Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

Determination of myrislignan levels in BALB/c mouse plasma by LC-MS/MS and a comparison of its pharmacokinetics after oral and intraperitoneal administration.

BACKGROUND: Myrislignan is a natural product from Myristica sp. with diverse pharmacological activities. Recently, the anti-Toxoplasma gondii (T. gondii) activity of myrislignan has been proposed, and in vivo studies of its pharmacokinetics in BALB/c mice are necessary to further evaluate the clinical effects of myrislignan. RESULTS: In this study, a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify myrislignan levels in mouse plasma using dehydrodiisoeugenol as an internal standard (IS) in positive ion mode. Chromatographic separation of the analytes was achieved using an ACE Ultracore Super C18 analytical column (2.5 µm, 2.1 × 50 mm) at 30 °C. A gradient mobile phase consisting of water (0.1 % formic acid) and acetonitrile (0.1 % formic acid) was delivered at a flow rate of 0.4 mL/min. Myrislignan and the IS eluted at 1.42 and 1.71 min, respectively. A good excellent linear response across the concentration range of 1-1000 ng/mL was achieved (r2 = 0.9973). The lower limit of quantification (LLOQ) was 1 ng/mL, and the inter- and intra-day accuracy and precision of the method showed relative standard deviations (RSDs) less than 10 %. The method was applied to examine the pharmacokinetics of myrislignan in mouse plasma following a single oral administration of 200 mg/kg or intraperitoneal administration of 50 mg/kg myrislignan, and the bioavailability (F) of orally administered myrislignan was only 1.97 % of the bioavailability of intraperitoneally administered myrislignan. CONCLUSIONS: A rapid and sensitive LC-MS/MS method has been was developed, validated and successfully used to determine myrislignan levels in mice after oral or intraperitoneal administration. This study is the first to report the pharmacokinetic parameters of myrislignan in mice and to compare its pharmacokinetics after oral and intraperitoneal administration, which will be useful for further research on the administration of myrislignan in animals and humans.

Effect of lactoferrin on murine sperm apoptosis induced by intraperitoneal injection of lipopolysaccharide.

Genital bacterial infection is one of the most important causes of infertility, however, bacteria frequently exist in seminal fluid. Sperm express Toll-like receptors (TLRs) on their cell surfaces and bacterial recognition by TLRs induces sperm apoptosis. In this study, we examined the lactoferrin (LF) potentiality on sperm apoptosis induced by bacterial lipopolysaccharide (LPS). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay indicated that TUNEL-positive sperm cells were scarce in the group treated with LF and LPS (LF/LPS group) compared to the group treated with LPS only (LPS group). In addition, real-time RT-PCR detected lower mRNA expression levels of apoptosis-associated genes in the LF/LPS group compared to the LPS group. These results indicate that LF treatment of semen might decrease LPS-induced apoptosis of sperm.

Intraperitoneal administration of lidocaine or tramadol alone or in combination on postoperative pain after ovariohysterectomy in dogs.

The present prospective randomized experimental study aimed to assess the intraperitoneal (ip) administration of lidocaine or tramadol, alone or in combination, on postoperative pain management following ovariohysterectomy in dogs. Eighteen healthy female mixed-breed dogs, aged 1-2 years, weighed 16.7 ± 3.8 kg, were used. Animals were sedated with acepromazine (0.1 mg/kg, intramuscular). Forty minutes later, anaesthesia was induced through intravenous titration with diazepam (0.5 mg/kg) and ketamine (10 mg/kg) and maintained with isoflurane 1.5%. Afterwards, ovariohysterectomy was performed, and prior to the closure of the linea alba, animals received lidocaine containing epinephrine (8.8 mg/kg, ip) in group L, tramadol (4 mg/kg, ip) in group T and lidocaine containing epinephrine (8.8 mg/kg, ip) plus tramadol (4 mg/kg, ip) in the LT group. Cortisol, vital signs and pain scoring systems were evaluated at different time points. Vital signs did not change among the groups. Cortisol level in the LT group significantly decreased compared to the L and T groups one, three and six hours after surgery. Pain scores also did not change among the groups based on Sammarco and Simple descriptive (SDS) scoring method. However, pain scores in the LT group were higher than the two other groups according to the University of Melbourne pain scale (UMPS) and the short form of Glasgow pain scale (CMPS-SF). According to the obtained results, the combination of lidocaine and tramadol seemed to be able to provide better analgesia compared with their separate administration. Therefore, combined intraperitoneal administration of lidocaine (8.8 mg/kg) and tramadol (4 mg/kg) with a final volume of (0.2 ml/kg) following ovariohysterectomy is recommended.

