Detection of lysozyme in llama, sheep, and cattle tears

OBJECTIVE: To determine whether the tears of llamas, sheep, and cattle contain lysozyme and compare lysozyme concentrations in tears among these species. ANIMALS: 40 llamas, 5 sheep, and 36 cattle. PROCEDURE: Electrophoresis, western blot immunoassay for lysozyme, a spectrophotometric assay to detect tear lysozyme by its ability to lyse a suspension of Micrococcus lysodeiticus, and a microtiter plate colorometric assay were performed. RESULTS: A 13.6-kd protein band was detected by use of electrophoresis and western blot immunoassay in llama and sheep tears but not cattle tears. Results of spectrophotometric assay suggested that llama and sheep tears had high concentrations of lysozyme, whereas cattle tears had low concentrations. Results of the microtiter plate colorometric assay suggested that llama tears had high concentrations of lysozyme, whereas concentrations in sheep and cattle tears were lower. CONCLUSIONS AND CLINICAL RELEVANCE: Lysozyme concentrations in tears may vary among species and this variability may contribute to differing susceptibilities to ocular diseases such as infectious keratoconjunctivitis.     

Lysozyme concentrations in the tears of cattle, goats, and sheep

Tear samples were collected from 1 eye of each of 40 cows, 27 sheep, 5 goats, and 5 human beings. Additionally, 10 bovine tear samples were pooled and concentrated. Spectrophotometric assays, using Micrococcus lysodeikticus, were performed on each sample to detect lysozyme activity expressed in hen egg lysozyme (HEL) equivalents. Lysozyme activity was not detected in tears of cows, but 158.8 +/- 159.3 mg of HEL/ml was detected in tears of sheep, 220.7 +/- 37.5 mg of HEL/ml in tears of goats, and 216.3 +/- 86.2 mg of HEL/ml in tears of human beings. In pooled bovine tear samples, lysozyme activity was not detected on plate assay and lysozyme protein was not detected on polyacrylamide gel electrophoresis, column chromatography, or immunoelectrophoresis with rabbit anti-bovine tear antibodies. On the basis of these observations, we concluded that the basic ocular protective mechanism in bovine tears is not lysozyme. Other anti-bacterial proteins such as lactoferrin, transferrin, complement, or beta-lysin may, therefore, be of primary importance in protecting the bovine eye.     

Evaluation of lysozyme and lactoferrin in lacrimal and other ocular glands of bison and cattle and in tears of bison

OBJECTIVE: To evaluate lactoferrin and lysozyme content in various ocular glands of bison and cattle and in tears of bison. SAMPLE POPULATION: Tissues of ocular glands obtained from 15 bison and 15 cattle and tears collected from 38 bison. PROCEDURE: Immunohistochemical analysis was used to detect lysozyme and lactoferrin in formalin-fixed, paraffin-embedded sections of the ocular glands. Protein gel electrophoresis was used to analyze ocular glands and pooled bison tears by use of a tris-glycine gel and SDS-PAGE. Western blotting was used to detect lactoferrin and lysozyme. RESULTS: Immunohistochemical staining for lactoferrin was evident in the lacrimal gland and gland of the third eyelid in cattle and bison and the deep gland of the third eyelid (Harder's gland) in cattle. Equivocal staining for lactoferrin was seen for the Harder's gland in bison. An 80-kd band (lactoferrin) was detected via electrophoresis and western blots in the lacrimal gland and gland of the third eyelid in cattle and bison, Harder's glands of cattle, and bison tears. An inconsistent band was seen in Harder's glands of bison. Lysozyme was not detected in the lacrimal gland of cattle or bison with the use of immunohistochemical analysis or western blots. Western blots of bison tears did not reveal lysozyme. CONCLUSIONS AND CLINICAL RELEVANCE: Distribution of lactoferrin and a lack of lysozyme are similar in the lacrimal gland of cattle and bison. Differences in other tear components may be responsible for variability in the susceptibility to infectious corneal diseases that exists between bison and cattle.     

