Radioimmunoassay of Bovine,Ovine and Porcine Luteinizing Hormone with a Monoclonal Antibody and a Human Tracer

Forsberg, M., R. Tagle, A. Madej, J.R. Molina and M.-A. Carlsson. Radioimmunoassay of bovine, ovine and porcine luteinizing hormone with a monoclonal antibody and a human tracer. Acta vet. scand. 1993, 255-262.– A radioimmunoassay for bovine (bLH), ovine (oLH) and porcine (pLH) luteinizing hormone was developed using a human ¹²⁵ ILH tracer from a commercial kit and a monoclonal antibody (518B7) specific for LH but with low species specificity. Standard curves demonstrated similar binding kinetics when bLH, oLH and pLH were incubated with tracer and antibody for 2 h at room temperature. A 30-min delay in the addition of the tracer gave sufficient sensitivity when analysing pLH. Separation of antibody-bound LH from free hormone was achieved by using second antibody-coated micro Sepharose beads. The assay was validated and the performance compared with that of an RIA currently in use for determination of bLH and oLH (coefficient of correlation: 0.99 and 0.98). Regardless of the standards used, intra-assay coefficients of variation were <10% for LH concentrations exceeding 1 µg/L. The inter-assay coefficients of variation were <15%. The assay was used for clinical evaluation demonstrating the pre-ovulatory LH surge in two cyclic cows, LH pulsatility in an oophorectomized ewe and LH response to GnRH injection in a boar.     

Recombinant Equine Luteinizing Hormone Stimulates Production of Progesterone from Murine Leydig, Ovine Small Luteal, and Equine Granulosal Cells

Recombinant equine luteinizing hormone (reLH) was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA-10), equine granulosal, and ovine small luteal cells (SLC). The response to reLH was compared with that obtained with human chorionic gonadotropin (hCG), equine chorionic gonadotropin, ovine luteinizing hormone, and equine luteinizing hormone (eLH). Media were collected hourly for 6 hours and assayed for progesterone content through radioimmunoassay. In MA-10 cells, production of progesterone was stimulated above baseline by reLH and hCG (P < .05). Ovine SLC responded to treatment with eLH, reLH, ovine luteinizing hormone, and hCG by increasing production of progesterone above that stimulated by vehicle control (P < .05). Production of progesterone in equine granulosal cells was maximally stimulated by treatment with hCG (P < .05), followed by reLH and eLH (P < .05). In conclusion, reLH elicited a progesterone response in MA-10, ovine SLC, and equine granulosal cells. Thus, reLH stimulates the production of progesterone in cell lines from three species.     

Serum Profiles of Luteinizing Hormone,Oestradiol and Cholesterol and Ovarian Functions in Layer Poultry Birds (Gallus domesticus) Fed Diets Containing Furazolidone

This study was carried out to assess the serum profiles of luteinizing hormone (LH), oestradiol, cholesterol and ovarian functions in layer poultry birds (Rhode Island Red: Gallus domesticus) fed a diet containing various concentrations of furazolidone (FZ). A total of 40 birds were randomly assigned to receive FZ 0, 200, 400 or 800 mg/kg feed (ppm) daily during the pre-laying age, i.e. 13-18 weeks (for 5 weeks). Blood samples were collected at weekly intervals. Concentrations of LH and oestradiol in serum were estimated at alternate weeks using radioimmunoassays. Serum cholesterol levels were analysed by an enzymatic calorimetric method. Furazolidone administration was terminated at the 18th week of age. The birds were sacrificed at 22nd week of age and ovarian tissues were processed for morphometric studies. Serum LH, oestradiol and cholesterol levels were affected by age (p < 0.001) and FZ dose (p < 0.001). Serum LH and oestradiol levels were lower (p < 0.05) in birds receiving FZ 800 mg/kg feed daily compared with the controls, whereas serum cholesterol profiles were lower (p < 0.05) in all FZ-administered groups than in the control group. The mean weight of ovaries having no yolky follicles observed in the group receiving FZ 400 or 800 mg/kg feed per day was reduced (p < 0.05) compared with the control group. Dosing FZ at 800 mg/kg feed per day reduced (p < 0.05) the mean volume of ovaries having no yolky follicles compared with the control group. In birds receiving FZ 800 mg/kg feed per day, the mean length of the oviduct was reduced (p < 0.05) as compared with the control group. Morphometric studies revealed that the mean number of oocytes with diameter in the range 401-800 microm decreased (p < 0.05) in birds fed FZ 400 or 800 mg/kg feed per day. Initial egg production was affected by age (p < 0.001) and dose (p < 0.001) of FZ. The mean number of eggs laid by different groups revealed that egg production was reduced (p < 0.05) in birds receiving FZ 800 mg/kg feed per day as compared with the controls. The present data suggest that FZ causes suppression in serum profiles of LH, oestradiol, cholesterol and ovarian functions in Rhode Island Red layer poultry birds. Therefore, great care must be taken with use of FZ in layer poultry birds (Gallius domesticus) with regard to dosage and duration of administration.     

