Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin

OBJECTIVE: To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT). SAMPLE POPULATION: Partially purified LKT from a wild type P. haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant Phaemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. PROCEDURE: Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation. RESULTS: Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host.     

Bovine viral diarrhoea virus: its effects on ovarian function in the cow

Bovine viral diarrhoea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. In this paper we investigate how BVDV interacts with the ovary. The viruses' tropism for the pre-ovulatory oocyte was studied by indirect immunohistochemistry. Two monoclonal antibodies, raised against the non-structural protein NS3 and the envelope glycoprotein E2 were used to probe cryo-sections cut from the ovaries of three persistently infected heifers. NS3 and E2 antigens were widely distributed within the ovarian stroma and follicular cells. NS3 was also localised within the proportion of oocytes. Overall 18.7% of the oocyte population had detectable levels of NS3. What is more, the proportion of antigen positive oocytes remained constant (P>0. 05) throughout the different stages of oocyte maturation.In a subsequent study seven cows were challenged with non-cytopathogenic BVDV (strain Pe515: 5x10(6) TCID(50)) to determine the oestradiol and progesterone responses to an acute infection. The sensitivity of the endogenous luteolytic mechanism was also established by analysing plasma prostaglandin F2alpha metabolite (PGFM) levels following an exogenous oxytocin (50 IU) challenge. The inoculation was given 2 days before a synchronised oestrus and was timed to ensure that viraemia occurred during the initial stage of corpora luteal development. Seven cows inoculated with non-infectious culture medium served as control animals and remained BVDV naive throughout the study. The BVDV challenge was followed by leucopenia, viraemia and sero-conversion. The virus also significantly (P<0.01) reduced plasma oestradiol levels between day 6 and day 11 post-inoculation (i.e. between day 4 and day 9 post-oestrus). However, the infection did not alter (P>0.05) progesterone secretion throughout the oestrous cycle or the plasma concentration of PGFM. These data indicate that bovine follicular cells and oocytes are permissive to BVDV at all stages of follicular development. They also show that a transient fall in oestradiol secretion may accompany an acute infection. In conclusion, this work has identified two potential routes through which BVDV can reduce fertility in the cow, namely impairment of oocyte quality and disruption of gonadal steroidogenesis.     

Desflurane anaesthesia in the horse: minimum alveolar concentration following induction of anaesthesia with xylazine and ketamine

The minimum alveolar concentration (MAC) of desflurane was measured in 6 ponies (aged 1 or 2 years) following induction of anaesthesia with iv xylazine (1.1 mgkg bwt) followed by ketamine (2.2 mgkg bwt). The stimulus utilised was electrical; pulses of 50 volts, 10 dsec duration delivered at 5 Hz, applied for 60 s across needles inserted 1 cm apart into the buccal mucosa (n = 2) and/or the coronary band (n = 6). Stimulation was not commenced until at least 1 h following anaesthetic induction, and the time of the final MAC measurement ranged from 2.5–3.5 h. The mean (± sd) MAC of desflurane under these circumstances was 7.0% (± 0.85) with a range from 5.8%-8.3%. One pony behaved aberrantly following a positive response to the stimulus and the MAC measurement was repeated 10 weeks later. At the end of the anaesthetic period xylazine (0.2 mgkg bwt) was administered iv. Mean time to standing was 13.3 (± 2.9) min, range 9–17 min, and the quality of recovery was excellent.     

Combined use of detomidine with opiates in the horse

The effects of administration of one of four opiates (pethidine 1 mg/kg bodyweight (bwt), morphine 0.1 mg/kg bwt, methadone 0.1 mg/kg bwt, and butorphanol 0.05 mg/kg bwt) given intravenously to horses and ponies already sedated with detomidine (10 micrograms/kg bwt) were investigated. Behavioural, cardiovascular and respiratory effects of the combinations were compared with those occurring with detomidine alone. Addition of the opiate increased the apparent sedation and decreased the response of the animal to external stimuli. At doses used, butorphanol produced the most reliable response. Side effects seen were increased ataxia (greatest following methadone and butorphanol) and excitement (usually muzzle tremors and muscle twitching). Following pethidine, generalised excitement was sometimes seen. Marked cardiovascular changes occurred in the first few minutes after morphine or pethidine injection, but within 5 mins cardiovascular changes were minimal. Following morphine or pethidine there was a significant increase in arterial carbon dioxide tension. Fourteen clinical cases were successfully sedated using detomidine/butorphanol combinations.     

