A study on cow comfort and risk for lameness and mastitis in relation to different types of bedding materials.

The aim was to obtain data regarding the effects of 4 freestall bedding materials (i.e., box compost, sand, horse manure, and foam mattresses) on cow comfort and risks for lameness and mastitis. The comfort of freestalls was measured by analyzing the way cows entered the stalls, the duration and smoothness of the descent movement, and the duration of the lying bout. The cleanliness of the cows was evaluated on 3 different body parts: (1) udder, (2) flank, and (3) lower rear legs, and the bacteriological counts of the bedding materials were determined. The combination of the cleanliness of the cows and the bacteriological count of the bedding material provided an estimate of the risk to which dairy cows are exposed in terms of intramammary infections. The results of the hock assessment revealed that the percentage of cows with healthy hocks was lower (20.5 ± 6.7), the percentage of cows with both damaged and swollen hocks was higher (26.8 ± 3.2), and the severity of the damaged hock was higher (2.32 ± 0.17) on farms using foam mattresses compared with deep litter materials [i.e., box compost (64.0 ± 10.4, 3.5 ± 4.7, 1.85 ± 0.23, respectively), sand (54.6 ± 8.2, 2.0 ± 2.8, 1.91 ± 0.09, respectively), and horse manure (54.6 ± 4.5, 5.5 ± 5.4, 1.85 ± 0.17, respectively)]. In addition, cows needed more time to lie down (140.2 ± 84.2s) on farms using foam mattresses compared with the deep litter materials sand and horse manure (sand: 50.1 ± 31.6s, horse manure: 32.9 ± 0.8s). Furthermore, the duration of the lying bout was shorter (47.9 ± 7.4 min) on farms using foam mattresses compared to sand (92.0 ± 12.9 min). These results indicate that deep litter materials provide a more comfortable lying surface compared with foam mattresses. The 3 deep litter bedding materials differed in relation to each other in terms of comfort and their estimate of risk to which cows were exposed in terms of intramammary infections [box compost: 17.8 cfu (1.0(4)) ± 19.4/g; sand: 1.2 cfu (1.0(4)) ± 1.6/g; horse manure: 110.5 cfu (1.0(4)) ± 86.3/g]. Box compost had a low gram-negative bacterial count compared with horse manure, and was associated with less hock injury compared with foam mattresses, but did not improve lying behavior (lying descent duration: 75.6 ± 38.8s, lying bout duration: 46.1 ± 18.5 min). Overall, sand provided the best results, with a comfortable lying surface and a low bacterial count.

Lawsonia intracellularis-associated proliferative enteritis in weanling foals in the Netherlands.

Equine proliferative enteropathy (EPE) is an emerging infectious enteric disease caused by the obligate intracellular gram-negative bacterium Lawsonia intracellularis. EPE was tentatively diagnosed in six weanling foals, aged between 5 and 7 months. Clinical signs included depression, anorexia, ventral oedema, and weight loss. Plasma biochemistry consistently revealed severe hypoproteinaemia. The ante-mortem diagnosis of EPE was based on clinical signs, hypoproteinaemia (6/6), the detection of moderate-to-high titres of L. intracellularis antibody (6/6), and severe thickening of the small intestinal wall on ultrasonography (2/2), or L. intracellularis detected in faeces by PCR (I/2). The first foal died despite treatment and at post-mortem examination the tentative diagnosis was EPE. Three foals from the same farm, which showed similar clinical symptoms were treated with azithromycin and rifampicin; two survived. Post-mortem examination of the foal that died confirmed the tentative clinical diagnosis of EPE on the basis of the lesions found and the detection of L. intracellularis--DNA in the ileum and jejunum. The fifth foal died despite intensive treatment and the post-mortem examination revealed lymphohistiocytic enteritis, typhlitis, and widespread thrombosis in several organs. The sixth foal recovered completely after treatment. This report confirms the presence of clinical L. intracellularis infection in weanling foals in the Netherlands and shows the difficulty in reaching a definitive ante-mortem diagnosis.

Aspects of the epidemiology, research, and control of lentiviral infections of small ruminants and their relevance to Dutch sheep and goat farming.

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

Localised pyogranulomatous dermatitis due to Mycobacterium abscessus in a cat: a case report.

A case of pyogranulomatous dermatitis, caused by Mycobacterium abscessus, an unusual opportunistic Mycobacterium spp., is described in a cat. Histopathological examination of the affected skin confirmed the diagnosis and Ziehl-Neelsen staining revealed acid-fast rods. A rapidly growing mycobacterium was found after culture on a Löwenstein-Jensen medium. Real-time polymerase chain reaction for the 16S rDNA (434bp) sequence and the sequence of the rpoB gene (359bp) revealed 99% and 100% matches, respectively, with M. abscessus. This is the first report of a feline infection caused by this organism in Europe.

Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction.

Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5' untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.

Repeated high dose imidocarb dipropionate treatment did not eliminate Babesia caballi from naturally infected horses as determined by PCR-reverse line blot hybridization.

Imidocarb treatment of horses infected with Babesia caballi is supposed to eliminate the infection, but data on the efficacy of this treatment is scarce. The study presented here concerns four Paso Fino horses, which were imported into the island of Curacao on the basis of a piroplasmosis negative complement fixation test (CFT). Upon re-testing with an indirect fluorescent antibody test immediately after arrival in Curacao, two horses appeared to have antibodies to B. caballi and all horses had antibodies to Theileria equi. Subsequent testing with polymerase chain reaction combined with a reverse line blot yielded positive results for both agents in all four horses. Treatment with five consecutive doses of imidocarb dipropionate (4.7 mg/kg BW im q 72 h), temporarily resulted in negative results, but B. caballi and T. equi were detected again in the samples taken at 6 and 18 weeks after completion of the treatment. These results confirm that the CFT is not a suitable test for pre-import testing and that even high dose treatment with imidocarb may not be capable of eliminating B. caballi and T. equi infections from healthy carriers.

Presence of pro-lentiviral DNA in male sexual organs and ejaculates of small ruminants.

To be able to predict sexual transmissibility of small ruminant lenti viruses (SRLV), it is necessary to know whether or not the virus is excreted in the semen, and under what circumstances. Thus, this research focussed on establishing the presence of proviral DNA of SRLV in semen and in the male genital tract of small ruminants. After initial results established the presence of SRLV in serum, the emergence of proviral DNA of SRLV in semen and presence in blood in a group of naturally SRLV-infected individuals (13 rams and 4 bucks), was followed temporally using real-time polymerase chain reaction (PCR). The same animals were also systematically serologically monitored by enzyme-linked immuno sorbent assay (ELISA) during the breeding season (August-February). A triple monocyte-macrophage count was performed on both blood and semen using a specific monoclonal antibody in conjunction with flow cytometry. The finding that epididymal semen and tissue samples of the testes, epididymides, ampullary, vesicular, prostate and bulbo-urethral glands all tested positive for the presence of proviral DNA indicates that various male sexual organs may contribute directly to shedding of proviral SRLV DNA in ejaculated semen. Our results suggest that small ruminants show intermittent shedding of proviral SRLV DNA into epididymal as well as ejaculated semen. They also demonstrate that a single PCR-negative semen sample cannot be used as a diagnostic tool to predict that subsequent ejaculates will be SRLV-free. No significant relationship was found between numbers of monocytes and/or macrophages in blood or semen and the detection of proviral SRLV in ejaculates.

Isolation of Mycobacterium mageritense from the lung of a harbor porpoise (Phocoena phocoena) with severe granulomatous lesions.

Post-mortem investigation of a harbor porpoise (Phocoena phocoena) found dead on the beach of the island of Vlieland, The Netherlands, revealed severe granulomatous changes in the right lung lobe. Ziehl Neelsen staining demonstrated relatively large acid-fast rods. Mycobacterial culture yielded a fast-growing mycobacterium, which was identified by molecular biological methods as Mycobacterium mageritense. Autolysis prevented histopathology. It was tentatively concluded that the granulomatous changes were the cause of porpoise's death and that M. mageritense was the causative agent. This is the first report of the isolation and molecular identification of this mycobacterium in a nonhuman animal species and the first association with the marine environment.

[Diagnostic aspects of Borrelia-infections in dogs]. / Diagnostische aspecten van Borrelia-infecties bij de hond.

This paper discusses the problem of diagnosing borreliosis (Lyme disease) in dogs. A prospective cohort study in the Kempen district, a known Borrelia focus in The Netherlands, showed that dogs with the presumptive symptoms of borreliosis, episodic malaise and lameness, had significantly higher and longer lasting anti-Borrelia IgG titers than asymptomatic dogs. A small part of these dogs also had antibodies directed against the IR6 (C6) antigen which indicates persistent active Borrelia infection. A few typical case histories are presented. Dogs with episodic malaise and lameness with persistent high IgG titers are suspect of suffering from borreliosis. IR6 antibodies make this diagnosis likely. Initially, such patients should be treated with doxycyclin (10 mg/kg 1dd) for 10 days. If the symptoms recurr within a few months, a longer treatment (eg 6 weeks) should be considered. Bernese mountain dogs were strongly over-represented among the borreliosis patients in the cohort study and most high titered samples among those submitted for--diagnostic--serology appear to come from this breed, which suggests that these dogs have difficulties with clearing this tick-borne infection.

