Optimization of fermentation medium for enhanced production of milbemycin by a mutant of Streptomyces bingchenggensis BC-X-1 using response surface methodology

A new strain Streptomyces bingchenggensis BC-X-1, in which one of the impute was disappeared had been screened by N-methyl-N’-nitroso-N-nitroso-guanidine (NTG) mutation. In order to improve the yield of milbemycin by the new strain, response surface methodology was employed to optimize the composition of fermentation medium. The Plackett-Burman design indicated that yeast extract, soybean flour, KH2PO4, FeSO4 and CaCO3 had significant effects on milbemycin production. The concentrations of these five components were investigated using Box-Behnken design and a polynomial model related to medium components concentration effect on milbemycin yield had been established. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) value of 0.9701, which indicated that good agreement between the experimental and predicted values of milbemycin yield. Student’s T-test of each coefficient showed that all the linear and quadratic terms effect were significant. The optimum concentrations of components: yeast extract, soybean flour, CaCO3, FeSO4, KH2PO4 were 2.58, 2.58, 0.40, 0.0058 and 0.088%, respectively. The yield of milbemycin reached  1110±98 μg/ml, which was 2.61- fold of the initial yield.Key words: Milbemycin, Streptomyces bingchenggensis, medium optimization, response surface methodology

Model applications of decision support systems in meat hygiene programs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University

Current systems to ensure safety of meat are to a large degree based on a "procedural" approach which specifies what inspection actions will be taken to protect human health. Both knowledge and disease priorities have changed substantially over recent decades, and moreover the scale of the problems created by any breakdowns in protection has escalated greatly, as food trading and consumption patterns have changed. It is now recognized that meat hygiene needs to focus primarily on ways by which the risk that product will represent a hazard to human health can be effectively reduced, rather than merely ensuring compliance with a defined set of procedures. In addition to human food safety, meat inspection has the potential to contribute information to improve animal health on a national and a local scale. This thesis examines example issues in order to identify possible approaches to the development of decision support systems which assist in protecting meat consumers and improving the health of livestock on farms. The main areas which were explored for this purpose were respiratory disease in lambs and chemical residues in slaughter animals. A literature review of pneumonia and pleurisy in lambs showed that numerous factors have been proposed as predisposing causes for these diseases, but there was surprisingly little valid experimental or observational research evidence to support such statements. A hazard analysis was performed for the micro-organisms which have been isolated from pneumonic lungs. The major commonly detected organisms did not appear to cause a risk to healthy people. However there were a number of micro-organisms which are isolated on occasion from pneumonic and sometimes from healthy sheep lungs that might cause human disease. A case-control study was carried out as an exploratory means to identify risk factors and to generate hypotheses about causal processes. A number of risk factors were initially identified at univariate level. At the second stage the importance of some of these risk factors was quantified in a logistic regression model. Finally a third stage analysis showed the interactions between the factors in a logistic path model, which consisted of three clusters. One cluster included characteristics of the farm and paddocks, one cluster included the yards and practices in the yards, and a third cluster included the types and number of animals on the farm. Two intervention studies were subsequently carried out to evaluate the effect of making various management modifications on the prevalence of pneumonia and pleurisy at slaughter. One intervention study evaluated the time lambs spent in the yards after weaning and the use or oral or injectable drenches. The second intervention study evaluated the use of oral versus injectable drenches and the use of a shower dip versus a wand. The intervention studies showed an effect of time in the yards on pneumonia. There was some association between time in the yards and acute localised pleurisy but none between the other measures tested and respiratory disease. The studies showed clear temporal patterns with regard to pleurisy and pneumonia and enabled comparisons to be made between farms. A study of inspection for pleurisy at slaughterhouses was analysed. The analysis identified the temporal patterns of certain types of pleurisy. Comparisons were made between four participating premises. The sensitivity and specificity of meat inspection for the various types of pleurisy was analysed. The pleurisy data over an eleven year period of the entire country were analysed. Differences were shown between islands and regions. The potential for development of components of a decision support system for pneumonia and pleurisy was illustrated with a number of examples. An important component was to determine how farmers could be assisted in improving the health of their lambs with regard to pleurisy. Ideas to improve farmer involvement were developed. The principles of a decision support system which evaluated the issue of cross-contamination due to handling of product by the inspector were developed. Epidemiological principles of chemical residues in slaughter animals were investigated. A number of statistical quality control tests were applied to known data sets to evaluate what sample sizes would be required to detect changing trends or spatial paterns. Temporal simulations were performed to determine how well clusters in time could be detected. The Moving Average approach was used and it appeared that with the given data set sample sizes well beyond those feasible to achieve would be required. Spatial analyses with a number of different statistics were performed. In this case also, large sample sizes were required for reliable results. It was concluded that use of a risk analysis model to define a risk-reduction strategy targeted to avoid any significant risk to the consumer offered a much more effective tool than a fixed sampling system. This model combines a range of possible risk reduction measures in various mixes, and determines whether or not each of the tested strategies achieves the goal of making it very improbable that a consumer would be exposed to sufficient levels of chemical residues in food to even constitute some minimal public health risk

