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Abstract: We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. [Copyright &y& Elsevier]

Abstract: Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal–anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx 2, eae-γ1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in⩽2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle. [Copyright &y& Elsevier]

While mammary gland tissues (MGTs) are difficult to sample without risks for cow's health or milk production, milk analysis are used in routine to assess dairy cow udder's health. This study aimed to identify, quantify, compare the milk and MGTs microbiota of macroscopically healthy dairy bovine mammary glands (MG) in order to evaluate their degree of similarity. We harvested 13 couples of milk and MGTs samples, originated from the same quarter at culling. 16S rDNA Amplicon Sequencing was performed, showing Corynebacterium as the main bacterial genus in both types of samples but generally found in the milk in higher proportions than in tissues. Species evenness was higher in MGTs while species richness was higher in milk samples. Beta diversity was significantly different between both matrices suggesting the presence of a resident microbiota in MGTs of dairy cows at time of culling partially reflected by the milk microbiota from the same quarter., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the capacity of cells from colostrum to modulate the intestinal microbial colonization, the activity of the inflammatory response, and for their influence on the development of diarrheal disease in calves. Twenty calves were distributed into two groups: COL+ (n = 10) receiving fresh whole colostrum; COL- (n = 10) receiving pooled frozen colostrum, containing no viable cells. All assessments were made before colostrum intake (D0), the next day (D2), and weekly on the 7th (D7), 14th (D14), 21st (D21) and 28th (D28) day of age. Diarrhea was assessed using a fecal score, and the systemic inflammatory status was assessed using a combination of temperature, anemia, total serum iron level, total haptoglobin concentration and the need for systemic antimicrobial treatment. The number of indicator bacteria present in the fecal population was estimated using qPCR. However, COL- calves presented more frequent signs of systemic inflammatory response including, fever at D7 (P = 0.011); indicator haptoglobin levels on D7 and D14, and lower levels of iron on D7, D14. Anemia was detected more often in the COL- calves on D21 (P = 0.043) and D28 (P = 0.016). COL- calves had a 1.66 greater chance of having elevated haptoglobin and a 1.8 greater chance of needing treatment with antimicrobials than COL+. A lower number of DNA copies of Clostridium perfringens were detected in COL+ calves on D2 (P = 0.088) and D7 (P = 0.040). Similarly, a low number of DNA copies was observed for Escherichia coli and Lactobacillus spp. (P = 0.012) in the fecal samples of COL+ calves on D7., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The vaginal microbiota has been studied in animal reproduction and fertility, in particular little information of vaginal microbes in reference to bovine reproduction and pheromone production is known. The vaginal mucosa in healthy cow is colonized by an equilibrated and dynamic composition of aerobic, facultative anaerobic and obligate anaerobic microbes. Cervico-vaginal mucus (CVM) composition, viscosity and volume vary with the cyclicity and health status of the reproductive tract. In addition, CVM contains pheromones, volatile compounds, and proteins that attract males for coitus. Commensal microbiota plays a key role in protection of the genital tract from pathogenic microbes by competition effect. In the bovine species, the microbial composition, its abundance and diversity in the female gut, vagina, urine, saliva, and feces, and the associated chemical communication remains poorly documented. The impact of microbes in the reproductive tract of cow, buffalo and certain mammals are discussed in this review. Since the microbial population diversity of CVM is modified during estrus phase it presumes that it may have a role for pheromone production in conspecific. Herein, we would like to critically discuss the current state of knowledge on microbially produced signals in animals and the role of genital and CVM microbiota in estrous cycle and pregnancy., (Copyright © 2021. Published by Elsevier Ltd.)

