Ovulation and fertility response to commercially available GnRH products in lactating cows synchronized with the Double-Ovsynch protocol.

This study was designed to evaluate whether commonly used gonadorelin products that are commercially available in the United States results in comparable ovulation and pregnancy per AI (P/AI) in synchronized lactating dairy cows. A total of 1411 Holstein cows receiving a Double-Ovsynch protocol (DOV) for conducting the first postpartum AI were randomized to receive one of the following GnRH products throughout the Double-Ovsynch: 1) Cystorelin® (CYS, gonadorelin diacetate tetrahydrate, n = 484); 2) Factrel® (FAC, gonadorelin hydrochloride, n = 482) or; 3) Fertagyl® (FER, gonadorelin diacetate tetrahydrate, n = 515). A subgroup of cows (n = 487) received ovarian ultrasound exams and collection of blood samples for progesterone (P4) analysis. Proportion of cows ovulating following the 3rd GnRH of DOV tended (P = 0.07) to differ between GnRH salts (hydrochloride = 61.5% vs. diacetate = 72.7%) but was similar for GnRH products (FER = 74.1% vs. FAC = 61.5% vs. CYS = 72.2%). Interestingly, a logistic regression analyses that considered the circulating P4 at the time of GnRH treatment indicated lower ovulation responses to FAC compared to FER and CYS; although greater circulating P4 decreased ovulation response to all GnRH products. Results for P/AI at 60 d post-insemination differed between GnRH salts (P = 0.02) as well as GnRH products (FER = 47.8% vs. FAC = 42.0% vs. CYS = 49.8%; P = 0.04). In conclusion, fertility following use of the Double-Ovsynch was less following a hydrochloride-based GnRH product likely due to lesser ovulatory responses throughout the synchronization protocol.

Profile of LH release in response to intramuscular treatment with kisspeptin in Bos indicus and Bos taurus prepubertal heifers.

The main objective of this study was to evaluate the effect of intramuscular (I.M.) administration of different doses of kisspeptin (Kp) on the pattern of luteinizing hormone (LH) release in Bos taurus and Bos indicus prepubertal heifers. Holstein heifers weighing 215.1 ±â€¯38.6 kg (n = 24; aged 5-8 mo) and Gyr heifers weighing 215.4 ±â€¯31.4 kg (n = 24; aged 6-10 mo) were enrolled in this study. The animals were confirmed as non-cyclical by absence of a corpus luteum (CL) as determined by ultrasound scanning of the ovaries and low circulating P4 concentrations (<1.0 ng/mL) evaluated at a 10-day interval (on day -10 and day 0). For each genetic group, heifers were randomly assigned to one of four treatments: Kp at 2.5 µg/kg body weight (Kp2.5), Kp at 5.0 µg/kg (Kp5), Kp at 10 µg/kg (Kp10), or gonadotropin-releasing hormone (GnRH) agonist (0.01 mg of buserelin acetate per heifer), all administered by I.M. injection. All animals responded to the treatments with an LH surge (P < 0.01). There was an effect of breed (P < 0.01) on induced LH release, with Holstein heifers having a greater area under the curve for LH (AUC; P < 0.01) and greater LH peak amplitude (P < 0.01) than Gyr heifers. Nevertheless, greater AUC for LH occurred in Kp10 heifers for both breeds. There was no effect of breed on LH AUC or LH peak amplitude after GnRH agonist treatment. For both breeds, heifers treated with Kp displayed an earlier (P < 0.01) LH peak with a reduced amplitude of the LH peak (P < 0.01) and reduced LH AUC (P < 0.01) compared to heifers treated with GnRH agonist. Thus, both zebu and taurus immature heifers treated with I.M. Kp injection responded with a rapid and dose-dependent LH surge, although even large doses of the native Kp-10 did not mimic the magnitude or duration of the LH surge produced by the GnRH agonist. The early onset of the LH surge after Kp treatment and the short duration suggest that the effects of Kp were likely due to pituitary rather than hypothalamic action. Finally, there seems to be a greater responsiveness to Kp in Bos taurus than in Bos indicus prepubertal heifers, although LH release after GnRH agonist treatment was similar for the two breeds.

