Properties of the avocado oil extracted using centrifugation and ultrasound-assisted methods.

The aim of this work was to evaluate the technologies effect of cold extraction by centrifugation (CE) and ultrasound-assisted (US-CE) methods without adding water, on the avocado oil yield, nutritional composition, physicochemical characteristics, oxidative stability (oxidation temperature and time, besides activation energy) and accelerated shelf life regarding hexane extraction (control). The US-CE improved the physicochemical properties such as acidity, peroxides, and iodine indexes regarding CE and Control. US-CE improved the yield, nutritional quality of fatty acids, oxidative stability, shelf life, and ω-6/ω-3 ratio regarding CE. Furthermore, US-CE improved the ratio yield/time extraction of the oil and increased the oxidation temperature regarding control. The main advantage of oils extracted using CE and US-CE concerning control was higher oxidative stability. The most representative polyunsaturated fatty acids identified in all treatments were γ-linolenic and conjugated α-linolenic acids. α-linolenic acid was only detected in US-CE and control. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00940-w.

Comparison of host cell invasion and proliferation among Neospora caninum isolates obtained from oocysts and from clinical cases of naturally infected dogs.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P<0.0001). A correlation was found between the IRs and TY (ρ>0.885, P<0.033), as well as between the TY and both dam morbidity (ρ=0.8452, P<0.033) and pup mortality (ρ>0.8117, P<0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates.

Low rates of Neospora caninum infection reactivation during gestation are observed in both chronically and congenitally infected mice.

Endogenous transplacental transmission (EnTT) of Neospora caninum is the most common route of infection in cattle and occurs as a consequence of a reactivation of N. caninum infection that may lead to abortion or to the birth of congenitally infected calves. The reactivation of N. caninum infection was studied during the gestation of chronically infected dams and, for the first time, in their congenitally infected pups. BALB/c mice were infected with Nc-Spain 7 (Group 1) or Nc-Spain 3H (Group 2), high virulence isolates and low-to-moderate virulence isolates, respectively. The mice were mated after 90 days post-infection, and the morbidity, mortality, vertical transmission and humoral immune responses were recorded for 2 consecutive generations. In the first generation, higher morbidity and mortality rates were observed in G1 before mating than in G2 (P < 0·0001). In the second generation, low vertical transmission rates were observed in both inoculated groups (7·7% and 17·1% in G1 and G2, respectively) and were significantly diminished in the third generation (8·7% in G2 versus 0% in G1). Low rates of reactivation of N. caninum infection were induced in chronically infected mice and decreased in subsequent generations regardless of the isolate employed in the inoculations. Thus, further studies are needed to improve this reactivation mouse model.

Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene.

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.

Transgenic Neospora caninum strains constitutively expressing the bradyzoite NcSAG4 protein proved to be safe and conferred significant levels of protection against vertical transmission when used as live vaccines in mice.

At present, there is no effective treatment or vaccine to prevent vertical transmission or abortion associated with Neospora caninum infection in cattle. Different vaccine formulations have been assayed, and live vaccines have shown the most promising results in terms of protection. Previously, transgenic N. caninum tachyzoites expressing the bradyzoite stage-specific NcSAG4 antigen in a constitutive manner (Nc-1 SAG4(c)) were obtained and showed a reduced persistence of parasite in inoculated mice. Thus, the present study evaluates the Nc-1 SAG4(c)1.1 and Nc-1 SAG4(c)2.1 transgenic strains and the Nc-1 wild-type (WT) strain to determine their protective efficacy against vertical transmission and cerebral neosporosis in mice. Consequently, dams were immunized twice with 5 × 10(5) tachyzoites of each strain and challenged with 2 × 10(6) tachyzoites of a heterologous and virulent isolate at 7-10 days of gestation. The Nc-1 SAG4(c)1.1 strain offered less protection than the other transgenic strain (Nc-1 SAG4(c)2.1) or their ancestor (Nc-1 WT). Indeed, 40%, 7% and 5.6% of the postnatal deaths corresponded to pups from dams vaccinated with Nc-1 SAG4(c)1.1, Nc-1 SAG4(c)2.1 and Nc-1 (WT) strains, respectively. In comparison, the non-immunized challenge group had a 100% mortality rate. In addition, mice were protected against congenital transmission; vertical transmission rates were 45%, 11.1% and 10.8% in the Nc-1 SAG4(c)1.1, Nc-1 SAG4(c)2.1 and Nc-1 WT immunized groups, respectively, vs. 94.9% in the non-vaccinated infected group. However, this protection against the postnatal mortality and the vertical transmission was not associated with a consistent Th1 or Th2-type immune response. Nonetheless, the Nc-1 SAG4(c)2.1 strain appears to be the best candidate for use as a live vaccine, as evidenced by results demonstrating its high levels of protection against vertical transmission and its lower persistence in mice, making this transgenic strain safer than Nc-1 WT.

Tentative identification of the species of Balantidium from ostriches (Struthio camelus) as Balantidium coli-like by analysis of polymorphic DNA.

The characteristics of Balantidium from ostriches (Struthio camelus) are similar to those of Balantidium coli; however, the species Balantidium struthionis was proposed on the basis of the host species (ostriches) and the shape of the macronucleus (with a deep depression in one side). In the present work, we have performed morphological and genetic comparisons between isolates of Balantidium from ostriches and B. coli from pigs to determine the specific status of B. struthionis. The morphological characteristics of the trophozoites of Balantidium from ostriches were reviewed in 100 trophozoites from two isolates. The macronucleus' shape of the ostrich Balantidium was highly variable, thus the use of this criterion for diagnostic purposes is not reliable. Besides, very few trophozoites showed a deep depression in their macronucleus and almost all the trophozoites conform to the description of B. coli. The complete sequence of the DNA coding for the 18s-rRNA-ITS1-5.8s-rRNA-ITS2 regions were obtained by PCR from five pig and five ostrich isolates. The sequences corresponding to the 18s and 5.8s-rRNA genes were identical for the ostrich and pig isolates. Two clearly different genotypes were found in the analysis of the ITS1 and ITS2 regions of the pig isolates; the genotype A was identified in all isolates, while the genotype B was found in only two of them. Their sequences show clear differences from that published corresponding to a B. coli gorilla isolate, which we will consider as a different genotype, C. In our opinion, these different B. coli genotypes reflect the genetic variability of this organism, but further studies would be necessary to determine if it could have practical importance. The polymorphism of the ITS regions have been also found in the ostrich isolates. The same genotypes A and B have been identified, although not as mixed infections. The morphological characteristics and the genetic results suggest that the species name B. struthionis is a synonym of B. coli; however, until experimental infections are carried out to determine if the parasite is transmissible between pigs and ostriches, it would be preferable to tentatively designate it as B. coli-like.