Test sensitivity of a commercial serine protease digestion kit for the detection of Trichinella spiralis and Trichinella pseudospiralis larvae in pig muscle.

The reference method for Trichinella detection at meat inspection is the magnetic stirrer method (MSM) utilising HCl-pepsin for pooled sample digestion. Due to availability and quality issues with pepsin, alternative digestion methods are being offered, such as the Priocheck Trichinella AAD kit (T-AAD), based on serine endopeptidase digestion. In this study the T-AAD kit was compared to the reference method. Minced pork samples were spiked with T. spiralis muscle larvae (ML) with- and without capsule or T. pseudospiralis ML, and analysed with both tests. Test results of individually spiked test samples were analysed by generalised linear modelling. The T-AAD test kit was comparable to the reference method for the qualitative detection of T. spiralis in pigs, but not quantitatively. Overall, 94% of spiked T. spiralis were recovered using MSM against 75.2% when using T-AAD (p < 0.0001). Using the MSM 80.0% of spiked T. pseudospiralis were recovered against 20% with the T-AAD (p < 0.0001). Based on our experience with the T-AAD kit, we strongly recommend validating the method on site prior to introduction into routine diagnostic laboratories, but this will not alleviate the poor test sensitivity of the T-AAD for the detection of T. pseudospiralis.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

Frequencies and spatial distributions of Cryptosporidium in livestock animals and children in the Ismailia province of Egypt.

Faecal samples from 804 cattle and buffaloes and 165 diarrhoeal children of Ismailia province were investigated by an immunochromatographic screening test and PCR to determine prevalences and distributions of Cryptosporidium spp. Results were analysed statistically for clustering of animal and human cases. Cryptosporidium herd prevalence was 73·3% and individual animal prevalence 32·3%. C. parvum was the dominant species in animals (65·7%). Young calves watered with canal or underground water were at particular risk of infection. Detection rates were higher when calves showed diarrhoea, fever and dehydration. Human Cryptosporidium prevalence was 49·1%. C. hominis dominated in humans (60·5%), followed by C. parvum (38·3%). Living in villages, drinking underground water and having contact with animals were risk factors. Cluster analysis revealed differences in the distribution of infections between animals and humans and suggests different transmission dynamics.

Effect of high dietary zinc oxide on the caecal and faecal short-chain fatty acids and tissue zinc and copper concentration in pigs is reversible after withdrawal of the high zinc oxide from the diet.

Zinc oxide (ZnO) used in high ('pharmacological') levels to prevent diarrhoea in pigs is assumed to reduce copper (Cu) in tissues and inhibits large intestinal microbial fermentation. To test it, German Landrace pigs were weaned on d28 of age and fed diets containing either 100 (LowZinc, LZn, n = 10) or 3100 mg ZnO/kg (HighZinc, HZn, n = 10). The mixed feed (13.0 MJ ME, 18.5% crude protein) was based on wheat, barley, soya bean meal and maize. After 4 weeks, the HZn group was further fed 100 mg ZnO/kg for another 2 weeks. Caecal contents, faeces and tissues were collected after 4 weeks (n = 5 and n = 10 respectively) and 6 weeks (n = 5 and n = 5 respectively). Faeces and caecal content were analysed for dry matter (DM), pH, ammonia, lactic acid (LA) and short-chain fatty acids (SCFA) on native water basis. anova was performed to elucidate significant differences at p < 0.05. No diarrhoea occurred. After 4 weeks, the caecal contents' pH increased (p < 0.001) and butyric (p < 0.05) and valeric acid (p < 0.01) decreased in the HZn group in comparison with LZn. In faeces, a decrease of acetic (p = 0.009), butyric (p = 0.007) and valeric acid (p = 0.046), as well as reduced acetic:propionic acid (A:P) ratio (p = 0.025) was observed in the HZn group in comparison with LZn. Faecal ammonia decreased in HZn (p = 0.018). No differences (p > 0.05) were recorded in caecal contents after 6 weeks. In faeces, acetic acid remained lower in the HZn group in comparison with LZn (p = 0.006), as did the A:P ratio (p = 0.004). Zn concentration in liver, kidneys and ribs, and Cu concentrations in kidneys increased in HZn. Withdrawal of ZnO resulted in reversibility of the changes. The effect on butyric acid should be discussed critically regarding the energetic support for the enterocytes. High Zn and Cu tissue concentrations should be considered by pet food producers.

