Differential cell count as an alternative method to diagnose dairy cow mastitis.

Changes in relative cell proportions occurring in diseased mammary glands of dairy cows can be determined using differential cell count (DCC). The present study was carried out in 2 consecutive trials, with 2 goals: (a) to test the consistency of DCC results on subsequent days, and (b) to establish an effective cutoff value for the diagnosis of mastitis. In the first trial, quarter milk and blood samples were taken from 8 healthy cows for 5 consecutive days. Milk samples were tested by somatic cell count (SCC) and bacteriological analysis, and DCC was performed on blood and milk samples by flow cytometer. In the second trial, 16 animals were randomly selected from a different herd and quarter milk samples taken on 3 consecutive milkings. All samples were cyto-bacteriologically analyzed and DCC was performed on the second sampling. In the first trial, mean SCC was 77,770 cells/mL and 4 samples were bacteriologically positive. No fixed or random effect had a significant influence on percentages of individual cell populations or ratios in blood or milk. A cutoff value of 0.495 for logarithmic polymorphonuclear neutrophilic leukocyte:lymphocyte ratio was established. Mean SCC of milk samples collected in the second trial was 543,230 cells/mL, and infection was detected in 53.1% of quarters, mostly caused by Staphylococcus aureus. When the cutoff value was applied to the data along with SCC, sensitivity and specificity of the diagnostic method were 97.3 and 92.3%, respectively.

CD2/CD21 index: a new marker to evaluate udder health in dairy cows.

Lymphocytes play a significant role in the immunological processes of the bovine mammary gland and were found to be the dominant cell population in the milk of healthy udder quarters. The objective of this study was to investigate the quantitative relationship between CD2(+) T and CD21(+) B lymphocytes using flow cytometry. In a first study, quarter foremilk samples from apparently healthy udder quarters [somatic cell counts (SCC) ≤100,000 cells/mL; n=65] were analyzed and compared with diseased quarters (SCC >100,000 cells/mL; n=15). Percentages of CD2(+) T cells were significantly higher in milk samples with SCC ≤100,000 cells/mL than in those with SCC >100,000 cells/mL, whereas percentages of CD21(+) B cells developed in the opposite direction. As a result of this opposing trend, a new variable, the CD2/CD21 index-representing the percentages of CD2(+) cells per CD21(+) cells-was defined. Although diseased quarters with SCC >100,000 cells/mL and the detection of major pathogens revealed generally CD2/CD21 indices <10, values >10 were observed in apparently healthy quarters. Hence, a CD2/CD21 index cutoff value of 10 may be suitable to aid differentiation between unsuspicious and microbiologically suspicious or diseased udder quarters. To test whether CD2/CD21 indices <10 were primarily related to pathogens, quarters with SCC ≤100,000 cells/mL and >100,000 cells/mL with different bacteriological status (culture negative, or minor or major pathogens) were selectively examined in a second biphasic study. In the first trial, 63 udder quarters were analyzed and 55 of these quarters were able to be sampled again in the second trial carried out 14 d later. In both trials, results of the first study were confirmed. Indeed, CD2/CD21 indices <10 were also found in quarters showing SCC ≤100,000 cells/mL and containing minor or major pathogens at the time of the current or previous bacteriological analysis. The results of our examinations indicated a clear relationship between the CD2/CD21 index and the bacteriological status of the mammary gland. In combination with SCC, it offers a new marker for quick differentiation of unsuspicious and microbiologically suspicious or diseased udder quarters.

Comparison of joining and constant κ-light chain regions in different cattle breeds.

A comparative transcription analysis of Ig κ-light chains (IGK) of the cattle breeds Holstein Friesian (HF), German Black Pied (GBP), German Simmental (GS) and Aubrac (A) revealed three alleles coding for two putative allotypic variants of IGKC. The amino acid residues p.Asp100Asn and p.Thr116Ala were located at the outer edge of the constant domain as demonstrated by homology-based modelling. Alleles were distributed in unequal frequencies within the breeds examined. While cattle breeds HF, GS, and A possessed all alleles and allotypic variants, GBP exhibited alleles encoding allotypic variant IGKC(a) . All three IGKJ segments were detected in 320 sequences analysed. IGKJ1 was combined predominantly with IGKC. The ORF2 of IGKJ2 was detected for the first time on transcriptional level.

Flow cytometric differential cell counts in milk for the evaluation of inflammatory reactions in clinically healthy and subclinically infected bovine mammary glands.

