Host and environmental reservoirs of infection for bovine digital dermatitis treponemes

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.     

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.     

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis.     

High prevalence of treponemes in bovine digital dermatitis-a molecular epidemiology

To validate the epidemiology of Treponema spp. associated with digital dermatitis (DD) a large number of DD samples (n=56) were examined by DNA-DNA dot blot analyses using oligonucleotide probes specific for phylogenetic group I-VII of oral treponemes and DD-associated phylotypes DDKL-4, DDKL-12 and DDKL-20 as well as for T. brennaborense and T. socranskii. Positive hybridisation results were obtained for phylogenetic groups I, II and IV and phylotypes DDKL-4 and DDKL-12. While phylotype DDKL-4 was detected in 100% of the samples treponemes belonging to phylogenetic group TRE I, TRE II and TRE IV were prevalent in nearly 80% of the samples and phylotype DDKL-12 was detected in 66.1% of the samples. Analysis of Treponema groups present concurrently in the same sample revealed that a combination of TRE I-TRE II-TRE IV-DDKL-4 was most prevalent and could be detected in up to 71% of the samples. These data indicate that this combination of different Treponema spp. seems to be the most important one in the pathogenesis of DD. In contrast, T. brennaborense originally isolated from DD material this treponeme was not detected in any of the samples clearly indicating that this species is not absolutely associated with DD and therefore may represent only an incidental treponeme. Fluorescence in situ hybridisation (FISH) obviously highlights the invasive character of DD-associated treponemes. Mainly treponemes belonging to phylogenetic group TRE I and phylotype DDKL-4 were detected in high numbers compared to the total number of bacteria and also in deeper layers of the epithelium at the transition of unaffected and affected tissue. Our results confirm a high prevalence and diversity of Treponema spp. in DD lesions. In addition, our data indicate that certain combinations of Treponema spp. are detected much more frequently than others. Furthermore, Treponema spp. appears at the interface between healthy and diseased tissue underlining their importance for the pathogenesis of DD.     

Bovine digital dermatitis and severe virulent ovine foot rot: a common spirochaetal pathogenesis

A potential pathological role for spirochaetes in bovine digital dermatitis (bovine DD) and severe virulent ovine foot rot (SVOFR) has been considered and a treponeme isolate obtained from each disease in the UK. In this work, we have investigated the hypothesis that the two diseases may have a shared (common) spirochaetal aetiology. Experiments were designed to identify serological similarities and differences between the two spirochaetes; an enzyme-linked immunosorbent assay (ELISA) was developed to detect anti-treponeme antibodies in the sera of cows and sheep against the two-treponeme isolates. Sera were further tested for antigen reactivity by Western blotting. Cattle and sheep with bovine DD and SVOFR, respectively, had increased seropositivity rates to both treponeme isolates, with different patterns of reactivity between farms. In some cattle herds, significant correlations were shown between antibodies to bovine DD treponemes and SVOFR treponemes (P<0.001). In other herds, there was no apparent cross reaction, suggesting the presence of more than one treponeme in bovine DD on some farms. There was no significant correlation between the two treponeme isolates when ELISA-tested against 58 sheep sera from SVOFR cases (P>0.05); sheep showed strong evidence of reactivity to one or the other treponeme antigens, but never to both. Western blotting against both treponeme antigens showed that they frequently displayed different antigen epitopes, although some minor bands were common to both organisms. The data suggest that there are a number of spirochaetes in UK farms, which could be involved in the pathogenesis of either bovine DD or SVOFR.     

The hardness of horn in different segments of the bovine claw

Hardness of bovine hoof horn was tested as ball indentation hardness and as shore D hardness post mortem in different segments of the hoof wall, in the sole and the hard bulb of sound claws of 10 Austrian Holstein Friesian cows. Both methods of hardness determination showed corresponding results, with shore D hardness between 52.2 and 63.9 hardness units (hu) and ball indentation hardness between 11.2 N/mm2 and 24.3 N/mm2. Bovine hoof horn becomes significantly softer from the coronary band towards the weight bearing border (vertical decrease) and from the dorsal wall towards the heel (horizontal decrease). Decreasing hardness was associated with decreasing dry matter content. Measurements of the claw capsule showed thickness of the hoof wall increasing from the coronary border towards the sole. In dorsopalmar/-plantar direction, bovine hoof wall at the weight bearing border decreases towards the heel.     

Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle

A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes. An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting. Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B. burgdorferi and the treponemes. Significant correlations were shown between antibodies to B. burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes. In Western blotting of B. burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands. For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa. Polyclonal antibodies to Treponema denticola and T. vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen. Monoclonal antibodies to B. burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting. The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis.     

Keratinopathogenic mould fungi and dermatophytes in healthy and diseased hooves of horses

Specimens of hoof horn from 187 horses were examined for a possible relationship between clinically affected hooves and the occurrence of pathogenic fungi. Specimens were taken from the coronary band and from the stratum externum and medium of the coronary horn and transferred on to Sabouraud dextrose agar, with and without cycloheximide, and incubated at 28 degrees C. Dermatophytes and mould fungi were identified by their macroscopic and microscopic characteristics. The 732 isolates could be assigned to 26 species of moulds, two different species of the dermatophyte Microsporum and three different species of the dermatophyte Trichophyton. Depending on their pathogenic potential they were assigned to three groups: (i) fungi known to be keratinopathogenic (Acremonium blochii, Alternaria alternata, Alternaria chlamydospora, Geotrichum candidum, Microsporum ferrugineum, Microsporum gypseum, Scopulariopsis brevicaulis, Trichophyton species, Trichophyton mentagrophytes, Trichophyton sch?nleinii, 57 isolates), (ii) a group of uncertain pathogenicity (223 isolates), and (iii) a group of non-pathogenic species (452 isolates). Eighty per cent of the samples from horses with hoof horn lesions and 66.7 per cent of the samples from horses with slightly affected hoof horn contained fungi of the keratinopathogenic group, whereas only 8.9 per cent of the samples from horses with healthy hoof horn contained fungi of this group. There were no significant correlations between the clinical data and the age, sex or breed of the horses or their bedding and hygiene. Twelve species of fungi were isolated from the air in the horses' stables, but none of them belonged to the keratinopathogenic group.     

Locomotion scores and lying behaviour are indicators of hoof lesions in dairy cows

Locomotion scoring, lying behaviour and lesion recording during hoof trimming are all ways of evaluating hoof health in dairy cows. The objective of this study was to evaluate the relationship between these measures in a random sample of 1340 cows from 42 Danish dairy herds. The hypothesis was that locomotion scoring and/or the monitoring of lying behaviour could be used as tools to identify cows with hoof lesions, either of the horn or of the skin. Cows were locomotion scored, lying behaviour recorded and data on hoof lesions seen during hoof trimming collected. The results were analysed using logistic regression with hoof lesion as the outcome and locomotion score (1-5), mean duration of lying bouts, parity and lactation stage as explanatory variables. This analysis was undertaken for all types of lesions, for hoof horn lesions only and for skin lesions only. Odds of all hoof lesions and of skin lesions increased with increasing locomotion score and increasing mean duration of lying bouts. Odds of horn lesions also increased with increasing locomotion score, but there was no significant association between horn lesions and the mean duration of lying bouts. It was concluded that locomotion scoring and duration of lying bouts may be used as tools in the management of hoof health in dairy herds.     

Detection and molecular characterisation of bovine polyomavirus in bovine sera in New Zealand

