Exhalomics as a noninvasive method for assessing rumen fermentation in dairy cows: Can exhaled-breath metabolomics replace rumen sampling?

Previously, we used secondary electrospray ionization-mass spectrometry (SESI-MS) to investigate the diurnal patterns and signal intensities of exhaled (EX) volatile fatty acids (VFA) of dairy cows. The current study aimed to validate the potential of an exhalomics approach for evaluating rumen fermentation. The experiment was conducted in a switchback design, with 3 periods of 9 d each, including 7 d for adaptation and 2 d for sampling. Four rumen-cannulated original Swiss Brown (Braunvieh) cows were randomly assigned to 1 of 2 diet sequences (ABA or BAB): (A) low starch (LS; 6.31% starch on a dry matter basis) and (B) high starch (HS; 16.2% starch on a dry matter basis). Feeding was once per day at 0830 h. Exhalome (with the GreenFeed System), and rumen samples were collected 8 times to represent every 3 h of a day, and EX-VFA and ruminal (RM)-VFA were analyzed using SESI-MS and HPLC, respectively. Furthermore, the VFA concentration in the gas phase (HR-VFA) was predicted based on RM-VFA and Henry's Law (HR) constants. No interactions were identified between the types of diets (HS vs. LS) and the measurement methods on daily average VFA profiles (RM vs. EX or HR vs. EX), suggesting a consistent performance among the methods. Additionally, when the 3-h interval VFA data from HS and LS diets were analyzed separately, no interactions were observed between methods and time of day, indicating that the relative daily pattern of VFA molar proportions was similar regardless of the VFA measurement method used. The results revealed that the levels of acetate sharply increased immediately after feeding, trailed by an increase in the acetate:propionate ratio and a steady increase for propionate (2 h after feeding the HS diet, 4 h for LS), and butyrate. This change was more pronounced for the HS diet than the LS diet. However, there was no overall diet effect on the VFA molar proportions, although the measurement methods affected the molar proportions. Furthermore, we observed a strong positive correlation between the levels of RM and EX acetate for both diets (HS: r = 0.84; LS: r = 0.85), RM and EX propionate (r = 0.74), and RM and EX acetate:propionate ratio (r = 0.80). Both EX-VFA and RM-VFA exhibited similar responses to feeding and dietary treatments, suggesting that EX-VFA could serve as a useful proxy for characterizing RM-VFA molar proportions to evaluate rumen fermentation. Similar relationships were observed between RM-VFA and HR-VFA. In conclusion, this study underscores the potential of exhalomics as a reliable approach for assessing rumen fermentation. Moving forward, research should further explore the depth of exhalomics in ruminant studies to provide a comprehensive insight into rumen fermentation metabolites, especially across diverse dietary conditions.

Implementation of an HACCP model in foot and mouth disease control programmes.

The organisation and structure of the official Veterinary Services (OVS) are designed to meet a specific aim--the health certification of animal health, welfare and food safety in the production and processing stage. Disease prevention and control calls for programmes and projects that, depending on the characteristics of each disease, may involve any branch of the OVS, from the laboratory to field activities. For the purpose of this work, the model used is that of a country that is 'free from foot and mouth disease with vaccination' in accordance with the conditions stipulated in Chapter 8.8. of the World Organisation for Animal Health Terrestrial Animal Health Code. These conditions state that, to maintain this health status, a programme of monitoring and continuous control of the relevant variables must be implemented. This is achieved by applying good practice and identifying the critical control points in all processes, using a checklist that simplifies the task. The system that is developed can also serve as a guide for internal or external programme audits.

Quantitative risk assessment of FMDV introduction in a FMD free country through bone-in beef and offal importation from a FMD free with vaccination country/zone.