Ruminal biohydrogenation and abomasal flow of fatty acids in lactating cows fed diets supplemented with soybean oil, whole soybeans, or calcium salts of fatty acids.

Ruminants have a unique metabolism and digestion of unsaturated fatty acids (UFA). Unlike monogastric animals, the fatty acid (FA) profile ingested by ruminants is not the same as that reaching the small intestine. The objective of this study was to evaluate whole raw soybeans (WS) in diets as a replacer for calcium salts of fatty acids (CSFA) in terms of UFA profile in the abomasal digesta of early- to mid-lactation cows. Eight Holstein cows (80 ± 20 d in milk, 22.9 ± 0.69 kg/d of milk yield, and 580 ± 20 kg of body weight; mean ± standard deviation) with ruminal and abomasal cannulas were used in a 4 × 4 Latin square experiment with 22-d periods. The experiment evaluated different fat sources rich in linoleic acid on ruminal kinetics, ruminal fermentation, FA abomasal flow, and milk FA profile of cows assigned to treatment sequences containing a control (CON), with no fat source; soybean oil, added at 2.68% of diet dry matter (DM); WS, addition of WS at 14.3% of diet DM; and CSFA, addition of CSFA at 2.68% of diet DM. Dietary fat supplementation had no effect on nutrient intake and digestibility, with the exception of ether extract. Cows fed fat sources tended to have lower milk fat concentration than those fed CON. In general, diets containing fat sources tended to decrease ruminal neutral detergent fiber digestibility in relation to CON. Cows fed WS had lower ruminal digestibility of DM and higher abomasal flow of DM in comparison to cows fed CSFA. As expected, diets containing fat supplements increased FA abomasal flow of C18:0 and total FA. Cows fed WS tended to present a higher concentration of UFA in milk when compared with those fed CSFA. This study suggests that under some circumstances, abomasal flow of UFA in early lactation cows can be increased by supplementing their diet with fat supplements rich in linoleic acid, regardless of rumen protection, with small effects on ruminal DM digestibility.

Effect of dose and timing of prostaglandin F2α treatments during a Resynch protocol on luteal regression and fertility to timed artificial insemination in lactating Holstein cows.

Our objective was to evaluate the effect of a second PGF2α treatment (25 mg of dinoprost) or a double dose of PGF2α (50 mg of dinoprost) during a Resynch protocol on luteal regression and pregnancies per artificial insemination (P/AI) in lactating dairy cows. Lactating Holstein cows (n = 1,100) were randomly assigned at a nonpregnancy diagnosis to receive (1) Ovsynch (control: 100 µg of GnRH; 7 d, 25 mg of PGF2α; 56 h, 100 µg of GnRH), (2) Ovsynch with a second PGF2α treatment (GPPG: 100 µg of GnRH; 7 d, 25 mg of PGF2α; 24 h, 25 mg of PGF2α; 32 h, 100 µg of GnRH), or (3) Ovsynch with a double dose of PGF2α (GDDP: 100 µg of GnRH; 7 d, 50 mg of PGF2α; 56 h, 100 µg of GnRH). All cows received timed artificial insemination (TAI) approximately 16 h after the second GnRH treatment (G2). Pregnancy diagnosis was performed by transrectal palpation 39 ± 3 d after TAI, and pregnancy status was reconfirmed 66 d after TAI. Blood samples collected from a subset of cows in each treatment at the first PGF2α treatment (n = 394) and at G2 (n = 367) were assayed for progesterone (P4). Data were analyzed by logistic regression using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC). At 39 d after TAI, GPPG cows tended to have more P/AI than control cows [35% (137/387) vs. 31% (107/349)], whereas P/AI for GDDP cows [32% (118/364)] did not differ from that for control cows. Pregnancy loss from 38 to 66 d did not differ among treatments and was 8% (30/362). The percentage of cows with complete luteal regression (P4 <0.4 ng/mL at G2) tended to differ among treatments and was greater for GPPG cows than for GDDP and control cows (94% vs. 88% vs. 88%, respectively). Overall, cows with P4 <1 ng/mL at the first PGF2α treatment had fewer P/AI than cows with P4 ≥1 ng/mL (27% vs. 38%), whereas cows with P4 ≥0.4 ng/mL at G2 had fewer P/AI than cows with P4 <0.4 ng/mL (15% vs. 38%). We conclude that adding a second PGF2α treatment 24 h after the first within a Resynch protocol tended to increase the proportion of cows undergoing complete luteal regression and P/AI, whereas treatment with a double dose of PGF2α at a single time did not.

