A probabilistic Poisson-based model to detect PRRSV recirculation using sow production records.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease associated with a decrease in the number of born alive piglets (NBA) and an increase in the number of lost piglets (NLP) per farrowing. Under practical conditions, it is critical to assess whether a farm is suffering PRRSV recirculation in the sow herd as soon as possible. The aim of this research work was to develop a new method to detect potential PRRSV recirculation in sow production farms. Sow reproductive performance records from one farm (farm T) were used to set up the method and records from ten additional farms (farms V1 to V10) were used for validation. A conditional Poisson model of NLP on NBA was proposed to fit the data. A three-step procedure was implemented to detect potential PRRSV recirculation: (i) computation of the maximum-likelihood estimates of the expected values of NBA and NLP in a PRRSV non-recirculating scenario; (ii) calculation, for each farrowing, of the p-value associated with the probability of jointly observing deviations towards decreased NBA and increased NLP. The detection of a potential PRRSV recirculation was based on (iii) the combined p-value resulting from weighing the p-values of the last N farrowings by the chi-square-inverse method. In order to gain specificity, a displacement on the expected non-recirculating NBA and NLP values was used for tuning purposes. With this approach, two PRRSV circulating periods were detected in farm T, which were confirmed with standard laboratorial diagnostic techniques. The method was subsequently validated in farms V1 to V10, where ten PRRSV-recirculating time episodes had been diagnosed. The method proposed here was able to detect the ten PRRSV recirculations using a relatively small set of contiguous farrowings, with only two mismatched weeks, one as a false negative, in farm V1, and one as a false positive, in farm V4. It is concluded that a conditional Poisson-based model of NLP on NBA can be a useful tool for routinely detecting PRRSV recirculation in sow herds.

Intraspecific variability of growth and ochratoxin A production by Aspergillus carbonarius from different foods and geographical areas.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of food commodities throughout the world. Aspergillus carbonarius is the most important source of OTA in food commodities such as wine, grapes and dried vine fruits and is also responsible for the formation of OTA in coffee. The aim of this study was to determine the simultaneous effect of three culture media (Czapek Yeast Extract Broth (CYB); Synthetic Grape Juice Medium (SGM) and White grape juice (WGJ)) at three water activity (aw) levels (0.90; 0.95 and 0.98-0.99), and three incubation temperatures (15 °C, 25 °C and 35 °C) on the growth and OTA production by 16 strains of A. carbonarius. The strains were mainly isolated from grapes from areas with a Mediterranean climate. All the strains were confirmed for identity by sequencing of the calmodulin gene. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA after 1, 2, 4 and 10 days of incubation. No significant differences were observed in absorbance values between the strains. The highest absorbance values were recorded in CYB at 0.99 aw and at 0.95 aw after 10 days of incubation at 25 °C and 35 °C. None of the strains were able to grow at 0.90 aw and 15 °C in any culture media after 10 days of incubation. OTA concentration was statistically higher at 15 °C than at 25 °C or 35 °C. The highest significant OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was CYB, followed by WGJ and SGM. While strains isolated from Mediterranean climate foods had a similar behavior despite being isolated from different geographical areas, OTA concentration produced by one Robusta coffee strain from Thailand was statistically higher at 25 °C than at 15 °C. This would suggest that the type of food matrices and consequently the adaptation of A. carbonarius strains to different climatic conditions would have a greater influence on the ecophysiology of the strains than only their geographical origin.