Somatic cell counts (SCC) are generally used as an indicator of udder health. In Germany, a cutoff value of 100,000 cells/mL is currently used to differentiate between healthy and diseased mammary glands. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed evaluation of the udder health status. The aim of this study was to differentiate immune cells in milk of udder quarters classified as healthy based on SCC values of <100,000 cells/mL. Twenty cows were selected and 65 healthy udder quarters were compared with a control group of 15 diseased udder quarters (SCC>100,000 cells/mL). Cells were isolated from milk of all quarters to measure simultaneously percentages of lymphocytes, macrophages, and polymorphonuclear neutrophilic leukocytes (PMNL) by flow cytometric analysis. The bacteriological status of all 80 quarters was also determined. Differential cell count patterns of milk samples (n = 15) with extreme low SCC values of ≤ 6,250 cells/mL revealed high lymphocyte proportions of up to 88%. Milk cell populations in samples (n = 42) with SCC values from >6,250 to ≤ 25,000 cells/mL were also dominated by lymphocytes, whereas DCC patterns of 6 out of 41 milk samples with SCC values from ≥ 9,000 to ≤ 46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL (56-75%). In 13 of 15 milk samples of the diseased udder quarters (SCC >100,000 cells/mL), PMNL were categorically found as dominant cell population with proportions of ≥ 49%. Macrophages were the second predominant cell population in almost all samples tested in relation to lymphocytes and PMNL. Further analysis of the data demonstrated significant differences of the cellular components between udder quarters infected by major pathogens (e.g., Staphylococcus aureus; n = 5) and culture-negative udder quarters (n = 56). Even the percentages of immune cells in milk from quarters infected by minor pathogens (e.g., coagulase-negative staphylococci; n = 19) differed significantly from those in milk of culture-negative quarters. Our flow cytometric analysis of immune cells in milk of udder quarters classified as healthy by SCC <100,000 cells/mL revealed inflammatory reactions based on DCC.

Somatic cell counts and bacteriological status in quarter foremilk samples of cows in Hesse, Germany--a longitudinal study.

Somatic cell counts (SCC) are generally used as an indicator of udder health. Currently in Germany, 100,000 cells/mL is the threshold differentiating infected and noninfected mammary glands. The aim of our study was the detailed analysis of udder health in a representative part of the dairy cow population in Hesse, Germany. Between 2000 and 2008, 615,187 quarter foremilk samples were analyzed. In addition to evaluation of distribution of SCC and prevalence of mastitis pathogens, pathogen prevalence was also calculated depending on SCC. The data indicated that 38% of all samples had SCC >100,000 cells/mL and 62% showed SCC ≤ 100,000 cells/mL; 31% of all samples revealed SCC ≤ 25,000 cells/mL. Coagulase-negative staphylococci were the dominant pathogens in the Hessian quarter foremilk samples (17.17% of all samples) followed by Corynebacterium spp. (13.56%), Streptococcus uberis (8.7%), and Staphylococcus aureus (5.01%). Mastitis pathogens were detected in 83% of all samples with SCC >100,000 cells/mL. However, the prevalence of mastitis pathogens in the SCC range from 1,000 to ≤ 100,000 cells/mL was 8.5% (5.51% minor pathogens, 2.01% major pathogens, and 0.98% other pathogens). For farms producing high quality milk, exceptional hygiene management is compulsory. One of the farms randomly selected showed clearly different results from the Hessian survey. Fifteen percent more samples lay in the SCC range ≤ 100,000 cells/mL with a lower prevalence of mastitis pathogens of 1.91% (1.03% minor pathogens, 0.83% major pathogens, and 0.05% other pathogens). Based on these results, inflammatory processes can obviously be detected in mammary glands of udder quarters healthy according to the current definitions. However, we argue that such inflammation can be detected by examination of the relationship of immune cells in milk.

Clinical and immune response to undiluted and diluted smallpox vaccine.

QUESTION UNDER STUDY: To assess clinical reactions, immune responses and adverse events to undiluted, three- and sixfold diluted Lister strain vaccine stockpiled in Switzerland. METHODS: A prospective, triple-blinded, randomised, parallel group clinical trial was performed. RESULTS: From 2001 to 2007 104 persons with an indication for vaccinia vaccination were recruited. They had a median age of 33 years (range 18-65), 56 (53.8%) were re-vaccinees and 48 (46.2%) primary vaccinees. There was no statistically significant variation in the proportion of revaccinees between diluted and undiluted vaccine groups (75% vs 51%, p = 0.118). With an overall clinical take rate (major reaction) of 97.1% the majority of the vaccinia-naïve participants exhibited an at least fourfold increase of neutralising antibody titres (32/38, 84.2%) post-vaccination. Interestingly this proportion was lower among re-vaccinees (29/46, 63.0%, p = 0.048). No significant difference was observed in the take rate or at least fourfold seroconversion rate between the threefold and sixfold diluted vaccine doses. Adverse events were reported by 98 (94.2%) participants, not accounting for itching at the vaccination site. CONCLUSION: Subjects requiring immunisation were successfully (re-) vaccinated with undiluted as well as with three- or sixfold diluted vaccinia vaccine. Our findings complement previous studies with respect to the clinical take rate and immune response. The rate of adverse events was substantial but not unexpected and no severe adverse events occurred. In conclusion, the existing smallpox vaccine stockpile might be expanded by administering three- or sixfold diluted vaccine doses combined with a careful pre-vaccination screening and extensive instructions to vaccinees.