Association of changes among body condition score during the transition period with NEFA and BHBA concentrations, milk production, fertility, and health of Holstein cows.

Our objective was to evaluate the association between body condition score (BCS) change during the transition period with fertility, non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) concentrations, milk yield, and health problems of Holstein cows in a retrospective cohort study. Holstein cows (n = 232) were assessed for BCS (5 point scale; 0.25 point increments) and had blood collected at 21 and 7 d before, on the day of, and 7 and 21 d after calving. Blood samples were assayed for NEFA and BHBA concentrations. All cows received a timed artificial insemination (TAI) at 65 ± 3 days in milk (DIM) following a Presynch-Ovsynch protocol with a progesterone implant during the Ovsynch protocol. Cows were grouped based on BCS change after calving as to whether they: 1) lost (L), 2) maintained (M), or 3) gained (G) BCS. Data were analyzed by logistic regression with GLIMMIX and ANOVA with repeated measures using the MIXED procedures of SAS. Both NEFA and BHBA concentrations after calving differed (P < 0.01) for cows that lost, maintained, or gained BCS from 21 d before to 21 d after calving (NEFA: 0.51 ± 0.01; 0.45 ± 0.01; 0.42 ± 0.01 mmol/L; BHBA: 0.73 ± 0.02; 0.70 ± 0.02; 0.68 ± 0.02 mmol/L; respectively; mean ± SEM). By design, BCS change after calving differed (P < 0.01) among groups and was -0.38; 0.00; and 0.35 for cows in groups L, M, and G, respectively. At 21 d before and 21 d after calving, BCS differed (P < 0.01) among groups and was [before (2.97, 2.70, and 2.57) and after (2.54, 2.70, and 2.90)] for cows in groups L, M, and G, respectively. Between evaluated days, higher circulating NEFA and BHBA concentrations were observed 7 d after calving. Change in BCS affected (P < 0.01) pregnancy/AI (P/AI), days to first ovulation, and percentage of cyclic cows at 50 DIM. At 32 d after TAI, P/AI differed (P < 0.01) for cows that lost [18% (11/84)], maintained [33% (26/80)], or gained [47% (32/68)] BCS. Cows that lost BCS during the transition period had more health events (P < 0.01), than cows that gained or maintained BCS. In conclusion, changes in BCS during the transition period affected NEFA and BHBA concentrations, fertility, and occurrence of health problems during the lactation.

Effects of whole flaxseed, raw soybeans, and calcium salts of fatty acids on measures of cellular immune function of transition dairy cows.

The objective of the current study was to evaluate the effects of supplemental n-3 and n-6 fatty acid (FA) sources on cellular immune function of transition dairy cows. Animals were randomly assigned to receive 1 of 4 diets: control (n=11); whole flaxseed (n-3 FA source; n=11), 60 and 80g/kg of whole flaxseed [diet dry matter (DM) basis] during pre- and postpartum, respectively; whole raw soybeans (n-6 FA source; n=10), 120 and 160g/kg of whole raw soybeans (diet DM basis) during pre- and postpartum, respectively; and calcium salts of unsaturated FA (Megalac-E, n-6 FA source; n=10), 24 and 32g/kg of calcium salts of unsaturated FA (diet DM basis) during pre- and postpartum, respectively. Supplemental FA did not alter DM intake and milk yield but increased energy balance during the postpartum period. Diets containing n-3 and n-6 FA sources increased phagocytosis capacity of leukocytes and monocytes and phagocytosis activity of monocytes. Furthermore, n-3 FA source increased phagocytic capacity of leukocytes and neutrophils and increased phagocytic activity in monocytes and neutrophils when compared with n-6 FA sources. Supplemental FA effects on adaptive immune system included increased percentage of T-helper cells, T-cytotoxic cells, cells that expressed IL-2 receptors, and CD62 adhesion molecules. The results of this study suggest that unsaturated FA can modulate innate and adaptive cellular immunity and trigger a proinflammatory response. The n-3 FA seems to have a greater effect on phagocytic capacity and activity of leukocytes when compared with n-6 FA.

