Quantitative evaluation of a transport-enrichment medium for Campylobacter fetus

The enrichment feature of a selective serum-based transport medium for Campylobacter fetus was quantitatively examined. Preputial samples from artificial insemination bulls were spiked with known numbers of C fetus strains and inoculated into transport-enrichment medium (TEM). The survival and multiplication of these strains in TEM under different incubation periods and temperatures were assessed by plate counts. Mean enrichment values of 3.72 log and 4.42 log were observed after incubation at 37 degrees C for two and four days, respectively. There was no significant difference in the enrichment values between the C fetus subspecies venerealis strains and a C fetus subspecies fetus strain. Incubation of inoculated TEM vials at room temperature for up to two days neither improved the growth of C fetus nor affected its subsequent enrichment when the vials were reincubated at 37 degrees C. Comparison of the survival of C fetus with and without the use of TEM under simulated transport conditions demonstrated the superiority of TEM.     

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.     

Investigation of bovine venereal campyloacteriosis in beef cow herds in New Zealand

AIMS: To determine regional prevalences of beef cow herds in New Zealand positive for Campylobacter fetus subsp venerealis antibodies in samples of vaginal mucus tested using an immunoglobulin (Ig) A enzyme-linked immunosorbent assay (ELISA), and to examine the suitability of the IgA ELISA for detecting infection with C. fetus subsp venerealis under field conditions in New Zealand. METHODS: Vaginal mucus samples (n=1,230) collected from beef cow herds (n=125) throughout New Zealand (approximately 10 samples/herd) were tested for antibodies to C. fetus subsp venerealis using an IgA ELISA. Test results were compared between herds classified as having low, medium and high fertility based on pregnancy test results interpreted in relation to the duration of the mating period used. In addition, a small number of samples were collected from dairy cows that were mated using artificial insemination (AI) and had no contact with breeding bulls. The influence of putative risk factors for the spread of venereal disease and the effect of sample quality on the status of herds according to test results was assessed using multivariate logistical regression. Preputial washings from 54 bulls from nine herds classified as low fertility in which mucus samples from > or =3 cows were IgA antibody-positive were cultured for the presence of Campylobacter spp, and isolates of C. fetus subspecies were characterised using a polymerase chain reaction (PCR) test. RESULTS: One or more mucus samples was positive to the IgA ELISA in 70% of all herds tested. The prevalence of IgA antibody- positive individuals was >20% in most regions of New Zealand and did not differ significantly for cows from herds classified as high, medium or low fertility (28%, 26% and 23%, respectively; p=0.39). No relationship was found between mucus antibody status and age of breeding group, herd size, herd fertility, number of herds that female replacements or breeding bulls were sourced from, whether a serving ability test (SAT) was used to assess bulls, or the quality of samples submitted to the laboratory. Campylobacter fetus subsp venerealis was not cultured from any of the 54 bulls sampled. Four other species of Campylobacter and related organisms were cultured, viz Arcobacter cryaerophilus, Campylobacter jejuni, Campylobacter fetus subsp fetus and Helicobacter cinaedi. CONCLUSIONS: The specificity of the IgA ELISA as a diagnostic test for C. fetus subsp venerealis was found to be unsatisfactory under New Zealand conditions. It is possible that an immunological response by cows to Campylobacter species other than C. fetus subsp venerealis caused cross-reactivity in the IgA ELISA. The results do not support the hypothesis that C. fetus subsp venerealis is widespread in New Zealand.     

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.     

Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium.

One hundred and five bulls from an artificial insemination unit were tested for the presence of Campylobacter fetus subspecies venerealis. The method involved the inoculation of preputial samples into a new transport enrichment medium prior to culture and immunofluorescence tests. Seventeen bulls (16%) were found to be either positive or suspected carriers of C. fetus at one or more sampling times. The average age of these 17 bulls was about two years greater than the average age of all the bulls in the unit. A combined treatment of vaccination and dihydrostreptomycin sulfate injection suppressed or eliminated the organism from carrier bulls. The use of transport enrichment medium has increased our capability and effectiveness to monitor the presence of C. fetus in artificial insemination bulls.     