Quantitative risk assessment was used to estimate the risk of introducing foot-and-mouth disease (FMD) through bone-in beef from Argentina (FMD-free with vaccination status) into other FMD-free countries. A stochastic model was built to characterize all the steps from primary production to bone-in beef export and introduction into an FMD-free country. The probability that bone-in beef from at least one animal infected with the FMD virus (FMDV) was exported during a year was 5.27 × 10-3 (95% CI <10-10 - 5.19 x 10-2) or in other words one case in 190 years. The risk of FMDV introduction was sensitive to the probability of an outbreak occurring in Argentina (r [Spearman´s rank correlation] = 0.99) and the number of herds affected during an outbreak (r = 0.10). Additionally, the probability that susceptible animals in the importing country came into contact with infective material (bones) and generated an outbreak was 6.16 × 10-4 (95% CI <10-10 - 6.20 ×10-3) or one FMD outbreak every 1623 years on average. Based on the quantitative risk assessment results, the probability of FMDV introduction into a FMD-free country where vaccination is not practiced from a FMD-free country where vaccination is practiced associated with bone-in beef trade from Argentina was negligible. The risk of an FMD outbreak caused by the potential introduction of the FMDV was associated with the existing conditions in the country. Thus, maintaining the FMD-free status with or without vaccination would not be relevant.

Localized SDF-1α Delivery Increases Pro-Healing Bone Marrow-Derived Cells in the Supraspinatus Muscle Following Severe Rotator Cuff Injury.

To examine how the chemotactic agent stromal cell-derived factor-1alpha (SDF-1α) modulates the unique cellular milieu within rotator cuff muscle following tendon injury, we developed an injectable, heparin-based microparticle platform to locally present SDF-1α within the supraspinatus muscle following severe rotator cuff injury. SDF-1α loaded, degradable, N-desulfated heparin-based microparticles were fabricated, injected into a rat model of severe rotator cuff injury, and were retained for up to 7 days at the site. The resultant inflammatory cell and mesenchymal stem cell populations were analyzed compared to uninjured contralateral controls and, after 7 days, the fold-change in anti-inflammatory, M2-like macrophages (CD11b+CD68+CD163+, 4.3X fold-change) and mesenchymal stem cells (CD29+CD44+CD90+, 3.0X, respectively) was significantly greater in muscles treated with SDF-1α loaded microparticles than unloaded microparticles or injury alone. Our results indicate that SDF-1α loaded microparticles may be a novel approach to shift the cellular composition within the supraspinatus muscle and create a more pro-regenerative milieu, which may provide a platform to improve muscle repair following rotator cuff injury in the future.

Advances in scrapie research.

The bovine spongiform encephalopathy (BSE) crisis clearly demonstrated the need to keep animal transmissible spongiform encephalopathies (TSE) under control in order to protect animal and human health. Scrapie is the most widespread TSE of livestock in the world. For this reason, health authorities in different countries have elaborated plans that aim towards scrapie eradication. The unusual nature of the scrapie agent and the fragmented status of scientific knowledge about it, along with the limitations of currently available diagnostic tools, make it unlikely that the objective of eradication will be achieved in the near future. Scientific research is focused on acquiring the knowledge that will improve the efficiency of these efforts.

International organisations and their role in helping to protect the worldwide community against natural and intentional biological disasters.

Preventing the spread of disease through international movements is one of the key objectives of the World Organisation for Animal Health (OIE). One of the ways it seeks to achieve this is by publishing international standards and guidelines aimed at, inter alia, preventing the importation of pathogens that are dangerous for animals and humans and strengthening Veterinary Services so that they can improve their surveillance and response systems. The OIE works in close partnership with the Food and Agriculture Organization of the United Nations (FAO), and together the two organisations have developed a joint initiative - the Global Framework for the Progressive Control of Transboundary Animal Diseases (GF-TADs). Member Countries of these organisations could increase their capacity to manage the risks of disease occurrences, whether natural or deliberately introduced, if they would all strictly implement existing OIE international standards. Compliance with these standards greatly depends on the political willingness of national policy-makers and on a successful transfer of resources to developing countries in support of good governance and appropriate policy implementation. A United Nations Resolution obliging its Member Countries to implement OIE standards could prove invaluable in this respect.

The translation into Spanish of the OIE Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees): problems, solutions and conclusions.