Fast and accurate identification of Mycoplasma bovis in cattle samples is of great importance for rational treatment and control of pneumonia, arthritis and mastitis. However, which growth conditions will allow the fastest identification of M. bovis with MALDI-TOF MS remains unclear. Therefore, growth conditions and incubation time were investigated to optimize identification of M. bovis with MALDI-TOF MS and an in-house library was constructed. Nine different M. bovis strains were inoculated in triplicate in three liquid media (B1-3). Basic broth (B1) consisted of pleuropneumonia-like organism broth, enriched with 25% horse serum and 0.7% yeast extract. B2 and B3 were additionally supplemented with 0.5% pyruvate or 520 μg/mL ampicillin, respectively. Protein extraction was performed after 24, 48, 72, 96 and 120 h of incubation (37 °C, 5% CO 2 ) and processed with Autoflex III smartbeam. Identification scores ≥1.7 were interpreted as reliable. The present study showed reliable identification of M. bovis with MALDI-TOF MS as early as 24 h after inoculation, and in broth supplemented with pyruvate, up to 120 h after inoculation. Serial dilutions showed improved survival of M. bovis in broth with pyruvate. The addition of ampicillin to prevent contamination, did not impair identification of M. bovis and state-of-the-art in-house libraries contributed to higher identification scores for M. bovis with MALDI-TOF MS., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Abstract: Primary amoebic meningoencephalitis is an acute disease of the central nervous system of humans and animals caused by Naegleria fowleri. This report describes a case of meningoencephalitis caused by N. fowleri in a crossbred, one-year-old bovine with progressive neurological signs. At necropsy there was thickening of the meninges and multifocal areas of malacia in the thalamus, caudal and rostral colliculi, parietal and occipital cortex, and cerebellum. Histologically there was multifocal necrosupurative meningoencephalitis associated with areas of malacia containing amoebic trophozoites. Immunohistochemistry of the brain was positive for N. fowleri. The disease should be included among the differential diagnosis of diseases of the central nervous system in cattle in areas where animals are exposed to hot, stagnant water. [Copyright &y& Elsevier]

