Milk Containing Mycobacterium bovis as a Source of Infection for White-Tailed Deer Fawns (Odocoileus virginianus)

Setting:White-tailed deer represent the first wildlife reservoir of Mycobacterium bovis in the United States. The behavior of does with nursing fawns provides several potential mechanisms for disease transmission. Little information exists concerning transmission between doe and fawn, specifically transmammary transmission. Objective: Determine if fawns can become infected by ingestion of milk replacer containing M. bovis, thus simulating transmission from doe to fawn through contaminated milk. Design: Seventeen, 21-day-old white-tailed deer fawns were inoculated orally with 2x108 CFU (high dose, n=5), 2.5 x 105 to 2.5 x 106 CFU (medium dose, n=5), and 1x104 CFU (low dose, n=5) of M. bovis in milk replacer. Dosages were divided equally and fed daily over a 5-day period. Positive control fawns (n=2) received 1x105 CFU of M. bovis instilled in the tonsillar crypts. Fawns were euthanized and examined 35-115 days after inoculation and various tissues collected for bacteriologic and microscopic analysis. Results: All fawns in the tonsillar, high oral and medium oral dose groups developed generalized tuberculosis involving numerous organs and tissues by 35-84 days after inoculation. Three of five fawns in the low-dose oral group had tuberculous lesions in the mandibular lymph node, and one of five had lesions in the medial retropharyngeal lymph node when examined 115 days after inoculation. Conclusion: White-tailed deer fawns can become infected through oral exposure to M. bovis. Therefore, the potential exists for fawns to acquire M. bovis while nursing tuberculous does.

VS - Bovine Tuberculosis (Mycobacterium bovis) Surveillance Standards 11/2001

Bovine tuberculosis (TB) is an infectious and communicable granulomatous disease caused by the acidfast bacilli bacteria of Mycobacterium bovis (M. bovis). It is commonly a chronic, debilitating disease, but occasionally may assume an acute, rapidly progressive course. M. bovisis a widespread zoonosis that is global in magnitude and affects nearly all species of vertebrates (cattle, sheep, goats, pigs, bison, buffalo, and camelids.) Disease is spread by direct contact, inhalation of infected droplets expelled from infected lungs, and ingestion of contaminated feed or milk. In most countries, TB is a notifiable disease. Overall, TB has an important world-wide impact on animal industries and human health. Control measures are based on prevention and eradication. Surveillance is a key element for management of preventions and control programs. Surveillance for TB serves the purpose of enabling Veterinary Services to obtain an accurate picture of the scope of the disease in the US livestock populations; in the event of a disease outbreak, the course TB follows in livestock and wildlife populations for a given area over time; and permits timely intervention if the trend observed deviates from what is expected.

Characterization of Staphylococcus aureus isolates in milk and the milking environment from small-scale dairy farms of Sao Paulo, Brazil, using pulsed-field gel electrophoresis

This research aimed to evaluate the occurrence of Staphylococcus aureus isolates in milk and in the milking environment of 10 small-scale farms (<400 L/d) located in the regions of Franca and Ribeirao Preto, state of Sao Paulo, Brazil. Two-hundred twenty samples of milk were collected from individual cows, along with 120 samples from bulk tank milk, 389 samples from milking equipment and utensils (teat cups, buckets, and sieves), and 120 samples from milkers' hands. Fifty-six Staph. aureus strains were isolated from 849 analyzed samples (6.6%): 12 (5.5%) from milk samples of individual cows, 26 (21.7%) from samples of bulk tank milk, 14 (3.6%) from samples collected from equipment and utensils, and 4 (3.3%) from samples from milkers' hands. Pulsed-field gel electrophoresis typing of the 56 Staph. aureus isolates by SmaI restriction enzyme resulted in 31 profiles (pulsotypes) arranged in 12 major clusters. Results of this study indicate a low incidence, but wide distribution of Staph. aureus strains isolated from raw milk collected from individual cows and surfaces of milkers' hands and milking equipment in the small-scale dairy farms evaluated. However, the high percentage of bulk milk samples found with Staph. aureus is of public health concern because raw, unprocessed milk is regularly consumed by the Brazilian population.

Effects of in vitro selenium supplementation on blood and milk neutrophils from dairy cows

The study was designed to assess the effects of in vitro selenium addition on intracellular hydrogen peroxide production by neutrophils from the milk and blood of dairy cows. Blood from 10 dairy cows and 20 milk samples from five dairy cows were incubated with 0 mg (control) or 10μM of sodium selenite. Then, milk and blood neutrophils were submitted for evaluation of intracellular hydrogen peroxide production by flow cytometry using 2',7'-dichlorofluorescein diacetate as a probe. The selenium status of the animals was evaluated by determination of the blood glutathione peroxidase activity. The results of the present work showed that in vitro selenium supplementation leads to an enhancement in intracellular hydrogen peroxide production, which indicates an improvement in the bactericidal effects of blood and milk neutrophils even in cows with a selenium-adequate status. Thus, the present study showed that in vitro Se supplementation leads to an enhancement in intracellular hydrogen peroxide production, indicating an improvement in the bactericidal effects of blood and milk neutrophils in cows with Se-adequate status.