Inhibitory effect of high-dosage zinc oxide dietary supplementation on Campylobacter coli excretion in weaned piglets.

AIMS: This study investigated the impact of zinc oxide (ZnO) on Campylobacter coli by in vivo and in vitro assays. METHODS AND RESULTS: By in vitro growth inhibition assays, a high susceptibility of Camp. coli against ZnO could be observed. At concentrations ≥ 2.6 mmol l(-1) ZnO, a decline in cell numbers occurred. Quantitative real-time PCR assays demonstrated an up-regulation of the main oxidative stress gene (katA) in response to ZnO treatment. The expression level of katA was increased by fivefold after ZnO treatment. An experiment was carried out in pigs to elucidate the impact of ZnO as feed supplement on Camp. coli faecal excretion. Feeding a high-dosage ZnO concentration (3100 mg kg(-1) ) to piglets significantly reduced the faecal excretion of Camp. coli by up to 1 log CFU g(-1) as compared to animals receiving a low (40 mg kg(-1) ) or medium (100 mg kg(-1) ) ZnO diet. CONCLUSION: In vitro assays showed a high susceptibility of Camp. coli against ZnO. Adding high levels of ZnO to the diet of weaned piglets reduced Camp. coli excretion significantly. There is evidence for the induction of an oxidative stress response by ZnO supplementation in Camp. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Supplementation of a high-dosage ZnO diet to piglets can reduce the Camp. coli load, potentially leading to a lower contamination risk of meat during slaughter.

Molecular typing of Giardia duodenalis isolates from German travellers.

Giardia duodenalis isolates from German travellers returning from tropical areas were characterised by PCR amplification and sequencing of fragments of the beta-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. Assignment of isolates to specific G. duodenalis assemblages was found to differ according to the marker used. Indeed, at the bg locus, assemblages A and B were identified, with a higher prevalence of the former over the latter, whereas at the tpi and gdh loci, most samples were classified as assemblage B. In agreement with previous studies, sequence analysis showed that assemblage B isolates have a higher genetic polymorphism than assemblage A isolates, and novel variants were described. The degree of polymorphism was shown in a graphical representation of the polymorphic sites generating a novel sequence, the heterogeneous positions common to assemblages A and B (double peaks), that may represent mixed assemblage infection and the heterogeneous positions detected at random sites. Notably, assemblage D, which is considered to be adapted to dogs, was found at the gdh locus in two samples originating from southern Asia, as novel genotypes. By comparing the geographical origin of the infected cases and the number of German travellers visiting the areas considered, India and west Africa appeared to be the areas associated to the highest risk of acquiring Giardia infection. The analysis of the geographical distribution of the genotypes did not suggest any particular geographical clustering pattern, but it may be useful to evaluate these results with a higher number of isolates. Most of the samples typed at the three markers could not be assigned unequivocally to either assemblage A or B, and this was confirmed also by a real-time PCR assay, using a set of assemblage-specific primers. The results of this study reinforce the notion that genetic exchanges and allelic sequence heterogeneity represent major obstacles towards understanding the epidemiology of giardiasis and that exposure to Giardia parasites in endemic areas often results in mixed infections in returning travellers.

Pulmonary abnormalities in dogs with leptospirosis.