Several factors are known to affect the intestinal microbiota of cattle. However, how these changes occur over time is poorly understood. This study aimed to investigate the consequences of entrance into a feedlot and the effects of virginiamycin used as a growth promoter on the bovine fecal microbiota. Two batches of beef cattle (B1, n=50 and B2, n=36) entering a feedlot operation were randomly divided into two pens: one receiving virginiamycin and one group not receiving antibiotic (control group). Fecal samples were collected at arrival, mid feedlot and at exit to slaughter. The V4 region of 16S rRNA gene was amplified and sequenced. Escherichia coli strains isolated in samples from arrival and exit of B2 were also isolated and used as indicators of antimicrobial susceptibility. Marked changes in membership and structure of fecal microbiota occurred following entrance into the feedlot. At mid feedlot, virginiamycin affected bacterial community membership, but not structure, suggesting that the antibiotic had a stronger effect on the rare, but not on the most abundant species. The use of virginiamycin had no demonstrable effect on antibiotic resistance in E. coli. The differences found between batches are suggestive that variations in study conditions are important and can strongly affect the intestinal microbiota., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) caused by infection with Mycobacterium bovis (M. bovis) and other closely related members of the M. tuberculosis complex (MTC) infects many domestic and wildlife species across Europe. Transmission from wildlife species to cattle complicates the control of disease in cattle. By determining the level of TB hazard for which a given wildlife species is responsible, the potential for transmission to the cattle population can be evaluated. We undertook a quantitative review of TB hazard across Europe on a country-by-country basis for cattle and five widely-distributed wildlife species. Cattle posed the greatest current and potential TB hazard other cattle for the majority of countries in Europe. Wild boar posed the greatest hazard of all the wildlife species, indicating that wild boar have the greatest ability to transmit the disease to cattle. The most common host systems for TB hazards in Europe are the cattle-deer-wild boar ones. The cattle-roe deer-wild boar system is found in 10 countries, and the cattle-red deer-wild boar system is found in five countries. The dominance of cattle with respect to the hazards in many regions confirms that intensive surveillance of cattle for TB should play an important role in any TB control programme. The significant contribution that wildlife can make to the TB hazard to cattle is also of concern, given current population and distribution increases of some susceptible wildlife species, especially wild boar and deer, and the paucity of wildlife TB surveillance programmes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A pilot study was implemented in the Veneto Region of Italy, aimed at classifying dairy farms which produce milk to be commercialised unpasteurised on the basis of their risk of faecal contamination of milk, which is directly correlated to the probability of a foodborne pathogen, if present in the herd and eliminated through faecal excretion, to contaminate the raw product. Factors considered to be relevant in the definition of the risk of pathogens potentially present in animal faeces to be transmitted to milk, were hierarchically structured, weighted through the application of experts elicitation methods (Analytic Hierarchy Process, Delphi) and used to categorise farms through the application of a herd questionnaire. The probability of faecal contamination of milk, and thus the risk of pathogens transfer appears to be modulated more by farm management than by the structure of the farm or the health status of the herd. Such a method, combined with the microbiological evaluation of the prevalence of faecal excretion of such pathogens, can be used to implement a risk-based surveillance programme and to apply targeted control measures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Before weaning, dairy calves are susceptible to many pathogens which can affect their subsequent performance. The use of lactic acid bacteria (LAB) has been identified as a tool to maintain the intestinal microbial balance and to prevent the establishment of opportunistic pathogenic bacterial populations. However, a consensus has not been reached as to whether probiotics may be effective in reducing the prevalence of gastrointestinal diseases in young calves. The aim of this meta-analysis was to assess the effect of probiotics on diarrhea incidence and the intestinal microbial balance. LAB supplementation has been shown to exert a protective effect and to reduce the incidence of diarrhea (relative risk, RR=0.437, 95% confidence interval (CI) 0.251-0.761). In the subanalysis, this protective effect of the probiotics against diarrhea was observed only in trials that used whole milk (RR=0.154, 95% CI 0.079-0.301) and trials that used multistrain inocula (RR=0.415, 95% CI 0.227-0.759). Probiotics did not improve the fecal characteristics (standardized mean difference, SMD=-0.4904, 95% CI -1.011-0.035) and were unable to change the LAB:coliforms ratio (SMD=0.016, 95% CI -0.701-0.733). Probiotics showed a beneficial impact on the LAB:coliforms ratio in the subanalysis that included trials that used whole milk (SMD=0.780, 95% CI 0.141-1.418) and monostrain inocula (SMD=0.990, 95% CI 0.340-1.641). The probability of significant effects (probiotic positive effect) in a new study was >0.70 for diarrhea and fecal consistency. Whole milk feeding improved the action of the probiotic effect on the incidence of diarrhea and LAB:coliforms ratio. The probability to find significant effects in the diarrhea frequency and LAB:coliforms ratio was higher (P>0.85) if the new studies were conducted using whole milk to feed calves. This paper defines the guidelines to standardize the experimental designs of future trials. LAB can be used as growth promoters in calves instead of antibiotics to counteract the negative effects of their widespread use., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The objective of this study was to determine the efficacy of 16 active compounds and 11 commercial disinfectants against Campylobacter jejuni. Two reference strains (one of avian origin and the other isolated from bovine) and two avian field strains were tested in suspension test in the presence and absence of serum. Chloramine-T, povidone-iodine (1% available iodine), cetylpiridinium chloride, ethanol, isopropanol, chlorhexidine digluconate, formaldehyde, phenol, and 10 of the 11 commercial formulations (eight of them based on quaternary ammonium compounds) showed an excellent disinfectant capability, resulting in the highest level of reduction (>6-log(10)) in colony-forming units of the four C. jejuni strains compared regardless of the presence or absence of organic material. These compounds might be helpful in the adoption of environmental control measures against C. jejuni., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The purpose was to determine IFN-g release as a response to vaccination against tuberculosis in dairy heifers under commercial settings. Four-hundred pregnant heifers from ten herds were randomly allocated into four groups: (1) unvaccinated, (2) BCG vaccinated, (3) BCG vaccinated plus a CFPP400 μg+polygen boost, and (4) BCG vaccinated plus a CFP200 μg+polygen boost, under a completely randomized blocks design. A dose of 106CFU of BCG was delivered SC in the neck, then blood samples were taken at days 0, 30, 120, 210, 300 and 720 to estimate IFN-g release in response to bovine-PPD antigen. No significant difference (P > 0.05) was observed in IFN-g release between groups at days 0 and 120. At days 30 and 210, vaccinated groups show higher IFN-g release than the control group but only difference of group 3 was significant (P < 0.05). At day 300, group 1 showed significantly higher IFN-g release. No significant difference was observed at day 720. Using IFN-g release as a surrogate for vaccine efficacy, BCG plus a boost with CFP or CFPP combined with an adjuvant that improves cellular immune response has the potential to protect cattle against tuberculosis for moderate periods of time in vaccinated cattle under commercial settings., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We studied throat swabs and corresponding serum samples collected from 1067 protein purified derivative (PPD)-tuberculin skin test (TST) positive cattle from different regions of China. The 1067 throat swabs were inoculated onto modified Löwenstein-Jensen medium for the isolation and culture of Mycobacteria. Acid-fast bacilli were identified using traditional biochemical methods, polymerase chain reaction (PCR) amplification and multiplex PCR. They were distinguished as Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains. An indirect Enzyme-Linked Immunosorbent Assay (ELISA) was applied to detect specific antibodies against bovine TB (bTB). Correlations among the ELISA, bacteriology and TST were analyzed and compared. Spoligotyping and variable number tandem repeats-mycobacterial interspersed repetitive unit (VNTR-MIRU) analysis were used to genotype the MTBC. In total, 111 strains of Mycobacteria were cultured from the 1067 throat swab samples, including 43 stains of MTBC (14 strains of Mycobacterium bovis and 29 of Mycobacterium tuberculosis) and 68 strains of NTM. Thirty-eight MTBC strains and four NTM strains were isolated from 72 throat swab samples that the ELISA determined were antibody positive; five MTBC strains and 64 NTM strains were isolated from 995 throat swab samples that were antibody negative on the ELISA. The positive isolation rates of MTBC and NTM were 38.7% (43/111) and 61.3% (68/111), respectively. The concordance rate of cultured MTBC with a positive result on the indirect ELISA for antibody was 52.8% (38/72), which was much higher than the positive rate for TST (4.0%; 43/1067). Genotyping of the 43 strains of MTBC isolated, using spoligotyping and VNTR-MIRU, showed that the 43 isolates had 26 genotypes; 16 strains had a unique genotype. Two groups of six strains and two strains, respectively, showed the same spoligotyping pattern, and belonged to the Beijing family and Beijing-like family, respectively. Combined application of spoligotyping and VNTR-MIRU typing would improve the molecular epidemiological investigation and monitoring of the etiology of bTB in China., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Microsporidia are a ubiquitous group of obligate intracellular parasites that infect all major animal groups. Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been reported worldwide in animals with importance in veterinary medicine (e.g., cats, dogs, horses, cattle and pigs). The identification of E. bieneusi in animals has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen, and the implications of the infection with this pathogen in infected animals. Considerable genetic diversity within E. bieneusi has been found with over 90 genotypes identified based on the ITS nucleotide sequence of E. bieneusi spores recovered from the feces of infected humans and animals. Both host-adapted E. bieneusi genotypes with narrow host ranges and potentially zoonotic genotypes with wide host specificity have been identified. The information presented in this review should be useful in understanding the taxonomy, epidemiology, zoonotic potential, and importance in public health of E. bieneusi., (Published by Elsevier India Pvt Ltd.)

