Efficacy of macrocyclic lactones for the control of larvae of the Old World screw-worm fly (Chrysomya bezziana)

OBJECTIVE: To assess the efficacy of four macrocyclic lactones for the control of larvae of the Old World Screw-worm Fly (OWS), Chrysomya bezziana, and to examine the effects of excreted residues on the dung fauna. ANIMALS: 100 heifers were divided into five groups of 20 animals. One group remained untreated, whereas the other groups were treated respectively with pour-on formulations of moxidectin, eprinomectin or doramectin, or a sustained-release bolus of ivermectin. PROCEDURES: At intervals of 1 to 15 weeks after treatment, five cattle from each group were challenged with newly-laid eggs of OWS. The efficacy of each treatment was determined 48 h later by comparing the number of myiases in the treated and untreated groups. Abundance of fly larvae in naturally-voided dung pads and the survival of a species of dung beetle, Onthophagus sagittarius, were used to assess the effects of drug residues on the dung fauna. RESULTS AND CONCLUSIONS: Moxidectin showed no activity against larvae of OWS during the first 14 days after treatment. Eprinomectin provided protection for 3 days after dosing, but failed at days 7 and 14, whereas doramectin was effective at day 7, but not at days 14 or 21. In contrast, no myiases were established on bolus-treated cattle from 14 to 102 days after treatment. Faecal residues of moxidectin had no effect on the survival of larvae of dung-feeding flies, whereas those of eprinomectin and doramectin reduced survival for 1 to 2 weeks. Dung voided by bolus-treated cattle inhibited fly breeding and had adverse effects on the development and survival of O sagittarius for up to 15 weeks after treatment.     

The use of irradiated embryos of Chrysomya bezziana for the detection of the Old World screw-worm fly

Irradiation of Chrysomya bezziana embryos 1 h before hatching with doses less than or equal to 7 kilorad (kr) had a significant effect on percentage egg hatch, weights and survival of larvae. Doses greater than or equal to 1 kr allowed larval development to the end of the 3rd instar stage in vitro, but prevented normal pupal development. Cattle with wounds infested with 1st instar larvae derived from irradiated embryos had 3rd instar larvae present after 3 d but these failed to pupate. Thus it would be feasible to use such larvae for wound infestation for the enhanced detection of screw-worm fly in areas where the release of fertile flies is undesirable.     

Field assessment of synthetic attractants and traps for the Old World screw-worm fly, Chrysomya bezziana

The performance of newly developed trapping systems for the Old World screw-worm fly, Chrysomya bezziana has been determined in field trials on cattle farms in Malaysia. The efficacy of non-sticky traps and new attractants to trap C. bezziana and non-target flies was compared with the standard sticky trap and Swormlure. The optimal trap was a modified LuciTrap(?) with a new attractant mixture, Bezzilure-2. The LuciTrap/Bezzilure-2 caught on average 3.1 times more C. bezziana than the sticky trap with Swormlure (P<0.05) and provided selectivity for C. bezziana against Chrysomya megacephala and Chrysomya rufifacies with factors of 5.9 and 6.4, respectively. The LuciTrap also discriminates with factors of 90 and 3.6 against Hemipyrellia sp. and sarcophagid flesh flies respectively, compared to the sticky trap. The LuciTrap/Bezzilure-2 system is recommended for screwworm fly surveillance as it is more attractive and selective towards C. bezziana and provides flies of better quality for identification than the sticky trap.     

Validation of a portable device (iSperm®) for the assessment of stallion sperm motility and concentration

The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm^® Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm^® Equine (Aidmics Biotechnology), Androvision^® (Minitube) and NucleoCounter^® SP‐100? (ChemoMetec). The total motility and progressive motility were determined by the iSperm^® Equine and the Androvision^® using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above‐mentioned devices. There was a significant correlation between the concentrations measured with iSperm^® and NucleoCounter^® at all the measured dilutions. Moreover, <10% difference in concentrations was observed between the iSperm^® and NucleoCounter^® using the Bland–Altman test. There was also a significant correlation between iSperm^® and Androvision^® for total and progressive motility. In the second experiment, the parameters used in the Androvision^® were modified to match those of the iSperm^®. Total motility and progressive motility of frozen/thawed semen samples (n = 10) were determined, and the similarity between the Androvision^® and iSperm^® was confirmed by correlation studies and Bland–Altman test. The results of these experiments demonstrate that the iSperm^® offers a reliable and practical alternative for the semi‐automated measurement of concentration and motility of stallion semen in the field. The iSperm^® enables the practitioner to obtain objective and repeatable measurements on a variety of semen types (fresh, cooled and frozen) in the field at the time of insemination and thus acquire more insight into the quantity and quality of the provided insemination doses. This mare‐side diagnostic tool may help practitioners in identifying presumed subfertility problems more rapidly and act accordingly.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Methicillin‐resistant Staphylococcus aureus in urban Norway rat (Rattus norvegicus) populations: Epidemiology and the impacts of kill‐trapping

