Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model.

PURPOSE: To test the hypothesis that beneficial effects of Cyclosporin A (CsA; Sandimmune; Sandoz, Basel, Switzerland) in treating keratoconjunctivitis sicca (KCS) include an effect on the mucin-producing conjunctival goblet cells independent of CsA's effect on lacrimation. METHODS: Keratoconjunctivitis sicca was induced bilaterally in six dogs after removal of orbital and nictitans lacrimal glands. Two weeks after induction of KCS, either 2% CsA or vehicle was applied twice daily to each surgically altered eye until 6 weeks after KCS induction. Eyes of three control dogs without surgically altered eyes were treated twice daily with vehicle only. Incisional biopsy specimens of ventral fornix conjunctiva were collected before gland removal (baseline) and at 2, 4, and 6 weeks after KCS induction. At each sampling time, eyes were photographed, and color images were subsequently graded for degree of conjunctivitis and characteristics of ocular discharge. Intracellular mucin stores in conjunctival epithelia were estimated using computer-assisted morphometry of biopsy specimen cross sections, and clinical and morphometric findings were correlated. RESULTS: Lacrimal gland removal resulted in induction of KCS in dogs by 2 weeks, with mean Schirmer tear test (STT) values of 5 mm/min or less occurring in surgically altered eyes compared with STT values of 22.5 mm/min before surgery and 22.9 mm/min in unaltered control eyes at 2 weeks. In surgically altered eyes, STTs remained low during the 6-week study, independent of topical treatment. Intracellular mucin stores were quantified from conjunctival samples collected from each eye at baseline and 2, 4, and 6 weeks. At 4 and 6 weeks (after 2 and 4 weeks of topical treatment), intraepithelial mucin quantities were significantly greater (P: < 0.05) in CsA-treated KCS eyes (14.4 and 13.1 microm(2)/microm, respectively) compared with pretreatment KCS (7.4 microm(2)/microm) eyes and vehicle-treated KCS eyes (7.3 and 8.5 microm(2)/microm, respectively). KCS eyes treated with CsA had lower conjunctivitis and ocular discharge scores than did vehicle-treated KCS eyes. CONCLUSIONS: Topical 2% CsA restored in vivo conjunctival mucin stores to control levels over a 4-week period, determined by computer-assisted morphometry of sequential conjunctival biopsy specimens from eyes of dogs with surgically induced KCS. Degree of conjunctivitis and severity of mucus discharge were decreased in KCS eyes treated with CsA. Because lacrimal tissues were removed from animals in this study, conjunctival responses occurred independent of lacrimogenic effect(s). These results indicate that restoration of conjunctival goblet cell mucin production, i.e., the balance between synthesis and secretion of mucin glycoproteins, may play an important role in the beneficial effect of CsA in treating KCS.

Prevalence of Neospora hughesi and Sarcocystis neurona antibodies in horses from various geographical locations.

Parasite-specific antibody responses to Neospora antigens were detected using the immunofluorescent antibody test (IFAT) and immunoblot analysis in select equine populations. For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated Neospora caninum horse were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 208 samples was evaluated. The equine populations were derived from five distinct geographic regions. Locations were selected based on distribution of Didelphis virginiana, the native North American opossum which serves as the definitive host for S. neurona. Only 11% of the samples that had positive titers of 1:100 using the IFAT were also positive for antibodies by immunoblot analysis in this study. Overall, there was a 2% seroprevalence for Neospora antibodies in all horses tested based on immunoblot analysis described. The seroprevalence for S. neurona antibodies varied from 0% (New Zealand and Montana) to 54% (Missouri). We concluded that, in testing for antibodies against Neospora antigens using either IFAT or immunoblot analysis, as described, positive results should not be attributed to the presence of antibodies to S. neurona.

Estimated prevalence of paratuberculosis in Missouri, USA cattle.

An absorbed ELISA for detection of antibodies against Mycobacterium paratuberculosis was performed on serum samples obtained from the Missouri Animal Health Laboratory. Samples from 1954 Missouri cattle representing 89 herds were randomly selected from samples submitted for brucellosis testing. The apparent seroprevalence of paratuberculosis in dairy cattle (8 +/- 3%) was similar to that in beef cattle (5 +/- 2%). When herds were classified as dairy or beef, 74% (14 of 19) of dairy herds and 40% (27 of 68) of beef herds were positive.

