Establishment of loop-mediated isothermal amplification assay for detection of parasitic ciliate Ichthyophthirius multifiliis in cyprinid fish

The objective of this research was to establish loop-mediated isothermal amplifications (LAMP) that could be used to detect parasitic ciliate Ichthyophthirius multifiliis (I. multifiliis) in freshwater cyprinid fish. Primers were developed from the distinguishing fragments of 18S ribosomal RNA of I. multifiliis and the LAMP test was then used to evaluate and optimize various concentrations of chemicals, time and temperature. The results indicated that LAMP required 1.6 μM of FIP primers and BIP primers, 0.2 μM of F3 and B3, 2 mM of Mg2+, 1 M of Betaine, and 0.6 mM of dNTP. This assay was able to detect parasite DNA within a 40 min period of incubation and at a constant optimal temperature of 64oC. The positive sample appeared as a clear ladder like pattern on gel electrophoresis, while a yellowish green color appeared with SYBR Green I under ultraviolet light with the use of a heating block. The LAMP test was determined to be more sensitive than conventional PCR in the detection of I. multifiliis. In conclusion, we have presented a sensitive and specific rapid detection system for I. multifiliis based on isothermal DNA amplification. Importantly, this system could then be employed as an alternative and effective diagnostic method in place of other molecular techniques

Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii

Background and Aim: Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. Materials and Methods: To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. Results: The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. Conclusion: Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples

The current state of biosecurity and welfare of ornamental fish population in pet fish stores in Chiang Mai Province, Thailand

Aquaculture has undergone extensive development in recent decades due to its use as a source of protein for human consumption, as models for scientific research, and as pets. The purpose of this study was to survey ornamental fish stores in Chiang Mai province, Thailand to identify practices which affect life quality and welfare of pet fish including general management, biosecurity management, and knowledge about fish disease and drug usage of ornamental fish store owners. The results show that most pet fish stores have poor husbandry in terms of fish health based on the observed incidence of skin erosion and fin rot (92.86%) and white spots on the skin (78.57%) in store fish. Moreover, treatment of these health conditions were performed by experienced people working in the stores without consulting a veterinarian. The top three drugs used for treatment included malachite green oxalate, trichlorfon, and formalin. Interestingly, oxytetracycline and chlortetracycline were also used frequently to treat fish disease. Despite a lack of formal training in fish management, fish health, and drug usage in ornamental fish, the knowledge of owners about fish disease and antibacterial agent usage was determined to be of an intermediate level based on testing. The information gained from this study can be used in future studies to identify stressors that affect pet fish welfare and to investigate biosecurity and ornamental fish welfare in the other sectors of the ornamental fish trade supply chain