One hundred and twenty seven Escherichia coli isolates from bovine mastitis were examined to detect the phylogenetic group/subgroups and a selection of virulence associated genes. Forty nine (38.58%) isolates belonged to group B1 the remaining isolates fell into four phylogenetic subgroups: A(0) (18.11%), A(1) (26.77%), D(1) (6.29%) and D(2) (10.23%). None of the isolates belonged to B2 group. Forty seven (37.00%) isolates were positive for at least one virulence gene, among them f17A was the most common gene, found in 20.47% of the isolates. Among the E. coli isolates, 11.81% had iucD, 9.44% f17c-A, 9.44% cnf2, 7.87% f17b-A, 6.29% afaD-8 and afaE-8, 3.14% f17d-A, 0.78% cnf1 and 0.78% clpG genes. All of the detected virulence genes were present alone or in combination with each other except clpG and f17d-A genes that were only found alone. None of the isolates contained the genes for F17a-A, intimin, P or S fimbriae., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The protective efficacy of Mycobacterium bovis BCG (1 x 10(6) single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNgamma PPD-B and IFNgamma ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future.

One hundred and two isolates of Escherichia coli O157:H7 derived from cattle were characterised by random amplification of polymorphic DNA (RAPD). Four different RAPD profiles were observed. Based on the combined results of RAPD typing and toxin genotyping, the isolates could be divided into six distinct groups.

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product. The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15-17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-fingerprinting. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms. Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.