Physiochemical properties of oviductal fluid from rabbits exposed to heat stress.

Oviductal fluids (OF) were collected at 24 hour intervals from 27 does. Fluids were collected from all does during estrus and subsequent pregnancy or pseudopregnancy. After collecting OF for 15 days from all does maintained at 21 degrees C and 45% relative humidity (RH), approximately one-half of the does were then subjected to heat stress (33.20+/-0.75 degrees C and 65.4+/-2.3% RH). Oviductal fluid was then collected for 5 days from heat stressed estrous does (HSER), after which all does (both control and HSER) were mated to fertile bucks. Human chorionic gonadotropin was administered to each doe to insure ovulation. Fluids were compared between HSER and heat stressed pregnant rabbits (HSPR) and control estrous (NHER) and pregnant rabbits (NHPR) for volume, pH, various protein concentrations, and total protein content. Fluid volume decreased due both to heat stress and pregnancy. Heat stress increased pH, whereas pregnancy resulted in a decrease. Protein concentration of OF from HSER declined due to heat stress. Protein concentration of OF from NHPR increased prior to breeding, peaking at day 3 of pregnancy, then declined to initial levels. While from HSPR there was also a tendency to have a similar increase, the peak in protein concentration was on day 4 and then decreased on day 5. Total protein content (concentration x volume) of OF from HSER decreased due to heat stress. Total protein content from HSPR and NHPR had similar changes with or without heat stress, although the total protein content from OF of HSPR was less (except for day 3) for the entire 5 day period studied. Relative percentages of albumin decreased and postalbumin increased during the first day of pregnancy (NHPR). These trends were reversed (a relative increase in albumin and a decrease in postalbumin) in OF from HSPR.

Partitioning the mortality risk associated with inadequate passive transfer of colostral immunoglobulins in dairy calves.

This study developed a method to partition the risk of mortality in dairy calves in the 1st 16 weeks of life. Observed population mortality and the relative risk of mortality in each serum protein concentration stratum were used to determine the population baseline mortality rate and the mortality rate due to inadequate passive transfer of colostral immunoglobulin. A total of 3,479 calves were studied, 8.2% of which died before 16 weeks of age. The population baseline mortality rate was 5.0% and the mortality rate due to inadequate passive transfer was 3.2%. Thirty-nine percent of the observed mortality was attributed to inadequate passive transfer. This partitioning of risk between passive transfer-related and unrelated sources should prove useful in conducting investigations of calf mortality problems in dairy herds.

Case control and historical cohort study of diarrhea associated with administration of trimethoprim-potentiated sulphonamides to horses and ponies.

Trimethoprim-potentiated sulphonamides (TPS) are among the most frequently administered antimicrobials in equine medicine. Anecdotally, TPS has been implicated as a cause of mild to moderate diarrhea in horses. The purpose of this study was to document the prevalence of diarrhea in horses receiving TPS, to characterize the severity of the diarrhea, and to identify any other factors associated with the development of diarrhea. A 2-part study was designed to identify the prevalence of diarrhea associated with TPS in our clinic population. Part I was a case-control retrospective study of 135 records over a 10.5-year period from January 1, 1980 through June 30, 1990. Part II was a historical cohort study of 784 records over a 37-month period from July 1, 1990 through July 31, 1993. There was no significant difference in the occurrence of diarrhea associated with TPS therapy in either study. The occurrence of diarrhea was 21% and 3% in parts I and II, respectively. Significant factors identified in association with diarrhea in part I were duration of hospital stay, and antibiotic therapy other than TPS or penicillin. Significant factors identified in part II included other antibiotic therapy, penicillin therapy, and combined penicillin and TPS therapy. Diarrhea does occur after the administration of antibiotics, most likely because of the alteration of the patient's normal intestinal flora. Diarrhea was noted in association with administration of TPS in this study; however, its prevalence was not significantly different than that in horses receiving other antibiotics, such as penicillin and its derivatives.