The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin.

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.

Plasma and Peritoneal Ceftriaxone Concentrations After Intraperitoneal Administration in Horses With Septic Peritonitis.

Intraperitoneal ceftriaxone administration in healthy horses results in high and prolonged peritoneal concentrations. Recent findings suggest that intraperitoneal ceftriaxone might increase survival rates in horses affected by peritonitis. The present study aimed to evaluate plasma and peritoneal concentrations of ceftriaxone after intraperitoneal administration in horses with septic peritonitis. Twenty-six horses presenting clinical, laboratorial, and sonographic findings compatible with the disease were included. All horses received daily intraperitoneal ceftriaxone (25 mg/kg bwt) in addition or not with other antibiotics and support therapies. High-performance liquid chromatography was used to determine plasma and peritoneal ceftriaxone concentrations before and after 12 and 24 hours of ceftriaxone administration. Mean plasma concentrations 12 and 24 hours after administration were, respectively, 1.84 ± 0.43 and 0.37 ± 0.07 µg/mL, and mean peritoneal concentrations were 5.7 ± 2.84 and 0.42 ± 0.13 µg/mL. Ceftriaxone concentration was lower in comparison with previous studies in healthy horses and presented under the minimal inhibitory concentration for enterobacteria (≤1 µg/mL) and for gram-positive isolates (≤0.5 µg/mL) at 24 hours. The variation of the results obtained between healthy horses and with septic peritonitis demonstrated that pharmacokinetics/dynamics are different between these patients and suggests the use of an interval of dose of 12 hours.

Intracoelomic- and Intramuscular-Injection Challenge Model of Piscine Streptococcosis in White Sturgeon Fingerlings.

Streptococcus iniae is a zoonotic pathogen and one of the major aetiologic agents of streptococcosis. In White Sturgeon Acipenser transmontanus, S. iniae infection typically presents as a necrotizing and heterophilic myositis, causing 30-50% mortality in infected fish. To gain a better understanding of the pathogenesis of streptococcosis in White Sturgeon, and to identify the experimental route of infection that most closely mimics the natural disease, fingerlings were challenged with a single dose of 1.3 × 108  cells/fish of S. iniae that was administered via intracoelomic/intraperitoneal (IC) or intramuscular (IM) routes. Acute mortalities were present only in the IM-challenged fish, with first mortality occurring 4 d postchallenge and the mortality rate reaching 18.3% after 9 d. The challenged fish presented erratic swimming, ulcerative skin lesions, and hemorrhages in the liver and swim bladder. Streptococcus iniae was recovered from the kidney and brain tissues of moribund and dead fish. Histopathologic analysis of fish that died acutely revealed massive proliferation of bacteria in the muscle at the injection site and within vascular organs such as the heart and spleen, with variable amounts of tissue necrosis including a necrotizing myositis. Fish that died closer to 9 d postchallenge demonstrated more pronounced multifocal to locally extensive granulomatous inflammation of skeletal muscle at the injection site, liver, kidney, and spleen. No mortality, clinical signs, or gross changes were observed in the control or IC-challenged fish. Postmortem evaluation of 10 survivors in each treatment was performed to determine carrier status in the brain and posterior kidney tissues. The prevalence of S. iniae in survivors was 10% and 0% in the IM- and IC-challenged groups, respectively. The results from this study suggest that IM-injection challenge methods are suitable for inducing streptococcosis in White Sturgeon, and they may be the preferred method for studying the pathogenesis of the naturally occurring disease in this species.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria).

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Cytochrome P450s regulates aloperine-induced pathological changes in mouse liver and kidney.