Two new plasmid encoded beta-lactamase enzymes produced by a strain of Escherichia coli and a strain of Citrobacter freundii isolated from calf faeces have been characterised. Both enzymes were similar to TEM-1 in terms of substrate and inhibition profiles and physical properties but differed from TEM-1 in being far less susceptible to the beta-lactamase inhibitors clavulanic acid or tazobactam. In each case transfer of the plasmid E coli K12 rendered it clinically resistant to the combination of amoxycillin and clavulanic acid. The beta-lactamase from the E coli had an iso-electric point (pI) of 5.4 and was encoded on a plasmid of 95 Kbp which also mediated resistance to tetracycline, sulphonamides, apramycin, streptomycin and gentamicin. The beta-lactamase from the C freundii had a pI of 5.2 and was encoded on a 75 Kbp plasmid which also mediated resistance to trimethoprim, chloramphenicol, apramycin, gentamicin and tobramycin.

The activities of danofloxacin, a novel fluoroquinolone, and two other antimicrobials were determined in vitro against field isolates of seven Mycoplasma species of veterinary importance isolated from cattle, swine and poultry in five European countries. The minimum inhibitory concentrations (MIC) of danofloxacin, tylosin and oxytetracycline were determined against a total of 68 isolates. Danofloxacin showed excellent activity against isolates of all Mycoplasma species (range 0.008 to 0.5 microgram ml-1), but in some isolates there was evidence of reduced sensitivity to tylosin (range 0.008 to 2.0 micrograms ml-1) and oxytetracycline (range 0.008 to over 16.0 micrograms ml-1). Danofloxacin was more active than other antimicrobials against M hyopneumoniae, M dispar and M bovigenitalium, and showed activity similar to that of tylosin against M bovis and M gallisepticum. Tylosin was the most active against M synoviae and M hyosynoviae. Generally, oxytetracycline showed the poorest activity, but was superior to tylosin against M bovigenitalium. A second (final) MIC reading was taken for all isolates 14 or 21 days after the initial reading, and MIC values rose during that time. However, the increase seen in danofloxacin values (typically one to two dilutions) was less than that seen for tylosin and oxytetracycline. It is concluded that danofloxacin is highly active in vitro against all of the Mycoplasma species tested, and thus shows great potential for the treatment of respiratory and other infections caused by Mycoplasma species in cattle, pigs and poultry.

During examination of the half-lives in cattle of a series of 5-substituted diaminobenzyl-pyrimidines, it was found that replacement of the phenyl ring of trimethoprim (TMP) by bicyclic structures, particularly a quinolyl group, led to increases in half-life. The presence of a dimethylamino group on the quinolyl ring of the compound baquiloprim (BQP) conferred a half-life of about 10 hours. In contrast to TMP (half-life about one hour), BQP was well absorbed from the gastrointestinal tract in all ages of cattle, plasma concentrations reaching a plateau on the day after dosing followed by a slow decline. BQP showed the same high broad spectrum antibacterial activity as TMP, with marked synergy with sulphonamides. Its differential binding of the dihydrofolate reductases of Escherichia coli and rat liver predicted a margin of safety similar to that of TMP. The results of efficacy studies in mice in comparison with TMP showed that the longer half-life of BQP was associated with greater efficacy, and therapeutic properties superior to those of TMP in cattle were therefore predicted for BQP.

Staphylococcus aureus strains originating from humans, cows, poultry, pigs, dogs and pigeons were characterised according to the biotyping scheme of Hájek and Marsálek (1971). All strains obtained from poultry, dogs and pigeons and the majority of bovine, human and porcine strains were classifiable as belonging to different biotypes. Two types were found to be present among poultry strains isolated in Europe and Japan. The porcine strains formed a heterogenic collection. One biotype predominated in the other host species. The characteristic S aureus wall teichoic acid (beta-N-acetylglucosaminyl ribitol teichoic acid) was present in nearly all poultry and pig strains. Strains from dogs and pigeons were found to present several properties which were not in agreement with the species description given for S aureus. They did not produce acetoin from glucose and their capacity to produce acid from mannitol in anaerobic conditions was very weak or absent. They were often negative in the clumping factor (slide coagulase) test and usually did not produce hyaluronidase. The production of acid from glucose in anaerobic conditions was slower and less intensive in these strains than in the S aureus strains from other origins. The results of this study support the concept of subdividing the species S aureus into biotypes or ecotypes.

