Developing an Automatic Color Determination Procedure for the Quality Assessment of Mangos (Mangifera indica) Using a CCD Camera and Color Standards.

Color is one of the key sensory characteristics in the evaluation of the quality of mangos (Mangifera indica) especially with regard to determining the optimal level of ripeness. However, an objective color determination of entire fruits can be a challenging task. Conventional evaluation methods such as colorimetric or spectrophotometric procedures are primarily limited to a homogenous distribution of the color. Accordingly, a direct assessment of the mango quality with regard to color requires more pronounced color determination procedures. In this study, the color of the peel and the pulp of the mango cultivars "Nam Dokmai", "Mahachanok", and "Kent" was evaluated and categorized into various levels of ripeness using a charge-coupled device (CCD) camera in combination with a computer vision system and color standards. The color evaluation process is based on a transformation of the RGB (red, green, and blue) color space values into the HSI (hue, saturation, and intensity) color system and the Natural Color Standard (NCS). The results showed that for pulp color codes, 0560-Y20R and 0560-Y40R can be used as appropriate indicators for the ripeness of the cultivars "Nam Dokmai" and "Mahachanok". The peels of these two mango cultivars present two distinct colors (1050-Y40R and 1060-Y40R), which can be used to determine the fruit maturity during the post-ripening process. However, in the case of the cultivar "Kent", peel color detection was not an applicable approach for determining ripeness; thus, the determination of the pulp color with the color code 0550-Y20R gave promising results.

Suitability of Slower Growing Commercial Turkey Strains for Organic Husbandry in Terms of Animal Welfare and Performance.

Intensive turkey production with fast growing strains is often critically discussed regarding animal welfare problems. Studies evaluating the welfare status of both organic and less intensive selected turkey strains are limited, except in the slightly slower growing Kelly Broad Breast Bronze (Kelly). The aim of this study was to assess the welfare of turkeys from two strains with further decreased growth rate, Hockenhull Large Bronze (HoBr) and Hockenhull Black (HoBl), in comparison to Kelly under commercial organic conditions with 100% organic feed. Altogether 844 non-beak-trimmed male turkeys (274-288 per line) were reared and fattened in three replications with each six groups. On group level, use of resources in the 7, 16, and 25th week of life, mortality and feed conversion were recorded. Each bird was assessed with regard to plumage and skin condition as indicators of agonistic interactions, cannibalism and feather pecking, with regard to leg health, footpad, breast skin condition and, as performance indicators, live and carcass weight, utilization, daily weight gain and weights of valuable meat parts. The significantly slower growing HoBl showed slightly fewer malposition of the legs, reduced injury rates and less breast buttons, but a higher susceptibility to footpad dermatitis than Kelly turkeys. HoBr with a similar growth rate compared to Kelly had slightly more problems concerning walking ability and plumage damage, but also less breast buttons than Kelly turkeys. However, effect sizes were negligible (Φ < 0.10), except for the higher occurrence of footpad dermatitis and the reduced number of breast buttons in HoBl with small effect sizes (Φ = 0.20-0.24). Use of resources, prevalence of breast blisters and mortality, were not statistically different, although mortality rate was numerically lower in HoBl. Thus, for none of the studied strains clear benefits or disadvantages in terms of the birds' predisposition for welfare problems could be identified. Overall, prevalences of animal welfare problems were mostly lower than in comparable studies while performances were comparatively high. Therefore, turkeys from the studied strains appear to be suitable for organic rearing and fattening with 100% organic feed, given a good management.

Occurrence of selected zoonotic food-borne parasites and first molecular identification of Alaria alata in wild boars (Sus scrofa) in Italy.

Wild boar is a source of human infections with zoonotic pathogens, including food-borne parasites. With the aim of a characterization of the human exposure risk, a survey on wild boars intended for human consumption was planned, selecting three pathogens, Toxoplasma gondii, Alaria alata, and Trichinella spp., as markers of meat infection. Diaphragm muscle samples from 100 wild boars hunted in Piedmont region (Northern Italy) in two hunting seasons (2015-2016) were collected. Concerning T. gondii, a combined approach of antibody detection and molecular techniques with genotyping was performed. For the detection of A. alata and Trichinella spp., the larva migration technique and the magnetic stirrer method were employed, respectively; in addition, molecular confirmation of the morphological identification of the recovered specimen was performed. Anti-T. gondii antibodies were found in meat juice samples (43.3%) and T. gondii DNA (type II) was detected in three animals (7.1%) out of 42 seropositive examined. In none of the sampled wild boars (0%), Trichinella spp. larvae were found, whereas one animal (1%) scored positive to A. alata mesocercariae. The molecular diagnosis proved the morphological identification of the trematode. This is the first finding of A. alata in Italian wild boar population. The present study confirmed the role of wild boars as a source of parasitic zoonotic diseases and thus the risk derived for humans posed by the consumption of game meat. Considering the zoonotic implications, the results underline the importance of monitoring and surveillance of zoonotic parasites in Italian wild boar populations.

