Genomic Characterization of Mycoplasma arginini Isolated from a Housefly on a Dairy Farm and Comparison with Isolates from Bovine Milk and Lung Tissue.

Mycoplasma mastitis can be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes of Mycoplasma spp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, a Mycoplasma spp. was found in the gut of a housefly captured in the sick pen and identified as M. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed an in silico virulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the housefly M. arginini isolate was highly similar to the milk isolates; interestingly, the similarity was highest with M. arginini isolated from milk on the same dairy farm where the housefly was captured. The housefly and milk M. arginini isolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers of Mycoplasma spp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless, M. arginini pathogenicity will need to be investigated with dedicated studies. IMPORTANCE It is critical to control the spread of bovine mastitis caused by Mycoplasma spp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the same Mycoplasma species found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.

A randomized controlled trial assessing the effect of intermittent and abrupt cessation of milking to end lactation on the well-being and intramammary infection risk of dairy cows.

The objectives were to compare the effects of an intermittent milking schedule with a thrice daily milking schedule during the final week of lactation on the well-being, udder health, milk production, and risk of culling of dairy cows. We hypothesized that cows subjected to an intermittent milking schedule would experience less udder engorgement and pain, lower concentrations of fecal glucocorticoid metabolites (11,17-dioxoandrostanes; 11,17-DOA concentration) after dry-off, lower risk of an intramammary infection during the dry period, higher milk production and lower somatic cell count in the subsequent lactation, and lower culling risk compared with herd mates milked 3 times daily and dried off by abrupt cessation. In a randomized controlled field study, Holstein cows (n = 398) with a thrice daily milking schedule were assigned to treatment and control groups. The treatment consisted of an intermittent milking schedule for 7 d before dry-off (gradual cessation of milking, GRAD). Gradual-cessation cows were milked once daily until the day of dry-off, whereas cows in the control group (abrupt cessation of milking, APT) were milked 3 times daily until the day of dry-off. Udder firmness and pain responses of the udder 3 d after dry-off, as well as the percentage change in fecal 11,17-DOA concentration (3 d after dry-off compared with the dry-off day), were used to assess the well-being of the animals. Compared with cows in the GRAD group, the odds [95% confidence interval (CI)] of udder firmness were 1.55 (0.99-2.42) for cows in the APT group, and the odds of a pain response were 1.48 (0.89-2.44) for cows in the APT group. The least squares means (95% CI) of the percentage change in 11,17-DOA concentration were 129.3% (111.1-150.4) for the APT group and 113.6% (97.5-132.4) for the GRAD group. Quarter-level culture results from the periods before dry-off and after calving were compared, to assess the likelihoods of microbiological cure and new infection. Cows in the APT group had lower odds of a new intramammary infection in the dry period [odds ratio, 95% CI: 0.63 (0.37-1.05)], whereas we observed no meaningful differences in the microbiological cure likelihood among groups. The least squares means (95% CI) for somatic cell counts (log10-transformed) were 4.9 (4.8-5.0) in the APT group and 4.9 (4.8-5.0) in the GRAD group. The odds (95% CI) of clinical mastitis in the first 30 d postcalving were 1.32 (0.53-3.30) in the APT group compared with the GRAD group. We observed no meaningful differences in milk production at the first test date postcalving or the culling risk among groups. We conclude that the gradual-cessation protocol tested herein failed to significantly improve animal well-being, udder health, milk production, and survival in the tested study cohort. However, the observed differences in udder firmness, as well as the numerical differences in udder pain and the percentage change in fecal 11,17-DOA concentrations suggest that this line of research may be useful. Future research is needed to develop drying-off strategies that are appropriate for lowering milk production at the end of the lactation and improve animal well-being without compromising udder health.

Pathogens associated with houseflies from different areas within a New York State dairy.

