Quantification of Ehrlichia ruminantium by real time PCR

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.     

Cryptic Metabolites from Marine-Derived Microorganisms Using OSMAC and Epigenetic Approaches

Marine microorganisms have proven to be a source of new natural products with a wide spectrum of biological activities relevant in different industrial sectors. The ever-increasing number of sequenced microbial genomes has highlighted a discrepancy between the number of gene clusters potentially encoding the production of natural products and the actual number of chemically characterized metabolites for a given microorganism. Homologous and heterologous expression of these biosynthetic genes, which are often silent under experimental laboratory culture conditions, may lead to the discovery of new cryptic natural products of medical and biotechnological interest. Several new genetic and cultivation-based strategies have been developed to meet this challenge. The OSMAC approach (one strain—many compounds), based on modification of growth conditions, has proven to be a powerful strategy for the discovery of new cryptic natural products. As a direct extension of this approach, the addition of chemical elicitors or epigenetic modifiers have also been used to activate silent genes. This review looks at the structures and biological activities of new cryptic metabolites from marine-derived microorganisms obtained using the OSMAC approach, the addition of chemical elicitors, and enzymatic inhibitors and epigenetic modifiers. It covers works published up to June 2021.     

Untersuchungen zum Voltinismus der Rotgelben Kiefern-BuschhornblattwespeNeodiprion sertifer (Geoffroy) in Südtirol und Trentino (Hym., Diprionidae)

Zusammenfassung  Bei einem Massenauftreten vonN. sertifer in Brentonico (am Monte Baldo im Trentino, N-Italien), in 1400–1600 m Seeh?he, wurden 1996 und 1997 Untersuchungen über den Voltinismus dieser Kurztags-Diprionide durchgeführt, durch Aufzucht von über 2000 Kokons (Abb. 1; Tab. 1–4). Dazu waren L4–5 Larven vonN. sertifer aus den verschiedenen H?henlagen nach Denno (430 m) und nach Brixen (550 m) transferiert und dort unter Freilandbedingungen aufgezogen worden. Die Untersuchungen wurden erg?nzt durch Populationsbeobachtungen aus Südtirol von St. Ulrich Gr?den (1200 m) sowie mittelitalienischen Herkünften aus Riolo Terme (150 m) in Brixen (Abb. 2). Diese vergleichenden Untersuchungen ergaben bei allen Populationen einen korrelativen Zusammenhang zwischen dem Zeitpunkt der Kokonbildung und dem Schlüpfen der Blattwespen. Es zeichnete sich dabei das Grundprinzip ab: je sp?ter die Kokonbildung—desto früher erfolgt das Schlüpfen der Blattwespen. Frühe Einspinner (z. B. Mai/Juni) schlüpften erst sp?t im Oktober, hingegen sp?te Einspinner (z. B. Anfang Aug.) bereits ab Ende August. Bei sp?ter Kokonbildung reduziert sich somit die obligatorische sommerliche Kokondiapause, bis sie schlie?lich bei Kurztagsbeginn (mit 15 Stunden Helligkeitsphase), Anfang August, zur Subitanentwicklung führt. Durch diese unterschiedlichen Kokonbildungszeiten kommt es auch beiN. sertifer —?hnlich wie bei anderen Diprioniden—zu verschiedenen Schlüpfwellen W1–W4, die auch aus den Pheromonfallenf?ngen ersichtlich sind (Abb. 2: 3); die m?glichen variablen Kombinationen dieser Schlüpfwellen werden diskutiert. Es wird aufgezeigt, da? der Voltinismus und das überliegerverhalten vonN. sertifer ma?geblich durch die Photoperiode und korrelierte klimatische Faktoren bestimmt werden. Die festgestellte niedrige überliegerrate von nur 1–2% h?ngt mit der südlichen und w?rmeren Lage des Befallsgebietes Brentonico (45° 49’ N) zusammen. Dabei spielt auch die au?ergew?hnliche Langlebigkeit der Imagines, die einige Wochen betragen kann, eine wichtige Rolle.

