A field trial of autogenous Moraxella bovis bacterin administered through either subcutaneous or subconjunctival injection on the development of keratoconjunctivitis in a beef herd

Abstract

The primary purpose of these experiments was to evaluate an autogenous Moraxella bovis bacterin administered through 2 separate routes of inoculation. An autogenous bacterin was manufactured by using M. bovis recovered from the herd. The bacterin was administered by subcutaneous injection or subconjunctival injection. In each experiment, unvaccinated animals served as controls. Random selection methods were used to place calves into a vaccination or control group. There was no statistical difference in development of infectious keratoconjunctivitis between the vaccinated and unvaccinated calves. There was a statistically significant difference between the sexes; heifers had a higher rate of keratoconjunctivitis.

Introduction

Infectious bovine keratoconjunctivitis (IBK) is a potentially devastating ocular disease. The clinical manifestations range from mild conjunctivitis to corneal ulceration, abcessation, and potential blindness (1,2). Aside from the individual's pain and suffering, there is a significant financial loss. The economic impact of IBK results from a loss in production, increased treatment costs, reduced weight gain, decreased milk production, and devaluation due to eye disfigurement and blindness (2). Although IBK has been reported as a contagious disease since the late 1800s, its treatment and control are still difficult. Moraxella bovis is identified as a cause of IBK; however, numerous other factors, including ultraviolet light, concurrent disease status, and other ocular bacterial organisms influence the disease (3,4). Varied susceptibility between various breeds of cattle and ages of animals has been reported (3,4,5). Infectious bovine keratoconjunctivitis is treated with systemic and topical antibiotics, with variable results (6). Fly control and vaccination against M. bovis remain the most commonly used methods of attempted prevention of IBK.

Varied serovars, different pili forms that may or may not result in infection, and varied toxins produced by M. bovis have been reported (4). Vaccines are normally produced against the various serovars of the organism. New vaccines based on M. bovis toxins are under investigation (7,8,9,10,11). Autogenous vaccines are usually made from a single serovar isolated from the herd that is affected. Autogenous vaccines have produced mixed results (8). Research on vaccines is usually carried out under controlled settings; however, these are not always applicable in field settings.

Vaccines have routinely been administered as subcutaneous (SC) or intramuscular (IM) injections. Better protection may be achieved if the antigen stimulates a mucosal immune response. Other methods of mucosal vaccination include application of the antigen to the affected site, such as by inhalation and deposition on the subconjunctival space (12).

The primary purpose of this study was to evaluate in a field setting the efficacy of 2 methods of inoculation of an autogenous bacterin of M. bovis in cattle.

Materials and methods

A herd of Angus and amerifax cattle with a history of endemic IBK was selected for these experiments. Numerous cattle within this herd had been identified as being affected with IBK on multiple occasions over a 5-year period immediately proceeding these studies, and many of these animals had been identified as being affected with M. bovis through conjunctival swabs submitted for bacterial culture (unpublished observations). The herd had been vaccinated with a commercial IBK vaccine (Piliguard Pinkeye-1; Schering-Plough, Union, New Jersey, USA), according to manufacturer's instructions, throughout this 5-year period.

The yearling calves used in this study were pasture raised and managed on a ranch located in north central Kansas. A creek or windmill with water tank provided a constant source of fresh water, while trees and brush provided shade. The pasture grass was predominately bluestem. All animals received mineral supplementation ad libitum and occasional grain feeding. During the peak fly season, the animals were treated with a commercial insect repellent (Saber Pour On or Boss Pour On; Schering-Plough) at approximately 21-day intervals. Calves were vaccinated against infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, bovine respiratory syncytial virus, and parainfluenza-3 virus (CattleMaster 4; SmithKline Beecham, West Chester, Pennsylvania, USA).

In the spring of 1998, conjunctival swabs were taken from eyes affected with keratoconjunctivitis and submitted for bacterial culture. Moraxella bovis was isolated and identified by using standard microbiologic techniques (13,14). A biologic manufacturing company (ImmTech, Bucyrus, Kansas, USA) was contracted to produce an autogenous vaccine from the M. bovis (hemolytic) organisms. Yearling calves (8 to 15 mo of age) were selected for vaccination or no vaccination (control) by systematically assigning (15) them to a group while they were being processed for routine herd health examinations (experiment 1). Vaccinated and control animals were pastured together. Animal tag numbers identified calves. Records were maintained on vaccination status, including date(s) of vaccination, ocular disease development, and treatment. The eyelid, conjunctiva, and cornea were examined to ensure that external eye disease was not present prior to vaccination. Calves with preexisting ocular disease were excluded from the study. A total of 441 yearling calves of approximately the same age were included. A total of 213 calves were vaccinated and 228 served as unvaccinated controls (Table 1). Of the 213 vaccinated calves, 109 were heifers and 104 were bulls; 70 animals (6 heifers and 64 bulls) received 1 vaccination, while the remaining 143 animals (105 heifers and 38 bulls) received 2 vaccinations between 2 and 4 wk apart. In the control group, there were 113 heifers and 115 bulls. All animals were monitored by routine herd inspection approximately every 2 d. The clinical examiner was unaware of the vaccination status of each calf. If a calf was observed to have excessive tearing, blinking, or corneal opacity, it was separated from the herd for closer examination and potential treatment. Keratoconjunctivitis was diagnosed when a corneal ulcer or a cloudy or hazy cornea with hyperemic conjunctival tissue was present. The severity of the ocular disease was not identified. Treatment consisted of a systemic injection of oxytetracycline (Liquamycin LA-200; Pfizer, New York, New York, USA), 9 mg/calf, or a subconjunctival injection of a penicillin and dexamethasone combination (Azimycin; Schering-Plough, Union, New Jersey, USA), 1mL/eye, and some animals received an eye patch. Treated calves were returned to the herd.