Effects of flaxseed, raw soybeans and calcium salts of fatty acids on apparent total tract digestibility, energy balance and milk fatty acid profile of transition cows.

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NEL) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NEL intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.

High levels of whole raw soya beans in dairy cow diets: digestibility and animal performance.

The aim of this study was to evaluate the effects of high levels of whole raw soya beans in the diets of lactating cows. Twelve Holstein dairy cows were used, randomized in three 4 ×  4 balanced and contemporary Latin squares and fed the following diets: (i) control (C), without including whole raw soya beans; (ii) 80 g/kg in DM of whole raw soya beans (G80); (iii) 160 g/kg in DM of whole raw soya beans (G160); and (iv) 240 g/kg in DM of whole raw soya beans (G240). There was significant reduction (p < 0.05) in dry matter intake (kg/day) in cows supplemented with G240 compared with C (23.8 vs. 25.3 respectively). G240 diets presented lower crude protein digestibility (g/kg) (p < 0.05) in comparison with C diet (683 vs. 757 respectively). There was significant effect of experimental rations in nitrogen balance (p < 0.05), G240 diet presenting significant reduction in comparison with the other diets, and faecal excretion of nitrogen was higher for G240 diet. The concentration of ruminal ammoniacal nitrogen was significantly higher (p < 0.05) for cows receiving control diet, compared to other diets. G240 diet resulted in significantly lower milk and protein yield (p < 0.05) in comparison with C diet. Significant C18:2 cis fatty acids were observed in milk concentrations (p < 0.05) for G240 diet. The use of high level of whole raw soya beans in dairy cow diets improves the unsaturated fatty acid profile in milk, and the diets (G80 and G160) led to minor alterations in the digestive processes and animal metabolism.

Effect of raw soya bean particle size on productive performance and digestion of dairy cows.

Differing soya bean particle sizes may affect productive performance and ruminal fermentation due to the level of fatty acid (FA) exposure of the cotyledon in soya bean grain and because the protein in small particles is more rapidly degraded than the protein in large particles, which influence ruminal fibre digestion and the amounts of ruminally undegradable nutrients. The objective of this experiment was to investigate the effects of raw soya bean particle size on productive performance, digestion and milk FA profile of dairy cows. Twelve Holstein cows were assigned to three 4 × 4 Latin squares with 21-day periods. At the start of the experiment, cows were 121 days in milk (DIM) and yielded 30.2 kg/day of milk. Cows were fed 4 diets: (i) control diet (CO), without raw soya bean; (ii) whole raw soya bean (WRS); (iii) cracked raw soya bean in Wiley mill 4-mm screen (CS4); and (iv) cracked raw soya bean in Wiley mill 2-mm screen (CS2). The inclusion of soya beans (whole or cracked) was 200 g/kg on dry matter (DM) basis and partially replaced ground corn and soya bean meal. Uncorrected milk yield and composition were not influenced by experimental diets; however, fat-corrected milk (FCM) decreased when cows were fed soya bean treatments. Soya bean diets increased the intake of ether extract (EE) and net energy of lactation (NEL ), and decreased the intake of DM and non-fibre carbohydrate (NFC). Ruminal propionate concentration was lower in cows fed WRS than cows fed CS2 or CS4. Cows fed cracked raw soya bean presented lower nitrogen in faeces than cows fed WRS. The milk of cows fed WRS, CS2 and CS4 presented higher unsaturated FA than cows fed CO. The addition of raw soya bean in cow diets, regardless of the particle size, did not impair uncorrected milk yield and nutrient digestion, and increased the concentration of unsaturated FA in milk. Cows fed cracked raw soya bean presented similar productive performance to cows fed whole raw soya bean.