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During an outbreak ofN. sertifer in Brentonico (on Monte Baldo, Trentino, North Italy), in altitudes of 1,400–1,600 m, surveys were made in 1996 and 1997 on the voltinism of these short-day-Diprionide, wherely over 2,000 cocoons were reared (fig. 1; tab. 1–4). Therefore L4–5 larvae ofN. sertifer from different altitudes were transferred to Denno (430 m) and to Brixen (550 m) and were reared there under outdoor conditions. These surveys were completed with further observations of populations from South Tyrol, of St. Ulrich Val Gardena (1,200 m) and italian provenances from Riolo Terme (150 m) in Brixen (fig. 2). These comparative studies revealed a correlative connection between the time of cocoonspinning and adult emergence. They showed the following principle: the later the cocoonspinning, the earlier the adult emergence. Early spinners (e. g., May/June) emerge only late in October, while late spinners (e. g., early August) emerge as soon at the end of August. In the case of late cocoon spinning the obligatory summer cocoon diapause is so reducted, eventually—at the start of the short-day phase (with only 15 hours brightness), in early August—leading to a subitan development. These different cocoon spinning periods cause varying flywaves, W1–W4, ofN. sertifer, similarly to other Diprionidae; this seems evident also by pheromon-trap catches (fig. 2: 3). The possible variable combinations of these fly-waves are discussed. It is shown that voltinism and diapause behaviour ofN. sertifer are essentially conditioned by the photoperiod and correlated climatic factors. The determined low diapause rate, of just 1–2%, is connected with the southern and warmer location of the outbreak area of Brentonico (45° 45’ N). The exceptional longevity of adults, which can amount to several weeks, also plays an important role.

     

Haematological and biochemical analysis of healthy neonatal puppies during the immediate foetal‐to‐neonatal transition

For the neonatal patient, precocity of diagnosis is crucial for effectiveness of medical approach. However, the newborn has its own physiological peculiarities due to ongoing adaptive mechanism for extrauterine life and deserves special attention in order to underline a specific management or clinical approach. The objective of this work was to verify clinical adaptations and biochemical balance of neonates during immediate period, with special reference to haematological, renal and metabolic functions. Neonatal puppies (n = 51) were physically examined for vitality and rectal temperature at birth, 5 and 60 min post‐birth. Blood was collected at birth and 60 min post‐birth for analysis of glucose, sodium, potassium, chlorine, blood urea nitrogen (BUN), haematocrit and haemoglobin. Neonatal vitality was lower at birth compared with 5 min and 60 min post‐birth. Progressive decline in rectal temperature (36.5 ± 0.3°C, 34.2 ± 0.2°C, 32.3 ± 0.5°C) was observed at birth, 5 min and 60 min post‐birth, respectively. Puppies presented slight hyponatremia (140.7 ± 0.5 mmol/L) at birth and hypopotassemia (3.5 ± 0.1 mmol/L) and blood urea nitrogen (13.1 ± 0.7 mg/dl) during the first hour, and high haematocrit (45.1 ± 1.0%) and haemoglobin (15.3 ± 0.3 g/dl) concentration. In conclusion, puppies had rapid evolution of vitality. Marked decrease in rectal temperature occurred at 5 min post‐birth. Haematological values of neonates immediately after birth reflected mainly the dam's blood status, not being useful for a blood panel at this time point. The peculiar pattern of BUN, sodium and potassium observed during transition period, suggested that specific reference range should be considered for neonatal puppies.     

Bartonella and Rickettsia in arthropods from the Lao PDR and from Borneo, Malaysia

Rickettsioses and bartonelloses are arthropod-borne diseases of mammals with widespread geographical distributions. Yet their occurrence in specific regions, their association with different vectors and hosts and the infection rate of arthropod-vectors with these agents remain poorly studied in South-east Asia. We conducted entomological field surveys in the Lao PDR (Laos) and Borneo, Malaysia by surveying fleas, ticks, and lice from domestic dogs and collected additional samples from domestic cows and pigs in Laos. Rickettsia felis was detected by real-time PCR with similar overall flea infection rate in Laos (76.6%, 69/90) and Borneo (74.4%, 268/360). Both of the encountered flea vectors Ctenocephalides orientis and Ctenocephalides felis felis were infected with R. felis. The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsia detected in two Boophilus spp. ticks collected from a cow in Laos may be a new species. Isolation and further characterization will be necessary to specify it as a new species. Bartonella clarridgeiae was detected in 3/90 (3.3%) and 2/360 (0.6%) of examined fleas from Laos and Borneo, respectively. Two fleas collected in Laos and one flea collected in Borneo were co-infected with both R. felis and B. clarridgeiae. Further investigations are needed in order to isolate these agents and to determine their epidemiology and aetiological role in unknown fever in patients from these areas.     

Secretory patterns of growth hormone in dogs: circannual, circadian, and ultradian rhythms

The objective was to characterize the circannual, circadian, and ultradian secretory patterns of growth hormone (GH) in intact crossbred and purebred dogs. In all experiments, blood samples were collected with minimal stress by direct peripheral venipuncture and GH was measured in plasma by a homologous radioimmunoassay. For circannual studies, samples were collected monthly from 6 male dogs between 15:00 and 17:30 h over a 1-year time span. For circadian studies, blood samples were collected at 145-minute intervals from 09:00 to 06:45 h of the following day in 14 female dogs. In ultradian experiments, blood samples were collected at 15-minute intervals for 2.5 h (15:00 to 17:30 h) in 7 males and 7 females. Plasma GH in male dogs remained without change in summer, autumn, and winter but declined (P ≤ 0.01) in spring (LSM ± SEM; 6.9 ± 0.5; 6.0 ± 0.5; 6.3 ± 0.5; 4.3 ± 0.5 ng/mL, respectively). No plasma GH circadian rhythmicity was detected. Nor was any ultradian pattern evident in either males or females. No gender-related differences were observed in ultradian GH plasma profiles. It is concluded that, while basal GH levels show seasonal fluctuations in dogs, neither circadian nor ultradian GH secretory fluctuations were present in the dogs assessed.     

