Management-related risk factors for M. paratuberculosis infection in Michigan, USA, dairy herds

A cross-sectional study was conducted from June through December 1996 to identify management-related risk factors for herd-level M. paratuberculosis infection. Data were collected from 121 participating herds. A two-part questionnaire was administered to gather data on current and previous management practices and herd productivity. A random sample of cows aged ≥24 months was selected from each herd and tested for antibodies to M. paratuberculosis using the IDEXX Antibody ELISA (sensitivity 64%, specificity 96%). A positive herd was one in which ≥2 animals tested positive for antibodies to M. paratuberculosis. A negative herd was one in which no animal tested positive. Herds in which only one animal tested positive were dropped from statistical analysis to reduce the risk of including false-positive herds in the statistical analyses.

There were 80 herds with one or more positive animals and 41 herds with no positive animals in the sample (66% herd-level prevalence). Twenty-six herds (21%) were dropped from further analyses because they had only one positive cow. Twelve herds (10%) were dropped from analysis because of missing data. The resulting sample used for statistical modeling included 46 positive herds and 37 negative herds (55% herd-level prevalence). A multi-variable logistic-regression model was used to evaluate the results. The variable ‘use of an exercise lot for lactating cows' was associated with a three-fold increase in odds of a herd being positive for M. paratuberculosis infection (O.R.=3.01, C.I.=1.03–8.80); ‘cleaning of maternity pens after each use' was associated with a three-fold reduction in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.28, C.I.=0.08–0.89); ‘application of lime to pasture areas in 1993' resulted in a ten-fold decrease in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.10, C.I.=0.02–0.56).     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

Differences in the kinetics of T cell accumulations in C3H/HeN (Bcg-resistant) and C57BL/6 (Bcg-susceptible) mice infected with Mycobacterium paratuberculosis

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and -resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, γδ TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or γδ T cells. Significant increases in accumulation of CD8+ and γδ T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or γδ T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and γδ T cells may play a role in the increased susceptibility of C57BL/6 mice to infection with M. paratuberculosis.     

Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on the infecting bacterial genotype

In this study we investigated the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows. We tested the hypotheses that infection status of cows affects macrophage killing ability and that survival of M. paratuberculosis in macrophages is dependent on the strain. Peripheral blood mononuclear cells (PBMC) were obtained from Johne's disease-positive (n = 3) and age and stage of lactation matched Johne's disease-negative (n = 3) multiparious cows. Following differentiation, MDMs were challenged in vitro with four M. paratuberculosis strains of different host specificity (cattle and sheep). Two hours and 2, 4, and 7 days after infection, ingestion, and intracellular survival of M. paratuberculosis strains were determined by fluorescence microscopy. There was no effect of the origin of MDMs (Johne's disease-positive or control animals) on phagocytosis, survival of bacteria, or macrophage survival. In contrast, important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which may make it important to include strain typing when designing control programs.     

Immunosuppression in goats by dexamethasone and cyclophosphamide

The influence of dexamethasone and cyclophosphamide on the goat immune system was investigated. Seven goats, with a previous contact with caprine herpesvirus type 1 (CHV-1), were used. All had been vaccinated with live Mycobacterium paratuberculosis vaccine.

Six goats were injected intravenously (i.v.) with dexamethasone daily for 5 days (2.5–4 mg/kg BW per day). Three also received 25 mg/kg BW of cyclophosphamide on day 0. The seventh goat was not treated.

Dexamethasone alone caused depression, slight lymphopenia and fall in tuberculin reaction. Dexamethasone plus cyclophosphamide caused a severe clinical reaction, marked leukopenia (lymphopenia), fall in tuberculin reaction and significant increase in CHV-1 neutralizing antibody titres. M. paratuberculosis antibody reaction was variable and thus difficult to be assessed. CHV-1 was not isolated.     

Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of mycobacterium paratuberculosis

Reference strains and 31 clinical isolates of M. paratuberculosis, mainly from goats, were analysed for restriction fragment length polymorphism (RFLP). Restriction digests of bacterial DNA were hybridized with a repetitive insertion sequence, IS900, to obtain banding patterns for comparison of strains. Twenty-five of the 31 field-strains hybridized with IS900, and five hybridization patterns were identified. It was not possible to identify specific patterns for goat strains of M. paratuberculosis. Four hybridization patterns were similar, whereas the fifth pattern of a sheep strain diverged considerably in position and number of band. Six goat strains failed to hybridize with IS900, and the absence of IS900 was verified by the polymerase chain reaction and hybridization with an oligonucleotide probe. The six IS900-negative goat strains had diverging phenotypic properties, and the identification of these strains is discussed. The present study shows that M. paratuberculosis strains infecting goats are genetically similar to cattle strains and that IS900 is a specific genetic element for identification of M. paratuberculosis.     

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10⁹ organisms in four divided doses of 1.5 × 10⁹ organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy (p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing (M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.     

The serological response of pigs experimentally infected with a species of Taenia from Taiwan

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody in pigs infected with a possibly new species of Taenia isolated in Taiwan is described. The test antigen ThFAS was fractionated from the cyst fluod of a heterologous cestode Taenia hydatigena. In lightly infected pigs ( 4 recovered cysts at necropsy 17 weeks post-inoculation), antibody was detected as early as 3 weeks post-inoculation. In more heavily infected pigs (6–72 recovered cysts at necropsy 32 weeks post-inoculation), antibody was still detectable at the time of necropsy. Cysterci were found only in the livers of the infected pigs. This ELISA should be highly useful for detecting infection of pigs with this larval cestode in regions where the presence of Taenia solium is unlikely.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds on the New York State Paratuberculosis Control Program

An extensive questionnaire was developed and used to collect data from 33 herds that were on the New York State Paratuberculosis Control Program, to study farm factors associated with the presence of Mycobacterium paratuberculosis infection in dairy herds. The results of the last whole herd paratuberculosis fecal culture were used to indicate presence of infection in a herd, with herds having one or more animals positive classified as ‘infected’. The average prevalence within herds was 5.2%. Fourteen herds were uninfected and 19 herds had prevalences ranging from 0.7%–28.2%. Data on 31 continuous and 67 categorical risk factors were collected by questionnaire. Ten factors were significantly associated with prevalence risk of infection in the univariable logistic regression. These factors were: the type of farm operation (commercial/registered or both); earlier diagnosis of the disease before entering the control program; number of clinical cases in the previous year; whether clinical cases were raised or purchased animals; typical signs in clinical cases; exposure of calves 0–6 weeks of age to feces of adult cows; contact of young stock with adult animal feces from using the same equipment to clean the housing for both groups of animals; spreading feces on fields from which forage is later harvested and fed to animals of any age group; what is done with animals that are suspected of having paratuberculosis or test positive on culture; and frequency of cleaning the cow barn. Stepwise logistic regression was used to determine the significance of each risk factor while controlling simultaneously for the effect of other factors. The significant factors were the type of farm operation, clinical signs, and exposure of calves to feces of adult cows. Commercial herds, presence of clinical signs typical of paratuberculosis in animals, and exposure of calves 0–6 weeks old to feces of adult cows all indicate a higher likelihood that a herd is infected with M. paratuberculosis.     

Indirect ELISA for the detection of antibodies against Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1790) in foxes

Serological investigations focused on the detection of specific opisthorchiid liver fluke antibodies in silver foxes (Vulpes vulpes fulva). Animals were experimentally infected with Opisthorchis felineus (nos. 1 and 2) and Metorchis bilis (nos. 3–8) by feeding fish with a counted number of metacercariae. Four foxes remained as non-infected negative controls (nos. 9–12). For the indirect ELISA, an excretory–secretory antigen was produced by in vitro cultivation of O. felineus and M. bilis adults isolated from livers of experimentally infected hamsters.

Immunoglobulin G (IgG) seroconversion against homologous antigen took place between weeks 2 and 6 postinfection (p.i.) and foxes remained seropositive up to the end of the trial at week 41 p.i. In contrast, IgG titres against heterologous antigen remained significantly lower and stayed near the cut-off. All infected animals excreted opisthorchiid eggs, starting between weeks 2 and 4 p.i. The number of liver flukes found at necropsy was relatively low, except in one fox that was sacrificed at the week 11 p.i. These results suggest that the ELISA is a suitable tool for the detection of specific O. felineus and M. bilis antibodies in the fox.     

A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds

Sixteen litters of seven pigs from each of nine Danish farrow-to-finish herds were followed to investigate the serological patterns caused by natural infection with Mycoplasma hyopneumoniae, Pasteurella multocida toxin and Actinobacillus pleuropneumoniae serotypes 2, 5–7, 12. In seven of the herds, pigs were followed as two separate cohorts started 4 weeks apart, and in two herds only one cohort was followed.

A total of 999 pigs were included in the study. The pigs were blood sampled at weaning and subsequently every fourth week until slaughter. All pigs were examined for antibodies against M. hyopneumoniae (enzyme-linked immunosorbent assay), P. multocida toxin (enzyme-linked immunosorbent assay) and A. pleuropneumoniae serotypes 2, 5–7, 12 (complement-fixation tests). The most-common pattern (28%) of seroconversion was that of pigs first seroconverting to A. pleuropneumoniae serotype 2, followed by seroconversion to M. hyopneumoniae. Each herd had a dominant serotype of A. pleuropneumoniae to which most pigs seroconverted. Seroconversion to the respiratory pathogens occurred mainly in the growing-to-finishing units (8–24 weeks). The risk of seroconversion to the P. multocida toxin was very low (<20%) and occurred late.

None, four and seven herds tested seropositive to PRRS and to swine influenza virus subtypes H3N2 and H1N1, respectively, when testing 10 pigs per herd (selected randomly among the study pigs) at the age of 20 weeks.     

Seasonal Prevalence of Gastrointestinal] nematodes of goats in Pernambuco state, Brazil

During each of 36 1-month periods from April 1979 to March 1982, 3–4 goats selected from typical farms were necropsied and examined for gastrointestinal nematodes. The goats were male, 12 months old, born on the farm and raised without any anthelmintic application. At the beginning of each month, from April 1981 to March 1982, three 12-month-old male goats shown to be free of gastrointestinal nematodes after anthelmintic treatment were grazedwith a flock of naturally infected goats (tracer goats). At the end of each month, these goats were placed on a cementfloored pen and were maintained there for 4 weeks prior to necropsy and examination for gastrointestinal nem nematodes.

Every goat examined was found to be parasitized by more than one species of nematode. Haemonchus contortus, Strongyloides papillosus and Oesophagostomum columbianum were the most prevalent nematodes found. Total worm burdens present in the farm animals were highest during late rainy/early dry season (March-June) and lowest in mid-rainy season (January-February). The acquisition of nematodes by tracer goats occurred mainly from mid-rainy to early dry season (January-June).     

Simulation model of within-herd transmission of bovine tuberculosis in Argentine dairy herds

Transmission of bovine tuberculosis was quantified in three dairy herds located in south Santa Fe Province, Argentina. Using estimates of Mycobacterium bovis transmission (β) and a Reed–Frost simulation model, the prevalence of tuberculosis infection in the study herds over time was investigated. The Reed–Frost model was modified by incorporating randomness in both β and the incubation period () of M. bovis. The mean estimated herd β was 2.2 infective contacts per year and did not differ significantly between the study herds. Modeling as Poisson distributed (mean 24 months) best fit the observed prevalences. Infection was predicted by the model either to spread quickly (<10 years) within a herd and reach a high prevalence (>50%), or to persist at a low prevalence (<5–10%). The model was robust, predictions were realistic and the mean β estimated was consistent with previous studies of bovine tuberculosis.     

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log₂) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.     

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

Mycoplasma hyopneumoniae (M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio (R_n). This R_n-value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates.

Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R_n-values of the highly virulent and the low virulent isolates were estimated to be 1.47 (0.68–5.38) and 0.85 (0.33–3.39), respectively. No significant difference between the groups was found (P = 0.53). The overall R_n was estimated to be 1.16 (0.94–4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae, assessed for the first time by a reproduction ratio, shows that one piglet infected before weaning will infect on average one penmate during the nursery phase.     

Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4–7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5–9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.     

Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection

The studies reviewed here evaluated the role cellular immune system components play in control of brucellosis by conducting comparative studies with brucella-resistant C57BL/10 or C57BL/6 mice and susceptible BALB/c mice. We have shown by both in vitro and in vivo studies that activation of macrophages with interferon-gamma (IFN-γ) is an important factor for control of infection with B. abortus in the mouse model and that the mechanism of anti-brucella activity largely involved reactive oxygen intermediates. Differences in control of the organism by resistant and susceptible mice was not related to inherent differences in the ability of their macrophages to control infection either with or without IFN-γ activation nor was it attributable to NK cells since we found no role for them in control of brucellosis in either mouse strain. However, relative resistance to brucellosis did correlate with increased production of IFN-γ by CD4 T cells during the first weeks after infection while IL-10 contributed to susceptibility in BALB/c mice. Moreover, by 3 weeks post-infection splenocytes from the susceptible BALB/c mice failed to produce IFN-γ and relied on TNF- as well as CD8 T cells to control infection until the end of the plateau phase around 6 weeks post-infection when IFN-γ production resumed and clearance began. In contrast, IFN-γ was crucial for control throughout the infection in the more resistant C57BL/6 mice and the mice died in its absence by 6 weeks post-infection compared to 12 weeks for the more susceptible mice that relied on additional mechanisms of control. In contrast to the IFN-γ knock-out mice, both β2 microglobulin knock-out C57BL/6 mice, which do not express conventional MHC class I molecules and thus cannot present antigen to CD8 T cells, or perforin knock-out C57BL/6 mice, which have no T cell cytotoxic activity, controlled and cleared the infection as well as normal C57BL/6 mice. The hiatus of IFN-γ production in BALB/c mice correlated with very high levels of total IL-12 and it was postulated that the lack of IFN-γ was a consequence of p40 homodimer blocking activity. However, reduction of p40 IL-12 in vivo through administration of indomethacin reduced the infection without a concomitant measurable increase in IFN-γ. Current studies are aimed at elucidating the mechanism of the IFN-γ hiatus.     

Growth and interaction of Mycoplasma bovirhinis and Mycoplasma dispar in vitro

Two mycoplasmas were grown in pure and mixed cultures in glucose calf-serum broth with initial pH 7.8. Sensitivity to pH was also tested. The main data for Mycoplasma bovirhinis and Mycoplasma dispar, respectively, in pure cultures were as follows: lag phase, days: <1, 1–2; log phase: 1–2, 1–4; relatively stationary phase: 0–1, 2–4; decline phase (to the extent roughly logarithmic): 2–6, 5–10; maximal titers: 5 × 10⁷ to 10⁹, 5 × 10⁶ to 10⁸ color changing units per 0.2 ml; highest pH, approximately, at which decline started: 7.7 and 7.0; definitely toxic initial pH: 5.6, 6.8; relative production of acidity; less, more. Decline either shortly ended in loss of viability or, correlated with higher pH levels, led to a prolonged maintenance in lower numbers. The decline of M. bovirhinis was postulated to be essentially caused by an autotoxic product/products other than H⁺.

In mixed cultures an antagonistic effect due to the faster growing M. bovirhinis against M. dispar was recorded. The effect varied according to the initial numbers of organisms and their ratio. Two mechanisms seemed to be active: 1. decrease of pH somewhat below neutrality led to the death of the sensitive M. dispar; 2. M. dispar, when present in relatively high initial numbers, was inhibited by M. bovirhinis during the latter's logarithmic growth at pH-levels above 7.0, the inhibition ending shortly afterwards. A rapidly inactivated product/products of M. bovirhinis metabolism, inhibitory to M. dispar was posited. The results offer an improved insight into diagnostic practices for M. dispar.     

Factors associated with seroprevalence to Mycobacterium paratuberculosis in small-ruminant farms in the Madrid region (Spain)

A cross-sectional study was conducted in a population of small ruminants in the Madrid region (Spain) to determine the Mycobacterium paratuberculosis seroprevalence and to identify farm factors possibly associated with paratuberculosis (PTB). Farming-management information and sera were collected from 60 sheep or goat flocks. The relationship between seropositivity and the variables in the questionnaire was assessed by unconditional logistic regression, followed by random-effects logistic regression analysis to adjust for overdispersion between herds. The seroprevalence to M. paratuberculosis was 11.7% (64 out of 546) using agar–gel immunodiffusion assay (AGID). According to the sensitivity and specificity of the AGID test the true prevalence could be as high as 44%. A herd size of between 200–400 head and the presence of foreign breeds and their crosses were significantly associated with seropositivity (OR=4.05 and OR=4.32, respectively). A higher replacement rate was also associated with seroprevalence to M. paratuberculosis (24.2% in positive herds vs. 18.1% in negative). All these three factors were related to more intensive management in the surveyed area. In contrast, membership of a professional livestock association appeared to be a protective factor against PTB (OR=0.28). No variables related to veterinary assistance were associated with seroprevalence, probably reflecting the current lack of interest in PTB on the part of the animal-health administration and veterinary services in Spain.