Urban Norway rat (Rattus norvegicus) populations can carry the bacteria methicillin‐resistant Staphylococcus aureus (MRSA). There are numerous knowledge gaps in the epidemiology of MRSA in these populations that limit understanding of its ecology in urban environments. For example, fecal shedding of MRSA, which may increase environmental contamination, has been reported in other species; however, it is unknown whether Norway rats carry the bacteria rectally. Furthermore, while intermittent MRSA shedding has been shown in other species and may dictate when the risk of transmission is highest, duration of carriage has not been examined for Norway rats. Previous work has shown that lethal animal‐control methods may increase the level of pathogens within reservoir populations, possibly by disrupting ecological patterns. However, the impact of rodent‐control on potentially environmentally acquired pathogens like MRSA has not been tested. Using capture‐mark‐recapture methods in an inner‐city neighborhood in Vancouver, Canada, we show that rats intermittently carry MRSA both in the rectum and oropharynx. By assessing the prevalence of MRSA before and after enacting a pest‐control intervention, we report that kill‐trapping had no impact on the prevalence of carriage of this environmentally‐acquired agent.     

Distribution of alpha‐neoendorphin,ACTH (18–39) and beta‐endorphin (1–27) in the alpaca brainstem

Using an immunocytochemical technique, we have studied in the alpaca brainstem the distribution of immunoreactive structures containing prodynorphin (alpha‐neoendorphin)‐ and pro‐opiomelanocortin (adrenocorticotrophin hormone (18–39) (ACTH), beta‐endorphin (1–27))‐derived peptides. No peptidergic‐immunoreactive cell body was observed. Immunoreactive fibres were widely distributed, although in most of the brainstem nuclei the density of the peptidergic fibres was low or very low. In general, the distribution of the immunoreactive fibres containing the peptides studied was very similar. A close anatomical relationship occurred among the fibres containing alpha‐neoendorphin, ACTH or beta‐endorphin (1–27), suggesting a functional interaction among the three peptides in many of the brainstem nuclei. The number of fibres belonging to the prodynorphin system was higher than that of the pro‐opiomelanocortin system. A moderate/low density of immunoreactive fibres was observed in 65.11% (for alpha‐neoendorphin (1–27)), 18.18% (for ACTH) and 13.95% (for beta‐endorphin) of the brainstem nuclei/tracts. In the alpaca brainstem, a high density of immunoreactive fibres was not observed. The neuroanatomical distribution of the immunoreactive fibres suggests that the peptides studied are involved in auditory, motor, gastric, feeding, vigilance, stress, respiratory and cardiovascular mechanisms, taste response, sleep‐waking cycle and the control of pain transmission.     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.     

Real‐time RT‐PCR for the detection and quantitative analysis of equine rhinitis viruses

Reasons for performing study: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objectives: To develop a sensitive, rapid, real‐time RT‐PCR (rRT‐PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5′ UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT‐PCR were verified by virus isolation and ERBV positive samples were verified by rRT‐PCR using a different set of primers. Results: The detection limit of the rRT‐PCR for both viruses was 10–100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co‐circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT‐PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). Conclusions: The rRT‐PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. Potential relevance: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.     

Pharmacokinetics and bioavailability after intramuscular injection of the 5‐HT1A serotonin agonist R‐8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) in domestic goats (Capra aegagrus hircus)

To determine the bioavailability and pharmacokinetic properties of the serotonin 5‐HT_1A receptor agonist R‐8‐OH‐DPAT in goats, and 0.1 mg kg^?1 R‐8‐OH‐DPAT hydrobromide was administered intramuscularly (i.m.) and intravenously (i.v.) to six goats in a two‐phase cross‐over design experiment. Venous blood samples were collected from the jugular vein 2, 5, 10, 15, 20, 30, 40 and 60 min following treatment and analysed by liquid chromatography tandem mass spectrometry. Bioavailability and pharmacokinetic parameters were determined by a one‐compartment analysis. Mean bioavailability of R‐8‐OH‐DPAT when injected i.m. was 66%. The mean volume of distribution in the central compartment was 1.47 L kg^?1. The mean plasma body clearance was 0.056 L kg^?1 min^?1. All goats injected i.v. and two of six goats injected i.m. showed signs of serotonin toxicity. In conclusion, R‐8‐OH‐DPAT is well absorbed following i.m. injection and the observed pharmacokinetics suggest that administration via dart is feasible. Administration of R‐8‐OH‐DPAT hydrobromide, at a dosage of 0.1 mg kg^?1, resulted in the observation of clinical signs of serotonin toxicity in the goats. It is suggested that dosages for the clinical use of the compound should be lower in order to achieve the desired clinical effect without causing serotonin toxicity.     