Hematology and Clinical Pathology Data in Chronically Starved Horses

Hematology and serum/plasma biochemistry are important diagnostic aids to assess metabolic status in starved horses and to rule out pathologic conditions that present as emaciation. This article reviews the interpretation of these parameters, using 10 chronically starved horses as examples. The study was conducted on these 10 neglected horses belonging to both sexes, aged between 4 and 25 years. The animals were divided into two groups according to their body condition scoring (BCS): A (n = 6; BCS of 3 or lower) and B (n = 4; BCS higher than 3). A complete clinical examination was carried out, and venous blood samples were taken to perform hematological, biochemical, and serological determinations. The findings indicated that the most emaciated horses (group A) presented normocytic normochromic anemia, with higher numbers of both immature and mature neutrophils, monocytes, and eosinophils, and lower number of lymphocytes. Furthermore, horses in group A showed lower serum concentrations of urea, triglycerides, bile acids, alkaline phosphatase, and a lower urea/creatinine ratio. Both groups of horses presented with hypomagnesemia. In conclusion, the knowledge of the laboratorial findings in emaciated horses is useful in scoring the intensity of the emaciation, and in establishing a prognosis and a plan to recover the health status of these animals.     

Prevalence of dog erythrocyte antigens in retired racing Greyhounds

Background: Blood groups in dogs are designated as dog erythrocyte antigen (DEA) 1.1, 1.2, 3, 4, 5, 7, and Dal. There is limited information about the frequency of different antigens in Greyhound dogs, despite their frequent use as blood donors. Objectives: The aims of this study were to determine the frequencies of DEA 1.1, 1.2, 3, 4, 5, and 7 in Greyhounds, to compare the frequencies with those of non‐Greyhound dogs, and to evaluate the presence of naturally occurring anti‐DEA antibodies. Methods: Blood was collected from 206 Greyhound and 66 non‐Greyhound dogs being screened as potential blood donors. Blood‐typing was performed at Animal Blood Resources International by tube agglutination utilizing polyclonal anti‐DEA antibodies. Results: Of the Greyhound dogs, 27/206 (13.1%) were positive for DEA 1.1, and this frequency was significantly lower (P<.0001) than for non‐Greyhound dogs of which 40/66 (60.6%) were DEA 1.1‐positive. The frequency of positivity for both DEA 1.1 and 1.2 was also lower in Greyhounds (P<.0001). There were no significant differences between Greyhounds and non‐Greyhounds for DEA 1.2, 3, 4, 5, or 7. All 137 dogs (113 Greyhounds and 24 non‐Greyhounds) that were evaluated for naturally occurring anti‐DEA antibodies in serum were negative. A higher percentage of Greyhound dogs (57.3%, 118/206) were considered “universal donors” (negative for all DEAs except DEA 4) compared with non‐Greyhound dogs (28%, 13/46). Conclusion: The frequency of positivity for DEA 1.1 in our population of Greyhounds was significantly lower than previously reported for dogs. Furthermore, a large majority of Greyhounds met the criteria for universal donors.     

Economical and financial analysis of lamb finishing fed with diets formulated according to the NRC (1985) and the NRC (2007)

This study compares both versions of the nutritional requirement system determined by the National Research Council (NRC) version 1985 (NRC85) and NRC version 2007 (NRC07), for finishing lambs in feedlots. Nineteen crossbred lambs were divided in four groups representing four experimental treatments: one diet according to NRC85 and three diets according to NRC07. The diets recommended by NRC07 considers crude protein intake relative to ruminal undegradable protein at 20, 40, and 60 % levels (NRC07/20, NRC07/40, and NRC07/60). Diets were composed of Brazilian semi-arid native grass silage, soybean meal, corn, annatto byproduct, and limestone. Purchases and sales of lambs were done according to average market prices in Brazil. The economic indicators considered pointed that all treatments were viable but NRC07/20 and NRC07/60 were more profitable with similar net present values (NPVs) and internal return rates (IRRs). NRC07/20 was the best option showing an IRR of 17.20 % and a payback period (PP) of 5.07 considering a fixed annual interest rate of 6 %. Sensitivity analysis considering a 10 % raise in variable costs showed negative NPVs, IRRs inferior to the opportunity cost rates adopted and PPs that exceeded the planning horizon of 7 years for both NRC85 and NRC07/40.