BACKGROUND: Leptospirosis in dogs is a multiorgan disease affecting mostly kidneys and liver. OBJECTIVES: The objective was to characterize prevalence, clinical, and radiological features and outcome of dogs with leptospirosis and pulmonary abnormalities. ANIMALS: Fifty dogs with leptospirosis. METHODS: Medical records of dogs diagnosed with leptospirosis at the Small Animal Clinic, Berlin, were reviewed. Diagnosis was based on microscopic agglutination test, blood or urine polymerase chain reaction, and histopathology. Based on clinical and/or radiological signs, patients were grouped into dogs with lung abnormalities (group 1) or without (group 2). Severity of respiratory distress was scored as mild to moderate (grade 1) or severe (grade 2). Thoracic radiographs were scored based on pulmonary changes and location as grade 1 (caudal interstitial pattern), 2 (generalized mild to moderate reticulonodular interstitial pattern), or 3 (generalized severe reticulonodular interstitial pattern with patchy alveolar consolidations). Results of CBC and biochemistry were compared between groups. RESULTS: Thirty-five dogs had radiological pulmonary changes (grade 1: 5; grade 2: 14; grade 3: 16); 31 of them had pulmonary distress (grade 1: 13, grade 2: 18). Sixty-seven percent of the dogs with dyspnea grade 2 were mainly euthanized because of respiratory distress. Fifteen percent of the dogs with dyspnea grade 1 and 21% without clinical respiratory signs were euthanized because of acute renal failure or sepsis. CONCLUSIONS AND CLINICAL IMPORTANCE: In 70% of dogs with leptospirosis pulmonary changes were detected. Lung involvement represented a severe complication causing increased case fatality depending on the severity of respiratory distress.

Validation of a Western Blot for the detection of anti-Trichinella spp. antibodies in domestic pigs.

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA.

[Tasks and duties of veterinary reference laboratories for food borne zoonoses]. / Aufgaben der veterinärmedizinischen Referenzlaboratorien im Bereich der lebensmittelbedingten Zoonosen.

Reference laboratories are of central importance for consumer protection. Field expertise and high scientific competence are basic requirements for the nomination of a national reference laboratory. To ensure a common approach in the analysis of zoonotic hazards, standards have been developed by the reference laboratories together with national official laboratories on the basis of Art. 33 of Directive (EG) No. 882/2004. Reference laboratories function as arbitrative boards in the case of ambivalent or debatable results. New methods for detection of zoonotic agents are developed and validated to provide tools for analysis, e. g., in legal cases, if results from different parties are disputed. Besides these tasks, national reference laboratories offer capacity building and advanced training courses and control the performance of ring trials to ensure consistency in the quality of analyses in official laboratories. All reference laboratories work according to the ISO standard 17025 which defines the grounds for strict laboratory quality rules and in cooperation with the respective Community Reference Laboratories (CRL). From the group of veterinary reference laboratories for food-borne zoonoses, the national reference laboratories are responsible for Listeria monocytogenes, for Campylobacter, for the surveillance and control of viral and bacterial contamination of bivalve molluscs, for E. coli, for the performance of analysis and tests on zoonoses (Salmonella), and from the group of parasitological zoonotic agents, the national reference laboratory for Trichinella.

Comparative examination and validation of ELISA test systems for Salmonella typhimurium diagnosis of slaughtering pigs.

The most frequently isolated Salmonella serotype from pork in Germany is S. typhimurium, especially phagetype DT 104. The monitoring programs on Salmonella in swine are based on enzyme-linked immunoadsorbent assay (ELISA) detecting antibodies in serum or meat juice. These serological results are used to classify swine herds in three categories to assess the hygienic status of farm regarding Salmonella infection in pigs. The object of this study was the comparative evaluation of four indirect Salmonella ELISA tests approved in Germany to detect Salmonella typhimurium infection of swine. Three tests (A-C) are based on LPS-antigen and directed against specific IgG-antibodies. The fourth test (D) bases on a whole-cell-lysate antigen and discriminates between Salmonella specific IgA-, IgM- and IgG-antibodies. In a longitudinal study sixteen 6 weeks old weaning pigs were orally infected with S. typhimurium DT 104. During an observation period of 138d clinical and bacteriological parameters were monitored and serum samples obtained at regular intervals as well as meat juice samples taken at slaughter were examined by the respective ELISA systems. Study results reveal that all tested ELISA systems are able to detect S. typhimurium infection in pigs in both sample matrices, blood serum and meat juice whereas test D showed the highest sensitivity to detect Salmonella antibodies in pigs. The sensitivity to detect Salmonella antibodies varied between tests A and C according to the used cut-off (test specific cut-off vs. recommended surveillance cut-off) resulting in a change of seroprevalence and hence may influence the Salmonella status of the farm.

Distribution of Cryptosporidium parvum subtypes in calves in Germany.