Canine Angiostrongylus vasorum

The French heartworm Angiostrougylus vasorum is a parasitic nematode that lives in the pulmonary vessels and the heart of canids. Transmission occurs through ingestion of infected intermediate hosts, such as snails and slugs. There are increasing reports of autochthonous infections in our neighbouring countries. Clinical signs usually relate to the respiratory system, coagulopathy and the neurologic system. Anorexia, gastrointestinal dysfunction and weight loss are also frequently observed. Diagnosis is not straightforward, but abnormalities detected by thoracic radiography, echocardiography, magnetic resonance imaging (MRI) or computed tomography (CT) scan can be helpful. Eosinophilia, regenerative anemia and thrombocytopenia with or without abnormalities in the coagulation profile can occur. Definitive diagnosis is made by demonstrating the parasite in the cerebrospinal fluid, in faeces (Baermann technique) and/or in bronchoalveolar lavage fluid. Treatment consists of anthelmintic drugs and supportive care if necessary

Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis

Additional file 3: Figure S3. Diagrams of site-directed mutation of Walker A and Walker B motifs in MilR. A: Mutation in Walker A motif. The first line shows the wild-type Walker A sequence. From the second to the eighth line, red words indicate the Ala or Arg substitution was performed in the corresponding position. B: Mutation in Walker B motif. The first line shows the wild-type Walker B sequence, from the second to the third line, blue words indicate the Ala substitution was carried out to replace Asp in the corresponding position

Survey-based pilot study into the chosen therapy and prophylaxis used by UK primary care veterinary surgeons against canine angiostrongylosis

Canine Angiostrongylosis (CA), a gastropod-borne parasitic infection caused by the metastrongyloid nematode Angiostrongylus vasorum, is an important cause of significant morbidity to domestic dogs across the UK as well as in other European countries. This study aimed to ascertain the frequency at which particular drugs were used by primary care practitioners in the UK for therapy against and prophylaxis for CA. Primary care veterinary clinicians were surveyed using an online questionnaire and face-to-face or telephone interviews. Eighty-six veterinary surgeons responded. The majority of practices (n = 52) included lungworm in their standard anthelmintic protocols; moxidectin was the most common drug used for prophylaxis (n = 71). Fenbendazole was the most frequently selected drug, by 45% of vets, for treatment of confirmed cases of CA despite it being unlicensed for this purpose in the UK and the absence of a clear treatment protocol. The results of this pilot study provide an initial insight into the approach taken by primary care practitioners in their approach to CA. This provides an important starting point for future studies investigating the decision-making for CA amongst UK veterinary surgeons, particularly to clarify whether in a larger cohort an unlicensed drug remains the treatment of choice. The absence of a clear protocol for fenbendazole means that treatment of dogs affected by CA may be suboptimal, increasing the risk of morbidity and mortality

Effectiveness of the spot-on combination of moxidectin and imidacloprid (Advocate®) in the treatment of ocular thelaziosis by Thelazia callipaeda in naturally infected cats