Development of a new selective medium for isolation of methicillin-resistant Staphylococcus aureus

A new selective medium containing cephem antibiotics was developed for isolation of methicillin-resistant Staphylococcus aureus (MRSA). MRSA colonies on a medium containing ceftazidime (CAZ) were most easily identifiable and a medium containing cefoperazone (CPZ) was superior in suppressing the growth of other bacteria. With the medium containing a couple of CAZ and CPZ, MRSA and methicillin-resistant coagulase-negative staphylococci (MRCNS) were detected from 2 and 1 of 15 chicken meat samples respectively. The MRSA and MRCNS recovery test showed that the medium was effective for MRSA isolation, suppressing the growth of other bacteria efficiently. These results suggested that the medium containing a couple of CAZ and CPZ was useful for MRSA detection from foods and animals.     

Control of Campylobacter fetus in artificially contaminated bovine semen by incubation with antibiotics before freezing

Fresh, diluted semen containing 1.55 X 10(6) cfu/ml of Campylobacter fetus subsp. venerealis was incubated with 500 iu of penicillin, 500 micrograms of streptomycin, 160 micrograms of lincomycin and 300 micrograms of spectinomycin per ml at 35 degrees C for 0, 1, 2, 5, 10, 20 or 40 minutes. The semen was cooled to 5 degrees C, packaged in 0.25 ml French straws and then frozen in liquid nitrogen for 2 weeks. Immediately after thawing and removal of the antibiotics by centrifugation semen samples from each of the seven treatment groups were cultured as for C. fetus. Semen samples were also examined by in-vitro tests for sperm motility prior to and post-freezing. Incubation with the antibiotics for 5, 10, 20 or 40 min prior to freezing reduced the numbers of C. fetus in the semen to non-detectable levels in 38%, 69%, 88% and 100% of samples respectively. The incubated semen showed no significant reduction of sperm motility although fertility trials have not been done.     

Detection of toxoplasmosis in experimentally infected goats by PCR

PCR was used to diagnose toxoplasmosis in two pairs of Barbari goats infected by oral administration of doses of either 10(4) or 10(5) oocysts of Toxoplasma gondii. Blood and lymph node aspirates were collected from the infected goats and control goat at intervals, and tissues were also collected from a fetus that was aborted and a doe that died during the trial. Both processed and unprocessed samples were used for the PCR, using primers directed to the multicopy B1 gene. None of the blood samples was positive, but a specific signal was obtained from the lymph node aspirates after partial DNA extraction. Direct PCR of the lung, muscle and mesenteric lymph node of the doe and lung tissue of the aborted fetus yielded the target fragment. The simplified PCR protocols, including partial DNA extraction and direct assay of lung tissue, were effective for the diagnosis of toxoplasmosis.     

Evaluation of Feed Mixtures Amended with Processed Food Waste as Feedstuffs for Finishing Lambs

This study was undertaken to evaluate the acceptability, growth performance, and diet digestibility of feed mixtures containing food wastes that were further heat processed for growing-finishing lambs. The first experiment consisted of two feeding trials in which 36 lambs in each trial were fed 1) total mixed rations (TMR) containing extruded feed mixtures that were amended with pulped plate waste (PW) collected from university dining centers or 2) control TMR containing corn, soybean meal, and ground hay in Trial 1 (T1) or soybean hulls in Trial 2 (T2). On a DM basis, the treatment TMR in T1 contained 23.9% PW, and the treatment TMR in T2 contained 24.1% PW. The PW used in this study contained 37.5 ± 9.4% DM, 33.0 ± 8.2% CP, 14.7 ± 5.5% ether extract (EE), and 12.6 ± 5.2% ADF. During T1, lambs fed the control TMR (compared with lambs fed PW) had greater (P<0.05) ADG (299.6 ± 36.3 vs 259.0 ± 54.5 g), less (P<0.05) ADFI (811.1 ± 64.0 vs 2,113.6 ± 748.6 g), and greater (P<0.05) gain:feed (0.37:1 vs 0.24:1 g). During T2, no differences between the control lambs and lambs fed PW were observed in terms of ADG (mean = 231.6 ± 40.9 g), ADFI (mean = 1,766.1 ± 168.0 g), and gain:feed (mean = 0.12:1 g). The second experiment consisted of three digestibility trials (T3, T4, and T5). In T3, 24 wethers were fed an extruded mixture (EF) containing soybean hulls, rolled shelled corn, and 24.5% pulped PW (DM basis). In T4, 28 wethers were fed a dehydrated mixture (DF) containing wheat middlings and ground food waste originating from retail groceries in a 75:25 (DM basis). In T5, 28 wethers were fed an extruded mixture (EFM) containing soybean hulls, rolled corn, and 42.3% pulped PW (DM basis). Apparent DE of EF was determined to be 3.22 ± 1.50 Mcal/kg. Apparent digestibility coefficients of EF were 54.50 ± 2.68% DM, 42.60 ± 4.39% ADF, 60.30 ± 15.68% CP, and 76.23 ± 3.77%EE. Apparent DE of DF was determined to be 3.17 ± 0.24 Mcal/kg. Apparent digestibility coefficients of DF were 64.98 ± 4.76% DM, 74.99 ± 4.91%protein, 15.70 ± 10.84% ADF, and 79.39 ± 5.07 EE. Apparent DE of EFM was determined to be 2.66 ± 0.18 Mcal/kg. The EFM apparent digestibility coefficients were 54.95 ± 4.16% DM, 62.61 ± 7.25% CP, 27.21 ± 7.77% ADF, and 79.78 ± 6.67% EE. This study indicates that amending traditional diets with dehydrated or extruded mixtures containing food waste is a practical processing method for including food wastes in sheep diets. However, acceptability of these diets by individual sheep may vary, and inconsistent growth performance levels and feed efficiencies may occur.     