Cryptosporidium DNA was extracted from 134 faecal specimens from pre-weaned calves from different German Federal States (age range, 3-15 days old), which tested positive for oocysts by microscopic analysis. The 18S rDNA gene and the oocyst wall protein gene (COWP) were used as targets for PCR and RFLP techniques. Cryptosporidium species were identified by using SspI, MboII and RsaI endonucleases for the digestion of 18S rDNA and COWP amplified fragments, respectively. In all samples, restriction patterns corresponding to Cryptosporidium parvum were identified, which is in agreement with abundant literature data indicating C. parvum as the most common species in pre-weaned calves. In order to estimate the genetic heterogeneity among C. parvum calf isolates, 53 samples chosen to represent different German Federal States were successfully subtyped by sequence analysis of the highly polymorphic 60-kDa glycoprotein gene. All isolates belonged to the allele IIa (with seven subtypes), with the exception of one isolate that belonged to the allele IId. Moreover, three novel subtypes of the allele family IIa have been found. This study confirms the utility of genotyping and subtyping tools in characterizing the transmission of Cryptosporidium spp. This is the first molecular epidemiological report about subtyping of Cryptosporidium bovine isolates in Germany.

Liver flukes in dogs and treatment with praziquantel.

Opisthorchiid eggs were found in 6 out of 27 (22%) huskies while an ELISA detected antibodies against Opisthorchis felineus and Metorchis bilis in 24 (89%) and 25 (93%) sera taken from these dogs, respectively. All dogs showed an increased activity of glutamate dehydrogenase while aspartate aminotransferase was normal in all samples. Ten weeks after treatment with praziquantel no fluke eggs were found in the faeces from any animal and antibody titres dropped in all animals.

Evaluation of clinical, laboratory, imaging findings and outcome in 99 dogs with leptospirosis.

OBJECTIVE: To report clinical, laboratory and diagnostic imaging features and prognostic factors in dogs with leptospirosis from North-East Germany. MATERIALS AND METHODS: Medical records of dogs diagnosed with leptospirosis from 2006 to 2013 were evaluated retrospectively. RESULTS: The study included 99 dogs. At initial presentation, the most common clinical signs were lethargy (96%), anorexia (88%), vomiting (85%), painful abdomen (39%), diarrhoea (38%), oliguria (27%) and tachypnoea (26%). Abnormal laboratory findings included anaemia (63%), thrombocytopenia (63%), leucocytosis (57%), increase of plasma urea (84%) and creatinine concentrations (81%), increased liver enzyme activities (80%), hyperbilirubinaemia (69%), hyperphosphataemia (67%), hyponatraemia (64%), hypoalbuminaemia (55%) and hypokalaemia (29%). Radiological pulmonary changes were detected in 57% of the dogs initially or during the course of disease. Severe dyspnoea, oliguria, azotaemia, hyperbilirubinaemia and severe radiological pulmonary changes were more often found in dogs that did not survive. There was renal, hepatic and pulmonary involvement in 95, 92 and 58% of the dogs, respectively, and multi-organ lesions in 98 dogs (98%); 32 dogs died or were euthanased. CONCLUSION: Several clinical and laboratory abnormalities were associated with a negative outcome; severe lung involvement was specifically associated with high mortality.

Molecular Survey on Brucellosis in Rodents and Shrews - Natural Reservoirs of Novel Brucella Species in Germany?

Brucellosis is a widespread zoonotic disease introduced from animal reservoirs to humans. In Germany, bovine and ovine/caprine brucellosis were eradicated more than a decade ago and mandatory measures in livestock have been implemented to keep the officially brucellosis-free status. In contrast, surveillance of wildlife is still challenging, and reliable data on the prevalence of brucellae in small mammal populations do not exist. To assess the epidemiology of Brucella spp. in rodents and shrews, a molecular survey was carried out. A total of 537 rodents and shrews were trapped in four federal states located throughout Germany and investigated for the presence of Brucella. Using a two-step molecular assay based on the detection of the Brucella-specific bcsp31 and IS711 sequences in tissue samples, 14.2% (n = 76) of the tested animals were positive. These originated mainly from western and south-western Germany, where preliminary analyses indicate population density-dependent Brucella prevalence in voles (Myodes glareolus) and mice (Apodemus spp.). recA typing revealed a close relationship to a potentially novel Brucella species recently isolated from red foxes (Vulpes vulpes) in Austria. The molecular detection of brucellae in various rodent taxa and for the first time in shrew species shows that these animals may be naturally infected or at least have a history of exposure to Brucella spp.