Alaria spp. mesocercariae in Eurasian badger (Meles meles) and wild boar (Sus scrofa) from the Bialowieza Forest, north-eastern Poland.

Alaria spp. mesocercariae are commonly found in wild boar and other omnivorous mammals. In Europe, the number of cases presenting Alaria mesocercariae infections has been on the rise in the last years. From October to December 2016, samples of muscle from tongue, neck, and mandibular regions were collected from 1 Eurasian badger (Meles meles) and 14 wild boars (Sus scrofa) hunted in the Bialowieza Forest, north-eastern Poland. Using the Alaria migration technique (AMT), Alaria mesocercariae were isolated and morphologically identified in one badger and one wild boar. To the authors' knowledge, this is the first report of Alaria mesocercariae in paratenic hosts from the Bialowieza Forest.

Slaughter of pregnant cattle in German abattoirs--current situation and prevalence: a cross-sectional study.

BACKGROUND: The slaughter of pregnant cattle and the fate of the foetuses are relatively new subjects in the field of animal welfare. The Scientific Committee on Veterinary Measures relating to Public Health (SCVPH), however, does not believe this topic to be a critical issue because of the hitherto supposed rare occurrence of this practice. Some previous studies though, contradict this assessment, emphasising its relevance to animal welfare. With regard to the heterogeneous study design of previous investigations, the objective of this study is to evaluate the current situation concerning the slaughter of pregnant cattle in different German abattoirs. Additionally, the prevalence was assessed semi-quantitatively on the basis of a cross-sectional, voluntary and anonymous survey that was conducted amongst senior veterinary students of the University of Leipzig from 2010 until 2013. RESULTS: Of 255 evaluable questionnaires, 157 (63.6%) mention the slaughter of pregnant cattle, corresponding to 76.9% of all visited abattoirs. Slaughter of pregnant cattle is reported often (>10% of females) in 6 (3.8%), frequently (1-10% of females) in 56 (35.7%), and rarely (<1% of females) in 95 (60.5%) of all cases (n = 157) respectively. About 50% of these animals were reported to be in the second or third stage of gestation. 15 (10.6%) of 142 questionnaires providing information about the foetus, state that the foetus showed visible vital signs after the death of the mother, but in one case the foetus was euthanized subsequently. CONCLUSIONS: The results show that the slaughter of pregnant cattle is a common and widespread practice in German abattoirs. The SCVPH's assumption that pregnant cattle are only slaughtered in rare exceptional cases can no longer be maintained. The high proportion of foetuses in the second and third gestational stage must also be considered. In this context the implementation of suitable studies and detailed analysis of the current situation is indispensable to ensure the high standards in animal welfare in Germany and Europe.

Effect of temperature on the survival of Alaria alata mesocercariae.

Recent findings of Alaria alata mesocercariae in wild boars and other animals in Europe reinforced the concern about the public health risk posed by this parasite especially if the game meat is insufficiently heated during preparation. Cooking and freezing are effective methods for the inactivation of parasites in meat whereas refrigeration is considered as an essential part of the Good Hygiene Practice. Additionally, microwave dielectric heating may represent an equally effective tool for parasite inactivation. Therefore, isolated vital mesocercariae were examined with respect to their resilience against heating, refrigeration, freezing, and microwave heating. A. alata mesocercariae stored in Ringer's solution do not survive heating temperatures that exceed 60.0 °C. Similarly, exposure to microwave heating ensured an inactivation of all parasite developmental stages after 90 s of treatment. In contrast, the parasites' tolerance towards cold is far higher as the mesocercariae survived refrigeration temperatures (4.0 ± 2 °C) in Ringer's solution for up to 13 days. An effective inactivation by cold is therefore only guaranteed if the infested game meat is frozen to a core temperature of -13.7 °C for a minimum of 2 h at least. Game meat should be handled with the same or even higher caution than meat of husbandry animals since wild animals may be infected with parasites or other zoonotic agents that are not common in livestock. It is therefore of crucial importance that appropriate temperature time protocols are used for the reliable inactivation of these zoonotic agents.