The lactoperoxidase system which had been previously shown to kill Gram-negative organisms (eg, coliforms, salmonellae, pseudomonads) in vitro, was found to be activated in vivo. The lactoperoxidase was provided by the milk and the thiocyanate either by the milk or by its secretion in the abomasum. The third factor was provided either by a H2O2 generating system (glucose oxidase and glucose) or by H2O2 producing lactobacilli. The latter occur naturally in large numbers in the abomasum of the calf.

Procedures used to prepare and test frozen vaccine against bovine tick fever are described. Blood from splenectomised calves infected separately with Babesia bovis, Babesia bigemina and Anaplasma centrale was diluted in the ratio of 3:1 with 8 M dimethyl sulphoxide in phosphate buffered saline and cooled at rates between 5 degrees C and 110 degrees C per minute to -196 degrees C. After varying periods of storage up to 369 days, blood was thawed by immersing containers in a 40 degrees C water bath and its infectivity tested in a total of 150 nonsplenectomised cattle. Subcutaneous inoculation of the cryopreserved blood, and 10-fold and 50-fold dilutions prepared from it, showed that high infectivity of all three parasites was retained during storage. The use of frozen vaccine exported from Australia to Trinidad and Tobago is briefly discussed.

Thiamineless dwarf-colony variants of Staphylococcus aureus (D strains) were compared with their homologous normal-colony variants and with normal S aureus. The majority of D strains tested in vitro exhibited biological features typical of S aureus. D strains, however, produced lower titres of alpha-haemolysin and this toxin was also produced by a smaller proportion of these strains. D strains and S aureus strains were equally susceptible to disinfectants used in dairy farming and were isolated with similar frequency from the skin of the udders of infected cows. Attempts at inducing and selecting D strains from normal S aureus by exposing the latter to antibiotics in vitro and in vivo have so far failed.

The cultural characteristics of Moraxella bovis and Neisseria ovis from eyes of cattle and sheep were examined to determine which tests precisely identified the isolates. The elongation test to distinguish the bacillary M vovis from the coccal N ovis, the nitrate reduction and the litmus milk tests were found to be the most reliable.

The pathogenic characteristics of thiamineless dwarf-colony variants of Staphylococcus aureus (D strains) were compared with those of normal S aureus isolated from the bovine udder. Intravenous injection of D strains into embryonated hen's eggs and intracranial inoculations into mice produced similar results to those with normal S aureus. Intramammary infusion of D strains into normal cows resulted in mastitis which was indistinguishable from the type produced by infusion of S aureus. Intramuscular injection of thiamine HCl into cows with chronic mastitis due to D strains did not alter the course of the disease, although normal sized S aureus colonies were isolated several days after thiamine injection.

A commercially available modified live reovirus-like (rotavirus) vaccine proved innocuous in calves deprived of colostrum and protected one out of three calves against challenge 72 h after vaccination. The vaccine was evaluated in two dairy herds in the 1977 calf season. No significant differences were observed in the incidence rates or severity of undifferentiated neonatal calf diarrhoea or rotavirus-associated late diarrhoea between calves given a placebo (76) and vaccinated (74) calves in these herds. Samples of colostrum contained specific antibodies against rotavirus and neutralisation of the vaccine virus by colostral antibody in the intestinal tract was considered the major reason for the failure of the vaccine to reduce the incidence of neonatal diarrhoea in these herds.

Nasopharyngeal swabs were taken from four groups of veal calves at intervals throughout their growth and the aerobic bacteria cultivated from the swabs counted. The calves were kept under three different husbandry systems; naturally ventilated straw-yards, fan-ventilated crates and crates with a controlled climate. The numbers of bacteria isolated varied in a complex manner; however, in one group of calves a significant proportion (P less than 0.01) of the variation in weekly bacterial counts was associated with the changes in vapour pressure and temperature which took place between two and four days previously. In calves kept at a constant temperature of 16 degrees C, the bacterial populations in the nasopharynx were at a minimum between 65 and 75 per cent relative humidity and tended to rise at humidities outside this range. There was a temporal relationship between nasopharyngeal bacteria and lung lesions. In three groups the numbers of bacteria in calves at nine weeks old were positively correlated (P less than 0.05) with lung damage observable at 16 to 18 weeks old.