Ketoprofen pharmacokinetics of R‐ and S‐isomers in juvenile loggerhead sea turtles (Caretta caretta) after single intravenous and single‐ and multidose intramuscular administration

Ketoprofen is a nonsteroidal anti‐inflammatory and analgesic agent that nonselectively inhibits cyclooxygenase, with both COX‐1 and COX‐2 inhibition. Recent studies on COX receptor expression in reptiles suggest that nonselective COX inhibitors may be more appropriate than more selective inhibitors in some reptiles, but few pharmacokinetic studies are available. The goal of this study was to determine single‐ and multidose (three consecutive days) pharmacokinetics of racemic ketoprofen administered intravenously and intramuscularly at 2 mg/kg in healthy juvenile loggerhead turtles (Caretta caretta). The S‐isomer is the predominant isomer in loggerhead sea turtles, similar to most mammals, despite administration of a 50:50 racemic mixture. Multidose ketoprofen administration demonstrated no bioaccumulation; therefore, once‐daily dosing will not require dose adjustment over time. S‐isomer pharmacokinetic parameters determined in this study were C_max of 10.1 μg/ml by IM injection, C₀ of 13.4 μg/ml by IV injection, AUC of 44.7 or 69.4 μg*hr/ml by IM or IV injection, respectively, and T½ of 2.8 or 3.6 hr by IM or IV injection, respectively. Total ketoprofen plasma concentrations were maintained for at least 12 hr above concentrations determined to be effective for rats and humans. A dose of 2 mg/kg either IM or IV every 24 hr is likely appropriate for loggerhead turtles.     

Intralesional 5‐fluorouracil (5‐FU) for the treatment of eyelid squamous cell carcinoma in 5 horses

Eyelid squamous cell carcinoma in equine patients often presents a therapeutic challenge to practitioners due to the generally large area affected upon presentation. Surgical excision can be curative if wide enough margins are achieved, but this is not often attainable without enucleation. Other alternatives have been examined including cryotherapy, radiotherapy, brachytherapy, intralesional chemotherapy and photodynamic therapy. Intralesional chemotherapy using cisplatin, mitomycin‐C and bleomycin have been shown to be successful in treating eyelid squamous cell carcinomas but may be prohibitive to some owners due to the cost of therapy. To the authors' knowledge, this is the first case series to illustrate the effectiveness of intralesional 5‐fluoruracil in treating large equine eyelid squamous cell carcinomas. Macroscopic reduction in the size of the affected areas was noted in each case together with regression of clinical signs associated with the mass. This, coupled with the relative inexpensive nature of the procedure, makes this technique an attractive therapy for either primary treatment of eyelid squamous cell carcinoma or as a cytoreductive technique prior to surgical excision.     

Attribution of human infections with Shiga toxin‐producing Escherichia coli (STEC) to livestock sources and identification of source‐specific risk factors,The Netherlands (2010–2014)

Shiga toxin‐producing Escherichia coli (STEC) is a zoonotic pathogen of public health concern whose sources and transmission routes are difficult to trace. Using a combined source attribution and case–control analysis, we determined the relative contributions of four putative livestock sources (cattle, small ruminants, pigs, poultry) to human STEC infections and their associated dietary, animal contact, temporal and socio‐econo‐demographic risk factors in the Netherlands in 2010/2011–2014. Dutch source data were supplemented with those from other European countries with similar STEC epidemiology. Human STEC infections were attributed to sources using both the modified Dutch model (mDM) and the modified Hald model (mHM) supplied with the same O‐serotyping data. Cattle accounted for 48.6% (mDM) and 53.1% (mHM) of the 1,183 human cases attributed, followed by small ruminants (mDM: 23.5%; mHM: 25.4%), pigs (mDM: 12.5%; mHM: 5.7%) and poultry (mDM: 2.7%; mHM: 3.1%), whereas the sources of the remaining 12.8% of cases could not be attributed. Of the top five O‐serotypes infecting humans, O157, O26, O91 and O103 were mainly attributed to cattle (61%–75%) and O146 to small ruminants (71%–77%). Significant risk factors for human STEC infection as a whole were the consumption of beef, raw/undercooked meat or cured meat/cold cuts. For cattle‐attributed STEC infections, specific risk factors were consuming raw meat spreads and beef. Consuming raw/undercooked or minced meat were risk factors for STEC infections attributed to small ruminants. For STEC infections attributed to pigs, only consuming raw/undercooked meat was significant. Consuming minced meat, raw/undercooked meat or cured meat/cold cuts were associated with poultry‐attributed STEC infections. Consuming raw vegetables was protective for all STEC infections. We concluded that domestic ruminants account for approximately three‐quarters of reported human STEC infections, whereas pigs and poultry play a minor role and that risk factors for human STEC infection vary according to the attributed source.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