ROLE OF LIPOSOLUBLE HORMONES AND MATERNAL mRNAs IN THE EPIGENETIC LONG-TERM MODULATION OF ZEBRAFISH DEVELOPMENT

PART I: Expression analysis of maternal and zygotic steroid hormone receptor mRNAs during zebrafish (Danio rerio) embryogenesis. I have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rates of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone (Pr), androgen (Ar), estrogen (Erα, Erβ1 and Erβ2), glucocorticoids (Gr), mineralocorticoids (Mr) and the membrane progestin receptor-α and β (mPrα and β). Gr mRNA was the most abundant maternal transcript in oocytes and early embryos, followed by erβ2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. Erβ1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. Pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of erα mRNA that initially was very low. Mprα and β mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription. PART II: Morpholino knockdown of glucocorticoid receptor in zebrafish during embryogenesis The glucocorticoid receptor (Gr) is involved in various physiological processes, including growth, osmoregulation and reproduction in various species of vertebrates. In this study, I have investigated the function of Gr during zebrafish embryogenesis using antisense Morpholino (MO) technology approach in order to block gr mRNA translation. I observed that Gr-morphants were characterized by small head and eyes, short body, pericardial edema, disrupted melanophore patterning, failure to inflate the swim bladder, tail curling, and abnormal swimming behaviour. The effectiveness of Gr knockdown was further verified by Western blotting and in vitro transcription and translation system, which showed marked reduction of the Gr protein in morphant embryos. Microarray analysis revealed that Gr functions prevalently as a negative regulator of gene transcription. Three genes found to be up-regulated following MO knockdown by microarray analysis, including caspase-8, igf2α and centaurin-1α, were evaluated by semiquantitative RT-PCR throughout the embryonic development. Furthermore, whole mount in situ hybridizations indicated that, whereas marker expression pattern was mostly unchanged in morphant embryos, three markers involved in neurogenesis, egr2b, emx1 and six3.1, were up-regulated. In conclusion, the knockdown of Gr in zebrafish revealed a requirement of both maternal and zygotic gr in multiple developmental processes involved in neurogenesis and gut and accessory organs formation. There are indications that the maternal receptor is critical in early embryogenesis, but more work is needed to elucidate the functional transition from the maternal to the zygotic gr and whether they affect distinctive components of the genetic machinery.PARTE I: Analisi dell’espressione degli mRNA materni e zigotici per recettori di ormoni steroidei durante lo sviluppo nel pesce zebrato (Danio rerio). Ho analizzato mediante qRT-PCR e/o RT-PCR la presenza ed il grado di degradazione degli mRNA materni codificanti per nove recettori di ormoni steroidei, e la loro possibile sostituzione da parte dei trascritti embrionali corrispondenti, sia negli ovociti ovulati che in embrioni di pesce zebrato raccolti a 0, 1, 2, 4, 8, 12, 24 e 48 ore dopo la fecondazione (hpf). Gli mRNA studiati sono quelli codificanti i recettori nucleari per il progesterone (Pr), gli androgeni (Ar), gli estrogeni (Erα, Erβ1 ed Erβ2), i glucocorticoidi (Gr), i mineralcorticoidi (Mr) ed i recettori di membrana α e β per i progestinici (mPrα e mPrβ). L’mRNA codificante la proteina Gr costituisce il trascritto materno maggiormente presente negli ovociti e nei primi stadi di sviluppo embrionale, seguito dagli mRNA codificanti i recettori Erβ2 ed Ar. Tutti questi trascritti diminuiscono durante le prime 8 ore di sviluppo, e vengono poi sostituiti dai rispettivi messaggeri embrionali. I livelli dei trascritti per erβ1 e mr sono bassi prima delle 8 hpf, ed aumentano costantemente, dopo l’inizio della trascrizione embrionale, passando dalle 24 alle 48 hpf. L’mRNA codificante il recettore Pr è stato rilevato soltanto negli ovociti ovulati e nei campioni corrispondenti a 24 e 48 hpf. A questi stadi di sviluppo c’è anche un leggero aumento dell’mRNA codificante la proteina Erα. I trascritti per i recettori mPrα e mPrβ sono presenti negli ovociti ovulati e persistono, a basse concentrazioni, fino alle 4 hpf. Non è stata evidenziata la presenza di trascritti embrionali codificanti per questi due geni. E’ interessante il possibile coinvolgimento degli mRNA codificanti Gr e i recettori degli ormoni sessuali nella programmazione dello sviluppo embrionale precoce del pesce zebrato, dal momento che sono presenti principalmente negli stadi di sviluppo in cui la replicazione genica predomina sulla trascrizione. PARTE II: Inattivazione genica del recettore dei glucocorticoidi di pesce zebrato mediante morfolino Il recettore dei glucocorticoidi (Gr) è coinvolto in numerosi processi fisiologici, tra cui la crescita, l’osmoregolazione e la riproduzione in molte specie di vertebrati. In questo studio, ho analizzato la funzione del Gr durante l’embriogenesi di pesce zebrato utilizzando la tecnologia degli oligonucleotidi antisenso morfolino per inibire la traduzione dell’mRNA codificante Gr. Ho osservato che i morfanti di Gr presentano occhi e testa di dimensioni ridotte, corpo corto, edema pericardico, riduzione della pigmentazione, mancata insufflazione della vescica natatoria, coda incurvata e comportamento natatorio anormale. L’efficacia dell’inattivazione genica del Gr sono stati verificati mediante Western blotting ed utilizzando un sistema di trascrizione e traduzione in vitro, che hanno dimostrato una riduzione della traduzione del Gr negli embrioni morfanti. Analisi di microarray hanno messo in evidenza che questa proteina funziona prevalentemente come repressore della trascrizione genica. I geni caspase-8, igf2α e centaurina-1α risultati sovraespressi mediante microarray dopo trattamento con morfolino, sono stati analizzati mediante RT-PCR semiquantitativa durante tutto il corso dello sviluppo embrionale. Inoltre, esperimenti di ibridazione in situ in toto indicano che, mentre in generale il modello di espressione dei marcatori utilizzati non cambia nei morfanti, tre marcatori coinvolti nel processo di neurogenesi, e cioè egr2b, emx1 e six3.1, sono risultati sovraespressi. In conclusione, i risultati ottenuti dall’inattivazione genica del gr di pesce zebrato rivelano la necessità dei trascritti di origine materna in molteplici processi di sviluppo, quali neurogenesi e formazione di intestino ed organi accessori. Si dovrà comunque analizzare più in dettaglio la transizione funzionale dai trascritti materni a quelli zigotici e stabilire se essi influenzino componenti distinti dei meccanismi a livello genomico