Houseflies (Musca domestica) are nonbiting muscoids of importance because they can be mechanical vectors of many kinds of pathogens such as bacteria, protozoa, viruses, and helminth eggs. This study aimed to evaluate the bacterial communities associated with houseflies captured in 3 different areas on a dairy farm located in New York State. Variations in the bacterial community were also evaluated based on the flies' sex and external or internal location where the bacteria were isolated. A total of 101 flies were collected: 27 flies from the sick pen, 42 from calf hutches, and 32 from the milking parlor. A total of 485 organisms were isolated, 233 (48.0%) from 53 female flies and 252 (52.0%) from 48 male flies. Most (74%) bacteria were found in the internal parts of the flies, with only 26% isolated from the external surfaces. The number of isolates detected per fly ranged between 1 and 11. A total of 392 bacteria were identified at the species level. We isolated 26 species reported to be bovine contagious or environmental mastitis pathogens. Within the group of organisms considered contagious, we isolated Staphylococcus aureus and Mycoplasma arginini. This was the first time that a Mycoplasma species was isolated from houseflies. We identified 5 organisms considered foodborne pathogens that affect human health: Salmonella spp., Escherichia coli, Staph. aureus, Bacillus cereus, and Bacillus subtilis. Four of the organisms isolated in this study were also linked with milk spoilage, including Pseudomonas aeruginosa, Bacillus cereus, Bacillus licheniformis, and Paenibacillus lactis. This study confirmed that houseflies carry a high bacterial diversity, including organisms associated with animal infections, organisms that could be a concern for public health, or organisms that could negatively affect milk quality.

Short communication: Comparative performance of 3 on-farm culture systems for detection of mastitis pathogens interpreted by trained and untrained observers.

On-farm culture (OFC) systems facilitate pathogen-based mastitis management and can facilitate antimicrobial stewardship on dairy farms. Interpretation of the results, however, may present a challenge for those with limited microbiology experience. Here, we compared results of 3 OFC systems interpreted by trained and untrained observers against results of a standard laboratory reference method (aerobic culture and mass spectrometry). Milk samples (280 quarter and 60 composite) were selected from submissions for routine diagnostic testing to Quality Milk Production Services (Cornell University, Ithaca, NY) between August 2017 and January 2018. Samples were cultured simultaneously using the standard laboratory reference method and 3 commercially available OFC systems that varied in detail of pathogen identification (provided in parentheses) as follows: (1) Minnesota Easy Culture System II Bi-plate (University of Minnesota Laboratory for Udder Health, St. Paul; gram-positive, gram-negative), (2) Minnesota Easy Culture System II Tri-plate (gram-positive, gram-negative, some genus level), and (3) FERA Diagnostics and Biologicals AccuMast plate (Ithaca, NY; genus level, some species level). After 18 to 24 h of incubation, OFC plates were interpreted by 1 trained observer (>10 yr of experience in milk microbiology) and 6 untrained observers with no previous milk microbiology training, using only the manufacturers' instructions for guidance. Strength of agreement (κ) between observer groups and the reference method was determined for the available outcomes of each system. Interpreted by the trained observer, agreement was moderate for identifying gram-positive organisms (Bi-plate, κ = 0.56) and substantial for Streptococcus spp. (Tri-plate, κ = 0.64, AccuMast κ = 0.61). Interpretation by untrained observers resulted in fair agreement (κ = 0.29-0.37) for these organisms. Moderate agreement (κ = 0.43-0.59) was found across all 3 OFC for the identification of gram-negative organisms (Bi-plate), non-aureus staphylococci (Tri-plate and AccuMast), Lactococcus spp., and Enterococcus spp. (AccuMast) when interpreted by the trained observer, and fair to moderate agreement was found (κ = 0.31-0.53) among untrained observers. Across all 3 OFC, agreement was almost perfect (κ = 0.80-0.89) for Staphylococcus aureus for the trained observer, and moderate to substantial (κ = 0.56-0.61) for untrained observers. We concluded that all 3 OFC appeared suitable to support pathogen-based mastitis management when operated by trained observers. Training beyond the instruction manual is a prerequisite to make OFC systems useful for pathogen-based mastitis management.