Background: The present study evaluated the therapeutic effectiveness of moxidectin 1.0% (w/v) and imidacloprid 10% (w/v) (Advocate® spot-on solution for cats, Bayer Animal Health) against natural infections with the eyeworm Thelazia callipaeda in cats. This study was conducted as a GCP, negative-controlled, blinded and randomised field study in privately owned cats living in an area in southern Italy where T. callipaeda is enzootic. Methods: The study was carried out in 30 cats (19 females and 11 males, aged from 8 months to 5 years, weighing 1.2-5.2 kg) of different breeds, naturally infected by T. callipaeda. At study inclusion (Day 0), animals were physically examined and the infection level was assessed by examination of both eyes for clinical score and live adult T. callipaeda count. Each cat was weighed and randomly assigned to one of the treatment groups (G1: Advocate, G2: untreated control). Clinical assessments and T. callipaeda adult counts were performed on Day 14. At the study completion visit on Day 28, clinical assessments and counts of T. callipaeda adults and larvae were performed. All cats were daily observed by their owners and general health conditions were recorded during the entire period of the study. Results: The primary effectiveness variable was the percentage of animals in G1 group (Advocate) showing a complete elimination (parasitological cure) of adult eye worms at Day 14 and Day 28. The effectiveness of the treatment in the G1 group was 93.3 and 100% at Day 14 and Day 28, respectively, when compared to group G2. Total worm count reduction from both eyes for Advocate was 96.3% on Day 14 and 100% on Day 28. Clinical data were confirmed by the examination of conjunctival pouch flushing. An overall reduction in the number of cats with lacrimation and conjunctivitis was observed following treatment despite the fact that in a few cats treated with Advocate clinical signs persisted due to the chronic nature of the disease. Conclusions: Based on the results of the present trial, a single dose of Advocate was found to be safe and highly effective in the treatment of natural T. callipaeda infection in cats

Clinical spirocercosis in a dog in the UK

A 2-year-old female neutered crossbreed dog was presented for evaluation of a 3-day history of haematemesis, melaena and hyporexia. The dog had been imported from a rescue centre in Hungary 4 months prior to presentation. Abdominal CT revealed the presence of a 3 cm×3 cm×4 cm diameter heterogenous intraluminal mass lesion in the gastric cardia, surrounding the ostium of the cardia and continuous with the distal oesophagus. The mass lesion was subsequently surgically resected. Histopathology of the gastric mass lesion was consistent with a Spirocerca lupi granuloma. The patient unfortunately developed a pyothorax and suffered cardiopulmonary arrest resulting in death 2 days postoperatively. To the authors’ knowledge, this is the first reported case of clinical spirocercosis reported in a dog in the UK

Field clinical study evaluating the efficacy and safety of an oral formulation containing milbemycin oxime/praziquantel (Milbemax®, Novartis Animal Health) in the chemoprevention of the zoonotic canine infection by Dirofilaria repens

Dirofilaria repens is the causative agent of subcutaneous dirofilariosis of dogs, other animals and humans. This nematode is transmitted by mosquitoes of Aedes, Anopheles and Culex genera. In dogs, the parasite may cause subclinical infection or cutaneous signs. Recently, D. repens has emerged and spread in different geographical areas, with an increase of cases in dogs and humans. Chemoprevention in dogs in endemic areas is the most reliable approach for controlling this infection. This paper describes a randomized, blocked and multicentric clinical field study investigating the efficacy of an oral, chewable formulation containing milbemycin oxime/praziquantel (Milbemax®, Novartis Animal Health) in the chemoprevention of subcutaneous dirofilariosis in dogs. METHODS: This study was conducted in endemic areas of Italy. A total of 249 dogs, at two sites, negative for D. repens, were allocated into two groups (i.e. Treated -T1 vs Untreated-T2) with a ratio of 1:1, and subjected to clinical visits and blood sampling once monthly until the end of the study. All blood samples were microscopically and genetically examined. Animals belonging to T1 group received a minimum target dose of 0.5 mg/kg bodyweight of milbemycin oxime and 5 mg/kg of praziquantel in commercial tablets (Milbemax®) according body weight once every 4 weeks. Animals of group T2 were not treated with Milbemax® but received, when necessary, specific parasiticide treatments. The study duration was 336 ± 2 days for each dog. RESULTS: A total of 219 dogs completed the study (i.e. 111 in T1 and 108 in T2), while 30 dogs (i.e. 13 in T1, 17 in T2) were withdrawn for a variety of reasons unrelated to administration of Milbemax®. The percentages of animals not showing microfilariae of D. repens were 100% (111 animals) in T1 and 94.7% (108 animals out of 114) in group T2. Milbemax® was shown to be safe in treated dogs. CONCLUSIONS: The results of this study confirm that the monthly use of Milbemax® in dogs is effective and safe for the prevention of subcutaneous dirofilariosis in endemic areas