[Post-mortem diagnosis of leptospirosis in two dogs]. / Twee postmortaal vastgestelde gevallen van leptospirose bij de hond.

Leptospirosis was diagnosed post-mortem in a 2-year-old male Dogo Argentino and a 7-week-old male Foxhound puppy. The two cases were unrelated. Clinical symptoms were mainly confined to the gastro-intestinal tract. Pathological lesions were suggestive of acute leptospirosis. Leptospires infection was confirmed by serological (indirect IgM/Ig6 ELISA and MAT) and immunohistochemical techniques.

Borrelia burgdorferi infections with special reference to horses. A review.

This review discusses the literature on B. burgdorferi infections in view of the rising incidence of this infection in general and the increasing concerns of horse owners and equine practitioners. Lyme disease, the clinical expression of Borrelia infections in man is an important health problem. The geographic distribution of B. burgdorferi infections in equidae should resemble that of human cases because the vector tick involved, Ixodes ricinus, feeds on both species and, indeed, the infection has been established many times in horses. However, a definite diagnosis of the disease "Lyme borreliosis" in human beings as well as in horses and other animals is often difficult to accomplish. Although a broad spectrum of clinical signs has been attributed to B. burgdorferi infections in horses, indisputable cases of equine Lyme borreliosis are extremely rare so far, if they exist at all.

An improved ELISA for the detection of antibodies to ovine and caprine lentiviruses, employing monoclonal antibodies in a one-step assay.

An improved ELISA for the detection of antibodies to ovine and caprine lentiviruses has been developed. The assay employs two monoclonal antibodies (Mabs) directed against the major core protein (p28) of the virus in a modified double antibody sandwich blocking procedure. The modification was necessary to circumvent the consequences of using low affinity Mabs and to lower the chance of false-negative results. A novel one-step concept was developed in which washing between steps was largely omitted. The new assay was designated complex trapping blocking (CTB)-ELISA. A range of sera from sheep and goats was tested comparatively in CTB-ELISA, in an indirect ELISA and in an agar gel precipitation test. The CTB-ELISA proved sensitive and highly specific and in addition reliable and easy to perform.

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.

Development of an ELISA for detection of antibodies to glycoprotein I of Aujeszky's disease virus: a method for the serological differentiation between infected and vaccinated pigs.

A blocking ELISA was developed to distinguish between Aujeszky's disease virus (ADV)-infected and vaccinated pigs, on the basis of presence or absence of serum antibodies to glycoprotein I (gI) of ADV. The gI-ELISA detects antibodies that block the reaction of monoclonal antibodies to one or two epitopes on gI of ADV. The ADV-gI antibody response appeared between one and two weeks post-infection and persisted at a high level for at least seven months. Five of the nine ADV-vaccine strains examined were found to be "gI-negative". Pigs vaccinated with a gI-negative vaccine did not develop an ADV-gI antibody response until they were challenge-exposed to a virulent strain of ADV. The gI-ELISA is highly specific, sensitive and suitable for large-scale sero-epidemiological studies to identify infected pigs in populations vaccinated with gI-negative vaccines. The gI-ELISA provides, therefore, a basis for ADV-eradication programmes, which introduces a novel concept in the control of animal virus diseases.

Immunoblot analysis of the antibody response to ovine lentivirus infections.

The antibody response of sheep to maedi-visna virus (MVV) infection was studied, using an immunoblotting technique that identified the four major viral structural proteins: the envelope glycoprotein gp135, and internal proteins p25, p16 and p14. In sequential serum samples of two inoculated sheep, antibodies to p25 appeared first, soon followed by antibodies to p16. In two sheep with natural infections, which were sampled with longer intervals, antibodies to p25 and p16 appeared simultaneously. Antibody response to p14, which was weak and inconsistent in most cases, appeared after the p16 response. Antibodies to gp135 were detected in only one sheep and appeared 7 weeks after those to p25. Antibodies to p25 were particularly persistent. Antibody recognition patterns of 21 necropsied sheep with naturally occurring infections were compared. Sheep with lesions lacked antibodies to p14 and p16 and sometimes even p25, although these antibodies had been present in earlier stages of infection. Since the time of onset of lesions was unknown, we could not determine whether this decline in antibody activity occurred before, together with, or after the onset of lesion development.