Modifications to Ovsynch improve fertility during resynchronization: Evaluation of presynchronization with gonadotropin-releasing hormone 6 d before initiation of Ovsynch and addition of a second prostaglandin F2α treatment.

Lactating Holstein cows (n=897) were randomly assigned to a 2×2 factorial arrangement of treatments to compare the main effects of presynchronization with GnRH 6 d before beginning an Ovsynch protocol (±GnRH) and a second PGF2α treatment 24h after the first (1 vs. 2 PGF2α) on pregnancies per artificial insemination (P/AI). This resulted in the following 4 treatments: (1) an Ovsynch protocol (GPG, control); (2) presynchronization with GnRH followed by an Ovsynch protocol (GGPG); (3) an Ovsynch protocol with a second PGF2α treatment (GPPG); and (4) presynchronization with GnRH followed by an Ovsynch protocol with a second PGF2α treatment (GGPPG). All cows were submitted for first timed artificial insemination (TAI) using a Presynch Ovsynch protocol, and cows detected in estrus after the second PGF2α treatment of the Presynch portion of the protocol were inseminated and removed from the experiment. Nonpregnant cows were resynchronized using an Ovsynch protocol initiated 32±3 d after artificial insemination. Blood samples were collected at the first GnRH treatment (G1), at the PGF2α treatment (PGF), and at the last GnRH treatment (G2) of the Ovsynch protocol and were assayed for progesterone (P4) concentrations. Overall, P/AI tended to be greater for cows receiving a second PGF2α treatment compared with cows not receiving the second PGF2α treatment (40 and 37% for GGPPG and GPPG treatments, respectively, vs. 33 and 32% for GGPG and GPG treatments, respectively). Interestingly, treatment effects on P/AI were detected only for resynchronized cows receiving second and greater TAI, but not for cows receiving first TAI. Fewer cows presynchronized with GnRH had low (<0.5ng/mL) P4 at G1 than cows not presynchronized (13 vs. 25%), and P4 was greater at PGF2α for cows presynchronized with GnRH (4.4 vs. 4.0ng/mL). Cows receiving 2 PGF2α treatments had lower P4 at G2 than cows receiving 1 PGF2α treatment (0.15 vs. 0.35ng/mL). Differences in P4 at PGF2α were detected only for resynchronized cows and not for cows submitted for first TAI. We conclude that presynchronization with GnRH 6 d before beginning an Ovsynch protocol tended to increase P4 at the first GnRH treatment of an Ovsynch protocol, and that a second PGF2α treatment 24h after the first decreased P4 at TAI, thereby increasing P/AI in cows resynchronized for second and greater TAI.

[Chronic-degenerative diseases in health care activities: data from health surveillance of Federico II University General Hospital in Naples]. / Patologie cronico-degenerative nelle attività assistenziali: i dati nell'Azienda Ospedaliera Universitaria Federico II di Napoli.

Recent average life increase, as well as new habits and lifestyles assumption, has determined a change in population health profiles, as a result of progressive increase in chronic-degenerative diseases prevalence. Among these latter; musculoskeletal and cardiovascular diseases are the most frequent ones. Health care workers also suffered from this change in health profiles. This study, performed thanks to the extension of Health Surveillance to all health care workers, aimed at evaluating chronic-degenerative diseases risk among three main welfare activities (medical, nursing and auxiliary). In conclusion, data showed that nurses, mainly the shift workers, are exposed to a higher risk of chronic-degenerative diseases. All other health care workers have not significant work risk factors, because of a greater burden of aging and lifestyles effects.

[Electromagnetic interference of electronic devices in workers with AICD]. / Interferenze elettromagnetiche dei dispositivi elettronici in lavoratori portatori di AICD.

The Implantable Cardioverter Defibrillators and the Pacemakers are devices that produce electric signals and consequently they are touchy to the electromagnetic interferences. This touchiness may cause a temporary or permanent device's malfunction both for short, but of high intensity, and prolonged exposures. Considered the wide diffusion in the job's environments of the technologies based on the electromagnetic waves, the aim of the study is identify the presence of electronic equipment's electromagnetic interferences in AICD bearing workers. The last aim is give to the AICD bearing workers more detailed information about the prevention of inappropriate AICD shocks.