A simple technique for mass cultivation of Campylobacter fetus.

Studies using 86 media for maximum growth of Campylobacter fetus for antigen production showed that a diphasic medium (solid base with liquid overlay) was most suitable. The solid base was double strength cystine heart agar. The liquid overlay was thioglycollate medium of Brewer (135-C) without agar. This medium yielded maximum growth of C. fetus in six days with good motility, less clumping and less filament formation than all other media tried.     

Isolation and characterisation of fetal bovine brain cells in primary culture

Primary cultures and cryopreservation procedure of bovine brain cells were established as in vitro experimental systems to study the responses of bovine brain cells to neuropathogenic agents. Brain cells were dissociated by papain from the cerebellum of a bovine fetus at 90 to 120 days old, and were cultured in different media. In a medium containing 1 per cent fetal bovine serum (FBS), neuronal cells were maintained and they formed clusters on glial and fibroblastic cell sheets. In a medium containing 10 per cent FBS, the proportion of neurones decreased, and fibroblastic and microglial cells dominated. In a serum-free medium containing epidermal growth factor, the highest neuronal proportion was obtained. Optimal cryopreservation condition for the brain tissues was investigated by changing the concentrations of DMSO and FBS. Brain cells could be cultured from cryopreserved tissue with only slightly reduced growth profiles and varying cell proportions in comparison to those prepared from fresh tissue.     

A comparative study of the growth of Campylobacter fetus strains in liquid media

The growth of C. fetus fetus, C. fetus venerealis and C. fetus venerealis biotype intermedius was examined in 10 liquid media. From the data obtained, a 10% inoculum size and an oxygen level of 6% seemed imperative for consistent growth, especially for the C. fetus venerealis strain. A lowered redox potential obtained by the addition of 0, 1% cysteine-HC1 to the media was stimulatory. The medium which yielded the best growth was the one described by Dennis & Jones (1959). The fastidious C. fetus venerealis strain yielded maximum values of 0,5% packed cell volumes after 48 h cultivation in a microaerophilic atmosphere on this medium. The other strains yielded higher values.     

Lung lesions in bovine fetuses aborted by Brucella abortus.

Considering the poor facilities available for microbiological diagnosis in some countries where Brucella abortus is a frequent cause of bovine abortion, a study was conducted to determine if isolation of B. abortus from an aborted bovine fetus could be predicted from a detailed histological study of the formalized lung. Thirty-nine samples of B. abortus positive and 20 negative fetal samples were examined for the presence of 14 different pulmonary lesions. Differences in the frequency of observed lesions between the positive and negative groups, were determined by odds ratios and chi square statistic. The confidence of the prediction was calculated by means of the logistic computer model. The frequency of eight lung lesions was found to be significantly (p less than 0.05) different between the groups; nevertheless, these lesions were not specific enough to be able to incriminate B. abortus as the cause of abortion.     

XX/XY mosaicism in lymphocyte cultures from a pig with freemartin characteristics

Sir. — Cultural confirmation of venereal campylobacteriosis in cattle can only be achieved if samples arc cultured on the day of collection or a suitable transport medium is used. Clark et al.⁽³⁾ have developed a selective enrichment medium for Campylobacter fetus venerealis which is also an effective transport medium ⁽⁸⁾. However, this transport medium is very cumbersome to prepare in that it requires a special gas mixture and large quantities of fresh bovine serum. This letter reports on the evaluation of Clark’s ⁽⁸⁾, cooked meat medium with antibiotics (CMMA), Weybridge ⁽⁴⁾, modified EMJH ⁽⁵⁾, Stuart’s (Oxford) and Cairy Blair (Oxoid) media, as transport media for C. fetus veneralis.     