Foot-and-mouth disease virus in the llama (Lama glama): diagnosis, transmission, and susceptibility

Foot-and-mouth disease virus (FMDV) was shown to be transmitted from either cattle to llamas, llamas to swine (interspecies), or llamas to llamas (intraspecies). Response to FMDV varied greatly in the 6 llamas studied; 3 llamas developed generalized clinical disease with mild pyrexia, 2 after intradermolingual inoculation, and 1 after exposure to a calf infected with FMDV serotype A24. Another contact llama developed vesicular lesions on all 4 extremities but no oral lesions. Two contact llamas, in separate study groups, did not seroconvert or develop clinical signs of FMDV infection. All 4 llamas showing clinical disease developed virus-neutralizing antibodies against FMDV A24 and antibodies against the virus-infection-associated antigen. Virus-neutralizing antibody titers remained elevated for over 200 days postinoculation or exposure. Antibodies to virus-infection-associated antigen were detected several days after virus-neutralizing antibody appeared and became weaker 100-125 days post-FMDV exposure in 3 of the 4 clinically affected llamas. One inoculated llama was still positive for virus-infection-associated antigen at 360 days after inoculation. Foot-and-mouth disease virus A24 was not detected from esophageal-pharyngeal fluid specimens beyond 8 days postexposure using in vitro techniques.     

Cross-reactivity between a monoclonal antibody that recognizes a tumor-associated antigen on bovine lymphosarcoma cells and blood lymphocytes from various mammalian species.

Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (MAB). By use of flow cytometry and radioimmunoprecipitation, 1 of the MAB (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (BL) from various mammalian species. By use of flow cytometry, the c143 MAB reacted with 10 to 49% of BL derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 MAB with BL from horses, pigs, human beings, and llamas ranged between 1:6.0 x 10(4) and 1:5.3 x 10(5); titer associated with BL of goats and sheep was 1:1.6 x 10(6); and that associated with BL of cattle was 1:4.3 x 10(7). The c143 MAB specifically immunoprecipitated 3 homologous proteins from cell extracts of caprine, ovine, and bovine BL (32-, 34-, and 36- to 37-kDa bovine proteins; 31-, 32-, and 36- to 37-kDa caprine proteins; and 31.5-, 33-, and 36- to 37-kDa ovine proteins), but none was immunoprecipitated from human, murine, canine, porcine, and llama BL. These results indicate that the avidity of the c143 MAB in binding to BL from ruminants (eg, goats, sheep, and cattle) is higher than that to BL from human beings, mice, dogs, horses, pigs, and llamas. In sheep, the c143 MAB could immunoprecipitate the aforementioned proteins from BL of the Suffolk breed, but not BL from the Corriedale breed, whereas the c143 MAB immunoprecipitated apparently identical proteins from BL of 4 breeds of cattle.     

Electrophoretic and immunoelectrophoretic analysis of feline serum proteins.

Serum from 28 clinically healthy cats was subjected to agarose gel electrophoresis and the migration distance relative to albumin was determined. The reference values for the relative and absolute concentrations of each protein fraction were determined and compared to previous reports. The immunoelectrophoretic, crossed immunoelectrophoretic and crossed line immunoelectrophoretic pattern, of a pooled sample of serum from clinically normal cats was determined. The cross-reactivity between goat and/or rabbit monospecific antisera to human proteins and feline serum was determined using immunoelectrophoresis and crossed-line absorption immunoelectrophoresis. Feline alpha-2-macroglobulin, haptoglobin, B1C-globulin, IgG, albumin and ceruloplasmin cross reacted strongly with the monospecific antisera. Alpha-2-macroglobulin migrated anodal to haptoglobin. Lipoproteins and ceruloplasmin were studied using staining procedures described in man. Feline transferrin was precipitated with Rivanol.     