AIM: To investigate the prevalence of bovine polyomavirus (BPyV) DNA in commercial batches of bovine serum products, cell lines and cattle in New Zealand and to characterise the viral DNA detected. METHODS: Two nested polymerase chain reaction (PCR) assays were applied to detect BPyV in bovine sera. One was used to screen for the VP1 gene of BPyV DNA in: 140 batches of commercial bovine serum products, including 66 batches of fetal bovine serum (FBS), 34 batches of calf serum, and 40 batches of adult bovine serum (ABS)/plasma; 112 individual adult bovine sera; and 16 cell lines of various species origin. Fifty batches of serum samples were also tested, using the second nested PCR assay that screened for the Large T gene. Restriction fragment length polymorphism (RFLP) was conducted with 36 PCR products amplified from the VP1 gene of BPyV using EcoRI. Five selected VP1 PCR products were subjected to DNA sequencing and phylogenetic analysis. RESULTS: BPyV DNA was detected in 46 (70%) batches of FBS, 11 (32%) batches of calf sera and two (5%) batches of ABS/plasma, an overall prevalence of 42%. None of 112 adult bovine sera was BPyV-positive. RFLP analysis demonstrated a uniform digestion pattern in the majority (31/36) of amplicons tested, while the remaining PCR amplicons did not show enzyme cleavage. Sequence analysis of the PCR products (a 263 base pair (bp) fragment of the VP1 gene) obtained from five batches of FBS showed 96.2-98.9% homology to that of published sequences of BPyV. CONCLUSION: BPyV is a frequent contaminant of commercial bovine serum in New Zealand. The incidence of BPyV in adult bovine serum products is much lower than in FBS and calf serum. Genomic variations exist among different viruses. The clinical significance of the high prevalence of BPyV DNA in bovine serum products is yet to be determined.     

Evidence for Parachlamydia in bovine abortion

Bovine abortion of unknown infectious aetiology still remains a major economic problem. In this study, we focused on a new possible abortigenic agent called Parachlamydia acanthamoebae. Retrospective samples (n=235) taken from late-term abortions in cattle were investigated by real-time diagnostic PCR for Chlamydiaceae and Parachlamydia spp., respectively. Histological sections of cases positive by real-time PCR for any Chlamydia-related agent were further examined by immunohistochemistry using specific antibodies. Chlamydophila abortus was detected only in three cases (1.3%) by real-time PCR and ArrayTube Microarray playing a less important role in bovine abortion compared to the situation in small ruminants in Switzerland. By real-time PCR as many as 43 of 235 (18.3%) cases turned out to be positive for Parachlamydia. The presence of Parachlamydia within placental lesions was confirmed in 35 cases (81.4%) by immunohistochemistry. The main histopathological feature in parachlamydial abortion was purulent to necrotizing placentitis (25/43). Parachlamydia should be considered as a new abortigenic agent in Swiss cattle. Since Parachlamydia may be involved in lower respiratory tract infections in humans, bovine abortion material should be handled with care given the possible zoonotic risk.     

Inter-observer agreement between two observers for bovine digital dermatitis identification in New Zealand using digital photographs

Aims: To assess the inter-observer agreement for detecting bovine digital dermatitis (BDD) lesions in digital colour photographs of the hind feet of cows, which had been taken while the animals were standing to be milked, between two trained observers.

Methods: Thirty-six photographs were selected from a total of 184 photographs held by the first author (R1), who had classified them as negative (n=11) or positive (n=25) for BDD. They were delivered to a technician (R2) who had previously visually inspected cattle for BDD lesions, and who then recorded the photographs as being either BDD-positive or BDD-negative. The percentage agreement between R1 and R2, and two other inter-observer agreement statistics, Cohen’s κ and Gwet’s first-order chance correction agreement coefficient (AC1), were calculated. The cumulative membership probabilities of Cohen’s κ and Gwet’s AC1 were then calculated for different benchmark ranges of κ.

Results: The percentage agreement between R1 and R2 was 33/36 (92%), Cohen’s κ was 0.80 (95% CI=0.57–1.0) and Gwet’s AC1 was 0.86 (95% CI=0.69–1.0). Based on the cumulative membership probabilities for Gwet’s AC1, there was 75% probability that the two observers had almost perfect agreement (κ≥0.81). For both Cohen’s κ and Gwet’s AC1, there was >95% probability that the two observers had at least substantial agreement (κ≥0.61).

Conclusions: The two trained observers had at least substantial agreement in identifying from a digital photograph as to whether BDD lesions were present or absent. Therefore results from the two could be used interchangeably.