Anti-Müllerian Hormone as a Diagnostic Marker for Equine Cryptorchidism in Three Cases with Equivocal Testosterone Concentrations

Cryptorchidism is a developmental disorder which can be diagnosed by a variety of different tests. Still, equine field veterinarians often rely on endocrine markers to detect retained testicular tissue. This report describes the value of anti-Müllerian hormone (AMH) as a diagnostic marker for cryptorchidism in three stallions suspected of cryptorchidism, with equivalent serum testosterone concentrations. A single measurement of AMH identified cryptorchidism, with inconclusive testosterone concentrations as either gelding or stallion. Furthermore, a human chorionic gonadotropin (hCG) stimulation test or surgery was performed to support the diagnostic value of AMH. To conclude, the endocrine panel for cryptorchidism should be expanded to include determination of serum AMH, as this determination can increase the diagnostic accuracy of a single blood sample.     

The Accuracy and Precision of an Equine In-Shoe Pressure Measurement System as a Tool for Gait Analysis

Normal horses with no clinical lameness were studied to test the hypothesis that Equine F-Scan sensors (EFS) would produce vertical force data that correlated highly with vertical force data from a force platform. Six horses were trotted across a force platform while wearing the EFS. Vertical force measurements were recorded from each system with data analyzed from hoof strikes that occurred simultaneously on both systems. Coefficients of variation (CV) were evaluated to test precision of the systems. The CV of the EFS was 10.2%, compared with 6.6% for the force platform. The accuracy of the pressure measurement system was tested by measuring agreement and a paired t test. The test for measuring agreement showed large differences between measuring devices, indicating an overall lack of agreement between measurements from the two systems, with differences ranging from 9 Newtons (N) to 1,200 N. Results from a paired t test, however, showed no significant difference between measurements from the two devices as noted by a P-value of .294, indicating a lack of significant differences because of assessment of averaged data. Because of the inability of the EFS to provide precise measurements of vertical ground reaction force as noted by high coefficients of variation and an overall lack of agreement between measurements from the two systems, this system should not be used to objectively measure vertical force in horses in its current format.     

Hormonal Response of Female Goats to Active Immunization against a Recombinant Human Inhibin α-subunit, and establishment of an Enzyme-linked Immunosorbent Assay for Caprine Follicle-stimulating Hormone

The effect of selective immunosuppression of endogenous inhibin in goats on FSH, LH, progesterone and estradiol-17β profiles was studied during the breeding and nonbreeding seasons. Eighteen adult female Boer goats were immunized against the recombinant human inhibin α-subunit (hINH-α). With the exception of estradiol, which was determined by radio-immunoassay (RIA), all plasma hormone concentrations were determined by ELISA. The ELISA for FSH presented in this paper was established in the authors' laboratory, based on an existing RIA. Mean basal concentrations of FSH were not affected by immunosuppression of endogenous inhibin, nor was there a difference in the amplitude of the pre-ovulatory FSH surge. Immunization against inhibin appears to eliminate the slight secondary rise of FSH occurring 12–20 h after the major surge associated with ovulation. The LH profiles of the immunized goats were characterized by lower basal concentrations both before and after the pre-ovulatory LH surge which itself was reduced by 50% in immunized does. By contrast, concentrations of circulating estradiol were significantly elevated after inhibin-immunization. Progesterone profiles were not affected. Extending immunization into the anoestrous season by a booster injection of hINH-α, implicating oestrus induction with a progestagen and eCG, produced no discernible differences in FSH and LH profiles in comparison with nonimmunized control goats. The findings suggest that in goats, paracrine factors may play a more significant role in controlling follicular activity than a feedback mechanism acting via the pituitary.     

Freezing of Maiden Stallion Semen - Motility and Morphological Findings in Sperm Cells Assessed by Various Staining Methods Including a Monoclonal Antibody with Reactivity Against an Antigen in the Acrosomal Ground Substance

Contents: From a group of thirteen 3-year-old Hanoveranian maiden stallions ejaculates were collected on 5 consecutive days and frozen each in 3 different packaging forms. Fresh, resuspended and frozedthawed semen was tested for motility and morphology with special regard to acrosomal integrity. Concerning packaging form no differences were found between 4 ml maxi straw, flattened 1.7 ml straw and 0.5 ml straw. The comparison of 3 direrent conventional staining techniques with an immunofluorescence test, employing a monoclonal antibody against an antigen in the acrosomal ground substance, revealed that staining sperm cells with the antibody yielded far more reproducible and consistent results - especially in diluted semen - than staining with Karras, moded Karras or Spermac®.     