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal

Objective To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears. Animal studied Domestic short‐haired cats (7–17 months; 2.6–5.2 kg) were used. Procedures Eye‐flush tears were collected periodically for up to 18 weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n = 4) or with nictitating membranes intact (n = 4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix‐metalloproteinase (MMP)‐9 measurements using sandwich enzyme‐linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP‐2 and ‐9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18 weeks were also investigated in histological sections using immunoperoxidase for visualization. Results Nictitating membrane removal did not significantly change TPC and MMP‐9 in tears within the first 4 weeks. MMP‐9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP‐2 and ‐9 activity in tears from eyes without nictitating membranes at week 1 and a decrease following week 2 post‐surgery. MMP‐2 and ‐9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18 weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes. Conclusions Based on this preliminary study, nictitating membrane removal appeared to cause long‐term changes in expression of tear proteins, including reduced MMP‐9 expression.     

Parasitism in llamas

Llamas in North America are infected with a number of helminth parasites, including: gastrointestinal nematodes, lungworms, meningeal worms, tapeworms, and flukes. Most of these helminths can be treated with the anthelmintic currently used to treat cattle and sheep. At least two protozoan parasites, coccidia and toxoplasma, have been reported in the llamas in North America. Coccidia can be controlled with the anticoccidial drugs used to control these organisms in other domestic animals. Toxoplasma has been reported uncommonly in llamas in North America and control is very difficult. Several external parasites occur in llamas in North America, including lice, mites, ticks, and the deer nasal bot. Except for the deer nasal bot, the other external parasites in the llama can be treated with pesticides approved for use in cattle.     

Characterization of erythrocytic indices and serum iron values in healthy llamas.

An electronic particle counter with attached particle-size analyzer was configured to directly determine concentration, mean cell volume, and volume distribution of erythrocytes in llama blood. Blood from 38 healthy llamas was used to characterize erythrocytic measurements and serum iron values for this species. Volume distribution curves for llama erythrocytes were similar in shape to those of other species. These curves had a unimodal, symmetric shape with a tail skewed to the right. Reference ranges for directly measured mean cell volume, erythrocyte concentration, hemoglobin concentration, and mean cell hemoglobin concentration were 21 to 28 fl, 11.3 x to 17.5 x 10(6) cells/microliters, 12.8 to 17.6 g/dl, and 43.2 to 46.6 g/dl, respectively. Reference ranges for serum iron concentration, total iron-binding capacity, and transferrin saturation were determined to be 70 to 148 micrograms/dl, 230 to 370 micrograms/dl, and 22 to 50%, respectively.     

Encephalitic listeriosis in two adult llamas (Lama glama): clinical presentations, lesions and immunofluorescence of Listeria monocytogenes in brainstem lesions

Encephalitic listeriosis was diagnosed in 2 adult llamas. Both had a multifocal suppurative encephalitis with mixed lymphocytic and neutrophilic perivascular infiltrates. Listeria monocytogenes was cultured from the brain stem of 1 llama using cold enrichment techniques; the other llama was culture negative. Formalin-fixed and paraffin embedded sections of brainstem lesions from both affected animals were labeled with a fluorescein-conjugated, anti-L. monocytogenes antibody. Using this technique, intralesional L. monocytogenes were identified in both llamas.     

Experimental equine herpesvirus-1 infection in llamas (Lama glama).

Three llamas (Lama glama) were experimentally infected intranasally with an isolate of equine herpesvirus-1 (EHV-1) from the brain of an alpaca that had experienced severe neurologic signs. Two of the 3 llamas developed severe neurologic disorders following inoculation; 1 died, and 1 was euthanized in a moribund state. The third llama showed only mild neurologic signs. The euthanized llama had preexisting antibodies to EHV-1, and the remaining 2 llamas were seronegative (virus neutralization titer less than 6) at the time of inoculation. One of the seronegative llamas died acutely without production of detectable antibodies, and the other developed antibodies typical of a primary immune response. The EHV-1 virus was recovered only from a sample of the thalamus of the llama that died acutely. Histopathologic lesions were observed in the brain and retina of the dead and euthanized animals. This study verifies the pathogenic potential of EHV-1 for llamas.     

