate that all four questionnaires make an important contribution in a clinical setting, and that the cat's behavioural response to pain during physical examination should be a parameter to take into account as a possible indication of chronic pain.

Market Basket Survey of Arsenic Species in the Top Ten Most Consumed Seafoods in the United States.

The study focused on the determination of arsenic species in the top ten most consumed seafoods in the United States. Fifty-four samples were collected from local supermarkets, and their species identities were confirmed by DNA barcoding. The total arsenic in the samples varied greatly in the range of 8-22200 ng/g (wet mass). Speciation analysis based on extraction of water-soluble and nonpolar arsenic showed that inorganic arsenic (iAs) was found only in clams and crabs, while arsenobetaine (AsB) predominates in most samples. Among the other arsenicals, trimethylarsoniopropionate (TMAP) was found in most matrices with higher concentrations in crabs, and arsenosugars existed in most clams and crabs. Nonpolar arsenic accounted for 1-46% of the total arsenic in the samples. The accuracy of the analytical results was evaluated using standard reference materials and spike recovery tests. The survey showed that the iAs concentrations in America's most consumed seafood products are much lower than the tolerable intake set by the Joint FAO/WHO Expert Committee, even at the highest levels found in this study.

Application of Multiantigen Profiling To Detect Pecan.

A problem often encountered in the detection and identification of undeclared tree nut food allergens is the lack of analytical methods. This problem is accentuated by the current trend, whereby the primary methods used to detect food allergens are antibody-based enzyme-linked immunosorbent assays (ELISAs) and the development of analyte-specific antibodies takes months. The recently developed xMAP food allergen detection assay (xMAP FADA) has the ability to generate multiantigen profiles with tree nuts, thereby providing a potential solution to this problem. The xMAP FADA includes 22 antibodies targeting peanut, soy, and nine tree nuts. The high number of antibodies to a diverse group of tree nuts and legumes and the propensity of tree nuts to cross-react have enabled the development of multiantigen profiling, whereby an analyte reacts with the various antibodies to generate a profile. Recently, a question arose regarding the possible presence of pecan dust at a manufacturer of pecan products that also stored fresh produce. The lack of suitable pecan ELISAs created an analytical challenge that was resolved using multiantigen profiling with the xMAP FADA. Pecan was detected on swab samples by using multiantigen profiling and confirmed by DNA analysis. The use of multiantigen profiling provided an analytical capability beyond what was possible with an analyte-specific analytical method.

A group-specific, quantitative real-time PCR assay for detection of crab, a crustacean shellfish allergen, in complex food matrices.

A real-time PCR assay was developed for detection of crab, a crustacean allergen, in food products. Group-specific primers and probes were developed to detect numerous species of crab. Method validation included tests of detection in complex food matrices, evaluation of commercial food products, and cross-reactivity testing on a wide variety of crustaceans. The method was able to detect several species of crab spiked into complex food matrices at levels ranging from 0.1 to 105 parts per million (weight/weight), worked equally well on different platforms, exhibited high specificity for crab over other types of crustaceans, and yielded much higher signals from commercial food products listing crab as an ingredient than from those containing other crustaceans.

Do cats with a cranial cruciate ligament injury and osteoarthritis demonstrate a different gait pattern and behaviour compared to sound cats?

BACKGROUND: Osteoarthritis (OA) is a common cause of chronic pain and dysfunction in older cats. The majority of cats with OA do not show signs of overt lameness, yet cats with orthopaedic disease are known to redistribute their body weight from the affected limb. OA can cause changes in the cat's behaviour, which is often misinterpreted as signs of aging. The aim of the present study was to investigate if cats with a previous cranial cruciate ligament (CCL) injury perform differently on the pressure mat and exhibit different behaviour compared to sound cats according to the owner´s subjective assessment. Ten cats with a previous CCL injury were assessed with a pressure mat system and their owners were asked to complete an assessment questionnaire. The results were compared to those of 15 sound cats, matched to have the same weight and body condition score. RESULTS: The front/hind limb index for peak vertical force (PVF) was significantly higher for CCL cats, and there was a decreased PVF and vertical impulse (VI) on the affected hindlimb compared to the unaffected one. The results indicate that cats with a previous CCL injury put less weight, on the affected hindlimb but for a longer time. There was a significantly higher owner assessment questionnaire score for the group of cats with CCL injury compared to sound cats. CONCLUSIONS: Cats with a previous CCL injury have a different gait pattern compared to sound cats and a different behaviour according to owner subjective assessment. It is of great importance that further studies are performed to investigate the long term effects of CCL injury as a cause of pain and physical dysfunction, and its role in the development of OA in cats. Improved assessment tools for chronic pain caused by OA in cats are needed, both to facilitate diagnosis and to evaluate pain-relieving treatment.

Gait and jump analysis in healthy cats using a pressure mat system.

Physical orthopaedic examination in cats does not always reveal signs of lameness and no objective gait analysis method has yet been standardised for use in cats. The aims of the present study were to define appropriate parameters for pressure mat analyses during walk and jump, and to define reference values for gait parameters of healthy cats. Further, the distribution of the vertical force within the paws and the influence of a non-centred head position were investigated. The hypothesis was that cats have a symmetrical gait, a front/hindlimb asymmetry similar to dogs, and that peak vertical force (PVF) and vertical impulse (VI) have high intraclass correlation coefficients, confirming the reliability of these parameters. Data for walking (n = 46) showed gait symmetry indices of close to 1.0, besides PVF front/hind (1.3 ± 0.2). The PVF front/hind for jumping cats (n = 16) was 1.7 ± 0.6. Results from the distribution of the vertical force within the paw (n = 39) showed that the main weight during a strike is transferred from the caudal towards the craniomedial part of the paw. The findings support the hypothesis that healthy cats have similar gait symmetry to healthy dogs and that PVF and VI are reliable gait parameters. In conclusion, the present study provides a reference interval for healthy cats. Further studies are needed to investigate gait parameters in cats with orthopaedic disease.