The Distribution of Bovine Trypanosomosis in Zimbabwe and an Evaluation of the Value of an Anti-trypanosomal Antibody Detection ELISA as a Tool for Monitoring the Effectiveness of Tsetse Control Operations

Tsetse have been cleared from large areas of Zimbabwe during the past 65 years. In most areas, they are prevented from re-invading cleared areas by barriers of odour-baited, insecticide-treated targets. A trypanosomosis survey was conducted to determine the effectiveness of such barriers against re-invasion and to confirm the absence of tsetse in areas where they had previously been eradicated. Parasitological diagnostic methods and an anti-trypanosomal antibody detection enzyme-linked immunosorbent assay (antibody ELISA) were used. The prevalence of trypanosomal infections in the tsetse-cleared areas was generally low. However, the prevalence of anti-trypanosomal antibodies was unexpectedly high in some areas. This high proportion of cattle with antibodies could, in most cases, be explained by recent or historic information on the distribution and density of tsetse. The results from the survey demonstrated the value of anti-trypanosomal antibody detection as an additional sensitive tool for monitoring the effectiveness of tsetse control operations.     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

Development,validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin

Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro–differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level.     

Results and complications of a novel technique for primary castration with an inguinal approach in horses

Reasons for performing study: Complications associated with equine castration can have medical and financial consequences. This retrospective study investigated a novel method of castration via an inguinal approach in mature stallions and compared the incidence of complications with other methods. Hypothesis: Castration via an inguinal approach has a low complication rate at the site of surgery compared with other castration techniques. Methods: Mature stallions (n = 238) were castrated under general anaesthesia in dorsal recumbency using an inguinal approach. The vaginal process was incised, the spermatic cord ligated twice and the testis removed. After suturing, the vaginal process and one or 2 layers of fascia, the subcutis and cutis were closed in a simple continuous pattern. Results: Five of 238 (2.1%) horses had post operative haemorrhage and a haematoma in the scrotal region, which required additional treatment. All horses made a full recovery. Five of 238 (2.1%) of the horses had a post operative respiratory infection, which resolved with antibiotic therapy. Sixteen of 238 (8.8%) had transient signs of colic shortly after surgery. Conclusion: This technique of castration with an inguinal approach had a low incidence of complications at the site of surgery compared with other methods. An inguinal approach and leaving the vaginal tunic in situ may cause less soft tissue trauma than a scrotal approach.     

Comparison of an antifungal agent with a mixture of antifungal, antibiotic and corticosteroid agents for the treatment of Malassezia species otitis in dogs

Twenty dogs with otitis externa in both ears and numerous Malassezia species yeasts on cytological examination were treated in one ear with a combination product containing clotrimazole, marbofloxacin and dexamethasone, and in the other ear with a topical antifungal containing miconazole. The effects of the treatments were analysed on the basis of the scores for pruritus, erythema and amount of cerumen, and the number of yeasts on cytological smears. There were reductions in the counts of Malassezia species after both treatments, but the combination product gave significantly greater reductions in erythema, cerumen and pruritus.     

Clinical comparison between a continuous Lembert pattern wrapped in a carboxymethylcellulose and hyaluronate membrane with an interrupted Lembert pattern for one-layer jejunojejunostomy in horses

Reasons for performing study: Although experimental studies have demonstrated differences in performance between methods for handsewn jejunojejunostomy in horses, information on clinical results after different methods of anastomosis are rare. Hypothesis: A continuous Lembert pattern wrapped in a carboxymethylcellulose and hyaluronate membrane would perform better than an interrupted Lembert pattern for jejunojejunostomy in horses. Methods: Data was reviewed on 32 horses that underwent jejunojejunostomy from 1993–2002. Kaplan‐Meier analyses and rates for post operative colic and death were used to compare outcomes after an interrupted Lembert pattern (15 horses with strangulating lesions and 5 horses with nonstrangulating lesions) and a continuous Lembert pattern with membrane (12 horses with strangulating diseases). Results: None of the 32 horses had post operative ileus or post operative endotoxaemia. One horse with a continuous pattern required a repeat celiotomy for anastomotic impaction. Short‐term survivals for the interrupted Lembert were 100% (nonstrangulating lesions) and 93% (strangulating lesions) and for the continuous pattern 92% (all strangulating). Long‐term rates for mortality and colic episodes were less for the continuous Lembert pattern with membrane compared with the interrupted Lembert for strangulating lesions (P<0.05) and were less for strangulating lesions than for nonstrangulating lesions (P<0.05). For strangulating lesions, Kaplan‐Meier analyses yielded a survival probability of 70% for up to 9 years after the interrupted Lembert pattern and 80% for up to 5 years for the continuous Lembert pattern. Conclusions and relevance: Both Lembert patterns performed well in clinical use, although the continuous pattern with the carboxymethylcellulose and hyaluronate membrane had superior long‐term outcomes with less colic and mortality from colic.     