Effects of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1.

OBJECTIVE: To determine the effects of various concentrations of L-lysine and L-arginine on in vitro replication of feline herpesvirus type-1 (FHV-1). SAMPLE POPULATION: Cultured Crandell-Reese feline kidney (CRFK) cells and FHV-1 strain 727. PROCEDURE: Uninfected CRFK cells or CRFK cells infected with FHV-1 were cultured in Dulbecco's modified Eagle's medium or in 1 of 7 test media containing various concentrations of lysine and arginine. Viral titer and CRFK growth rate were assessed in each medium. RESULTS: Media depleted of arginine almost completely inhibited viral replication, whereas 2.5 or 5.0 microg of arginine/ml of media was associated with a significant increase in FHV-1 replication. In media with 2.5 microg of arginine/ml, supplementation with 200 or 300 microg of lysine/ml reduced viral replication by 34.2 and 53.9%, respectively. This effect was not seen in media containing 5.0 microg of arginine/ml. Growth rates of CRFK cells also were suppressed in media containing these concentrations of amino acids, but they were not significantly different from each other. CONCLUSIONS AND CLINICAL RELEVANCE: Arginine exerts a substantial growth-promoting effect on FHV-1. Supplementation of viral culture medium with lysine attenuates this growth-promoting effect in media containing low concentrations of arginine. Analysis of data from this study indicates that high concentrations of lysine reduce in vitro replication of FHV-1 but only in media containing low concentrations of arginine. Clinical trials will be necessary to determine whether supplemental administration of lysine, with or without arginine restriction, will be useful in the management of cats with FHV-1 infections.

Comparison of milk with serum ELISA for the detection of paratuberculosis in dairy cows.

OBJECTIVE: To compare milk with serum ELISA for detection of antibodies to Mycobacterium paratuberculosis. DESIGN: Epidemiologic survey. ANIMALS: 821 Missouri dairy cattle of 12 herds that were brucellosis certified and Dairy Herd Improvement Association members. PROCEDURE: Milk and serum samples obtained concurrently from Missouri dairy herds were tested by use of a standard ELISA procedure. Concurrent collection of milk and serum samples controlled for interactions such as colostral antibodies and the effect of time. On the basis of milk and serum ELISA values, disease prevalence and correlation between milk and serum test results were determined. RESULTS: Correlation measures on individual animals indicated low correlation of milk and serum ELISA values. McNemar's chi 2 of 7.6 was significant (P = 0.05). Analysis correlation was low (kappa = 0.08), as was regression analysis (R2 = 0.02). CLINICAL IMPLICATIONS: Milk ELISA for the detection of exposure to M paratuberculosis lacked correlation with serum ELISA. Further evaluation to determine sensitivity and specificity of milk ELISA will augment the usefulness of milk ELISA as a herd screening test.

Risk factors for development of dysautonomia in dogs.

OBJECTIVE: To identify risk factors associated with dysautonomia in dogs. DESIGN: Case-control study. ANIMALS: 42 dogs with dysautonomia examined between October 1988 and January 2000 and 132 control dogs examined during the same period for an unrelated problem. PROCEDURE: Information was gathered from medical records and surveys mailed to owners of case and control dogs. RESULTS: 42 case and 132 control dogs were included; completed surveys were returned by owners of 30 case and 103 control dogs. Dogs with dysautonomia were significantly younger (median, 18 months) than control dogs (median, 60 months) and more likely to come from rural areas and to spend > or =50% of their time outdoors. Compared with rural control dogs that spent at least some time outdoors, affected dogs were more likely to have access to pasture land, farm ponds, and cattle, and to have consumed wildlife, at least occasionally. The largest numbers of dogs with dysautonomia were identified during February and April, with relatively few dogs identified during the summer and early fall. CONCLUSION AND CLINICAL RELEVANCE: Although the cause of dysautonomia is unknown, results suggest that dogs with dysautonomia were significantly more likely to live in rural areas and spend > or =50% of their time outdoors than were control dogs examined for unrelated diseases.

