Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Hemorrhagic and necrotizing hepatitis associated with administration of a modified live canine adenovirus-2 vaccine in a maned wolf (Chrysocyon brachyurus)

A 15-yr-old, female, maned wolf (Chrysocyon brachyurus) was euthanized after presenting semicomatose with severe, uncontrolled frank hemorrhage from her rectum 6 days following a routine physical examination and vaccination. Histopathology indicated severe hemorrhagic and necrotizing hepatitis with intranuclear basophilic inclusion bodies in the liver that were thought to be consistent with adenoviral infection. Further classification by polymerase chain reaction, immunohistochemical staining, virus isolation, and electron microscopy confirmed the etiologic agent to be canine adenovirus-2. A representative sample of the vaccine that had been used was submitted and sequenced along with the virus isolated from the maned wolf. The sequencing of the etiologic agent that had been isolated from the maned wolf was determined to be the same as the strain of virus used in the production of the modified live vaccine that had been administered 6 days prior to death. From this information, the diagnosis of vaccine-induced adenoviral hepatitis was made. This is the first confirmed case of vaccine-induced canine adenoviral hepatitis in a maned wolf.     

Assessment of ketamine/medetomidine anaesthesia in the New Zealand White rabbit

Objective To compare the characteristics of anaesthesia induced with four dose combinations of ketamine/medetomidine. Design Prospective randomized study. Animals Five female New Zealand White (NZW) rabbits of approximately 2.3 kg. Methods Rabbits were given one of four drug combinations (25/0.25; 15/0.5; 15/0.25 and 10/0.5 mg kg^?1 IM) on four successive occasions with a four day interval. Response to injection and then arterial blood gas and cardiovascular parameters were recorded at predetermined time points. Toe and ear pinch reflexes gave measures of total duration of surgical anaesthesia and total sleep time. Analyses used repeated measures analysis of variance. Results Induction was smooth with little reaction to injection and intubation achieved easily. Two combinations (15/0.25, 10/0.5) produced moderate hypoxaemia (mean pO₂ < 8.0 kPa) and two (25/0.25, 15/0.5) very marked hypoxaemia (mean pO₂ < 5.3 kPa). This was reversed within 15 minutes of oxygen administration and all rabbits recovered uneventfully. Heart rates fell in all cases, with only minimal effects on arterial blood pressure and no cardiac arrhythmias. Mean duration of surgical anaesthesia was significantly longer for dose groups 25/0.25 (57 ± 12 minutes) and 15/0.5 (59 ± 17 minutes, p = 0.01) compared to dose group 15/0.25 (27 ± 8 minutes). Only three animals in the 10/0.5 mg kg^?1 group achieved surgical anaesthesia. Mean duration of loss of the ear pinch reflex was similar between doses, being, respectively, 64 ± 13, 81 ± 7, 60 ± 22 and 62 ± 24 minutes. Sleep time was significantly longer for the 15/0.5 dose (112 ± 10 minutes) compared to 15/0.25 (86 ± 22 minutes, p = 0.04). Sleep times for the 25/0.25 and 10/0.5 mg kg^?1 doses were, respectively, 103 ± 23 and 108 ± 12 minutes. Conclusions Ketamine/medetomidine reliably produces smooth induction and recovery in the NZW rabbit, but due to the degree of hypoxaemia produced, should only be used with simultaneous provision of oxygen. Clinical relevance Currently recommended dose rates of ketamine/medetomidine for minor procedures such as ovariohysterectomy in rabbits (25 mg/0.5 mg kg^?1) are unnecessarily high; a dose of 15/0.25 mg kg^?1 should be adequate for 15–30 minutes of surgical anaesthesia.     

A study of periodontal disease in sheep over a twelve month period

Sheep affected by broken mouth periodontal disease (P.D.) were examined over a twelve month period for different clinical parameters. It is suggested that P.D. in sheep is an episodic phenomenon similar to human P.D., and that only a few animals with signs of P.D. may undergo clinically significant destruction over a yearly period. No single parameter could reliably predict future deterioration in other parameters.     

Blood thiamine levels in clinically normal goats and goats with suspected polioencephalomalacia

Abstract

AIMS: To identify and describe culture and antimicrobial resistance (AMR) patterns in bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory.

METHODS: Records from a veterinary diagnostic laboratory were examined for bacterial isolates cultured from canine urine samples between January 2005 and December 2012. Culture and susceptibility results were compiled with information on the age, sex and breed of dog. Repeat submissions were removed. Susceptibility was assessed using results of the Kirby-Bauer disk diffusion method, for a standard panel including amoxicillin-clavulanic acid (AMC), cefovecin (from 2010–2012), cephalothin, clindamycin, enrofloxacin and trimethoprim-sulphonamide (TMS).