Protection against respiratory infection with bovine virus diarrhoea virus by passively acquired antibody

Susceptibility to infection with bovine virus diarrhoea virus (BVDV) was compared for calves with varying amounts of specific antibody in their sera passively acquired from the ingestion of colostrum. Challenge consisted of intranasal exposure to a strain of BVDV isolated from an outbreak of respiratory disease. Resistance to infection, as judged by nasopharyngeal shedding of virus, was directly related to the titre of neutralizing antibodies in sera. Besides protecting against infection of the upper respiratory tract, passive antibody, which was mainly IgG1, also protected against viraemia and, to a lesser extent, leukopenia. In the presence of colostral antibody, neutralizing and IgG1 antibody responses were apparently inhibited, but a specific IgG2 response occurred.     

The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs.

The heterogeneity of Mycoplasma ovipneumoniae isolates from the lungs of sheep with chronic non-progressive pneumonia (CNP) from the same flock raised the possibility that multiple isolates derived from one lung were not all identical. To test this hypothesis, thirty isolates were obtained from each of six pneumonic sheep lungs at slaughter. Four lungs had relatively severe lesions and from each of these, three or four strains of M. ovipneumonia, distinguishable by REA and in most cases by SDS-PAGE, were detected. From the lungs of each of two sheep with mild lesions, two strains of M. ovipneumoniae were detected. Four isolates from one lung were further examined by restriction endonuclease analysis (REA) using many restriction endonucleases. Those which differed with EcoRI also differed when other restriction endonucleases were used. However, partial digests occurred mainly with those restriction endonucleases which recognise cytosine-rich sequences. The presence of multiple strains of one species of microorganism in individual lesions is an unusual concept which may not be limited to one disease or to one host.     

Vaccination of calves with a diaminopimelic acid mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the safety and efficacy of a diaminopimelic acid mutant of Salmonella typhimurium as a vaccine for calves. Transposon techniques were used to create a stable nonreverting diaminopimelic acid mutant of a virulent S. typhimurium strain. Calves were vaccinated at weekly intervals with the diaminopimelic acid mutant given as an oral dose of 10(10) organisms, followed by two subcutaneous doses of 10(9) organisms. The calves tolerated vaccination well and the vaccine strain was eliminated from the feces within four days. Of five vaccinated calves, three survived challenge with 5 X 10(9) organisms of the parent strain whereas all five unvaccinated calves that were challenged died. The surviving calves eliminated the challenge organism from the feces within three weeks.     

Effects of cryotherapy on the palmar and plantar digital nerves in the horse

The duration of anesthetic effect and the histopathologic changes resulting from a controlled freeze of the palmar and plantar digital nerves in the horse were evaluated. Two techniques were compared: (i) nerves were frozen by direct application of the cryoprobe after surgical exposure and (ii) nerves were frozen by percutaneous application of the cryoprobe to the overlying skin. Return of skin sensation and ability to detect a stimulus were used to determine return of nerve function. The duration of anesthetic effect was significantly (P less than 0.005) longer for nerves frozen after surgical exposure than for those frozen by the percutaneous technique (mean 156 days vs 47.5 days). At the termination of the study, horses were euthanatized and all nerves were examined histologically. Neuromas-in-continuity were observed in 10 of 28 frozen nerves. Classification was based on the involvement of the supporting fibrous connective tissues of the nerve, endoneurium, perineurium, and epineurium. The direct technique of freezing the nerve after surgical exposure was repeatable and could be used to provide temporary neurectomy in the horse. The percutaneous technique caused a temporary loss of pain perception, but could not be relied on to interrupt nerve function for longer than a few weeks.     

Effect of coccidiosis on reproductive maturation of male Japanese quail

The effects of coccidiosis on reproductive development of male Japanese quail were examined. Male Japanese quail were exposed to high (5 x 10(5) sporulated oocysts/quail) or low (5 x 10(3) sporulated oocysts/quail) doses of Eimeria uzura at 16 or 30 days of age and sampled at 37 days. Quail given high doses of coccidia had reduced testes weight and lowered circulating concentrations of androgen compared with control males. Low doses of coccidia did not affect testes weight but did result in elevated plasma androgen levels. There were no differences in average testes weights by 51 days; however, plasma androgen was still reduced in most groups. To study the effects of coccidiosis on egg production, males exposed to high doses of coccidia at 16 (16H) or 30 (30H) days of age were mated with control females, and control males were mated with control or 16H females. The onset of laying was delayed for 5 days in the control male: 16H female group. During the first week of production, eggs from females bred to 30H males had lower fertility and hatchability than those bred to control or 16H males. By the third week of production, levels of fertility were similar. Apparently, exposure of quails to coccidiosis before sexual maturation might result in long-term effects on later reproductive capability.