Antimicrobial resistance among Pasteurella spp recovered from Missouri and Iowa cattle with bovine respiratory disease complex.

A retrospective study was conducted to determine the prevalence of antimicrobial resistance among Pasteurella spp recovered from cattle with bovine respiratory disease complex. The study extended from January 1976 through May 1980, and included a review of the necropsy records of 386 beef cattle. Susceptibility or resistance of the Pasteurella isolants was determined by using the standard disk diffusion susceptibility test. Each isolant was tested for susceptibility with 15 different antimicrobial agents. A high prevalence of resistance (greater than 80%) was found when Pasteurella was tested with triple sulfonamides. For P haemolytica isolants, 57% to 70% were resistant to ampicillin (56/97), penicillin (58/101), and streptomycin (70/100); for unidentified Pasteurella spp isolants, 64% to 91% were resistant to ampicillin (83/129), penicillin (89/129), and streptomycin (118/129). For P haemolytica (21/100) and P multocida (34/146) isolants, 21% to 23% were resistant to tetracycline. Most of the P multocida isolants did not show marked antimicrobial resistance to 9 of the 15 drugs tested. However, 58% of the P multocida isolants (84/145) were resistant to streptomycin and 88% of them were resistant to three combined sulfonamides (126/144).

Uterine NK cells in murine pregnancy.

Murine uterine natural killer (uNK) cells are transient, short-lived, terminally differentiated lymphocytes found in decidualized endometrium. Cells expressing natural killer cell surface markers are present in uteri of infant mice. Terminal uNK cell differentiation coincides with mesometrial decidual development subsequent to blastocyst implantation and begins about gestation day 5. uNK cells proliferate rapidly and, within 3 days, senescent uNK cells appear in normal implantation sites. Mid-gestation, senescent cells become dominant and uNK cell numbers decline until term when remaining cells are shed with the placenta. Transplantable uNK cell progenitors occur outside the uterus, suggesting that blood cell homing augments any in-utero progenitors. Early in healthy pregnancies, uNK cells produce cytokines and angiogenic molecules. Their lytic capacity in normal gestation and in pregnancy failure is incompletely defined. A significant shift recently occurred in thinking about major uNK cell functions. Activated uNK cells are now considered critical for appropriate endometrial angiogenesis in early implantation site development and in non-gestational endometrium. Because analogous cells appear in the endometria of women during each menstrual cycle and become abundant in early pregnancy, studies involving experimental pregnancy termination in genetically manipulated mice continue to have great importance for understanding regulation at the human maternal-fetal interface.

Efficacy of aerosol, intranasal and intramuscular vaccination against selected bovine viral diseases.

An experiment was designed to compare the efficacy of an IM modified live vaccine, an IN temperature-sensitive vaccine, an IN modified live vaccine, and an aerosolized modified live vaccine (two concentrations) to stimulate specific serum antibody and protect calves from a known viral challenge. All vaccines except the IM vaccine contained IBR virus and PI3 virus. The IM vaccine and the IN vaccines provided excellent protection to an IBR challenge, but both concentrations of aerosolized vaccine provided only patial protection. The use of IN modified live vaccine resulted in IBR serum Ab titers in all the test calves (100%), while the IM preparation elicited a response in only 60% of test calves and the IN temperature sensitive vaccine resulted in serum Ab titers in 40% of test calves. The aerosolized vaccine induced IBR serum antibody titers in only 20% of the calves. The use of IN modified live and the aerosolized vaccine resulted in a greater immune response to the PI3 virus (60-80% sero-conversion) than did the temperature sensitive vaccine (20% sero-conversion).

Inhalation anesthesia in adult cattle.

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.

Escherichia coli O157:H7 restriction pattern recognition by artificial neural network.

An artificial neural network model for the recognition of Escherichia coli O157:H7 restriction patterns was designed. In the training phase, images of two classes of E. coli isolates (O157:H7 and non-O157:H7) were digitized and transmitted to the neural network. The system was then tested for recognition of images not included in the training set. Promising results were achieved with the designed network configuration, providing a basis for further study. This application of a new generation of computation technology serves as an example of its usefulness in microbiology.