Effects of in vitro conditions on the survival of Alaria alata mesocercariae.

The aim of this study was to determine the effect of different concentrations of table salt (NaCl) and ethanol (v/v) solutions on the viability of Alaria alata mesocercariae. Furthermore, the survival of A. alata mesocercariae during simulated human gastric digestion was evaluated. For this purpose, A. alata mesocercariae migration technique (AMT) was used for the isolation of the parasite from high-positive A. alata mesocercariae meat from wild boar, raccoon, raccoon dog, and badger meat. In total, we have studied the behavior of 582 larvae under different conditions (NaCl, ethanol, and artificial gastric juice) in three independent in vitro experiments. The larvae survived at a NaCl concentration of up to 2.0% until day 21 with a median survival time of 11 days. At 3.0% NaCl concentration, the larvae lost their vitality after less than 24 h. In addition, it was found that ethanol concentrations from 8.0 to 70.0% were effective at reducing survival of A. alata mesocercariae within a short period of time (<1 min). Finally, our studies have revealed that it required 120 min to reliably inactivate all A. alata mesocercariae within HCl-pepsin digestion solution with a pH of 1.5-2.0 at 37°C. Consequently, the results showed that 3.0% is the minimum concentration of NaCl in meat products recommended for human consumption because at lower NaCl concentration the parasite survived for a substantial period of time. Finally, the common concentrations of ethanol used for the disinfection of surfaces in household and/or laboratory, are sufficient for the inactivation of A. alata mesocercariae.

Assessing laboratory performance in Trichinella ring trials.

Trichinosis (Trichinellosis) is a zoonotic disease acquired by eating raw or not adequately processed pork or wild game infected with the larvae of the roundworm genus Trichinella. According to European regulations, animals susceptible to Trichinella have to be examined for infestation. To evaluate the performance of laboratories in Germany, inter-laboratory comparisons known as "ring trials" were introduced by the Federal Institute for Risk Assessment in 2004. The current method of analysis makes use of tolerance zones based on the number of larvae in the sample, but does not permit one to determine if a given lab can detect an infested sample reliably, as required by the quality assurance recommendations of the International Commission on Trichinellosis (ICT). A new way of analysing the ring trial data is presented here, which is based on Bayesian hierarchical models. The model implements the ICT requirement by providing an estimate for the probability that a given lab would fail to detect a sample containing, say, five larvae. When applied to the 87 labs that participated in Germany's 2009 ring trials, it turns out this probability is greater than 10% for 21 of them, although only 10 of these in fact returned a false negative result. Such a new method is required to abide by the ICT requirements and make ring trials effective.

Tenacity of Alaria alata mesocercariae in homemade German meat products.

A renewed interest in the pathogenic potential of Alaria alata mesocercariae emerged over the last 10years as a result of increased findings of this parasite in feral pigs during official examination for Trichinella spp. Cases of food associated human alariosis in North America suggest that a risk associated with the consumption of traditional raw cured products from infected wild boar meat cannot be neglected because the commonly applied preservation techniques may not necessarily kill the mesocercariae. In addition, changes in consumer behavior and new preparation methods for game meat (e.g. pink roasting and grilling) may increase the risk for food-associated parasitic infections. Thus, there is a strong need for the evaluation of the tenacity of A. alata mesocercariae against different physical and chemical influences as pertaining to common preservation and preparation techniques. Against this backdrop the aim of our work was a sound analysis of the survivability of A. alata mesocercariae during curing, fermentation, cold smoking and drying in raw cured meat products. Eighty three samples of traditional German meat products were prepared from naturally infected game meat and partly spiked with additional vital mesocercariae to achieve an adequate dose of infection. The resultant products were examined chronologically for dead and viable A. alata mesocercariae with the Alaria mesocercariae migration technique. After 24h of production, vital A. alata mesocercariae were still found in raw type sausages but no vital parasites were detected in the final products. Based on these results a possible risk for the consumer for an infection with A. alata mesocercariae through the consumption of contaminated raw cured products can be largely ruled out if the respective food technological procedures are carried out properly. However, a risk for the consumer cannot be excluded in cases of very early consumption of these products.

Alaria alata mesocercariae in raccoons (Procyon lotor) in Germany.