[Operational aspects of risk perception in the electromagnetic fields exposition]. / Aspetti applicativi della percezione del rischio nella valutazione dell'esposizione a campi elettromagnetici.

The increase of electromagnetic fields exposition is being associated with the increase of risk perception in the people exposed due to the uncertainty of the biological and sanitary effects. Research is being carried out on the symptomatology shown by the 45 people living near power-lines in the Benevento area and consequently exposed to electromagnetic fields. The measure of the magnetic and electric field was in the normal range while the people showed most symptoms in the subjective and behavioural sphere. The research findings suggest that risk assessement should take into account the subjectivity of the people exposed as shown in the special questionnaires with the aim of reducing the subjective and behavioural symptomatology for developing a new environmental medicine.

B-cell epitopes in the immunodominant p34 antigen of mycobacterium avium ssp. paratuberculosis recognized by antibodies from infected cattle.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) causes Johne's disease, a chronic and fatal enteritis in ruminants. In the last stage of the disease, antibody titres rise and levels of interferon-gamma decrease, suggesting that the host-immune response is switching from a T helper 1 (Th1) to a Th2 profile. In infected cattle, the membrane protein p34 elicits the predominant humoral response against M. paratuberculosis. To map the B-cell epitopes of this antigen, affinity-purified bovine antibodies against the carboxy-terminal region of p34 were used to screen a 12-mer phage display library. Several phage clones carrying peptides resembling fragments of p34 were affinity selected. Based on the predicted amino acid sequence, peptides were chemically synthesized, which demonstrated reactivity with serum from naturally infected and p34-vaccinated cattle. Immunization of mice with these peptides elicited an anti-p34 antibody response. Two B-cell epitopes were identified and characterized. Based on the reactivity and the type of immune response elicited, epitope A was determined to be conformational, whereas epitope B was demonstrated to be sequential. Both epitopes were shown to be present in p34 proteins from M. avium ssp. avium or M. paratuberculosis but absent from M. intracellulare, the other member of the M. avium complex. Furthermore, both epitopes were mapped to regions of p34 that display high variability when compared to homologous proteins from other mycobacterial species of public and animal health importance. We hypothesize that these variable regions of p34 may play a role in the immunobiology of M. paratuberculosis infections.

Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP.

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Identification of a secreted superoxide dismutase in Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, is an important animal pathogen that has also been implicated in human disease. The major proteins expressed by M. paratuberculosis were analyzed by two-dimensional gel electrophoresis, and a superoxide dismutase (Sod) was identified from this protein profile. The M. paratuberculosis Sod has a molecular mass of 23 kDa and an isoelectric point of 6.1. Sequence analysis of the corresponding sodA gene from M. paratuberculosis indicates that this protein is a manganese-dependent enzyme. We show that the M. paratuberculosis Sod is actively secreted, suggesting that it may elicit a protective cellular immune response in the host during infection.

Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine.

Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.

Development of a firefly luciferase-based assay for determining antimicrobial susceptibility of Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (Johne's disease) is a fatal disease of ruminants for which no effective treatment is available. Presently, no drugs against Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease, are approved for use in livestock. Additionally, M. paratuberculosis has been linked to a human chronic granulomatous ileitis (Crohn's disease). To assist in the evaluation of antimicrobial agents with potential activity against M. paratuberculosis, we have developed a firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used was M. paratuberculosis K-10(pYUB180), a clinical isolate carrying a plasmid with the firefly luciferase gene. The MICs determined by the broth macrodilution method were as follows: amikacin, 2 microg/ml; Bay y 3118, 0.015 microg/ml; clarithromycin, 1.25 microg/ml; D-cycloserine, 25 microg/ml; ethambutol, 20 microg/ml; and rifabutin, 0.5 microg/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferase assay were identical or fell within 1 doubling dilution. These results suggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment of M. paratuberculosis infections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We demonstrate that the luciferase drug susceptibility assay is reliable for M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.