In order to carry out the translation into Spanish of the Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees) ensuring full scientific and linguistic accuracy, its authors relied on coordination between three types of experts: linguistic, translational and veterinary. In this paper the planning, execution and quality control of such work, which was undertaken with the support and guarantee of the World Organisation for Animal Health (OIE), is reported. In the conclusions the authors describe what they view as necessary guidelines for the OIE to apply in the future regarding its linguistic policy. The working methodology reported in connection with the translation of the Terrestrial Manual into Spanish will be useful for the translation of the Terrestrial Manual or other texts into languages other than Spanish, whether or not they are among the official languages of the OIE.

Localization of foot and mouth disease virus RNA in tissue culture infected cells via in situ polymerase chain reaction.

Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice.

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.

Reconstitution of immunosuppression mice with mononuclear cells from donors sensitized to foot-and-mouth disease virus (FMDV).

Passive transfer experiments were performed to serve as a basis for analyzing the immune response of adult mice to FMDV infection. Animals were irradiated (750 rad: 1 lethal dose 50%) and reconstituted with allogeneic mononuclear cells from blood, spleen, thymus and peritoneal cavity from donors 2 and 8 days post-inoculation (p.i.). Donors were primed with 10 000 suckling mouse 50% lethal doses of FMDV strain O1 Campos. The following parameters were studied in recipient mice challenged with 10 000 suckling mouse 50% lethal doses of the same virus: (1) viremia; (b) FMDV neutralizing antibody titres; (c) sheep red blood cell (SRBC) hemagglutinating antibody titres. Viremia was substantially prolonged in irradiated control mice, which did not produce detectable antibodies to FMDV or SRBC. In contrast, the span of viremia was markedly shorter in animals reconstituted with cells obtained 8 days p.i. and its eclipse coincided with the onset of neutralizing antibody production. An equally efficient antibody response to the inoculation of SRBC was observed in these animals. No effect was detected after the transfer of cells obtained 2 days p.i. It is concluded that the humoral immune response plays a predominant role in the recovery from FMDV experimental infection in adult mice.

Reduction in morbidity due to diarrhea in nursing beef calves by use of an inactivated oil-adjuvanted rotavirus-Escherichia coli vaccine in the dam.

An outbreak of neonatal diarrhea occurred among beef calves (2000 animals) from one large Argentinian farm in 1985. Rotavirus was detected in 78% (106/136) and enterotoxigenic Escherichia coli in 1.5% of the samples (2/136) obtained from sick calves. In comparison rotavirus was identified in only 1.6% (1/63) of the samples from clinically healthy calves. The rotavirus strain responsible for the outbreak was characterized as serotype 6 belonging to group A. In the following three years the protective capacity of a combined rotavirus-E. coli inactivated vaccine administered to the dams during the last third of the gestation period was evaluated on this farm by comparison of morbidity due to diarrhea in calves from vaccinated vs. placebo cows within the same year. The morbidity due to diarrhea among calves from dams in the vaccinated and placebo groups was 34% and 77%, respectively in 1986; 23% and 47% in 1987, and 15% and 34%, in 1988. In 1987 morbidity of diarrhea in calves born from vaccinated heifers was 54% and 74% in calves from placebo heifers. In 1988 morbidity from diarrhea was 41% and 54%, respectively among calves in these two groups. In all experiments, calves from heifers showed significantly greater morbidity than calves from cows. Differences in diarrhea morbidity between the vaccinated and placebo groups were statistically significant (P less than 0.05). Additional studies showed that the diarrhea had a significant influence (P less than 0.05) on the average live weight of the calves at weaning (5 to 7 months) with an average weight loss of 7.8 kg per calf among the calves affected with diarrhea.

Immune response to foot-and-mouth disease virus in an experimental murine model. II. Basis of persistent antibody reaction.