Trichinella spiralis and Trichinella pseudospiralis mixed infection in a wild boar (Sus scrofa) of Germany.

A wild boar (Sus scrofa) from the island Usedom in Mecklenburg-Western Pomerania (north-east Germany) was detected as Trichinella-positive during routine meat inspection. Encapsulated and non-encapsulated larvae were detected in the muscle tissue by trichinoscopy. In the diaphragm, 922 larvae per g were detected by artificial digestion. Muscle larvae displayed two different sizes of about 700 and 1100 microm. By a multiplex PCR analysis, larvae with a large size were identified as Trichinella spiralis, whereas those of a smaller size were identified as Trichinella pseudospiralis. This is the first finding of a mixed infection of T. spiralis and T. pseudospiralis in a naturally infected animal and it supports the tendency of more frequent detection of the non-encapsulated species T. pseudospiralis in Europe.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures. This paper presents an overview of the current methods for Trichinella meat inspection and their implementation in the EU, listing advantages and disadvantages for each method, including some suggestions for specific points of improvement.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures.

Seroprevalence of Trichinella in slaughter pigs in Kathmandu Valley, Nepal.

The aim of the present study was to determine the Trichinella seroprevalence in slaughter pigs in Kathmandu Valley, Nepal. Serum samples were obtained from 400 pigs at 4 major slaughterhouses and tested for Trichinella antibodies by ELISA using larval excretory-secretory (E/S) antigen. Four were positive and one was equivocal, giving a Trichinella seroprevalence of 1% (95% CI: 0.27 - 2.54). On titration, all positive and equivocal samples had titers greater than 1:80. Upon re-examination the equivocal sample failed to give a positive ELISA result. The pigs were from four major areas of Nepal, Kathmandu Valley, eastern Nepal, Terai and adjoining areas of the valley. Positive results were found from only Kathmandu Valley and adjoining areas. There was no significant difference in the prevalence between areas (p = 0.43). All four positive samples were from indoor managed pigs. The Trichinella seroprevalence determined in this study deserves a direct demonstration of the parasites for proof of the presence of Trichinella in Nepal and to discover the species and infection sources.

Isolation and Identification of Brucella melitensis Biovar 3 from Vaccinated Small Ruminants: A Public Health Threat in Kosovo.

In 2011, a human brucellosis case with severe clinical symptoms was reported at the University Clinic for Infectious Diseases in Prishtina, Kosovo. A trace-back investigation was conducted to find the source of human infection. A total of 49 blood samples and 15 corresponding milk samples from sheep and goats raised on the patient's farm were taken for serological and molecular analysis. Serology using RBT and CFT revealed 11 positive animals. Twelve milk samples were PCR positive. A Brucella strain isolated from a goat's milk sample was classified as Brucella melitensis biovar 3, indicating the first ever isolation and report in Kosovo. The use of the Bruce-ladder PCR provided differentiation between the field strain and the vaccine strain. Hence, the accidental transmission of the vaccine strain Rev 1 that was previously used for the vaccination of the farm animals could be excluded. The findings of this study show that brucellosis is still a public health threat in Kosovo despite control measures.

Experimental studies in pigs on Trichinella detection in different diagnostic matrices.

A total of 72 specific pathogen-free (SPF) and Iberian pigs (three animals per group) were inoculated with 200, 1000 or 20,000 muscle larvae of T. spiralis, T. nativa, T. britovi and T. pseudospiralis. For each animal, the muscle larva burden was evaluated in nine muscle samples by digestion. The anti-Trichinella IgG kinetics in blood samples, taken twice prior and at days 5, 10, 15, 20, 25, 30, 40, 50 and 60 post-inoculation, and in muscle juice, obtained at necropsy, was evaluated by an ELISA using an excretory/secretory antigen. The mean larval recovery rate in SPF/Iberian pigs corresponded with the level of inoculum dose, and tongue, diaphragm and masseter were identified as predilection muscles. In SPF and Iberian pigs receiving 20,000 larvae of T. spiralis, an earlier seroconversion was detected from day 25 post-inoculation. At a 10-fold dilution, the muscle juice showed a good test agreement with blood serum.