A case of a dog refractory to different treatments for pulmonary capillariasis

open9siPulmonary capillariasis is a parasitic disease caused by the nematode Eucoleus aerophilus which affects wild and domestic carnivores. Currently, there are no anthelmintics approved for use in the treatment of dogs infected with E. aerophilus. The use of several anthelmintics has been reported in a few case reports and field efficacy studies in cats; much less is known on the treatment of dogs infected with E. aerophilus. The paper describes a case of a 4-month-old, mixed breed intact male referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Medical Science of the University of Bologna for a routine vaccination and tested positive for E. aerophilus. The dog has not been responding to three different administered treatments, such as moxidectin, fenbendazole, and milbemycin oxime. Eighteen months after the first fecal examination, owner has brought in the dog for a routine visit; a coprological examination was requested and performed resulting negative for parasites. Veterinary practitioners, parasitologists, diagnostic laboratories, and dog owners need to be aware of the increased danger of possible treatment failure when attempting to control parasitic infections for which there are no approved anthelmintics with established efficacies available for use.embargoed_20211026Morandi B.; Sabetti M.C.; Veronesi F.; Morganti G.; Pietra M.; Poglayen G.; Linta N.; Conboy G.; Galuppi R.Morandi B.; Sabetti M.C.; Veronesi F.; Morganti G.; Pietra M.; Poglayen G.; Linta N.; Conboy G.; Galuppi R

Therapeutic efficacy of milbemycin oxime/praziquantel oral formulation (Milbemax®) against Thelazia callipaeda in naturally infested dogs and cats

BACKGROUND: Over the last few decades, canine and feline thelaziosis caused by Thelazia callipaeda eye worms has gained the attention of the veterinary community due to the spread of this ocular infestation in geographical areas previously regarded as non endemic. The therapeutic efficacy of milbemycin oxime/praziquantel tablets (Milbemax®) against T. callipaeda was tested in naturally infested dogs and cats. METHODS: From January 2009 to July 2011 a placebo controlled and randomized field study was conducted in T. callipaeda endemic areas of Switzerland (CH) and Italy (ITA) involving client-owned animals. Dogs (n = 56) and cats (n = 31) were physically examined at enrolment Day 0 (D0) and twice afterwards (D7 and D14). Infested animals were orally treated with Milbemax® or with placebo tablets on D0 and, if an animal was found still infested with T. callipaeda, also on D7. On D14 nematodes were flushed from the conjunctiva, identified and counted. RESULTS: Out of 56 dogs, 43 were included in the statistical analysis, whereas 13 were excluded because the products under investigation were not administered with food, as required by the label. On D7 and D14, 72.7% and 90.9% of treated dogs were eye worm free, whereas in the placebo group 95.2% and 76.2% still harbored nematodes, resulting in a mean percentage worm count reduction for the Milbemax® group of 86.1% and 96.8%, respectively. Both results were significantly higher (p = 0.0001) than the placebo group. Out of the 31 cats included in the study at D7 and D14, 53.3% and 73.3% treated with Milbemax® were free of T. callipaeda, while 81.3% and 73.3 in the placebo group were still harbouring eye worms, resulting in a mean percentage worm count reduction for the treated group of 62.2% and 80.0%, respectively. Both results were significantly higher (p = 0.0106 and p = 0.0043) than the placebo group. CONCLUSIONS: The commercial formulation of milbemycin oxime at the minimal dose of 0.5 mg/kg and 2 mg/k in dogs and cats, respectively, showed a high therapeutic efficacy in curing T. callipaeda infestations. The advantages of an oral application are additionally increased by the large spectrum of activity of praziquantel and milbemycin oxime against Cestodes and Nematodes infesting dogs and cats