Alaria alata is a trematode of carnivores from Europe. The mesocercarial stage was recently identified in wild boar meat from Europe. Previous histopathologic studies showed the presence of unidentified parasitic cysts within the tongues of raccoons from northern Germany. For identification of the parasite species, tissue samples of 105 raccoons originating from a National Park in northern Germany and from Berlin metropolitan area were collected. Histological examination of cryotome sections of frozen as well as paraffin-embedded tongues were used to identify parasite cysts. These were located in the connective and adipose tissue and in close proximity to small arterioles, suggesting a hematogenous spread of the parasite. Often, cysts were surrounded with mild infiltration by inflammatory cells. Additionally, mesocercariae were isolated from defrosted tongue samples of 11 raccoons. Molecular-biology assays confirmed the parasite species as A. alata. Except for one positive raccoon from Berlin City, all other positive raccoons originated from the sylvan Müritz National park, indicating an abundance of intermediate hosts in this area. Our results show that raccoons can act as paratenic hosts for A. alata and extend the broad host range of this parasite to a species introduced into Germany.

First interlaboratory test for the detection of Alaria spp. mesocercariae in meat samples using the Alaria spp. mesocercariae migration technique (AMT).

Emerging and re-emerging zoonotic diseases affect both public and animal health and require the development and contemporary implementation of suitable detection methods. A growing number of findings of the mesocercarial stage of the digenean trematode Alaria alata in game inhabiting wetlands have necessitated the development of a specific detection method. With the Alaria spp. mesocercariae migration technique (AMT), a specific and sensitive detection method is now available. To make the method accessible to the official controls, method validation is necessary. In this context, interlaboratory tests (IT) are a key factor to demonstrate both (1) the suitability of the respective method and (2) the reference materials. In the first IT performed on this issue, 15 laboratories from nine German federal states took part. Every lab received two negative and four positive standard samples each as well as a standardized examination device for AMT, and a standard operating procedure. All participating laboratories showed very good results in terms of qualitative analysis: 96.7 % of the samples were assessed correctly positive or negative. An analysis of the qualitative performance shows that 263 (58.4 %) of 450 mesocercariae that were inserted in the meatballs were identified by the participants, and 5 (33.3 %) out of 15 labs were able to count at least 70 % of the Alaria spp. mesocercariae. A direct comparison with the results of the German Trichinella IT, which were conducted since 2004, shows that the overall sensitivity of the AMT is even higher than that registered for the reference method for Trichinella detection (e.g. 93 % in 2010). Also, in terms of quantitative analysis, AMT stands up to the comparison with the results from the German Trichinella IT. The refinement of the implementation protocol of this innovative, easy-to-use and cost-effective method harbours great potential for further optimization and successful implementation in the official controls.

Trichinella detection: identification and statistical evaluation of sources of error in the magnetic stirrer method for pooled sample digestion.

Proficiency testing (PT) is the use of inter-laboratory comparisons to determine the performance of individual laboratories for specific tests or measurements, and to monitor a laboratory's performance. Participation in proficiency testing provides laboratories with an objective means of assessing and demonstrating the reliability of the data they are producing. To ensure the reliability of Trichinella detection and meat hygiene within the European Union and afford optimal protection to the consumer, PT is conducted under the direction of the European National Reference Laboratories for Trichinella. Evaluation of data from the national PT showed that lab-internal shortcomings are frequent. These shortcomings are specifically related to: (1) improper sample collection and preparation; (2) incorrect transposition and application of the protocol as laid down in Annex I, Chapter I, Nr. 3 (a-g) of the Commission Regulation (EC) No. 2075/2005; (3) insufficient sedimentation times; and (4) improper equipment.(e.g. Prost and Nowakowski, 1990; Rossi and Pozio, 2008; Forbes and Gajadhar, 1999; Rossi and Pozio, 2008). To test the hypothesis that both method based errors as well as internal lab errors can influence the accuracy and precision of the magnetic stirrer method for pooled sample digestion (MSM), we initiated a study to evaluate the analytical uncertainty of the MSM. Results presented here are based on: (i) data from PT in Germany (2008, 2009, and 2010); (ii) within-lab performance conducting high volumes of MSM; (iii) larval recovery experiments; and (iv) statistical evaluation of data resulting from these procedures. Quantitative data from the PT show that on average only 60% of Trichinella larvae were detected. Even laboratories that showed relatively good performance (>80% larva recovery, no false negative or false positive results), frequently reported samples with an unexpectedly low larval count (loss of >2 larvae). In our own laboratory, high numbers of repeated analyses of standards and re-analyses of residual fluids indicated that these outliers could be described by a binomial distribution based on a laboratory-specific Trichinella-detection probability. Results of recovery experiments indicate that only a part of the total larval losses can be attributed to lab-internal shortcomings inasmuch as a significant number of L1 could be isolated from the residual and washing fluids.