Salmonella was sought in 100 normal, slaughtered cattle, most of which had been held for at least four days before slaughter. The organism was isolated from 76 cattle: from the rumen contents of 62, the ruminal lymph nodes of two and the mesenteric lymph nodes of 54. The mesenteric nodes of 35 of the cattle yielded salmonellae by direct plating; plate counts indicated that some nodes contained over 2500 organisms per gram. S typhimurium was the most prevalent serotype in the mesenteric nodes but not in the rumen; up to seven serotypes were isolated from one animal. In animals which have travelled or been held for several days before slaughter, the mesenteric lymph nodes may be a source of contamination for meat and edible offal in the abattoir.

Neonatal calf diarrhoea was studied in 115 calves of one dairy herd from January 1976 to June 1977. Two syndromes could be distinguished: a mild and short lasting 'early diarrhoea' within the first three days of life and a usually more severe 'late diarrhoea' from the fourth to the 14th day of life. The latter type of diarrhoea occurred almost exclusively during the first half of the year. Thirty-four out of 45 calves with late diarrhoea excreted rotaviruses, whereas only one of 34 calves with early diarrhoea excreted virus. In addition, rotavirus excretion was found in 11 calves that either remained healthy or had recovered from diarrhoea before virus was first detected. Similar findings were obtained in the spring of 1978 but in addition bovine coronavirus was recovered from four calves with late diarrhoea and from three healthy calves. One faecal sample obtained from a calf with diarrhoea on day 2 yielded K99+ Escherichia coli. Nearly all cows excreted rotavirus-specific antibodies in their colostrum but no relationship was found between the initial colostral antibody titre against rotavirus and the development of rotavirus-associated diarrhoea in the calf.

Staphylococcus hyicus (subsp hyicus) was found to be a frequently occurring inhabitant of the skin of cattle. Significant numbers of this bacterium were found to be present in mange lesions of cattle between one and three years old. Experimental inoculations revealed that S hyicus had a pathogenic effect on the superficially scarified skin of young cattle.

Sixty-eight methicillin-resistant Staphylococcus aureus strains were isolated from mastitis milk samples originating from 20 Belgian dairyherds. All these strains appeared to be representatives of one single strain which was probably of human origin. Evidence is presented indicating a rapid in vivo evolutionary change in this strain. The following characteristics were found to be variable: the production of beta haemolysin inversely connected with fibrinolysin (staphylokinase) activity; the production of lipase, enterotoxin B and delta haemolysin; the resistance to neomycin, chloramphenicol, tetracyclines, methicillin and spectinomycin associated with constitutive or inducible macrolide resistance.

The restriction endonuclease profiles of bacterial DNA from Brucella abortus isolates were evaluated. It was not possible to distinguish between vaccine strain 19 and virulent (biotype 1 and biotype 2) strains of B abortus. Restriction endonuclease analysis is therefore not a suitable epidemiological tool in bovine brucellosis investigations. The genetic homogeneity of the Brucella genus was reinforced by these findings.

Macrolide-lincosamide-streptogramin group B (MLS) resistant strains were found among enterococci isolated from caeca of poultry, pigs and cattle. The percentage of MLS resistance among the poultry strains was 70 per cent. The Streptococcus faecium strains were more susceptible than the other enterococcal strains to virginiamycin, a member of the streptogramin class of antibiotics. This was due to higher susceptibility of the S faecium strains to the virginiamycin component M which belongs to the streptogramin group A antibiotics. Ability to inactivate clindamycin, an antibiotic of the lincosamide class of antibiotics, was noted in an unclassified group D streptococcus strain isolated from a pig.

Chloramphenicol therapy of acute and chronic Salmonella typhimurium infection in mice did not lead to the development of chloramphenicol resistant mutants of this organism. However, chloramphenicol resistant organisms were readily produced in vitro. Transfer of chloramphenicol resistance from a donor strain of Escherichia coli X12 to a suitable recipient strain of S typhimurium 5235 occurred readily in the intestine of 15 out of 20 conventional mice, following oral administration of large doses of these strains supplemented by chloramphenicol therapy. When a similar system was used in untreated animals, only a small number of chloramphenicol resistant S typhimurium were isolated from three out of 18 mice. The virulence of chloramphenicol resistant S typhimurium produced in vitro and in vivo was similar to that of the sensitive parent strain.