Validation of the BioPRYN enzyme‐linked immunosorbent assay for detection of pregnancy‐specific protein‐B (PSPB) and diagnosis of pregnancy in American bison (Bison bison)

This study assessed the accuracy of the commercial BioPRYN^® ELISA for the detection of pregnancy‐specific protein‐B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time‐points during the gestation period, fall and spring, correlating with early‐ to mid‐term gestation (average 62.9 days post‐mating) and mid‐ to late‐term gestation (average 229.2 days post‐mating), respectively. Sensitivity of the test during early‐ to mid‐term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late‐term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single‐sample BioPRYN^® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post‐mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds.     

Reliability of the i‐STAT for the determination of blood electrolyte (K+, Na+, and CI−) concentrations in cattle

Background

Rapid determination of blood electrolyte concentrations can help determine electrolyte status and delivery of effective volume of electrolyte solutions in field conditions.

Objective

To evaluate reliability of the i‐STAT, a point‐of‐care (POC) device, in measuring blood K⁺, Na⁺, and CI ⁻ concentrations in cattle.

Animals

Ninety‐eight cattle with various diseases.

Methods

In this prospective study, blood samples collected from the jugular vein were processed for determination of K⁺, Na⁺, and CI ⁻ concentrations in blood and plasma using the i‐STAT and auto‐analyzer (Cobas C501), respectively. Blood and plasma electrolyte data were subjected to student t‐test for comparison, the concordance analysis for agreement, accuracy, and precision, the Passing‐Bablok regression and the Bland‐Altman plot for reliability, and receiver operating characteristics curves for sensitivity (Se) and specificity (Sp).

Results

Plasma concentrations of K⁺ (4.39 versus 4.2 mmol/L; P < .0001) and CI ⁻ (100.30 versus 99.4 mmol/L; P < .04) were greater than their concentrations in blood. Plasma and blood Na⁺ concentrations were similar (136.95 versus 136.8 mmol/L). The i‐STAT results were highly correlated with the Cobas C501 results (r = 0.970, 0.922, and 0.866 for K⁺, Na⁺, and CI ⁻, respectively). Regression equations fitting blood (Y) and plasma (X) concentration did not deviate from the identity line for K⁺ (Y = −0.10 + 0.98 × X), Na⁺ (Y = X), and CI ⁻ (Y = 3.04 + 0.96 × X). The mean bias (blood concentration ‐ plasma concentration) was −0.20 for K⁺ (P = .03), −0.16 for Na⁺ (P = .12), and −0.87 for CI ⁻ (P = .93). The i‐STAT had 76–100% Se and 87.7–100% Sp for assessing electrolyte statuses.

Conclusions and Clinical Importance

The i‐STAT yielded results that were in agreement with the auto‐analyzer, with negligible biases in measurement of plasma K⁺, Na⁺, and CI ⁻ concentrations. The i‐STAT is a reliable POC device and can be used in field condition to assess electrolyte status in cattle.     

Ontogeny of the stomach in yellow catfish (Pelteobagrus fulvidraco): detection and quantifictation of pepsinogen and H+/K+‐ATPase gene expression

Yellow catfish (Pelteobagrus fulvidraco) is an important commercial species with high aquaculture potential in China. To better understand the process of digestive functioning of gastric gland development during the larval from 1 dph (day post‐hatching) to 30 dph, real‐time PCR was used to detect and quantify the pepsinogen and H⁺/K⁺‐ATPase gene expression in P. fulvidraco. These data were also compared with the adult situation. The results showed that the expression of pepsinogen and H⁺/K⁺‐ATPase genes in P. fulvidraco larvae both started at 1 dph, though the expression level was very low until 3 dph. The quantification of pepsinogen gene expression increased significantly from 4 to 8 dph, increased fluctuantly from 8 to 23 dph and rose sharply from 23 to 30 dph. In comparison with adult fish, there were no significant differences with larvae at 5 and 23 dph. However, data of 10 and 30 dph larvae were obviously higher than those of adult group. H⁺/K⁺‐ATPase gene expression increased linearly from 1 to 30 dph. However, it was significantly lower than that of adult. The results show that P. fulvidraco larvae have an earlier functional stomach, though the function of the stomach is still not perfect. There is a gradual acidification environment within the stomach during the P. fulvidraco larvae development. Based on these results, we suggest that the weaning time for P. fulvidraco larvae would be much better after 23 dph.