New cholinesterase inhibiting bisbenzylisoquinoline alkaloids from Abuta grandifolia

The phytochemical study of the stem bark and wood of Abuta grandifolia (Mart.) Sandwith led to the identification of four bisbenzylisoquinoline alkaloids (BBIQs), namely (R,S)-2 N-norberbamunine (1), (R,R)-isochondodendrine (2), (S-S)-O4″-methyl, Nb-nor-O6'-demethyl-(+)-curine (3), and (S-S)-O4″-methyl, O6'-demethyl-(+)-curine (4), together with the aporphine alkaloid R-nornuciferine (5), all obtained by countercurrent distribution separation (CCD) and identified on the basis of their spectroscopic data. Alkaloids 3 and 4 were new. All the isolated compounds were tested for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. 1 was the most active against AChE, whereas 3 and 4 were the most potent against BChE. Interestingly, all tested alkaloids are more potent against BChE than against AChE. This selectivity of cholinesterase (ChE) inhibition could be important in order to speculate on their potential therapeutic relevance.     

In vitro induction of hexaploid plants from triploid hybrids of Pennisetum purpureum and Pennisetum glaucum

This study describes an efficient in vitro method using colchicine-treated triploid seedlings of Napiergrass and Pearl millet hybrids to produce hexaploid hybrids and their subsequent identification with flow cytometry. It also describes chromosome count and stomatal morphology and their use to ploidy analysis. Four-hundred and eighty triploid seeds, representing 12 different hybrids were sterilized and transferred to MS media to induce chromosome doubling. Surviving plants were analysed by flow cytometry. From six triploid (control) and hexaploid plants, the stomata sizes and frequency were analysed. Chromosome count was performed only in the plants identified as hexaploid. Seventeen plants were identified as hexaploid by flow cytometry analysis. Further confirmation of the hexaploid condition was performed with stomatal morphology (stomatal frequency reduction and stomatal length increase) and chromosome count (2n = 6x = 42). Chromosome doubling has numerous applications in Pennisetum breeding. It can be used to restore the fertility of interspecific hybrids and to improve seed size.     

Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5×10¹⁰ (group I), 3×10¹⁰ (group II) or 6×10¹⁰ (group III) infected erythrocytes mixed with STDCM® adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1×10⁸ infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses (P0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.     

Diabetic cataracts: different incidence between dogs and cats

Diabetes mellitus is one of the most common endocrinopathies in the dog and cat. Diabetic cataract primarily affects the canine species and is rarely observed in the cat. It has been proposed that the incidence of cataracts in diabetic dogs is high because many of these patients have significant hyperglycemia despite insulin therapy. Age, gender, levels of serum glucose (before and during insulin therapy) and cataract formation were evaluated, retrospectively, in 23 dogs and 22 cats with diabetes mellitus. In the canine population, the groups with the highest frequency of presentation were females and sexually intact animals. In contrast, males and neutered animals were the most prevalent groups in the feline diabetic population. Over 80% of diabetic cats and dogs were older than 7 years. Our results confirm the almost total lack of cataracts in diabetic cats, while they were present in more than half of the dogs. A relation between the incidence of cataracts and the correspondent level of hyperglycemia in the canine and feline species could not be established. The estimation of the relative risk for the development of cataracts in diabetic dogs shows that some population groups have a higher probability for suffering from this ocular alteration. A relation between relative risk and the correspondent level of hyperglycemia in the various groups was not found. This fact indicates that other factors are involved in the unequal appearance of diabetic cataracts in dogs and cats.     

Mortierella species from declining Araucaria araucana trees in Patagonia,Argentina

Since 2015, Araucaria araucana, an ecologically and economically important conifer native to Argentina and Chile, has suffered an unusual partial death of the crown throughout almost all of the distribution range in Argentina. No primary pathogen or pest was evident, associated with the phenomenon. Isolates of Mortierella, a poorly studied fungal genus in Patagonia, were obtained from the margins of necrotic phloem tissue of symptomatic trees. Five species of Mortierella were isolated from affected tissues. In inoculation tests, Mortierella alpina and M. aff. basiparvispora were pathogenic to A. araucana. These species caused necrosis of phloem, leading to chlorosis, foliar desiccation and eventually death, demonstrating that Mortierellales may play a role in the decline of the tree.     

Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.     

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.     

High Embryonic Recovery Rates with In vivo and Ex vivo Techniques in the Bitch

The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans‐surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.     

Erythrocytic pyruvate kinase deficiency and AB blood types in Australian Abyssinian and Somali cats

Objective   To determine the frequency of the mutant pyruvate kinase (PK) allele, haematological parameters and AB blood types of Abyssinian and Somali cats in Australia.
Design   Complete blood cell and reticulocyte counts, DNA PK mutation testing and blood typing were performed in all cats.
Results   A total of 60 cats (36 Abyssinians, 24 Somalis) were included (37 females, 23 males). For the mutant PK allele, three female Somalis were homozygous (affected, 5%), 17 cats were heterozygous (carrier, 28%) and 40 cats tested negative (normal, 67%). Pedigree analysis revealed common ancestry of affected and many carrier cats. Of affected cats, two had regenerative anaemias and all had reticulocytosis (range 64–390 × 10⁹/L; P < 0.001 compared with normal or carrier cats). The only consistent historical sign was lethargy. One affected cat was euthanased 18 months after testing, because of anaemia, neutropenia, anorexia and weight loss. The mutant allele frequency was 0.19 overall (0.29 in Somalis, 0.13 in Abyssinians). All cats had blood type A. The commercial blood typing card method incorrectly identified 12 cats as having type AB blood.
Conclusions    The frequency of the mutant PK allele is high in Australia. Screening for PK deficiency is indicated before mating and in individual cats of these breeds, even in the absence of anaemia and especially when there is reticulocytosis. Although all cats in the present study had blood type A, blood type B is common in these breeds worldwide. Retyping of any AB typed cats by a laboratory technique is recommended.