A murine model was used to study the mechanisms involved in the prolonged immune response to live and inactivated foot-and-mouth disease virus (FMDV). The antibody response elicited by the infection persisted throughout the entire life of the animal, while immunization with inactivated virus induced a transient response. The administration of inactivated virus in a water-in-oil emulsion increased antibody titres to values as high as those obtained by infection. There was a high correlation between neutralizing antibody titre and transfer of immunity with primed cells, and the protection afforded against challenge with infectious virus. It appears that the mechanism involved in the induction of prolonged immune memory in infected animals is not due to viral persistence. Nude mice infected with FMDV also evidenced a prolonged immune response, showing marked differences in antibody levels but equal effectiveness against challenge when nu/nu and nu/+ animals were compared. Furthermore, athymic and euthymic littermates were efficient in conferring protection when cells were transferred to irradiated animals. It is concluded that there is an effective, T-cell-independent, prolonged immune memory against FMDV in this murine model, and that the difference in the immune responses to live and inactivated virus is due mainly to differential antigenic processing rather than to a difference in the degree of sensitization of effector cells.

Detection of antibodies against foot-and-mouth disease virus using a liquid-phase blocking sandwich ELISA (LPBE) with a bioengineered 3D protein.

A liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA-3D) was developed to detect specific antibodies to the 3D protein in sera from foot-and-mouth disease (FMD) virus (FMDV)-infected animals. The assay uses a nonstructural 3D recombinant protein and two polyclonal antisera, one for capture (bovine) and the other for detector (guinea pig). The specificity of the assay was demonstrated by negative results with 101 sera of cattle from the FMD-free zone in Argentina and with bovine and porcine sera raised against various RNA and DNA viruses. The ELISA-3D was able to detect antibodies in cattle after natural or experimental infection with FMDV of A, O, or C types as early as 5 days postinfection and at later stages in persistently infected animals. Comparison of the results with those obtained with the routinely used agar gel immunodiffusion test and a previously described ELISA, both employing a partially purified virus-infection-associated antigen, shows that the ELISA-3D is highly sensitive and specific and gives reproducible results. Its use as a tool for monitoring viral activity and for certification of FMDV-free animals is recommended.

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines.

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of naïve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.

Avian adenoviruses--distribution of infection in commercial flocks of fowls in Argentina.

Group-specific avian adenovirus antibodies were surveyed in broiler, layer, and breeder chickens in three poultry-producing areas of Argentina: north, middle, and south. The method used was the agar double-immunodiffusion test. Of the flocks examined, 89% had antibodies to avian adenovirus, with the percentages higher in layers and breeders than in broiler chickens. A close relation was found between poorer hygienic condition and high adenovirus antibodies values.

Viral antibodies in bovine fetuses in Argentina.

In order to establish the prevalence of viral infections of the bovine fetus in Argentina, a serological survey for antibodies against viral agents currently affecting cattle in this country was conducted. Antibodies against foot-and-mouth disease virus (FMDV), bovine herpesvirus-1 (BHV-1), bovine leukaemia virus (BLV), bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 (PI-3) were investigated in a total of 315 fetal serum samples. Conventional techniques were used: indirect immunofluorescence (FMDV, BHV-1, BVDV and BCV), radial immunodiffusion (BLV), ELISA (BRV) and haemagglutination inhibition (PI-3). Antibodies against BHV-1, BVDV and PI-3 were detected in samples from fetuses in the second and third trimester of gestation, with a prevalence of 1.21 per cent (two of 165), 2.03 per cent (four of 197) and 5.08 per cent (nine of 177), respectively. Either antibodies or non-antibody factors able to bind to BRV and BCV antigens were detected with a prevalence of 2.44 per cent (five of 205) and 4.54 per cent (five of 110), respectively. In addition, 14.68 per cent of non-specific inhibitors of PI-3 mediated haemagglutination were found. No seropositives against FMDV and BLV were detected.

Nucleic acid hybridisation and polymerase chain reaction in the diagnosis of infectious animal diseases.

The authors describe and summarise the use of nucleic acid hybridisation and polymerase chain reaction (PCR) technologies in the diagnosis of animal diseases. PCR is a powerful biological tool which enables exponential enzymatic amplification in vitro of a given deoxyribonucleic acid sequence. This technique is currently used to study the molecular pathogenesis of many infectious diseases and also for diagnosis. PCR is usually more sensitive than conventional methods and does not require complex facilities, and will therefore soon become the preferred technology of laboratory diagnosticians, field veterinarians and health officers.