Clinical Relevance: Visual assessment for BDD lesions in the milking parlour can be subjective. However a high agreement between these two trained BDD inspectors means BDD prevalence reported from different regions in New Zealand by these two can be directly compared.     

Scanning electron microscopy and fungal culture of hoof horn from horses suffering from onychomycosis

Horn samples were taken from the hooves of eight horses with clinical signs of equine onychomycosis in at least one hoof capsule. None of the horses had a documented mycological history. The predominant alterations of the horn capsules were sand cracks, white line disease, brittleness (especially around the nail holes), parakeratosis and bruising. The horn samples were stored in sterile tubes for transportation and transferred onto Sabouraud Dextrose Agar and dermatophyte test agar for mycological examination within 6 h. Fungal cultures were incubated for 30 days at room temperature. Fungal identification was based on colonial morphology and microscopic examination of conidia. Horn samples were also stored at ?80°C until used for scanning electron microscopy (SEM). The fungal culture revealed that the hoof horn from all eight horses was infected with keratinophilic fungi. The keratinopathogenic fungi Trichophyton spp and Scopulariopsis brevicaulis were also detected in six horses. SEM revealed severe alterations of the horn structure in horn samples infected with keratinopathogenic fungi compared to horn samples from a sound hoof. The most evident changes were deterioration of the tubular structure of the horn wall, disruption of the horny layers, superficial lysis of cornified cells and the presence of fungal elements. Samples without dermatophyte or Scopulariopsis infection, in contrast, were similar to healthy hoof horn.     

Simultaneous occurrence of selected food-borne bacterial pathogens on bovine hides, carcasses and beef meat

The aim of this study was to determine the simultaneous occurence of Salmonella spp., L. monocytogenes, verotoxigenic E. coli (VTEC), and Campylobacter spp. in slaughtered cattle and in beef meat subjected for human consumption. A total of 406 bovine hides and 406 corresponding carcasses were used to collect the samples with a swab method after exsanguination and evisceration of animals, respectively. Furthermore, 362 beef meat samples were purchased in local retail shops over the same period of time as for the bovine samples. Food-borne bacterial pathogens were identified with standard ISO methods with some modification by the use of PCR for VTEC. The isolated bacteria were then molecularly speciated (Campylobacter), serotyped (L. monocytogenes) and characterized for the presence of several virulence marker genes (VTEC and Campylobacter). It was found that 49 hide (12.1%) and 3 (0.7%) carcass samples were contaminated with more than one bacterial pathogen tested. Most of the hides were positive for Campylobacter spp. and VTEC (27 samples) and Campylobacter spp. together with L. monocytogenes (12 samples). Eight bovine hides contained L. monocytogenes and VTEC while L. monocytogenes and Salmonella spp. were detected in one sample. Furthermore, 3 pathogens (Campylobacter spp., L. monocytogenes and VTEC) were simultaneously identified in one bovine hide tested. In case of bovine carcasses 2 samples contained Campylobacter spp. and VTEC whereas one carcass was positive for L. monocytogenes and VTEC. On the other hand, 10 out of 362 (2.8%) minced beef samples were contaminated with at least two pathogens tested. The majority of these samples were contaminated with L. monocytogenes and Salmonella spp. (6 samples). It was noticed that equal number of C. jejuni and C. coli were found, irrespective of the origin of the samples. Most of the strains possessed more than one pathogenic factor as identified by PCR. Molecular serotyping of L. monocytogenes revealed that the majority of the isolates (27 out of 31; 87.1%) belonged to 1/2a serogroup. It was found that most of the VTEC isolates possessed the Shiga toxin stx2 gene (12 strains) whereas only 2 strains were str1-positive. The eneterohemolysin and intimin markers were identified only in 7 and 2 isolates, respectively. PCR analysis revealed that 4 VTEC belonged to O91 serogroup, 2 strains were O145 and 1 isolate was identified as O113. None of the VTEC detected in the study was O157 serogroup.     