A critical assessment of antimicrobial treatment in uncomplicated Salmonella enteritis.

The human and veterinary literature on the effect of antimicrobials on the clinical and bacteriological cure in uncomplicated Salmonella gastroenteritis is reviewed. Comparison of data on the efficacy of conventional antimicrobials (chloramphenicol, neomycin, ampicillin, amoxycillin, tetracycline, trimethoprim/sulfonamide combinations) and the newer fluoroquinolones indicate that quinolones may shorten the course of clinical disease in contrast to the conventional antimicrobials. Postconvalescent excretion of Salmonella was not affected by the conventional antimicrobials whereas the data on the fluoroquinolones in this respect are conflicting. The fluoroquinolones are the drugs of choice in human medicine for severe Salmonella infections and for the elimination of the carrier state. These drugs have not been evaluated in this respect in veterinary medicine. Well designed prospective placebo-controlled studies regarding the effect of antimicrobials, especially the fluoroquinolones, on the clinical cure and the postconvalescent shedding of Salmonella in animals are imperative to develop optimal therapeutic strategies.

Preparation and evaluation of antigens used in serological tests for caprine syncytial retrovirus antibody in sheep and goat sera.

Methods used to prepare antigens from caprine syncytial retrovirus (CSR) for use in the agarose gel immunodiffusion test (AGID) or an indirect enzyme-linked immunosorbent assay (ELISA) are described. Caprine and ovine sera were tested for antibody to CSR using the AGID test and ELISA incorporating a caprine system (CSR antigen and rabbit anti-goat IgG) or an ovine system (maedi-visna virus antigen and rabbit anti-sheep IgG). Good correlation was achieved in the results of the 3 tests when sera were devoid of antibody or were strongly positive. Variations in the results on weakly positive sera were considered to be more a matter of interpretation than due to basic differences in the reagents employed.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are sensitized with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate. The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT). All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.

Maedi-visna control in sheep II. Half-yearly serological testing with culling of positive ewes and progeny.

In 1979 a field trial was started to study the feasibility of maedi-visna control in sheep by half-yearly serological testing (by ELISA) with culling of sero-positive ewes and their progeny. In 13 commercial flocks, with a mean initial incidence of serological reactors of 17%, the sero-positive ewes and all their progeny, those of preceding years included, were culled after each half-yearly test. The percentage of sero-positive sheep decreased gradually and at the end of the second year, at the 5th test, all flocks were sero-negative. Also the 6th and 7th test did not yield sero-positive sheep. At the 8th test, however, 3 sero-positive ewes were detected in one of the flocks. A definite conclusion as to the source of infection could not be drawn. The following flock test was negative. In 2 other commercial flocks, which had a mean initial incidence of sero-positive sheep of 53%, those sero-positive and only their suckling lambs were culled. Here too, a gradual decrease in the incidence of sero-positive sheep was observed at the 2nd and 3rd test, but at the 4th test a sharp increase occurred. The programme was continued and a decrease followed until 0% was reached at the 7th test (end of third year). Age analysis of the sero-positive sheep which caused this peak revealed that the majority had been born before the start of the trial. This suggests that a 'second wave' of sero-positive sheep may be prevented and a quicker result obtained if progeny of preceding years are culled as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Maedi-visna control in sheep. I. Artificial rearing of colostrum-deprived lambs.

A field trial to study the practicability and efficacy of maedi-visna control in sheep by artificial rearing of lambs was carried out during the lambing season of 1979. Lambs were immediately separated from the dams at birth, deprived of ovine colostrum, and reared isolated from the parent flock. Bovine colostrum was given instead of maternal colostrum. Eleven farms participated in the experiment. All flocks were severely infected with maedi-visna virus: 63-100% of the ewes were seropositive as demonstrated by ELISA. Artificially reared lambs were serologically tested and positives culled at the age of 6, 12, 18, 24, 30 and 36 months. Only very few positives were found: 1/389, 1/376, 0/337, 1/223, 1/192 and 0/144, respectively. The first two sero-positive lambs occurred in one flock, and it could be ascertained that both had mistakenly been given ovine colostrum probably containing maedi-visna virus. No explanation, other than sub-optimal hygiene and isolation, could be found for the two sero-positive sheep that turned up in another flock at 24 and 30 months of age although, transplacental infection cannot be entirely excluded. It is concluded that artificial rearing of ovine colostrum-deprived lambs is an effective and practicable method for the control of maedi-visna in sheep. The method appears particularly useful when valuable genetic material has to be salvaged.