[Swinepox--skin disease with sporadic occurrence]. / Schweinepocken--eine sporadisch vorkommende Hauterkrankung.

Swinepox virus infection results in an acute, mild or subclinical course and is characterised by typical poxvirus skin lesions in affected pigs. Additionally, sporadic vertical swinepox virus transmission leads to congenital generalised infection and subsequent abortion or stillbirth. The present report describes the occurrence of epidermal efflorescences in two piglets after intrauterine natural suipoxvirus infection. No clinical abnormalities of the gilt and littermates as well as in other pigs from this herd were present. One of the affected piglets was stillborn and submitted for necropsy, the other animal was alive at birth, but died 3 days later. Histologically, a proliferative to ulcerative dermatitis with epithelial ballooning degeneration and characteristic intracytoplasmatic inclusion bodies was observed. The pathomorphological and histopathological suspected diagnosis of a poxvirus infection was confirmed by electron microscopy. Furthermore, the agent was identified as suipoxvirus by polymerase chain reaction. As demonstrated here, obvious skin lesions in suipoxvirus infection leads to a suspected diagnosis in newborn piglets on macroscopic examination. However, further post mortem examinations, including electron microscopy as well as molecular techniques are essential for the identification of the aetiology and the exclusion of differential diagnoses. Because the disease only affected two pigs there was only a small economic loss. A valid diagnostic plays an important role in advising farmers and for herd health monitoring.

Distribution of Mycobacterium avium subsp. paratuberculosis in a Subclinical Naturally Infected German Fleckvieh Bull.

Although it has been known for years that Mycobacterium avium subsp. paratuberculosis (MAP) is detectable in the reproductive organs and semen of infected bulls, only few studies have been conducted on this topic worldwide. This study surveyed the MAP status of a bull, naturally infected due to close contact with its subclinically infected parents over a period of 4 years. From the age of 7 weeks to necropsy, faecal, blood and, after sexual maturity, semen samples were drawn repeatedly. Already at the first sampling day, MAP-DNA was detected in faeces by semi-nested PCR. True infection was confirmed by the detection of MAP-DNA in blood at the age of 40 weeks. In total, MAP-DNA was present in 25% faecal (34/139), 16% blood (23/140) and 5% semen (4/89) samples, including MAP-free intervals of up to 9 weeks. MAP genome equivalents (MAP-GE) of up to 6.3 × 106 /g faeces and 1.8 × 105 /ml blood were determined. Cultivation of MAP occurred only in three of 137 faecal and two of 109 blood, but never in semen samples. Over the whole period, the bull was a serological negative MAP shedder. During necropsy, 42 tissue samples were collected. Neither macroscopic nor histological lesions characteristic of a MAP infection were observed. Cultivation of MAP in tissue sections failed. However, MAP-DNA was spread widely in the host, including in tissues of the lymphatic system (7/15), digestive tract (5/14) and the urogenital tract (5/9) with concentrations of up to 3.9 × 106 MAP-GE/g tissue. The study highlighted the detection of MAP in male reproductive organs and semen. It supports the hypothesis that bulls may probably transmit MAP, at least under natural mating conditions. In artificial insemination, this might not be relevant, due to antibiotics included currently in semen extenders. However, the survivability of MAP in this microenvironment should be investigated in detail.

Cowpox virus infection associated with a streptococcal septicaemia in a foal.

Cowpox virus infection associated with a streptococcal septicaemia was diagnosed in a weak German Warmblood filly, born 29 days prematurely, and humanely destroyed on the sixth day of life. At necropsy, ulcerative lesions in the alimentary tract, colitis, polyarthritis and nephritis were observed. Transmission electron microscopical examination of specimens from ulcerative lesions revealed typical orthopox virions. Cowpox virus was unequivocally identified by virological and molecular-biological methods.

Characterization of a cowpox-like orthopox virus which had caused a lethal infection in man.