Enhancement of acaricide activity of citronella oil after microemulsion preparation

The objectives of this study were to evaluate the preparation of microemulsions for citronella oil and subsequently to compare their acaricide efficacy as against conventional citronella oil. The chemical analysis of the commercial citronella oil used was carried out using an Agilent 6890 gas chromatograph (GC) coupled to an electron impact ionization (EI, 70 eV) with an HP 5973 mass selective detector (MSD). In comparison among the emulsifier systems, the mixture of Tween 20 with propylene glycol in ratios 3 : 1, 2 : 1, 1 : 1, 1 : 2, and 1 : 3, the pseudoternary phase diagrams were conducted and used to define the most suitable system for preparing citronella oil microemulsions and using them in acaricide efficacy testing. The citronella microemulsions in concentrations ranging from 0.39-25% w/w were prepared and tested for their acaricide efficacies using Adult Immersion Test and Larval Package Test. The acaricide efficacies were determined by percentages of mortalities of larval and adult ticks and the values of LC50 and LC99. Results show that the most suitable emulsifier system was in a ratio of 3 : 1. The physicochemical characteristics indicated that the size of the microemulsions were in the range of 19.6 ± 0.4 nm to 47.3 ± 2.3 nm with moderate polydispersity index (0.3-0.7). The microemulsions had stronger acaricide efficacy than the conventional citronella oil, indicated by significant lower LC50 and LC99 values. The LC99 of the microemulsions against larval and adult ticks at 24 h were 0.78% w/w (0.56-1.02) and 28.4% w/w (23.27-37.43), respectively. These results demonstrated that microemulsion preparation can be used to improve the acaricide efficacy of citronella oil

Performance of Loop-Mediated Isothermal Amplification Technique in Milk Samples for the Diagnosis of Bovine Tuberculosis in Dairy Cattle Using a Bayesian Approach

This study aimed to estimate the sensitivity (Se) and specificity (Sp) of loop-mediated isothermal amplification (LAMP) and single intradermal tuberculin (SIT) tests for the diagnosis of bovine tuberculosis (bTB) in dairy cattle in Thailand using a Bayesian approach. The SIT test was performed in 203 lactating dairy cattle from nine dairy farms located in Chiang Mai province, Thailand. Milk samples were collected for the LAMP test. Kappa analysis was performed to determine the agreement between the two tests. A one-population conditional independence Bayesian model was applied to estimate the Se and Sp of the two tests. Of 203 dairy cattle, 2 were positive for the SIT test using standard interpretation, whereas 38 were positive for the LAMP test. A poor agreement (kappa = 0) was observed between the two tests. The median Se and Sp of the SIT test using standard interpretation were 63.5% and 99.1%, respectively. The median Se and Sp of the LAMP test were 67.2% and 82.0%, respectively. The estimated true prevalence of bTB was 3.7%. The LAMP test with milk samples can potentially be used as a non-invasive screening test for the diagnosis of bTB in dairy cattle