Comparison of Pulsed Wave and Color Doppler Myocardial Velocity Imaging in Healthy Dogs

Background: Tissue velocity imaging (TVI) is increasingly used in small animal cardiology. Tissue velocity of the myocardial wall can be measured by pulsed wave (PW) or color Doppler (CD) imaging methods. Currently, the same reference ranges are used for PW TVI and CD TVI methods. However, if and how both methods correlate, and whether they can be used interchangeably, have not been assessed in small animals. Objectives: To compare the results of PW TVI and CD TVI measurements. Animals: Seventy‐one healthy dogs. Methods: Longitudinal myocardial velocity profiles were recorded from the 4‐chamber left apical view. Peak maximal systolic (S), early (E), and late diastolic (A) velocities were measured off‐line in a blinded fashion in the septal and lateral left ventricular wall by PW TVI and CD TVI. Differences between peak PW TVI and CD TVI waves were analyzed by a paired t‐test. Regression analysis and Bland‐Altman difference plots also were used to assess agreement between methods. Results: There was a significant correlation between PW TVI and CD TVI (P < .001). However, S, E, and A waves measured by PW TVI were significantly higher than the CD TVI values (P < .001). Peak systolic and diastolic PW velocities were approximately 2.20 cm/s higher than corresponding mean CD TVI velocities. Conclusions and Clinical Importance: PW TVI measurements are significantly higher compared with CD TVI measurements. Theses differences are clinically relevant. These methods should not be used interchangeably, and different reference ranges for PW TVI and CD TVI should be used.     

The prevalence of bovine venereal campylobacteriosis in cattle herds in the Lake Chad basin of Nigeria

The prevalence of bovine venereal campylobacteriosis (BVC) was investigated in the Lake Chad basin of Nigeria. Preputial washings and cervico-vaginal mucus samples were obtained from 270 cattle presenting a history of abortion and lowered fertility, kept in traditional and institutional farms. All the samples investigated were cultured using standard bacteriological technique. Campylobacter fetus was isolated from six bulls and four cows. In all cattle sampled, the isolation rates were 2.2% for C. fetus subsp. venerealis and 1.5% for C. fetus subsp. fetus; the herd and within-herd prevalence rates for C. fetus were 22.2% and 3.4%, respectively, while the overall active infectivity rate was 3.7%. BVC probably contributes to lowered fertility and abortions found in cattle in the Lake Chad basin of Nigeria, associated more with C. fetus subsp. venerealis than C. fetus subsp. fetus.     

Chromosomal trisomy in an anormalous bovine fetus

This case report describes an anomalous male fetus of approximately six months gestation with abnormal karyotype containing an extra metacentric chromosome of medium size. The fetus had severe phenotypic abnormalities including anasarca, eye malformations, cardiac hypertrophy, pulmonary hypoplasia, adrenal dysplasia, and an umbilical hernia.     

Mummified Fetus in the Thoracic Cavity of a Domestic Short-haired Cat

This short communication describes the diagnosis, treatment, and clinical course of a domestic short-haired cat with diaphragmatic hernia in which the herniated structure in the thoracic cavity contained a mummified fetus. The cat was pregnant when rescued from the street and, days later, gave birth without abnormalities. Some months later, during an ovariohysterectomy, an abnormal localization of the uterus was observed, and at that time the case was referred to our center. A thoracic radiograph showed an abnormal thoracic mass cranial to the heart. The main suspicion was the presence of a thoracic hernia with the uterus herniated and containing a mummified fetus. A thoracotomy was performed to confirm the nature of the mass and reduce the diaphragmatic hernia. Although this clinical case is quite rare, a mummified fetus can be observed in thoracic hernias.     

The development of a transport and enrichment medium for Campylobacter fetus

A transport and enrichment medium was developed for Campylobacter fetus. From inocula of between 10 and 35 organisms the medium was able to support the multiplication of 19 of 21 strains of C. fetus if the medium was incubated immediately after inoculation; when incubation was delayed for 3 days after inoculation, only seven of the 21 strains multiplied. From inocula of 100-350 organisms all 21 strains multiplied following immediate incubation, and 20 of 21 when incubation was delayed for 3 days. From inocula of about 10(4) organisms all strains multiplied following immediate or delayed incubation. The medium restricted the growth of Proteus vulgaris and Pseudomonas aeruginosa.