Design,optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil

Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic bacterial species that can, alone or in combination, induce anemia in cats. The diagnostic test of choice for hemoplasma infections is PCR. Conventional PCR assays have been developed for the detection of Mycoplasma haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for CMtc. Although real‐time PCR assays have been reported for all of the feline hemoplasmas, the expense of necessary instrumentation precludes its use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a conventional PCR assay to diagnose CMtc using an internal control to detect false‐negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species‐specific primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10‐fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica‐based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl₂ concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the first time the prevalence of CMtc infection in domestic cats in Brazil.     

Isolation and immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini

Mycoplasma ovipneumoniae NCTC 10151(T) and four new isolates from UK sheep flocks were compared. Only glucose and pyruvate were used as energy sources by the five strains: glucose was the best energy source for the type strain, pyruvate supported better growth of the new strains. Whole cell protein patterns and antigenic profiles showed high similarity between all five strains. The new isolates fell into two groups in ELISA tests. Serum samples from 30 pneumonic sheep were assessed for M. ovipneumoniae infection and Mycoplasma arginini co-infection. Fourteen (out of 30) serum samples were positive for M. ovipneumoniae both by ELISA and immunoblotting. Twelve antigenic proteins of M. ovipneumoniae were detected in infected serum samples: the antigen patterns were unique, with between one and at least seven occurring in any one sample. All serum samples were designated as negative for M. arginini antibodies by both ELISA and immunoblotting.     

Clinical and histological evaluation of an analogue of palmitoylethanolamide, PLR 120 (comicronized Palmidrol INN) in cats with eosinophilic granuloma and eosinophilic plaque: a pilot study

Fifteen cats with eosinophilic granuloma or eosinophilic plaque were given PLR 120 at the dosage of 10 mg kg⁻¹ twice daily for one month. PLR-120 down-modulates mast cell degranulation via a receptor-mediated mechanism. No other drugs were permitted and cats were kept free of parasites throughout the study. A clinical evaluation and skin biopsies were performed before and after the treatment. Clinical improvement was assessed at 15 and 30 days. Mast cell numbers were counted and their granular content was assessed by densitometric analysis on toluidine blue-stained sections before and after the treatment. Ten of 15 (67%) cats showed clinical improvement of signs and lesions. There was no significant difference between mast cell numbers in skin biopsies taken before and after the trial, whereas the number of granules was significantly increased ( P < 0.009). This pilot study suggests that PLR-120 might be a useful drug for the treatment of eosinophilic granuloma and eosinophilic plaque.     

Suboccipital craniectomy, dorsal laminectomy of C1, durotomy and dural graft placement as a treatment for syringohydromyelia with cerebellar tonsil herniation in Cavalier King Charles spaniels

OBJECTIVE: To evaluate retrospectively the efficacy of the suboccipital craniectomy and dorsal laminectomy of C1 with durotomy and placement of a dural graft for treatment of syringohydromyelia (SHM) because of cerebellar tonsil herniation in Cavalier King Charles spaniels (CKCS). This technique is used with great success in human medicine. STUDY DESIGN: Four CKCS diagnosed by Magnetic resonance imaging (MRI) of SHM because of cerebellar tonsil herniation and not responsive to medical therapy underwent a suboccipital craniectomy and dorsal laminectomy of C1 (2 dogs) and of C1 and partial C2 (2 dogs) with durotomy and placement of a dural graft. Three dogs were evaluated neurologically 24 hours, 1 month, and 3 months postoperatively and evaluations were compared with preoperative neurological examination. Repeat MRI took place 3 months postoperatively. RESULTS: Neurological examinations showed neither improvement nor progression of clinical signs 3 months postoperatively. MRI showed no regression of syrinx size 3 months postoperatively. CONCLUSION: Improvement was not seen. Given the progressive nature of the disorder, evaluation over a longer period of time is necessary to detect if progression has stopped. Some modification to the surgical technique is needed to accomplish the same results as in human medicine. A study of a larger population is needed to attain more reliable information. CLINICAL RELEVANCE: Suboccipital craniectomy and dorsal laminectomy of C1 with durotomy and placement of a dural graft is a feasible technique in CKCS, but needs some modification to accomplish the same results as in human medicine.