Effect of beverages on bovine dental enamel subjected to erosive challenge with hydrochloric acid

This study evaluated by an in vitro model the effect of beverages on dental enamel previously subjected to erosive challenge with hydrochloric acid. The factor under study was the type of beverage, in five levels: Sprite® Zero Low-calorie Soda Lime (positive control), Parmalat® ultra high temperature (UHT) milk, Ades® Original soymilk, Leão® Ice Tea Zero ready-to-drink low-calorie peach-flavored black teaand Prata® natural mineral water (negative control). Seventy-five bovine enamel specimens were distributed among the five types of beverages (n=15), according to a randomized complete block design. For the formation of erosive wear lesions, the specimens were immersed in 10 mL aqueous solution of hydrochloric acid 0.01 M for 2 min. Subsequently, the specimens were immersed in 20 mL of the beverages for 1 min, twice daily for 2 days at room temperature. In between, the specimens were kept in 20 mL of artificial saliva at 37ºC. The response variable was the quantitative enamel microhardness. ANOVA and Tukey's test showed highly significant differences (p<0.00001) in the enamel exposed to hydrochloric acid and beverages. The soft drink caused a significantly higher decrease in microhardness compared with the other beverages. The black tea caused a significantly higher reduction in microhardness than the mineral water, UHT milk and soymilk, but lower than the soft drink. Among the analyzed beverages, the soft drink and the black tea caused the most deleterious effects on dental enamel microhardness.

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2±0.24) and the DP (12.5±0.22) as compared with during early lactation (10.2±0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9±24.8 and 49.0±4.96% of cows, respectively), basal (56.2±28.1 and 54.6±7.78% of cows, respectively), or entire cytoplasm (56.8±13.4 and 61.6±14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR

Bovine mastitis caused by Mycoplasma bovis is of great economic importance to the beef and dairy industry. Here we describe a new specific real-time PCR assay targeting the uvrC gene that was developed to directly detect M. bovis from milk and tissue samples without laborious DNA purification.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of beta- and alpha(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of beta-casein being faster than that of alpha(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of beta- and alpha(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from alpha(s1)- and beta-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

[Herd problem: udder health. Retrospective study of farms assessed by the Swiss Bovine Health Service (BHS) from 1999 to 2004]

Data from 59 farms with complaints of udder health problems and insufficient quality of delivered milk that had been assessed by the Swiss Bovine Health Service (BHS) between 1999 and 2004 were retrospectively analysed. Data evaluated included farm characteristics such as farm size, herd size, average milk yield, milking system and housing system, deficits of the milking equipment and the milking practices, and bacteriological results of milk samples from all cows in lactation. The average size of the farms assessed by the BHS was larger than the size of the were evaluated, 42 showed obvious failures which the farm managers could have noticed. Only 5 of the 57 milkers carried out their work according to the generally valid guidelines of the National Mastitis Council. More than 2 basic mistakes were observed in the milking practices of 36 milkers. In 51 farms, mixed infections with several problem bacteria (those present in at least 20 % of the tested cows on a farm) were found. Staphylococcus aureus proved to be the most common problem germ. As the bacteria responsible for the herd problem (the sole problem bacteria detectable on a particular farm) Staphylococcus aureus was detected in 4 farms. The current study revealed that education in the area of milking techniques and milking practices of farmers should be improved in order to reduce the incidence of udder health problems on herd level. Staphylococcus aureus is the most important problem bacteria involved in herds with udder health problems in Switzerland. Staphylococcus aureus might be used in practice as the indicator germ for early recognition of management problems in dairy farms.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties

Based on a former study from our group, one subtype of Staphylococcus aureus was associated with high within-herd prevalence of mastitis, whereas the other subtypes were associated with a low prevalence (sporadic intramammary infection). To confirm this hypothesis, a prospective study was done in 29 Swiss dairy herds. In particular, milk samples were collected from 10 herds with Staph. aureus herd problems (cases) and compared with samples from 19 herds with only sporadic cases of with Staph. aureus intramammary infection (controls). The isolates were tested for their virulence gene pattern and genotyped by PCR amplification of the 16S-23S rRNA intergenic spacer. The patterns and genotypes were then associated and compared with epidemiological and clinical data. Confirming the hypothesis, one particular subtype (genotype B) was associated with high within-herd and within-cow prevalence of intramammary infection, whereas the other subtypes were associated with low within-herd prevalence and infected single quarters. The gene patterns and genotypes were highly related, demonstrating the genetic diversity of the genotypes. The somatic cell counts were clearly increased in herds with a genotype B problem compared with herds with infections of other genotypes. Based on the different clinical properties and treatment consequences associated with these different genotypes found in Switzerland, we recommend subtyping Staph. aureus in other countries to determine if this finding is universally applicable.

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.