Reference ranges for hematologic and serum biochemical values in llamas (Lama glama)

Hematologic and serum biochemical values were determined in 174 llamas of all age groups and both sexes from ranches in California and Nevada. Compared with hematologic values for horses and cattle, llama erythrocytes were more numerous (10.1 to 17.3 x 10(6)/microliters), but the PCV was lower (25 to 45%) because the smaller elliptical cells pack tighter. The mean corpuscular volume was half that of horses and cattle (22 to 29.5 fl). The mean corpuscular hemoglobin concentration was higher (38.9 to 46.2 g/dl), and the mean corpuscular hemoglobin slightly lower (9.6 to 12.6 pg). Most serum biochemical values were similar to those of cattle and horses, with the exception of triiodothyronine (48 to 468 ng/dl) and thyroxin (9.8 to 30 micrograms/dl), which are up to 10 times higher than values for other domestic species.     

Pharmacokinetics of famciclovir and penciclovir in tears following oral administration of famciclovir to cats: a pilot study

Objective To validate a means of collecting tears from cats, develop an assay for quantifying famciclovir and penciclovir in tears, and to assess famciclovir and penciclovir concentrations and pharmacokinetics in the tears of cats being treated orally with famciclovir for suspected herpetic disease. Animals Seven client‐owned cats. Procedures Cats were treated orally with a median (range) dose of 40 (39–72) mg of famciclovir/kg three times daily for at least 24 h. At various time points following famciclovir administration, tear samples were collected using Schirmer tear test strips. Tear famciclovir and penciclovir concentrations were measured using liquid chromatography‐mass spectrometry, and concentration‐time profiles were analyzed noncompartmentally. The relationship between famciclovir dose and tear penciclovir concentration near its maximum was evaluated using least squares linear regression. Results Maximum tear famciclovir concentration of 0.305 μg/mL occurred at 2.64 h; elimination half‐life was 2.28 h. Maximum tear penciclovir concentration (0.981 μg/mL) occurred 2.25 h following oral administration of famciclovir; elimination half‐life was 2.77 h. A significant positive correlation was noted between famciclovir dose and tear penciclovir concentration at various time points between 0.5 and 3.75 h following drug administration (P = 0.025). Tear penciclovir concentration exceeded the concentration shown to have in vitro efficacy against feline herpesvirus (FHV‐1) (0.304 μg/mL) in about half of samples collected. Conclusions Oral administration of 40 mg of famciclovir/kg to cats resulted in a tear penciclovir concentration‐time profile that approximated the plasma penciclovir concentration‐time profile and frequently achieved a penciclovir concentration at the ocular surface likely to be effective against FHV‐1.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1alpha, IL-1beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1alpha, IL-1beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.     

Tear urea nitrogen and creatinine levels in horse and their correlation with serum values

The objective of this paper was to determine the physiological values of urea nitrogen and creatinine in tears, and to compare the results with those obtained from serum. Thirty healthy thoroughbred horses were included in the study. Tear fluid samples were obtained using a glass capillary tube placed in lower conjunctival cul-de-sac. Blood samples were taken from the jugular vein. Tear and serum urea nitrogen and creatinine levels were quantitatively analyzed by an enzymatic colorimetric method. Urea nitrogen values were 4.22+/-1.84 mmol/l in tears and 4.44+/-1.78 mmol/l in serum, whereas creatinine values in tears were 14.14+/-7.74 micromol/l and in serum 147.63+/-12.17 micromol/l. Statistical analysis confirmed a significant correlation between serum and tear urea levels (P<0001). However, there was no significant correlation between blood and tear creatinine values. Mean value of creatinine obtained from tears was 9.6% of the mean value from serum. Urea nitrogen and creatinine levels can be measured in tears. A significant correlation was found between serum and tears urea levels. This finding may permit development of a new alternative laboratory diagnosis of uremia based on the content of urea in tears.     

Coccidia of the llama, Lama glama, in Colorado and Wyoming.

Fecal samples were collected from 144 llamas, Lama glama, on four premises in northern Colorado and southern Wyoming. Feces were examined for the presence of coccidial oocysts; Eimeria alpacae was found in 55.6%, Eimeria lamae in 67.3% and Eimeria macusaniensis in 1.4% of the samples. An adult female llama was found to be infected with Eimeria macusaniensis at necropsy. This is the first report of these species of coccidia in the llama.     