Impact of some environmental factors on growth and ochratoxin A production by Aspergillus niger and Aspergillus welwitschiae.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for OTA contamination in grapes and derived products. This species has recently been split into A. niger and Aspergillus welwitschiae. Both species can not be distinguished by phenotypic or extrolite profiles and to date there is no ecophysiological information of A. welwitschiae. The aim of this study was to determine the effects of water activity (aw) (0.90; 0.95 and 0.98-0.99), culture media (Yeast Extract Sucrose Broth (YESB); Synthetic Grape Juice Medium (SGM); White grape juice (WGJ)) and temperature (15 °C, 25 °C and 35 °C) on the growth and OTA production of four strains of A. niger and six strains of A. welwitschiae. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA at 1, 2, 4 and 10 days. No significant differences were observed in absorbance and OTA values between the two species under study. The highest absorbance values were recorded in YESB, followed by SGM and WGJ. Absorbance values increased with increasing aw and temperature. The highest OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was YESB, followed by WGJ and SGM. The studied strains of A. niger produced the highest mean OTA level at 25 °C whereas A. welwitschiae strains produced the highest mean OTA concentration at 15 °C, although not differing significantly from concentration produced at 25 °C. To our knowledge, this is the first report on the impact of some environmental factors on growth and OTA production by A. welwitschiae.

Trichophyton erinacei in pet hedgehogs in Spain: Occurrence and revision of its taxonomic status.

Hedgehogs have increased in popularity as pets in Spain but there are no data of infection rates of this exotic animal with dermatophytes in our country. During the period of 2008-2011 a total of 20 pet hedgehogs (19 African pygmy hedgehogs and 1 Egyptian long-eared hedgehog) suspected of having dermatophytoses were studied. This is the first survey of the occurrence of T. erinacei in household hedgehogs in Spain. The T. erinacei infection rate was 50% (9 out of 19 African pygmy hedgehogs, and the one Egyptian long-eared hedgehog surveyed). Morphological identification of the isolates was confirmed by molecular analysis. All the strains had the same ITS sequence and showed 100% sequence similarity to T. erinacei type strain CBS 511.73 (AB 105793). The Spanish isolates were confirmed as T. erinacei urease positive. On the basis of ITS sequences, T. erinacei is a species close to but separate from the taxa included in the A. benhamiae complex. Review of the current literature on DNA-based methods for identification of species included in this complex has highlighted the urgent need to reach a consensus in species circumscription and classification system accepted by all mycologists.

Phylogenetic diversity of Fusarium incarnatum-equiseti species complex isolated from Spanish wheat.

Wheat is the most important cereal grown in the European Union and Spain is its fifth largest wheat producer. There is little information about Fusarium species associated with wheat in Spain. Phylogenetic diversity of 51 strains belonging to Fusarium incarnatum-equiseti species complex (FIESC) isolated from Spanish wheat was investigated using partial sequences of the translation elongation factor gene (EF-1α). Maximum-parsimony and Bayesian analysis of aligned DNA sequences resolved 18 haplotypes and 7 phylogenetic species. Strains morphologically identified as F. equiseti belonged to two different phylogenetic species, FIESC-5 and FIESC-14. Some correlation between phylogenetic species and geographical region was found. The present results highlight the potential contribution of FIESC to the mycotoxin contamination of Spanish wheat.

Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes.

Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. Besides, OTA production is a very consistent property of this species and for this reason atoxigenic isolates of A. carbonarius are very rarely found in natural environments. In the present study, for the first time, three nonochratoxigenic wild strains of A. carbonarius have been discovered, unambiguously identified, characterized in deep and compared to ochratoxigenic strains of the same species. In addition, polyketide synthase (pks) genes suggested to be involved in OTA biosynthesis were also screened in these strains. The identification of the strains was confirmed by ITS-5.8S rRNA, ß-tubulin and calmodulin gene sequencing. The three atoxigenic strains did not produce OTA in a conducive culture medium at any of the temperatures and times of incubation tested. Five ketosynthase domains from pks genes previously described in A. carbonarius were detected both in ochratoxigenic and in nonochratoxigenic strains. Atoxigenic strains of A. carbonarius could be useful as biotechnological agents to be used in food industry and as biological agents for control of OTA production in vineyards and other crops.

Mycobiota and mycotoxin contamination of maize flours and popcorn kernels for human consumption commercialized in Spain.