Table 1.

The following year, experiment 2 was conducted using the same autogenous bacterin. A herd of 331, 8- to 15-month-old yearling calves (153 heifers and 178 bulls) were used. Animals were systematically asigned into 1 of the 3 groups: group 1 (139 calves, 66 heifers and 73 bulls) received 2 vaccinations, SC; group 2 (121 calves, 56 heifers and 65 bulls) received 2 vaccinations, inoculated under the dorsal bulbar conjunctiva; and group 3 (71 calves, 31 heifers and 40 bulls) served as unvaccinated controls. Calves were pastured together. Herd inspection was conducted every 2 to 3 d for 5 mo. If a calf was observed to have excessive tearing and blinking or corneal opacity, it was separated from the herd, and treated for keratoconjunctivitis if it was observed to have a cloudy cornea, hyperemic conjunctival tissue, or a corneal ulcer. Affected calves were treated as described for experiment 1.

Differences in the occurrence of IBK among the different groups and between the sexes were analyzed by the χ² method (EPI Info 2000 Version 1.0; Centers for Disease Control and Prevention, Atlanta, Georgia, USA, June 14, 2000). Differences were considered significant when 95% confidence intervals were > 1.

Results

In experiment 1, 191 out of 441 total calves developed keratoconjunctivitis (Table 1). In the vaccinated group, 93 of 213 (44%), 68 heifers and 25 bulls, developed keratoconjunctivitis, of which 21 had received 1 vaccination and 72 had received 2 vaccinations. In the unvaccinated group, 98 (43%) calves (69 heifers and 29 bulls) developed keratoconjunctivitis. Statistical comparisons of the double vaccinated versus control and the single vaccinated versus control revealed no significant differences. Significantly more heifer than bull calves were affected, RR 2.5 (95% CI 1.94–3.23).

In experiment 2, 141 (42%) out of 331 calves developed keratoconjunctivitis. In group 1, 57 out of 139 calves (32 heifers and 25 bulls) were affected. In group 2, 56 out of 121 calves (30 heifers and 26 bulls) developed keratoconjunctivitis. In group 3, 28 out of 71 (14 heifers and 14 bulls) developed keratoconjunctivitis. The percentage of calves affected with IBK was 41% in group 1, 46% in group 2, and 39% in group 3. There was no statistically significant difference between the vaccinated and control groups. There was a slight difference between heifers and bulls, RR 1.36 (95% CI 1.06–1.75).

Discussion

This investigation revealed no preventative effects from this autogenous bacterin being given either subcutaneously or subconjunctivally. There are many plausible explanations for the vaccination failure. Perhaps there was an inadequate immune response to the bacterin; the titers of systemic or lacrimal immunoglobulins were not evaluated. Conflicting reports exist regarding protective titers and types of immunoglobulins necessary (4,16,17). Antigen presentation is critical in development of an adequate immune response. The development of specific IgA is best achieved through direct stimulation of the mucous membranes (12,18,19).

Another possible reason for vaccine failure was varied serogroups of the M. bovis in the herd. Serotyping of the bacterial cultures was not completed during this study. Field-testing in the manner completed in this study lacks control for changes in serovars. Also, alterations in environmental factors, including level of ultraviolet light, wind conditions, and concurrent disease status, could have influenced the occurrence of keratoconjunctivitis.

Heifers were affected with keratoconjunctivitis at a higher rate than bulls, regardless of the vaccination status of the calf. These data are in contrast to those in a previous report that suggested that bulls had a higher rate of infection than heifers (20), but in keeping with those from a study where, in IBK-affected calves, higher rates of bacteria were cultured from the conjunctival sacs of females than from those of males (21). This difference in bacterial culture results between sexes is not a common finding.

This study was not designed to determine the cause(s) for possible sex-related differences in immunity. Varied innate protection may include differences in tear film properties of heifers and bulls, differences in ocular functional anatomy, or environmental conditions. Several studies conducted on humans and laboratory animals have documented physiological differences in tears, related to hormonal influence (22,23,24,25,26,27,28). Prolactin and androgen may play a major role in lacrimal regulation (23,24,25,26). Sjó́gren's syndrome, a tear film disorder in humans, affects women at a much higher rate than men (27). Blinking is one of the primary mechanisms involved in clearing the eye of contaminates, and men blink at a different rate then women (29). Blink rate has not been investigated in cattle. Differences in social structure between heifers and bulls, allowing greater contact with flies at the primary sources of infection may also play a role. Further studies will be required to investigate these gender differences. CVJ

Table 2.