Aloperine is a major active component in Sophora alopecuroides L that plays diverse pharmacological properties. Recent studies have indicated the potential effect of aloperine against hypertension and cancers. However, possible toxicity of aloperine has not been carefully studied in vivo. The aim of this study was to assess the effect of intraperitoneal aloperine injection on mouse liver and kidney tissues and to investigate the role of CYP450 genes in aloperine-induced toxicity. 72 BALB/c mice were randomly divided into four groups: vehicle control group (normal saline), low-dose group (4 mg/kg), medium-dose group (8 mg/kg), and high-dose group (16 mg/kg). 18 mice in each group were intraperitoneally injected with aloperine daily for 4 weeks, and were then kept for another 1 or 4 weeks without aloperine treatment. Serum was colleted for analysis of serum biochemical indexes including ALT, AST, BUN and CRE. The liver and kidney were collected for analysis of histopathologic changes and CYP450 expression and activity. Vacuolization of cytoplasm in liver cells, swelling in kidney tubular cells, increased levels of ALT, AST, BUN, and CRE, and alteration in the expression and activity of CYP450 were observed in the high-dose group after 4 weeks of treatment. However, all aloperine-induced damages were recovered to a certain degree after maintained without aloperine for 1 week, and fully recovered after maintained without aloperine for 4 weeks. These findings suggested that aloperine regulated the expression of CYP450, which was possibly involved in aloperine-induced reversible toxicity in mouse liver and kidney tissues.

Association of intraperitoneal ceftriaxone, intravenous gentamicin and oral metronidazole in the treatment of an abdominal abscess and peritonitis in a horse: case report / Associação da ceftriaxona intraperitoneal, gentamicina intravenosa e do metronidazol oral no tratamento de abscesso abdominal e peritonite em equino: relato de um caso

Medical management of abdominal abscesses in horses requires prolonged antibiotic therapy and presents varied success rates. A 6-year-old male horse with a history of colic and multiple abdominal punctures to relieve gas was attended. At admission, tachycardia, tachypnea, hyperthermia, mucosal congestion, dehydration, and rigid gait were observed. The association of physical examination, laboratory and ultrasonographic findings allowed the diagnoses of peritonitis and abdominal abscess. Supporting treatment plus broad spectrum antibiotic therapy was performed: daily intraperitoneal ceftriaxone (25 mg/kg, 7 days); daily intravenous gentamicin (6.6 mg/kg, 7 days); per os metronidazole three times a day (15 mg/kg 12 days), followed by the same dose twice a day (15 mg/kg 33 days), totaling 45 days of treatment. Plasma fibrinogen and ultrasonographic examination were the most effective tools to evaluate abscess evolution. There was normalization of the physical examination 24 h after beginning the treatment, consecutive regression of the nucleated cell count in the peritoneal fluid, and regression of plasma fibrinogen and size of the abscess. On the 10th treatment day, the animal was discharged from the hospital, maintaining oral therapy with metronidazole every 12 h (15 mg / kg). When the animal returned on the 30th day, an abscess size regression was observed. However, there was no resolution, and therapy with metronidazole was maintained. On the 45th day of treatment, a new hospital evaluation was performed, where the abscess resolved, and metronidazole was suspended. It is highlighted that the therapeutic association used in the treatment of abdominal infection and abscess resulted in a rapid clinical response.(AU)

O tratamento conservativo dos abscessos abdominais em equinos requer antibioticoterapia prolongada e apresenta variadas taxas de sucesso. Foi atendido um cavalo de seis anos de idade, com histórico de cólica e múltiplas punções abdominais por agulha para esvaziamento de gás. Na admissão, foram observados taquicardia, taquipnéia, hipertermia, congestão mucosa, desidratação e marcha rígida. A associação do exame físico, achados laboratoriais e ultrassonográficos permitiu o diagnóstico de peritonite e abscesso abdominal. Foi realizado tratamento suporte e antibioticoterapia de amplo espectro: ceftriaxona intraperitoneal diária (25 mg/kg, 7 dias); gentamicina intravenosa diária (6,6 mg/kg, 7 dias); metronidazol oral três vezes ao dia (15 mg/kg, 12 dias), seguido de mesma dose duas vezes ao dia, por mais 33 dias, totalizando 45 dias de tratamento. O fibrinogênio plasmático e o exame ultrassonográfico foram os recursos mais eficazes para a avaliação da evolução do abscesso. Após 24 horas do início do tratamento foi constatada a normalização do exame fisico, regressão progressiva da contagem de células nucleadas no líquido peritoneal, do fibrinogênio plasmático e do tamanho do abscesso. No 10° dia de tratamento o animal recebeu alta hospitalar, mantendo-se a terapia oral com metronidazol a cada 12 horas (15 mg/Kg). Em retorno, ao 30° dia, observou-se regressão do tamanho do abscesso, entretanto, não houve resolução, tendo sido mantida a terapia com metronidazol. No 45º dia de tratamento, realizou-se nova avaliação hospitalar, onde foi observada a resolução do abscesso e a admnistração do metronidazol foi suspensa. Destaca-se, que a associação terapêutica utilizada no tratamento de infecção abdominal e abscesso resultou em rápida resposta clínica.(AU)

Melatonin regulates the ovarian function and enhances follicle growth in aging laying hens via activating the mammalian target of rapamycin pathway.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.05). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.05). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.

Immune responses induced by inactivated vaccine against Aeromonas hydrophila in pacu, Piaractus mesopotamicus.

Aeromonas hydrophila is responsible for outbreaks of a severe infectious disease in fish farms around the world and is one of the major causes of economic losses to the neotropical fish farmers. This study assessed the induction of immune responses and protection against A. hydrophila in pacu, Piaractus mesopotamicus, vaccinated through intraperitoneal and immersion route with inactivated virulent strain. Fish were randomly distributed in three vaccinated groups: intraperitoneal (i.p.) route; immersion; and immersion + booster; and control group (unvaccinated). All vaccination protocols used the concentration of 1.7 × 108 CFU mL-1 of inactivated A. hydrophila., and an oil adjuvant was used for vaccine prepararion for i.p. route vaccination. Blood and skin mucus from 9 fishes per treatment were collected at 14, 28, 42 and 84 days post-vaccination (DPV) for determination of lysozyme concentration in skin mucus, as well as antibodies anti-A. hydrophila in blood serum and skin mucus. Fish were challenged at 84 DPV with homologous and virulent strain of A. hydrophila for evaluation of resistance against bacterial infection. The results demonstrated that vaccination with inactivated A. hydrophila suspension by i.p. or immersion resulted in significant increase of skin mucus lysozyme and specific antibody levels in serum and skin mucus, at 28 and 42 DPV, and this increase in innate and adaptive immunity remained significant in pacu vaccinated through i.p. route up to 84 DPV. Although no significant differences were observed in the survival study, pacu vaccinated through i.p. route presented 31,33% of relative percentage survival (RPS) in LD50-96h when compared unvaccinated fish challenged at 84 DPV. The results observed in this study indicate that vaccination programs with inactivated A. hydrophila, including booster doses by i.p. or immersion routes, could result in more effective protection in pacu against this bacteriosis, by increasing innate and adaptive mucosal and systemic immune responses.

Comparison of intraperitoneal ropivacaine and ropivacaine-dexmedetomidine for postoperative analgesia in cats undergoing ovariohysterectomy.

OBJECTIVE: To investigate the intraperitoneal (IP) administration of ropivacaine or ropivacaine-dexmedetomidine for postoperative analgesia in cats undergoing ovariohysterectomy. STUDY DESIGN: Prospective, randomized, blinded, positively controlled clinical study. ANIMALS: A total of 45 client-owned cats were enrolled. METHODS: The cats were administered intramuscular (IM) meperidine (6 mg kg-1) and acepromazine (0.05 mg kg-1). Anesthesia was induced with propofol and maintained with isoflurane. Meloxicam (0.2 mg kg-1) was administered subcutaneously in all cats after intubation. After the abdominal incision, the cats were administered one of three treatments (15 cats in each treatment): IP instillation of 0.9% saline solution (group Control), 0.25% ropivacaine (1 mg kg-1, group ROP) or ropivacaine and dexmedetomidine (4 µg kg-1, group ROP-DEX). During anesthesia, heart rate (HR), electrocardiography, noninvasive systolic arterial pressure (SAP) and respiratory variables were monitored. Sedation and pain were assessed preoperatively and at various time points up to 24 hours after extubation using sedation scoring, an interactive visual analog scale, the UNESP-Botucatu multidimensional composite pain scale (MCPS) and mechanical nociceptive thresholds (MNT; von Frey anesthesiometer). Rescue analgesia (morphine, 0.1 mg kg-1) IM was administered if the MCPS ≥6. Data were analyzed using the chi-square test, Tukey test, Kruskal-Wallis test and Friedman test (p < 0.05). RESULTS: HR was significantly lower in ROP-DEX compared with Control (p = 0.002). The pain scores, MNT, sedation scores and the postoperative rescue analgesia did not differ statistically among groups. CONCLUSIONS AND CLINICAL RELEVANCE: As part of a multimodal pain therapy, IP ropivacaine-dexmedetomidine was associated with decreased HR intraoperatively; however, SAP remained within normal limits. Using the stated anesthetic protocol, neither IP ropivacaine nor ropivacaine-dexmedetomidine significantly improved analgesia compared with IP saline in cats undergoing ovariohysterectomy.

Phenotypic and Molecular Characterization of Lacinutrix venerupis Isolated from Atlantic Horse Mackerel Trachurus trachurus.

The aim of the present study was to characterize two gram-negative bacterial strains that were isolated from diseased Atlantic Horse Mackerel Trachurus trachurus in 2017. Based on the results obtained from the biochemical and chemotaxonomic characterization, the isolates were identified as Lacinutrix spp. The highest similarity of the 16S rRNA gene sequences was obtained with the strain L. venerupis CECT 8573T (99.1%), while other species showed similarities of 98% (L. jangbogonensis) and 97% (L. algicola and L. mariniflava). Molecular characterization by repetitive element (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as proteomic characterization by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), demonstrated heterogeneity between the strains from the Atlantic Horse Mackerel and the type strain, CECT 8573T . The virulence of one of the isolates for Turbot Scophthalmus maximus, European Sea Bass Dicentrarchus labrax, Senegalese Sole Solea senegalensis, and Rainbow Trout Oncorhynchus mykiss was assessed under experimental conditions. No mortalities were recorded after intraperitoneal injections with high doses of bacteria (1 × 109  CFU/mL). Thus, further studies are necessary to elucidate the impact of this bacterial species as a fish pathogen.

Therapeutic applications of adipose-derived mesenchymal stem cells on acute liver injury in canines.

In this study, canine adipose-derived mesenchymal stem cells (cADSCs) therapeutic potential was investigated in artificially induced acute liver injury model by CCl4 in canines. The primary cADSCs cells were cultured and then intravenously administered into the canine animal model. Six cross-breed dogs were divided into three groups including blank control group, CCl4 model group, CCl4 induced cADSCs transplantation group. The results showed that after intraperitoneal injection of CCl4 solution, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin (ALB) in peripheral blood of experimental canines confirmed the correct induction of acute liver injury. Moreover, the liver structure showed clear macroscopic damage. The cADSCs were homed in the liver of the administered animals. The AST, ALT and ALB in the peripheral blood rapidly decreased. H&E and PAS histological evaluation showed that both the structure of canine liver tissue and the ability to synthesize hepatic glycogen could be restored to the control level after cADSCs transplantation. Therefore, cADSCs can play a therapeutic role in the recovery of liver injury. Overall, this study demonstrates that the primary cADSCs transplantation into the acute liver injury model induced by intravenous injection can play a certain therapeutic role in the recovery of liver in canines. These results may provide a new treatment idea for acute liver disease in pets clinically.

Whole cell inactivated autogenous vaccine effectively protects red Nile tilapia (Oreochromis niloticus) against francisellosis via intraperitoneal injection.

Francisella noatunensis subsp. orientalis is a pathogen of tilapia and other warm-water fish for which no vaccines are commercially available. In this study, a whole cell formalin-inactivated vaccine was developed for the first time using the highly virulent isolate STIR-GUS-F2f7 and the oil-based adjuvant Montanide™ ISA 763A VG. The efficacy of the vaccine was assessed in red Nile tilapia via intraperitoneal (i.p.) injection using homologous experimental infection and correlates of protection such as seral antibody production and bacterial loads in the spleen. For immunization, fish were i.p. injected with 0.1 ml of the vaccine, the adjuvant alone or PBS. At 840 degree days post-vaccination, all fish were i.p. injected with 4.0 × 103 CFU/fish of pathogenic bacteria. The RPS at the end of the trial was 100% in the vaccinated group with significantly higher survival than in the adjuvant and control groups. The RPS in the adjuvant group was 42%, and no significant difference was seen in survival between this and the PBS group. Moreover, significantly higher antibody titres in the serum and significantly lower bacterial loads in the spleen were detected in the vaccinated fish by ELISA and qPCR, respectively. These findings highlight the potential of autogenous vaccines for controlling francisellosis in tilapia.

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions.

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 ß, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.

A comparison between intraperitoneal injection and intranasal and oral inoculation of mink with Aleutian mink disease virus.

Intranasal, with (INS) and without (IN) sedation, and oral inoculation were compared with intraperitoneal (IP) injection for establishing infection with a local isolate of Aleutian mink disease virus (AMDV) in 35 American mink. Blood samples were collected on 0, 21, 36 and 56 day post-inoculation (dpi). Antiviral-antibodies and viral DNA in plasma and tissues were measured by counter-immunoelectrophoresis (CIEP) and PCR, respectively. The presence of AMDV DNA was tested by PCR in saliva, rectal and fecal samples collected on 0, 6, 10, 15, 21, 28, 36 and 56 dpi. Animals were killed at 56 dpi, samples of six organs were tested for antibody and AMDV DNA, and samples of the lungs, liver, kidneys and heart were subjected to histology. Viral DNA was detected in the spleen, lungs and lymph nodes of all inoculated mink on 56 dpi, indicating that all inoculation routes caused infection in mink. Viral DNA and antibodies were detected in plasma of all IP and INS inoculated mink by 36 dpi, but some animals which were inoculated orally or via IN remained seronegative by 56 dpi. It was concluded that INS route was the most effective method for establishing infection in mink without breaking the integrity of the animals' anatomical barriers. Viremia was short-lived in some mink, whereas antibody production persisted in seroconverted animals during the duration of the experiment. Saliva, rectal and fecal samples did not accurately detect infection. Histologic lesions of AD were observed on the four organs of most mink.

Assessment of Pain Associated with the Injection of Sodium Pentobarbital in Laboratory Mice (Mus musculus).

The AVMA Guidelines for the Euthanasia of Animals considers injection of barbiturates to be an acceptable method of euthanasia in rodents but states there is a potential for pain when administered intraperitoneally. This study examined the potential for pain in mice by assessing visceral pain after intraperitoneal administration and acute pain by using a paw-lick test. Male and female mice (n = 160) intraperitoneally received a euthanizing dose of sodium pentobarbital at a concentration of 5, 50, or 390 mg/mL and were observed for writhing, peritoneum-directed behaviors (PDB), loss of righting reflex, and time to death. Writhing was not observed in any animal. There was no significant difference in the number of mice exhibiting PDB or in the rate of PDB for responders receiving either saline or the 390-mg/mL solution. There was a significant treatment effect on time, with greater concentration and dose resulting in more rapid loss of righting reflex and death. In the second set of experiments, the same solutions were injected subcutaneously into the plantar hindpaw of male and female mice (n = 84). The number of responders, latency until the first lick, and the number of licks per responder were recorded. The number of responders was increased in the 50-mg/mL group; however, there was no difference in latency or the number of licks per responder. These results show that intraperitoneal injection of sodium pentobarbital for euthanasia in mice did not result in increased behavioral signs of pain, and animals lose consciousness more rapidly than the onset of pain seen in the pawlick test. Therefore, although sodium pentobarbital is capable of inducing inflammation, euthanasia through intraperitoneal administration is rapid and does not result in overt signs of pain when compared with injection of saline.

Efficacy of Injectable and Immersion Polyvalent Vaccine against Streptococcal Infections in Broodstock and Offspring of Nile tilapia (Oreochromis niloticus).

A vaccine against streptococcosis, lactococcosis and enterococcosis in tilapia was formulated, ME-VAC Aqua Strept, as a polyvalent inactivated vaccine containing Streptococcus agalactiae, S. iniae, Lactococcus garvieae and Enterococcus faecalis along with a nano-particulate adjuvant. Use of ME-VAC Aqua Strept by injection or immersion resulted in an improved non-specific and adaptive immunity of broodstock and offspring. Intra-peritoneal vaccination of tilapia broodstock increased the total leukocyte count, phagocytosis, lysozyme activity, antibody titer, number of seeds/vaccinated broodstock, seeds quality and survival rates. Also, immersion mass vaccination of tilapia larvae provided a long period of protection up to three months, with a relative percent of survivability (RPS) not less than 60% at this time. To our knowledge, this vaccine may be the first to offer a combined protection against streptococcosis, lactococcosis and enterococcosis in tilapia. The results support the use of this vaccine as an effective tool for disease control and well-being of fish.