Mycobiota and co-occurrence of aflatoxins, citrinin, ochratoxin A and zearalenone in 30 samples of maize flours and 30 of popcorn kernels purchased in Spain for human consumption were determined. The mycotoxin-producing ability of Aspergillus, Fusarium and Penicillium spp. was also studied. Total fungal counts of maize flours ranged from <10 to 8.4 × 10(4) CFU/g and predominant mycobiota belonged to Aspergillus spp. and Penicillium spp. In popcorn kernels samples the most frequent species were Aspergillus spp., Mucorales, Fusarium spp. and Penicillium spp. Aflatoxins were produced by Aspergillus flavus and Aspergillus parasiticus, citrinin by Penicillium citrinum and Penicillium verrucosum, ochratoxin A by Aspergillus niger and patulin by Aspergillus clavatus and Penicillium griseofulvum. Identification of all the mycotoxin-producing strains as well as some Aspergillus spp. difficult to identify using phenotypic characters only was also performed by molecular methods. Aflatoxins were detected in 14 maize flours and 2 popcorn kernels samples, while ochratoxin A was detected in 4 maize flours and 10 popcorn samples. Co-occurrence of aflatoxins and ochratoxin A was found in the 4 ochratoxin-positive maize flour samples. Citrinin and zearalenone were not detected. This is the first report of aflatoxins and ochratoxin A contamination in maize flours and popcorn kernels commercialized in Spain.

Malassezia cuniculi sp. nov., a novel yeast species isolated from rabbit skin.

Members of the genus Malassezia have rarely been associated with lagomorphs. During the course of an investigation of the lipophilic mycobiota of rabbit skin, two lipid-dependent isolates which could not be identified were recovered on Leeming and Notman agar medium from different animals. No growth of Malassezia yeasts was obtained either on Sabouraud's glucose agar or modified Dixon agar media. In this study, we describe a new taxon, Malassezia cuniculi sp. nov., including its morphological and physiological characteristics. The validation of this new species was supported by analysis of the D1/D2 regions of the 26S rRNA gene and the ITS-5.8S rRNA gene sequences. The results of these studies confirm the separation of this new species from the other species of the genus Malassezia, as well as the presence of Malassezia yeasts on lagomorphs.

Chrysosporium guarroi sp. nov. a new emerging pathogen of pet green iguanas (Iguana iguana).

Chrysosporium guarroi sp. nov. represented by five strains isolated from cases of dermatomycosis in pet green iguanas (Iguana iguana) in Spain, is described and illustrated. This taxon is characterized by its ability to grow at temperatures from 15 to 37 degrees C and by the presence of arthroconidia and aleurioconidia. The latter are unicellular, smooth, pyriform or clavate, sessile or borne at the ends of narrow stalks. The analysis of the sequences of the D1/D2 and ITS regions confirm the separation of this new species from others of the genus Chrysosporium.

Dermatomycosis in a pet inland bearded dragon (Pogona vitticeps) caused by a Chrysosporium species related to Nannizziopsis vriesii.

A Chrysosporium sp. related to Nannizziopsis vriesii was isolated in pure culture from squames and biopsies of facial lesions in a pet inland bearded dragon (Pogona vitticeps) in Spain. The presence in histological sections of morphologically consistent fungal elements strongly incriminates this fungus as the aetiological agent of infection. Lesions regressed following treatment with oral ketoconazole and topical chlorhexidine and terbinafine until the lizard was lost to follow up 1 month later. The ITS-5.8S rRNA gene of the isolate was sequenced and a search on the GenBank database revealed a high match with the sequences of two Chrysosporium sp. strains recently isolated from green iguanas (Iguana iguana) with dermatomycosis, also in Spain. Phylogenetic analysis of the sequences revealed that all these strains are related to N. vriesii. This is the first report of dermatomycoses caused by a Chrysosporium species related to N. vriesii in a bearded dragon outside North America.

Mating triggers dynamic immune regulations in wood ant queens.