RESULTS: A total of 5,786 urine samples were submitted for analysis, and 3,135 bacterial isolates were cultured from 2,184 samples. Of these 3,135 isolates, 1,104 (35.2%) were Escherichia coli, 442 (14.1%) were Staphylococcus spp., 357 (11.4%) Proteus mirabilis and 276 (8.8%) were Enterococcus spp. The frequency of culture-positive samples increased with increasing age in both female and male dogs (p<0.001). The percentage of E. coli isolates resistant to AMC and cephalothin increased between 2005 and 2012 (p<0.001), as did resistance to enrofloxacin (p=0.022), but there was no change in resistance to TMS (p=0.696). Enrofloxacin was the antimicrobial with the least resistance shown by the four most common bacteria isolated during the course of the study.

CONCLUSIONS AND CLINICAL RELEVANCE: The results of this study provide important regional information regarding the prevalence of bacterial uropathogens and their susceptibility patterns. There was an increase in resistance to some commonly used antimicrobials in the treatment of urinary tract infections. Having access to regional antimicrobial susceptibility results is crucial when forming guidelines for the use of antimicrobials for the treatment of urinary tract infections. Given changes in practising habits and antimicrobial usage over time, ongoing monitoring and surveillance of resistance in pathogens is needed.     

Aspects on Hormonal Control of Normal and Induced Parturition in the Dog

Average length of gestation on the dog is 63 ± 2 days but may vary between 57-71 days due to the long period of receptivity at oestrus and the extended period of sperm survival in the female genital tract. In contrast to other domestic animals progesterone- and oestrogen concentrations are almost identical in pregnant and non pregnant bitches, except for their rapid decline immediately prior to parturition. Control of luteolysis still poorly understood. Experiments with indomethacin leading to a blockade of the prepartal PgF_2α increase, which commences with the decrease of progesterone, point toward a role of PgF_2α at this stage of pregnancy, which was extended for several days. At physiological conditions first visible signs of parturition were observed at peak-PgF_2α levels, 33.6 ± 17.6 hours after onset of luteolysis, which lasted over 16.8 ± 3.4 hours. Pulse-type releases of oxytocin were only observed after this point of time. To test for the effect of progesterone-withdrawal, four 51-57 days pregnant bitches were treated with the antiprogestin RU 38486 which inhibits the activity of progesterone at the receptor level. In all dogs first visible signs of parturition were observed 33.5 ± 7.5 hours after onset of treatment. However, the process of parturition came to an end after cervical opening and totally only one puppy was born. Different to a normal parturition no increase of PgF_2α was observed. Relaxin levels were not influenced by treatment. These observations suggested that treatment with antiprogestin followed by PgF_2α might be an adequate method to induce parturition in the dog; first experiences seem to confirm this conclusion.     

In vivo growth and genomic characterization of rickettsia‐like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89^T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 ^T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.     

Optimizing nitrogen use efficiency in wheat and potatoes: interactions between genotypes and agronomic practices

One approach to decrease the environmental impact of crop production and reduce costs is to optimize agronomic practices and genotypes so that nutrients are used more efficiently. In this study the effects of agronomic practices (rotations, crop protection, fertilization) on yields, nitrogen use efficiency (NUE) and associated parameters were studied in an experiment using two winter wheat genotypes (Cordiale and Scaro) in one season and two potato genotypes (Sarpo Mira and Sante) in two seasons. The wheat showed no varietal differences in yield and NUE; instead the fertilization regime was the main factor affecting yield and NUE with higher values observed when conventional fertilization was used. The exception was for wheat grown after three years grass/clover ley when there was no added yield benefit from conventional fertilization of the organically bred variety (Scaro). This demonstrates the potential for N fixing crops to provide sufficient N to high yielding cereals if grown for long enough prior to planting. The greatest gains in NUE were achieved by combining an N efficient genotype with conventional crop management in an organic rotation. Fertilization and genotypic variation were the main factors affecting potato tuber yield and NUE, with the late maturing Sarpo Mira displaying elevated yields and NUE compared with the early maturing Sante. The use of organic fertility sources resulted in lower NUE, but N release from organic sources may increase NUE of future crops. This highlights the need for long-term nutrient balance and modelling studies to assess NUE at the crop rotation scale.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.