Hematologic features of iron deficiency anemia in llamas.

Iron deficiency anemia was identified and characterized in three 14 to 29-month-old male llamas (llama Nos. 1-3) from separate herds in Colorado. The identification of iron deficiency anemia was based on hypoferremia (serum iron = 20-60 micrograms/dl), erythrocytic features, and hematologic response to iron therapy. The anemia was moderate and nonregenerative and characterized by erythrocyte hypochromia, microcytosis (mean cell volume = 15-18 fl), and decreased mean corpuscular hemoglobin concentration (36.0-41.0 g/dl). Morphologic features unique to llamas with iron deficiency anemia included irregular distribution of hypochromia within erythrocytes and increased folded cells and dacryocytes. The cause of iron deficiency was not determined. The llamas were treated with various doses and schedules of parenteral iron dextran. Two of the llamas were monitored for up to 14 months after the start of iron therapy and experienced increases in hematocrit and mean cell volume values. In one llama, progressive replacement of microcytic cells with normal cells was visualized on sequential erythrocyte volume distribution histograms following iron therapy.     

Long-bone fractures in llamas: six cases (1993-1998)

OBJECTIVE: To determine clinical and radiographic findings, treatment, and outcome for llamas with long-bone fractures. DESIGN: Retrospective study. ANIMALS: 6 llamas. PROCEDURE: Medical records of llamas admitted between 1993 and 1998 because of long-bone fractures were reviewed. Data collected included age, sex, type of fracture, method of fracture repair, and postoperative complications. The Fisher exact test was used to compare age and sex of the llamas with long-bone fractures with those of the hospital population of llamas. All owners were contacted by telephone to determine perceived postoperative problems and whether the llamas were able to perform as expected. RESULTS: Mean age was 160.8 days (range, 23 to 365 days). There was 1 male and 5 females. Fractures were more likely to occur in young llamas (< or = 1 year old) than in adults. Five of the fractures were attributed to traumatic episodes. Long bones affected included the tibia (n = 2), radius (2), femur (1), and humerus (1). Internal fixation with lag screws, plating, or both was performed on fractures of all llamas except 1; that llama was treated by use of confinement to a stall. None of the llamas had intraoperative complications, but postoperative complications were reported in 2 llamas. All fractures healed eventually, and clients were pleased with outcomes. CONCLUSIONS AND CLINICAL RELEVANCE: Long-bone fractures in llamas are uncommon. Several types of long bone fractures can be successfully repaired by use of internal fixation, resulting in few complications and minimal convalescent time.     

Flow cytometric analysis of an immunodeficiency disorder affecting juvenile llamas

The present study was undertaken to characterize the immune system of llamas and alpacas and establish the basis for an immunodeficiency disorder affecting juvenile llamas. Flow cytometric (FC) analysis of the immune system with a panel of monoclonal antibodies (mAbs) revealed the immune system of llamas and alpacas is similar in leukocyte subset composition to that in ruminants. Peripheral blood mononuclear cells in adults are comprised of surface immunoglobulin (sIg(+)) B-cells (31%+/-8 S.D.), alphabeta T-cells (27%+/-12 S.D.), WC1(+) gammadelta T-cells (16%+/-11 S.D.), and 5-16% monocytes. In contrast to cattle, goats, and sheep, however, the frequency of WC1(+) gammadelta T-cells is not high in juveniles but similar to the frequency in adults. Also, sIg(+) B-cells are present in high concentration in juveniles (43%+/-11 S.D. ). Expression of major histocompatibility class II molecules on resting T-cells was low or absent. Comparative analysis of peripheral blood lymphocyte composition in normal juvenile llamas and llamas presenting with the signs of the juvenile llama immunodeficiency syndrome (JLIDS) revealed the concentration of B-cells is extremely low (1-5%) in affected animals. The findings suggest JLIDS is attributable to an autosomal recessive genetic defect in the development of B-cells.