Mating can affect female immunity in multiple ways. On the one hand, the immune system may be activated by pathogens transmitted during mating, sperm and seminal proteins, or wounds inflicted by males. On the other hand, immune defences may also be down-regulated to reallocate resources to reproduction. Ants are interesting models to study post-mating immune regulation because queens mate early in life, store sperm for many years, and use it until their death many years later, while males typically die after mating. This long-term commitment between queens and their mates limits the opportunity for sexual conflict but raises the new constraint of long-term sperm survival. In this study, we examine experimentally the effect of mating on immunity in wood ant queens. Specifically, we compared the phenoloxidase and antibacterial activities of mated and virgin Formica paralugubris queens. Queens had reduced levels of active phenoloxidase after mating, but elevated antibacterial activity 7 days after mating. These results indicate that the process of mating, dealation and ovary activation triggers dynamic patterns of immune regulation in ant queens that probably reflect functional responses to mating and pathogen exposure that are independent of sexual conflict.

Occurrence of Penicillium verrucosum in retail wheat flours from the Spanish market.

In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins.

Ochratoxin A and citrinin producing species of the genus Penicillium from feedstuffs.

In Spain, low ochratoxin A (OTA) levels have been detected in several pork products but there is no information published about the fungi involved in this OTA contamination. It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe where levels of OTA generally seem to be lower or not detected. Much less information is available about citrinin (CIT) and CIT producing species in cereals and their by products. The aim of this study was to determine, identify and characterize the occurrence of potential OTA and CIT producing Penicillium spp. from mixed feeds and raw materials purchased in the Spanish market and used as feedstuffs. A total of 155 Penicillium spp. isolates belonging to 34 species were analyzed in order to know if they are able to produce OTA and/or CIT. From these isolates, 11 P. verrucosum which were characterized by RAPD analyses, produced OTA. Fourteen isolates were CIT producers, 10 isolates of P. verrucosum and 4 of P. citrinum. Although the occurrence and abundance of OTA and CIT Penicillium producing species have been low in our study, our results confirm the potential risk of OTA and CIT production in feeds if stored improperly. Our results also confirm the occurrence of P. verrucosum in South European countries and that it is the only OTA producing Penicillium species in these substrates.

Cutaneous hyalohyphomycosis caused by a Chrysosporium species related to Nannizziopsis vriesii in two green iguanas (Iguana iguana).

This report describes the first isolation of a Chrysosporium species as the etiological agent of dermatomycosis in two green iguanas (Iguana iguana). The ITS-5.8S rRNA gene of the two strains was sequenced and a search on the GenBank database revealed that the closest match was Nannizziopsis vriesii. Treatment with oral ketoconazole, in combination with topical 2% chlorhexidine solution and terbinafine resulted in clinical cure.

RFLP characterization of Aspergillus niger aggregate species from grapes from Europe and Israel.

In order to characterize by molecular methods the Aspergillus niger aggregate species involved in the ochratoxin A (OTA) contamination of European wine grapes and table grapes from Israel, a total of 173 strains were studied. The ITS-5.8S rDNA fragments of 173 A. niger agreggate strains from grapes included in this study were amplified and their PCR amplicons were RsaI digested in order to classify the strains in the RFLP types, N and T. All of the strains belonging to the A. niger aggregate were classified into the two RFLP types previously defined: type N (43%) and type T (57%). Twenty out of the 173 strains of A. niger aggregate produced OTA (0.1 to 10.5 mug g(-1)). All the OTA producing species belonged to the N-RFLP type.

Genetic typing of Malassezia pachydermatis from different domestic animals.

In the present study, random amplification of polymorphic DNA, which detects DNA polymorphism in fungal genomic DNA, was applied for genetic typing of Malassezia pachydermatis isolates. Fifty-five isolates from different domestic animals and body sites and the neotype strain CBS 1879 were characterized. Primers M13 and OPT-20 were used to analyse their genetic relatedness and similarity. This technique allowed us to distinguish four different genetic types. The predominant genetic type was observed in isolates recovered from different anatomical locations in all animals. It was the only genetic type found in cats, horse, goat and pig. The other three genetic types were observed only in isolates from external ear canals of dogs. Types II and IV were only recovered from external otitic ears and type III from healthy ears. An animal was colonised by more than one type of M. pachydermatis and different genetic types were detected in the same body site. Some genetic types were only isolated from diseased skin.