Bovine spongiform encephalopathy in South America: a regional preventive approach.

Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease of cattle caused by prions that was first described in the United Kingdom (UK) in 1986. The BSE epizootic that commenced in the UK in the 1980s has since spread into other countries in Europe and Asia through exports of contaminated meat-and-bone meal or infected cattle. Over the past few years, other emerging or reemerging diseases have spread into previously free countries or regions through international trade. This negative effect of globalisation means that to implement successful preventive and strategic programmes to safeguard animal health, such programmes must, as a priority, take a regional approach. Global thinking, regional planning and local performance constitute the key factors for the successful control of animal diseases. In South America, initial preventive actions against BSE were adopted in 1989. Further measures adopted since then and based on new scientific and technical findings, have led to the demonstration that the region is free of BSE. These early preventive actions have reliably protected the region from importing BSE-infected material. An integral part of the project to determine the BSE status of South America was the training of personnel, the incorporation of technology and the provision of updated information through close relationships with international organisations and prominent international researcher workers. Regional activities aimed at harmonising BSE prevention programmes, producing objective and transparent data on the equivalence of regional BSE status and facilitating regional and international trade have recently been launched. Maintaining the BSE-free status of the region must be given high priority by the beef agro-industrial sectors.

[Seroepidemiologic indicators for the evaluation of campaigns for the control of foot-and-mouth disease]. / Indicadores seroepidemiológicos para la evaluación de las campañas de control de fiebre aftosa.

A sero-epidemiological survey was conducted in two districts in Argentina between 1993 and 1995, to provide additional information on the epidemiology of foot and mouth disease (FMD) in Argentina and to assess the level of immunity in cattle populations, and the circulation of FMD virus. As part of the final stage of this survey, a comparison was made of the results obtained by the enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion techniques. Levels of population immunity against the four types of virus included in the vaccine increased progressively during the period of the survey until, in 1995, at the end of the vaccination period, the percentage of animals possessing adequate levels of protection was approximately 77% in yearlings, and more than 94% in cattle over one year old. During the three-year study, there was a clear tendency for viral activity to diminish, until in 1995 when between 3% and 0.6% were positive to the agar gel immunodiffusion test for the antigen associated with viral infection. By contrast, the ELISA detected antibody in about five times as many animals. The authors show how the increase in the level of population immunity was accompanied by a fall in viral activity.

[Prevention of and attention to emergencies in South America]. / Prevención y atención de emergencias en sudamérica.

The authors review the policies designed to prevent and deal with animal health emergencies which have been implemented in countries of South America. They describe the evolution of the epidemiological situation of the continent, the new arrangements for international trade in animals and products of animal origin arising from the creation of the World Trade Organization (WTO), and the consequences of such developments for livestock production in South America. Veterinary systems used to prevent and deal with emergencies in the eleven OIE Member Countries on the continent are described, together with emerging problems which confront the Veterinary Services of the continent, namely: exotic diseases, abnormal occurrence of endemic diseases subject to control programmes, faults in food-safety mechanisms, diseases which have an environmental impact, and problems connected with animal welfare. The emergencies which present the greatest risk to South America are foot and mouth diseases, transmissible spongiform encephalopathies, the porcine reproductive and respiratory syndrome, food poisoning, Newcastle disease and fowl plague. Other problems are the appearance of new strains of existing agents, and the presence of resistant individuals among species of bacteria or harmful arthropods. The authors emphasise the need to co-ordinate the prevention of emergencies with development work at the international level, particularly regional and international agreements, harmonization of procedures, progress in animal health and public health, risk analysis, etc. These systems and methods of prevention have a contribution to make in enhancing the potential of animal production in South America, and the adoption of stricter health and quality standards, according to criteria established by the WTO Agreement on the Application of Sanitary and Phytosanitary Measures.