Long-term observations on the dynamics of bovine digital dermatitis lesions on a California dairy after topical treatment with lincomycin HCl

The objective of this study was to observe the dynamics of clinical cure and recurrence of the lesions of bovine digital dermatitis for 11months after treatment with topical lincomycin HCl. The study was a clinical follow-up of 39 active bovine digital dermatitis lesions (from 29 cows). Cows with active, painful bovine digital dermatitis (BDD) lesions on the interdigital commissure of the rear feet were identified on day 0. On day 1, lesions in all cows were photographed and full-skin thickness 6mm punch biopsies were obtained for histological evaluation. All lesions on all cows were treated with topical lincomycin paste under a light bandage. On days 12 and 23, a subsample of 10 lesions was randomly selected, photographed, and biopsied. On day 37, all lesions on all cows were photographed and biopsied. After day 37, lesions were evaluated on a monthly basis. All lesions were photographed at each observation until day 341 (end of study) but only cows that had macroscopically active lesions were biopsied. Of the 39 lesions treated on day 1, 21 (54%) required re-treatment on at least one occasion before day 341. Macroscopic classification agreed well with histological classification when lesions were small, focal and active (M1 lesions) or large, ulcerative and active (M2), but agreement was variable for lesions that had healed macroscopically (M5) or that were chronic (M4). A transition model showed that M1 and M2 lesions were 27 times more likely to be an M2 lesion on the next observation than to be a healed (M5) lesion.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.     

How Structures in Bovine Hoof Epidermis are Influenced by Nutritional Factors

The structure of the hoof epidermis is the link between nutrition and horn quality. The aim of this study was to demonstrate the relationship of single structures in the process of keratinization and cornification of bovine hoof epidermis to certain nutritional factors such as lipids, minerals and vitamins. Furthermore, we wanted to show the structural changes in the dyskeratotic epidermis caused by an insufficient supply of keratinizing epidermal cells. For our study we used samples of hoof epidermis from 25 dual-purpose dairy cattle, with ages ranging between 2.5 and 4 years. We also obtained a complete set of hooves from a biotin-deficient calf. All samples were investigated by light and transmission electron microscopy, using routine methods as well as histochemical and enzyme-histochemical techniques. We focused on epidermal structures that have a major influence on horn quality and are known to be related to single nutritional factors. The strength of the keratin filament bundles is determined by their cross-linking via sulphur-containing amino acids. Essential fatty acids are required for the synthesis of an intercellular cementing substance connecting the horn cells and establishing a permeability barrier in the stratum corneum. Minerals, in particular calcium, are essential for activation of enzymes that are a prerequisite for physiological keratinization and cornification. Furthermore, vitamins such as biotin are essential in the metabolism of the keratinizing epidermal cells.     

Timing of seroconversion and acquisition of positive polymerase chain reaction assay results in calves experimentally infected with bovine leukemia virus

OBJECTIVE: To determine the interval to provirus and serum antibody detection (via PCR assay and ELISA, respectively) in calves after experimental inoculation with bovine leukemia virus (BLV). ANIMALS: 8 colostrum-deprived, BLV-negative Holstein bull calves (> or = 6 weeks old). PROCEDURES: Via IM injection, each calf received a fresh whole-blood inoculum (day 0) calculated to contain 2 x 10(6) lymphocytes. Blood samples for the ELISA and PCR assay were collected from calves immediately prior to inoculation and weekly thereafter for 7 weeks. Mean and median number of weeks to PCR-detected conversion of BLV status and seroconversion were calculated. Point sensitivity and cumulative sensitivity of the 2 assays were calculated at each sample collection. At each sampling time, the proportion of calves identified as infected by the cumulative weekly ELISA and PCR assay results was compared by use of a Fisher exact test. RESULTS: In 5 calves, conversion of BLV status was detected via PCR assay before seroconversion was identified. However, seroconversion preceded PCR-detected conversion in 2 calves. In 1 calf, both assays yielded positive results at the same test date. These differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: In experimentally inoculated BLV-negative calves, conversion of BLV status was detected via PCR assay more quickly than via ELISA; this difference was not significant and probably not clinically important. The PCR assay may be useful as a confirmatory test in animals of exceptional value; tests based on viral identification may become critically important if vaccines against BLV infection are developed and marketed.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.