Alaria alata in wild boars (Sus scrofa, Linnaeus, 1758) in the eastern parts of Germany.

Over the last decade, incidental findings of Alaria alata in stocks of German wild boar during the official Trichinella inspection have been increased. As early as 2006, the German Federal Institute for Risk Assessment pointed out the possible health risk to the consumer posed by this trematode. However, at that time, reliable data concerning the prevalence of the parasite in German wild boars and feral pigs were lacking especially because no appropriate detection method was available. The development of the A. alata mesocercariae technique (Riehn et al., Parasitol Res 107(1):213-220, 2010) now makes it possible to close the remaining gaps in knowledge in this field. Over a 2-year period, 286 retained samples of fresh meat from wild boars originating from different hunting areas in Brandenburg and Saxony-Anhalt, which were tested negative for A. alata during the official Trichinella inspection in the competent veterinary inspection offices, were reexamined with the A. alata mesocercariae migration technique (AMT). In 33 out of 286 retained meat samples (11.5%) with a preliminary negative report, the trematode was demonstrated during the follow-up examination using AMT. This result especially in connection with the highly heterogeneous distribution of the parasite within the hosts' body (Riehn et al., Parasitol Res 107(1):213-220, 2010; Moehl et al., Parasitol Res 105(1):1-15, 2009) shows clearly that a high number of unreported cases of alariosis in wild boars have to be assumed.

Development of a PCR approach for differentiation of Alaria spp. mesocercariae.

To date classification and differentiation of Alaria spp. are based largely on external characteristics and comparative morphology of adult flukes. The accurate differentiation between various Alaria spp. mesocercariae is indeed difficult because there are only few data on morphological and morphometrical features of the parasite's developmental stages. We established a conventional polymerase chain reaction (PCR) for a molecular-based diagnosis of Alaria alata mesocercariae that can aid in their identification. Twenty Alaria spp. mesocercariae specimens were collected from three different wild boars originating from different areas of eastern Germany. DNA from the prepared isolates was extracted, and a primer pair was selected to amplify a 303-bp region of the A. alata genome. The DNA preparations extracted from the field samples as well as A. alata positive controls were successfully amplified and yielded a single sharp band of the expected size. In all samples, molecular identification was consistent with morphological identification. With our new PCR assay, we present the first approach for identification and characterization of A. alata mesocercariae specimens using molecular methods. This practicable and reproducible protocol can be used for both diagnostic and epidemiological purposes.

A novel detection method for Alaria alata mesocercariae in meat.

Distomum musculorum suis (DMS), the mesocercarial stage of the trematode Alaria alata, can cause severe damages within their hosts, and since several reports about cases of human larval alariosis have been published, it became apparent that infected game animals and in particular wild boars are a potential source of infection for both humans and animals. A final statement concerning the health risks for consumers could not be given due to the lack of information about both the prevalence of DMS and the suitability of Trichinella inspection methods to detect this parasite in wild boar meat. Our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for DMS detection in wild boars, (2) development, optimization, and validation of methods, and (3) the distribution of the parasites within their paratenic hosts. A total of 868 individual samples/digests from 48 wild boars were analyzed by the reference method for Trichinella detection in meat samples according to regulation (EC) No. 2075/2005. In addition to the official protocol, a method modification with Pankreatin(c) and bile acid was applied for analysis of adipose tissue samples (n = 89). On the basis of our results, a new detection method based on a larvae migration technique was developed and used for detection of DMS in 574 single samples. Furthermore, the distribution patterns of DMS in wild boars in a total of 1377 single sample migrations/digestions from 35 positive animals were analyzed by application of all three methods. The official digestion method for Trichinella spp. in wild boars meat is inapplicable for the detection of A. alata mesocercariae as it shows shortcomings in both digestion and sampling. A direct comparison between the newly developed A. alata mesocercariae migration technique and the official digestion method for Trichinella spp. based on 574 single samples from 18 animals clearly shows that the sensitivity to detect A. alata developmental stages in tissues of wild boars of the new method is nearly 60% higher compared with the magnetic stirrer method for pooled sample digestion as laid down in regulation (EC) No. 2075/2005. Among other advantages, this method offers a simple, highly applicable, fast, and cost effective way to detect DMS in wild boars which is already applicable in routine veterinary inspection.