Lymph nodes and other tissues from a number of sites in the viscera, carcase and head were collected from 28 cattle which had been held for at least four days before slaughter. Salmoinella was isolated from 21 of these cattle, but in al except three of them it was confined to the gastrointestinal tract and the mesenteric lymph nodes. There was no evidence, in any of the animals, of spread beyond these nodes via the circulatory system. The distribution of infection suggested that the ileum and perhaps the caecum were the primary sites for invasion of the animal. Individual mesenteric nodes were collected from a further 85 cattle, and the number of salmonellae present in each was estimated by direct plating. Salmonella was isolated from 61 of these animals, and in each of 29 animals at least one node contained more than 5000 organisms per g. The predominant serotype in the mesenteric lymph nodes was Salmonella typhimurium.

Attempts to transmit malignant catarrhal fever (MCF) from 16 bovine cases of the 'sheep-associated' form of the disease are described. On two occasions disease was transmitted to bovine calves but transmission to red deer (Cervus elaphus) was not achieved. In addition, MCF was transmitted from one experimentally affected calf to a rabbit and on another occasion directly to rabbits with material from a field case which failed to transmit to a bovine calf or red deer. Subsequently each of these isolates was readily passaged through rabbits and one was also passaged to Syrian hamsters. Tissue from MCF-affected red deer consistently produced disease on inoculation into rabbits and deer but failed to cause disease in bovine calves. Contact infection between red deer occurred once and roe deer (Capreolus capreolus) were also shown to be susceptible to infection by inoculation. Passage of MCF in rabbits with an isolate from red deer failed to produce evidence of further adaptation even after 125 serial passages. Despite the failure to transmit disease from cattle to deer or from deer to cattle it is considered probable that there is only one sheep-associated agent which causes MCF in both species. The reasons for the anomalies in transmission of this form of the disease are discussed.

The sensitivity to 18 antimicrobial drugs was examined for 66 strains of Ureaplasma sp isolated from respiratory tracts of calves suffering from enzootic pneumonia, urinary tracts of bulls and eyes of cows suffering from infectious bovine kerato-conjunctivitis. Furamizole, tiamulin fumarate, erythromycin lactobionate, malidomycin C, doxycycline hydrochloride, kitasamycin tartrate, tylosin tartrate, T-2636C, tetracycline hydrochloride, oxytetracycline hydrochloride, chlortetracycline hydrochloride, oleandomycin phosphate, furazolidone, spiramycin adipate, chloramphenicol and thiophenicol showed strong inhibiting activity on all the test strains. Among them, furamizole, tiamulin fumarate and erythromycin lactobionate were most active. Kanamycin sulphate showed weak activity on all the strains tested. The differences in origin of the test strains did not affect their sensitivity to any of the drugs.

Thirty strains of Pasteurella haemolytica which were untypable by the indirect haemagglutination (IHA) test were examined serologically by rapid plate agglutination (RPA), agar gel diffusion (AGD), crossed immunoelectrophoresis (CIE) and counter current immunoelectrophoresis (CCIE) tests. Nine serogroups were identified by CCIE. Serogroup specificity, dependent on two antigens, was present in heated saline extracts of cells. Single representative strains from two serogroups were not pathogenic for specific pathogen-free lambs.

The T1 and KH3J attenuated strains of M mycoides, well-known in Africa for immunisation against contagious bovine pleuropneumonia, were recovered from the lymph nodes of cattle at intervals up to three months after vaccination. Virulence titrations in mice indicated that the organisms recovered from cattle were of no greater virulence than the strains used for inoculation. There was, therefore, no suggestion that the use of attenuated vaccines in the field might be associated with reversion to virulence after inoculation.

Clipping and shaving remove cell and lipid layers from the stratum corneum and also modify the effects of subsequent lipid collection and microbiological sampling techniques. The damage to the epidermis caused by solvents indicates that further evaluation of some sebum collection procedures and of the effects of dips and dressings applied to the skin is required.