Mycoplasma species isolated from bovine milk collected from US dairy herds between 2016 and 2019.

Determining the species of mycoplasma isolated from culture-positive milk samples is important for understanding the clinical significance of their detection. Between August 2016 and December 2019, 214,518 milk samples from 2,757 dairy herds were submitted to Quality Milk Production Services (QMPS) at Cornell University for mycoplasma culture. From these samples, 3,728 collected from 204 herds were culture positive. Based on the request of herd managers, owners, or veterinarians, 889 isolates from 98 herds were subjected to molecular identification by PCR and amplicon sequencing. The largest proportion of the identified isolates were from New York State (78.1%), while the others came from the eastern United States (17.8%), Texas (2.0%), and New Mexico (2.1%). As expected, Mycoplasma spp. were the most common (855 isolates, 96.2%) and Acholeplasma spp. accounted for the remainder (34 isolates, 3.8%). Mycoplasma bovis was the most prevalent Mycoplasma species (75.1%), followed by M. bovigenitalium (6.5%), M. canadense (5.9%), M. alkalescens (5%), M. arginini (1.7%), M. californicum (0.1%), and M. primatum (0.1%). A portion of the isolates were confirmed as Mycoplasma spp. other than M. bovis but were not identified at the species level (16 isolates, 1.8%) because further information was not requested by the manager, owner, or veterinarian. Mycoplasma bovis was the only species identified in 59 of the 98 herds. However, more than 1 Mycoplasma sp. was identified in 29 herds, suggesting that herd infection with 2 or more mycoplasmas is not uncommon. Moreover, a Mycoplasma sp. other than M. bovis was the only species identified in 8 herds. From the subset of 889 mycoplasma culture-positive isolates from 98 herds, we determined that over a third of the herds had either more than 1 Mycoplasma sp. or a Mycoplasma sp. other than M. bovis detected in their milk samples. In conclusion, we observed that M. bovis is the most common pathogenic Mycoplasma species found in mastitic milk, but other Mycoplasma species are not uncommon. Our results suggest that it is critical to test milk samples for mycoplasmas using diagnostic tests able to identify both the genus and the species.

Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Distribution of Lactococcus spp. in New York State dairy farms and the association of somatic cell count resolution and bacteriological cure in clinical mastitis samples.

We investigated the distribution of pathogenic non-agalactiae gram-positive, catalase-negative cocci (GPCN) in a convenience sample of New York State dairy farms. Our primary objective with the clinical mastitis (CM) GPCN samples was to evaluate somatic cell count (SCC) resolution and bacteriological cure of Streptococcus dysgalactiae or Streptococcus uberis versus Lactococcus lactis or Lactococcus garvieae in cows that received an approved intramammary treatment. In phase I, we assessed the distribution of the GPCN and SCC resolution. In phase II, we evaluated the SCC resolution and bacteriological cure in CM samples from the 4 farms with the highest prevalence of L. lactis or L. garvieae in phase I. In phase I, 8,868 CM and subclinical mastitis (SCM) milk samples were received from 143 farms. The GPCN samples identified by culture were confirmed with MALDI-TOF. From the 473 MALDI-TOF-confirmed GPCN samples, 155 were S. dysgalactiae (33%); 150, S. uberis (32%); 112, L. lactis (24%); 16, L. garvieae (3%); and 40, other GPCN (8%). From these, 277 were CM samples and 127 were eligible for the evaluation of SCC resolution, which was defined as SCC ≤200,000 cells/mL in a composite sample 15 to 60 d post-diagnosis. The odds of SCC resolution in CM samples was evaluated with multivariable logistic regression, and the odds were 6.1 [95% confidence interval (CI):2.7-13.9] times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. In phase II, a total of 1,662 CM and SCM samples were evaluated with microbiological methods as in phase I, of which 211 samples were confirmed by MALDI-TOF: 39% were S. dysgalactiae (n = 61) and S. uberis (n = 21); 55%, L. lactis (n = 114) and L. garvieae (n = 2); and 6%, other GPCN (n = 13). In total, 168 CM samples were eligible for analysis and 118 were included in the final SCC resolution model. Similar statistical methods as in phase I were performed, and the odds of SCC resolution were 2.4 (95% CI: 1.1-5.5) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Bacteriological cure was defined as having a different or negative culture on a quarter sample taken 14 to 28 d after initial diagnosis. The odds of bacteriological cure (n = 121) were 8.0 (95% CI: 2.5-25.6) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Differences in SCC resolution and bacteriological cure between these groups may dictate a different management approach.

Effects of repeated gram-positive and gram-negative clinical mastitis episodes on milk yield loss in Holstein dairy cows.

The objective of this study was to estimate the effects of recurrent episodes of gram-positive and gram-negative cases of clinical mastitis (CM) on milk production in Holstein dairy cows. We were interested in the severity of repeated cases in general, but also in the severity of the host response as judged by milk production loss when a previous case was caused by a similar or different microorganism. The results were based on data from 7,721 primiparous lactations and 13,566 multiparous lactations in 7 large dairy herds in New York State. The distribution of organisms in the CM cases showed 28.5% gram-positive cases, 31.8% gram-negative cases, 15.0% others, and 24.8% with no organism identified. Mixed models, with a random herd effect and an autoregressive covariance structure to account for repeated measurements, were used to quantify the effect of repeated CM and several other control variables (parity, week of lactation, other diseases) on milk yield. Our data indicated that repeated CM cases showed a very similar milk loss compared with the first case. No reduction of severity was present with increasing count of the CM case. Gram-negative cases had more severe milk loss compared with gram-positive and other cases irrespective of the count of the case in lactation. Milk loss in multipara (primipara) due to gram-negative CM was approximately 304 kg (228 kg) in the 50 d following CM. This loss was approximately 128 kg (133 kg) for gram-positive cases and 92 kg (112 kg) for other cases. The severity of a second case of gram-negative CM was not reduced by previous cases of gram-negative CM in multipara and only slightly less severe in a similar scenario in primipara cows. Similarly, a previous gram-positive case did not reduce severity of a second or third gram-positive case. Hence, our data do not support that immunological memory of previous exposure to an organism in the same generic class provides protection for a next case of CM with an organism in the same class.

Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts.

To better understand the emergence and transmission of antibiotic-resistant Streptococcus agalactiae, we compared phenotypic and genotypic characteristics of 52 human and 83 bovine S. agalactiae isolates. Serotypes found among isolates from human hosts included V (48.1%), III (19.2%), Ia and Ib (13.5% each), and II (5.8%). Among isolates from bovine hosts, molecular serotypes III and II were predominant (53 and 14.5%, respectively). Four and 21 different ribotypes were found among human and bovine isolates, respectively. A combination of ribotyping and serotyping showed that two bovine isolates were indistinguishable from human isolates. Resistance to tetracycline and erythromycin was more common among human (84.6% and 26.9%, respectively) than bovine (14.5% and 3.6%, respectively) isolates. tetM was found in all tetracycline-resistant human isolates, while tetO was the predominant resistance gene among bovine isolates. tet genes were found among various ribotypes. ermB, ermTR, and mefA were detected among erythromycin-resistant human isolates, while ermB was the only erythromycin resistance determinant among isolates from bovine hosts. For isolates from human hosts, erythromycin resistance genes appeared to be associated with specific ribotypes. We conclude that (i) human and bovine S. agalactiae isolates represent distinct populations; (ii) human host-associated S. agalactiae subtypes may occasionally be transmitted to bovines; (iii) while emergence of erythromycin and tetracycline resistance appears to largely occur independently among human and bovine isolates, occasional cross-species transfer of resistant strains or transmission of resistance genes between human- and bovine-associated subtypes may occur; and (iv) dissemination of antibiotic-resistant S. agalactiae appears to include both clonal spread of resistant strains as well as horizontal gene transfer.

The decreased replicative capacity of simian immunodeficiency virus SIVmac239Delta(nef) is manifest in cultures of immature dendritic cellsand T cells.

Transmission of simian immunodeficiency virus SIVmac239Delta(nef) (Delta(nef)) to macaques results in attenuated replication of the virus in most animals and ultimately induces protection against challenge with some pathogenic, wild-type SIV strains. It has been difficult, however, to identify a culture system in which the replication of Delta(nef) is severely reduced relative to that of the wild type. We have utilized a primary culture system consisting of blood-derived dendritic cells (DCs) and autologous T cells. When the DCs were fully differentiated or mature, the DC-CD4(+) T-cell mixtures supported replication of both the parental SIV strain, 239 (the wild type), and its mutant with nef deleted (Delta(nef)), irrespective of virus dose and the cell type introducing the virus to the coculture. In contrast, when immature DCs were exposed to Delta(nef) and cocultured with T cells, virus replication was significantly lower than that of the wild type. Activation of the cultures with a superantigen allowed both Delta(nef) and the wild type to replicate comparably in immature DC-T-cell cultures. Immature DCs, which, it has been hypothesized, capture and transmit SIV in vivo, are deficient in supporting replication of Delta(nef) in vitro and may contribute to the reduced pathogenicity of Delta(nef) in vivo.

Magnetic resonance imaging in the evaluation of inflammatory lesions in muscular and soft tissues: an experimental infection model induced by Candida albicans.

We have developed an experimental model to monitor inflammatory lesions in muscle and soft-tissues during the different stages of the disease by means of Magnetic Resonance Imaging (MRI). MRI of mice legs infected with Candida albicans was performed by standard two-dimensional spin echo and fast spin echo (RARE) using customized coils. The MRI findings were compared with pathologic examinations at the initial acute and established acute inflammatory stages, which provided accurate and detailed information on the evolution of the processes involved. The yeast caused inflammation within the first hours post-inoculation, appearing on T2-weighted images as an inhomogeneous mass with increased signal intensity. The presence of fungal hyphae was observed as hypointense signal areas in both T2 and T1 weighted images, with histologic confirmation. Areas of decreased signal intensity on T2 weighted images were apparent on the last experimental day and were attributed to the granulation tissue located within the capsule surrounding the abscess. The close correlation found between MRI and histopathology suggests that MRI is an ideal radiologic technique for monitoring the clinical and therapeutic follow-up of fungal infections in muscle and soft tissues.

Improvement of functional magnetic resonance images by pretreatment of data.

Functional magnetic resonance images of the brains of subjects performing the finger-tapping paradigm were made using a conventional technique. Two threshold values for the pixels were obtained by analysing pixel by pixel the distributions of the means and variances of each subject's images for 20 consecutive scans, both while performing the task and while at rest. Considerable signal improvement in the final images was achieved by removing from our data all pixels beyond these threshold values (mean < or = 16 and variance > or = 7).

Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice.

In contrast to immunity against some other facultative intracellular parasites, protective immunity against Brucella abortus is mediated in mice by antibodies as well as by cell-mediated immune responses. It was the purpose of this study to determine whether antibody alone would prevent infection with B. abortus. The majority (82%) of CD-1 outbred mice infected with 100 CFU of virulent B. abortus 2308 preincubated with graded quantities of an O polysaccharide-specific IgG2a monoclonal antibody (MAb) were free of infection 1. 2, 4, and 6 weeks later, based on detection limits of 13 brucellae per spleen and 39 per liver. Infection was present in 95% of control animals. Similar results were obtained with a challenge dose of 500 CFU, but with a challenge dose of 5,000 CFU, infection became established even with the highest concentration of MAb used (50 micrograms of MAb per 5,000 brucellae). Pretreatment with an O polysaccharide-specific IgG1 MAb or with convalescent-phase serum diminished but did not prevent establishment of infection by 100 CFU of B. abortus. A majority of culture-negative mice tested 6 weeks after infection were serologically negative, which could have signified either the absence of previous infection or the early elimination of infection. In an in vitro test system, all of the antibody preparations were efficient in opsonizing B. abortus. Effective killing of the organism by unelicited mouse peritoneal macrophages occurred in conventional but not in endotoxin-free medium, suggesting that activated macrophages were required for killing of opsonized B. abortus. These results emphasize the potential importance of antibodies in the immunoprophylaxis of brucellosis and suggest that the design of a successful vaccine will require the induction of antibodies not only of appropriate specificity but also of the optimal isotype for mediating protective functions.

Effect of bovine non-agglutinating antibodies on the blood clearance of 131I-labelled Brucella abortus strain 45/20.

Non-agglutinating anti-Brucella abortus S45/20 antibodies were isolated and purified from sera of immunized cattle by means of immunoadsorption and ion-exchange chromatography (DEAE-Sephadex A-50). They corresponded to the IgG1 isotype as shown by immunoelectrophoresis using monospecific anti-IgG1 and anti-bovine gamma globulin sera. These antibodies failed to agglutinate the antigen. They were detected by the anti-bovine gamma globulin test, showing higher titres than those of agglutinating antibodies during the whole period of the experiment. Blood clearance of 131I-S45/20 in mice, was slower in those groups which had received non-agglutinating antibodies than in the control group.

Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity.

Calves inoculated with Brucella abortus S45/20 produced, against surface antigens, non-agglutinating antibodies (NAAb) which were isolated and purified. A kinetic analysis was carried out of NAAb in antibody-dependent cell-mediated cytotoxicity (ADCC) using sheep red blood cells labelled with surface antigen from B. abortus S45/20 as target cells. Three parameters were examined: time of incubation, effector cell:target cell (E:T) ratio and NAAb dose. It was found that the NAAb were not able to mediate ADCC with bovine spleen cells.

Tears and aqueous humor from horses inoculated with Leptospira contain antibodies which bind to cornea.

An antigenic relationship between Leptospira interrogans and equine cornea was previously described by us. An enzyme-linked immunosorbent assay was employed in the present work to investigate the existence of anti-leptospira and anti-cornea antibodies in tears, aqueous humor and serum from horses inoculated i.m. with those antigens. Ten days after a booster by the same route, antibodies that bind to microtiter plates, coated with an homogenate of either equine cornea or leptospira, were detected in those fluids and in the sera. At the same time, the corneas of the horses began to develop a diffuse opacity. This finding of anti-leptospira antibodies in equine tears and aqueous humor shows the pathway along which they arrive at the cornea and bind to it.

Detection of antigenic fractions from Brucella abortus S45/20 which bind to non-agglutinating antibodies using electroblotting and enzyme-linked antibody probes.

Outer membrane antigens which bind to non-agglutinating antibodies (NAAb) elicited by smooth (S19) and rough (S45/20) Brucella abortus strains, were extracted from S45/20 by stirring in cold 2.5% NaCl and then analyzed by SDS-PAGE, electroblotting and enzyme-linked antibody test. Eight bands were observed in the gel stained with Coomassie blue. Seven antigenic fractions were transferred to nitrocellulose by blotting. A 27-kd band was recognized by bovine anti-S45/20 non-agglutinating serum and not by purified NAAb against surface antigens. Bands 10 kd and 14.3 kd bound to bovine anti-S45/20 NAAb from calves immunized with either S19 or S45/20. A 12.0-kd band was recognized by the serum and NAAb from calves immunized with S45/20 but not by those injected with S19. There are thus antigenic fractions shared by S19 and S45/20 which bind in vitro to NAAb.

Experimental demonstration of an antigenic relationship between Leptospira and equine cornea.

Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. They bound themselves to equine cornea in vivo and in vitro as was proved by immunofluorescence. This antigenic relationship may be in part responsible for pathogenesis of corneal opacity in leptospirosis of horses.

[Detection in bovine serum of non-agglutinating antibodies directed against occult antigens of Brucella abortus strain 19]. / Detección en suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos ocultos de Brucella abortus cepa 19.

Injection of different animal species with particulate T-dependent antigens induces the appearance of two antibody populations belonging to IgG class, one of them with agglutinating activity and the other lacking this capacity, that is, non-agglutinating. In this paper, 3 adult female Hereford were inoculated subcutaneously, fortnightly, four times, with 5 x 10(10) heat killed smooth S19 Brucella abortus cells. Blood samples were taken ten days after the last inoculation and were individually processed. Serum agglutinating titres against the homologous antigen and the Coombs test score were similar, indicating that nonagglutinating antibody titres directed against surface antigens of the smooth phase were not significant (Table 1). When sera were incubated with a suspension of Brucella abortus S45/20 (a rough strain) they showed poor agglutinating ability, but they contained non-agglutinating activity at high level, as determined by the anti-gamma-globulin test (Table 1). Similar results were obtained when the same sera were adsorbed with Brucella abortus S19 up to elimination of the antibodies directed against surface antigens, and then incubated with a suspension of strain 45/20 (Table 1). These antibodies were isolated from sera by immunoadsorption, using a thick suspension of S45/20 and then dissociated with glycine-CHl buffer 0.1M pH 3. These antibodies showed little agglutinating capacity for S45/20; however non-agglutinating antibodies were bound to the bacteria up to 1.5 micrograms g as demonstrated by Coombs test. Apart from the incomplete antibodies against external antigens of smooth Brucella abortus strains detected in long-term stimulated cattle, non-agglutinating antibodies with specificity for deeper antigens of the smooth bacteria were evidenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Detección en suero bovino de anticuerpos no aglutinantes dirigidos hacia antígenos ocultos de Brucella abortus cepa 19. / [Detection in bovine serum of non-agglutinating antibodies directed against occult antigens of Brucella abortus strain 19]

Injection of different animal species with particulate T-dependent antigens induces the appearance of two antibody populations belonging to IgG class, one of them with agglutinating activity and the other lacking this capacity, that is, non-agglutinating. In this paper, 3 adult female Hereford were inoculated subcutaneously, fortnightly, four times, with 5 x 10(10) heat killed smooth S19 Brucella abortus cells. Blood samples were taken ten days after the last inoculation and were individually processed. Serum agglutinating titres against the homologous antigen and the Coombs test score were similar, indicating that nonagglutinating antibody titres directed against surface antigens of the smooth phase were not significant (Table 1). When sera were incubated with a suspension of Brucella abortus S45/20 (a rough strain) they showed poor agglutinating ability, but they contained non-agglutinating activity at high level, as determined by the anti-gamma-globulin test (Table 1). Similar results were obtained when the same sera were adsorbed with Brucella abortus S19 up to elimination of the antibodies directed against surface antigens, and then incubated with a suspension of strain 45/20 (Table 1). These antibodies were isolated from sera by immunoadsorption, using a thick suspension of S45/20 and then dissociated with glycine-CHl buffer 0.1M pH 3. These antibodies showed little agglutinating capacity for S45/20; however non-agglutinating antibodies were bound to the bacteria up to 1.5 micrograms g as demonstrated by Coombs test. Apart from the incomplete antibodies against external antigens of smooth Brucella abortus strains detected in long-term stimulated cattle, non-agglutinating antibodies with specificity for deeper antigens of the smooth bacteria were evidenced.(ABSTRACT TRUNCATED AT 250 WORDS)