Molecular analysis of Malassezia sympodialis-related strains from domestic animals.

Recently, several new lipid-dependent species belonging to the genus Malassezia have been described. Some of them, such as Malassezia dermatis, Malassezia nana, and the tentatively named "Malassezia equi," have similar phenotypes and are genetically close to Malassezia sympodialis Simmons et Gueho 1990. DNA characterization by D1/D2 26S rRNA gene and internal transcribed spacer (ITS)-5.8S rRNA gene sequencing analysis of lipid-dependent strains from different animal species close to M. sympodialis is described and illustrated. Phylogenetic analysis of both the D1/D2 regions of 26S rRNA gene and ITS-5.8S rRNA gene sequences showed four distinct clusters. Cluster I included isolates from different animal species (horse, pig, and lamb) and the type culture of M. sympodialis. Cluster II included isolates from horses grouping close to the "M. equi" AJ305330 sequence. Cluster III comprised isolates mainly from goats. Cluster IV contained isolates mainly from cats grouping together with the M. nana AB075224 sequence. This last cluster included isolates from healthy and external otitic ears. All of these strains had identical 26S rRNA gene and ITS regions. It is not clear whether the value of these genetic differences is for the definition of species or whether they only demonstrate genetic variation among strains from different origins within M. sympodialis, which are in the course of differentiation and probably adaptation to specific animal hosts.

Selective efficacy of culture media recommended for isolation and enumeration of Fusarium spp.

Selective culture media, such as Nash and Snyder medium (NS), dichloran-chloramphenicol peptone agar (DCPA), modified Czapek-Dox agar (MCz), Czapek Dox iprodione dichloran agar (CZID), potato dextrose iprodione dichloran agar (PDID), or malachite green agar (MGA 2.5), have been developed for isolating and enumerating Fusarium spp. from natural samples. However, some of these culture media are not very selective because they allow the growth of many other fungal species. In this study, a comparison of the selective efficacy of these culture media, using different strains of Fusarium spp. (F. anthophilum, F. culmorum, F. dlamini, F. graminearum, F. napiforme, F. nygamai, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. subglutinans, and F. verticillioides) and natural samples has been carried out. Among the six recommended selective culture media assayed, no statistical differences were detected in colony counts of the Fusarium spp. strains tested, although the colony diameters in MGA 2.5 were significantly lower than in NS, MCz, DCPA, CZID, and PDID media. With natural samples, MGA 2.5 performs as a potent selective medium for Fusarium spp., whereas the other recommended selective media allow the growth of many other different fungal species including Zygomycetes and yeasts.

Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market.

Ochratoxin A (OTA) can occur in a wide range of foods, but unexpectedly high concentrations have been detected in dried vine fruits of various origins. The European Union has recently established a maximum OTA limit of 10 microg/kg for these foodstuffs. In order to determine the likely origin of OTA, a mycological study of 50 dried fruit samples (currants, raisins, and sultanas) representative of the Spanish market was conducted. Fungal contamination was detected in 49 of 50 (98%) samples. Black aspergilli were isolated from all of the positive samples. Aspergillus niger var. niger was isolated from 98% of the samples, and Aspergillus carbonarius was found in 58% of the samples. One hundred sixty-eight A. niger var. niger isolates and 91 A. carbonarius isolates were screened for their ability to produce OTA. Eighty-eight (96.7%) A. carbonarius isolates and one (0.6%) A. niger var. niger isolate were found to be OTA producers. Black aspergilli were the dominant fungi. Among black aspergilli, A. carbonarius has shown a consistent ability to produce OTA and is the most probable source of this mycotoxin in these substrates.

What is the source of ochratoxin A in wine?

During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine.