Serum pharmacokinetics and tissue and milk residues of oxytetracycline in goats following a single intramuscular injection of a long-acting preparation and milk residues following a single subcutaneous injection

Separate groups of goats were used to determine drug depletion patterns in serum (n=10), tissue (n=20) and milk (n=8) following a single intramuscular (i.m.) dose of 20 mg/kg of a long-acting oxytetracycline (OTC) formulation (Liquamycin LA-200). Milk residues were also determined following a subcutaneous (s.c.) administration of the same product at the same dose. Serum samples were taken for 24 h post-treatment and tissues (fat, liver, kidney, muscle and injection site) collected at 4, 7, 14, 21 and 28 days following injection. Milk from lactating goats was collected every 12 h for 8 days following both the i.m. and s.c. treatments utilizing an intervening 5-week washout period. Residues in serum and tissue were measured using a microbial inhibition assay, while milk residues were measured using both a microbial inhibition assay and a validated HPLC method. The serum pharmacokinetic parameters of OTC in goats were determined, with a mean AUC=67.4 microg h/mL, mean terminal half-life=14.4 h, and apparent clearance=0.33 L/kg h. Tissue half-lives could not be determined with confidence because the collection times provided only two points at which residues could be measured for most tissues. Oxytetracycline residues in all goat tissue samples measured less then cattle tissue tolerance by 96 h postdosing. One-compartment model describing milk depletion data for i.m. and s.c. dosing had terminal slope half-lives of 20.1 and 36.1 h, respectively. By 96 h post-treatment none of the milk samples contained OTC residues in excess of the cattle milk tolerance (0.3 p.p.m.). For both milk and tissue, the upper-bound 99% confidence intervals for the samples taken from goats 96 h postdosing were lower than approved cow milk and tissue tolerances.     

Mice immune responses to Brucella abortus heat shock proteins. Use of baculovirus recombinant-expressing whole insect cells,purified Brucella abortus recombinant proteins,and a vaccinia virus recombinant as immunogens

Brucella abortus resists the microbicidal mechanisms of macrophages, and the expression of its heat shock proteins (HSPs) such as GroEL, GroES and HtrA may play a role in this resistance. Bacterial HSPs can be very immunogenic, inducing protective immunity in various types of bacterial infections. However, the significance of immune responses directed against B. abortus HSPs in the protection against brucellosis is currently unresolved. To elucidate the role of these proteins in protection against Brucella challenge, individual, divalent or trivalent baculovirus (BV) recombinants of B. abortus GroEL, GroES and/or HtrA were injected into BALB/c mice either as protein-expressing whole cells or as purified proteins. The preparations were given to mice in combination with Freund's or Ribi adjuvant, respectively. In addition, some mice were primed with a vaccinia virus-GroEL recombinant, followed by inoculation with purified GroEL-Ribi adjuvant combination. Antibodies were observed against B. abortus GroEL and HtrA, but not against GroES. Cellular immune response was demonstrated by observing significant IFN-gamma release by lymphocytes of mice immunized with the purified HtrA-Ribi adjuvant combination. However, none of the mice inoculated with individual, divalent or trivalent HSP-expressing cells combined with complete Freund's adjuvant or inoculated with purified B. abortus HSPs combined with Ribi adjuvant, were protected against challenge with B. abortus virulent strain 2308. Priming with vaccinia virus-GroEL recombinant and boosting with GroEL-Ribi combination did not induce protective immunity. Based on the results obtained, we suggest that although humoral and cell-mediated immune responses are induced, but protective immune response is not induced by B. abortus HSPs.     

A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers.     

Assessment of enzyme linked immunosorbent assay based diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii in human serum

An enzyme linked immunosorbent assay (ELISA) using penicillinase was developed in the form of diagnostic kits (Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodies to Toxoplasma gondii. The performance of both the kits was compared with commercially available diagnostic kits, i.e. Enzygnost-Toxoplasmosis/IgG (Behring Co., Germany), TOXOTEK-G (Flow Lab., U.K.) and Toxoplasma IgM Microassay (Diamedix Corp., U.S.A.) by testing toxoplasma-suspected human serum samples. The results indicate a good reliability between these diagnostic kits. Toxokit-G has 86.66 and 96.05% sensitivity and specificity respectively. The main advantage of Toxokit-G is that the end result can be assessed visually without using sophisticated instruments. Toxokit-M has 100% sensitivity and specificity and test results were not affected by the presence of antitoxoplasma IgG antibodies, rheumatoid factor or antinuclear antibodies.     

Dysfibrinogenemia or afibrinogenemia in a Border Leicester lamb.

Hereditary fibrinogen deficiency is a rare condition in all species. Measurement of plasma fibrinogen should indicate low levels. Specific factor assays and pedigree analysis are essential in establishing a definitive diagnosis of the hereditary deficiency. Differentiation between afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia requires sophisticated techniques and assistance from a specialized laboratory.     

Laparoscopic Cryptorchid Castration in Standing Horses

Objective — This article describes a new technique for laparoscopic cryptorchid castration in standing horses. Study Design — Prospective study. Animals or Sample Population — Eight horses aged 11 months to 3 years and weighing between 300 and 643 kg. Methods — Food was withheld for 24 to 36 hours, and then horses were sedated with detomidine HC1 (0.02 to 0.03 mg/kg) and butorphanol tartrate (0.02 mg/kg). The paralumbar fossa region was desensitized with 2% mepivacaine in an inverted “L” pattern and caudal epidural anesthesia was administered with either xylazine (0.18 mg/kg diluted to 10 to 15 mL with 0.9% sodium chloride) or a combination of 2% mepivacaine and xylazine (0.18 mg/kg). Initial laparoscopic exploration was performed from the left flank; in three horses, right flank laparoscopy was needed to complete the procedure. The spermatic cord was ligated within the abdomen with one or two sutures of 0 polydioxanone suture, and the testis or testes removed through a flank incision. Results — In five horses with no palpably descended testes, standing laparoscopy was the only procedure performed, whereas in two horses, the abdominal testis was removed laparoscopically, and the descended testis was removed under short acting anesthesia. In one horse, with nonpalpable testes, it was determined by laparoscopic observation that the testes were in the inguinal canal, and castration was performed under general anesthesia. No surgical or postoperative complications were noted. The right side of the abdomen, and especially the right vaginal ring, could be easily observed from the left side by passing the laparoscope through a small perforation in the mesocolon of the descending colon or by elevating the descending colon with an instrument or by use of an arm in the rectum. Conclusions — The standing laparoscopic approach combined with or without short-acting anesthesia to remove the descended testis is easily performed. Clinical Relevance — This approach will provide surgeons with another option to castrate cryptorchid stallions.     

Relationship between milk fatty acid composition and dietary roughage source in dairy cows

According to a 2 × 2 crossover design, 14 Holstein dairy cows were fed two isoenergetic diets based on either grass hay (GH) or maize silage (MS). Milk samples were collected during the third week of each period, and fatty acid (FA) profiles were analyzed using gas chromatography. The data obtained were subjected to ANOVA. Dietary treatment had no effect on either dry matter intake or milk yield. Milk from animals fed the GH-diet contained lower concentrations of saturated FAs (61.9 vs. 63.4% of total FAs; P < 0.05) and higher levels of polyunsaturated FAs (PUFAs) (6.1 vs. 5.8; P < 0.01). Feeding additional hay also increased conjugated linoleic acid and n-3 FA levels and decreased C16:0 levels. Increases in both PUFAs and n-3 FAs resulted in lower (P < 0.01) atherogenic and thrombogenic indices in milk from animals fed the GH diet compared with those fed the MS diet. A complete substitution of GH for MS appeared to improve milk FA profiles, even using different types of concentrates to provide a balanced diet.

     

A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite

Background

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.

Methods

In the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.

Results

Tick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.

Conclusions

The present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.