In August 1990 an orthopox virus (OPV) had been isolated from a severe case of a generalized infection with lethal outcome in an immunosuppressed 18-year-old man. In this communication we present a detailed characterization of the causative virus strain. Based on distinct epitope configurations detected by various monoclonal antibodies the isolate could be differentiated from other OPV species and was classified as a cowpox virus (CP). This classification was confirmed by a species-specific PCR assay and by establishing physical maps for the restriction enzymes HindIII and XhoI. Based on serological data of neutralization assays, blocking-ELISAs and Western blotting analysis evidence is provided that the infection had been acquired from a stray cat.

Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces.

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.

A seroepidemiological survey for orthopox virus in the red fox (Vulpes vulpes).

703 blood samples from red foxes (Vulpes vulpes) were investigated to determine the prevalence of antibody against an orthopox virus (vaccinia virus strain Elstree). A blocking-ELISA based on a neutralizing monoclonal antibody was used. In this assay 46 sera (6.5%) were positive with titers of 1:2 to 1:16. ELISA-results were confirmed by the plaque reduction test with 44 of the 46 sera reacting positively. The specificity of antibodies in 21 selected sera was also demonstrated by Western blot analysis.

A monoclonal blocking-ELISA for detection of orthopoxvirus antibodies in feline sera.

A double sandwich blocking-ELISA using a genus-specific neutralizing monoclonal antibody (MAb) against the vaccinia virus 32 kD adsorption protein (D8L open reading frame: ORF) was developed to detect orthopoxvirus (OPV) antibodies in sera. A collection of 2173 feline serum samples was examined in an epidemiological study. The blocking-ELISA revealed 44 (2%) sera with positive titres of 1:2-1:256. ELISA results were confirmed by the plaque-reduction test. A close correlation between titres of both assays could be observed (r = 0.986). In general, the sensitivity of the blocking ELISA was two to four times higher. Neutralizing OPV-antibodies were found in nine sera with ELISA-titres > 1:4. Antibody specificity to OPV was also demonstrated by Western blotting analysis with selected feline sera. The epidemiographical distribution of the ELISA-positive sera and case histories of 37 seropositive cats available from the referring veterinarians are demonstrated. The blocking-ELISA enables a rapid serological diagnosis and can be used in veterinary and human medicine. It allows OPV-antibody screening in human and other animal species.

Interferon induction in peripheral blood mononuclear leukocytes of man and farm animals by poxvirus vector candidates and some poxvirus constructs.

Prototypes of three poxvirus genera--orthopoxvirus (OPV), parapoxvirus (PPV), avipoxvirus (APV)--and Newcastle disease virus (NDV) as a control, as well as three recombinant OPV strains and one recombinant APV strain, were incubated in vitro with peripheral blood mononuclear leukocytes (PBML) of man, sheep and swine. Antiviral activity was determined in PBML culture supernatants at different time intervals after virus cell interaction using a cytopathic effect inhibition bioassay. Additionally, supernatants derived from human PBML were screened for interferons (IFN) alpha and gamma as well as for tumor necrosis factor by enzyme-linked immunosorbent assay. IFN titers reached a maximum 24 h after PBML stimulation at a multiplicity of infection (MOI) greater than 1. IFN alpha/beta was found to be responsible for the antiviral effect. Using a MOI > or = 1 the highly attenuated strain MVA was the only representant of vaccinia virus (VV) that induced significant amounts of IFN also as a lacZ recombinant. Replicable virus from five well-known VV strains as well as the Chinese VV strain Tien Tan (VVTT) as a recombinant vaccine failed to induce leukocyte IFN. Inactivated VV strain Elstree and the recombinant TT strain induced high titers of leukocyte IFN. Supernatants derived from human, porcine and ovine PBML stimulated with replicable PPV, native VV MVA and MVA lacZ recombinant or native APV and APV lacZ recombinant virus regularly contained IFN alpha. In contrast to NDV, neither specific antisera nor monoclonal antibodies were able to block the INF induction by VV and PPV.

Seroprevalence of orthopox virus specific antibodies in red foxes (Vulpes vulpes) in the Federal State Brandenburg, Germany.

The prevalence of orthopox virus (OPV)-specific antibodies in 1,040 red foxes (Vulpes vulpes) was evaluated on a large scale in the German Federal State Brandenburg. Serum samples were selected from 809 communities within the study area from January 1991 to September 1994 by simple random sampling. Screening was carried out by an indirect enzyme-linked immunosorbent assay (ELISA). Orthopox virus-specific antibodies were found in 162 (16%) of the 1,040 fox sera. Furthermore 154 (15%) sera were considered suspect positive. The specificity of the antibodies detected in ELISA-positive and suspect positive sera was confirmed by Western blotting. Presence of OPV-antibodies occurred in 291 communities. No correlation of OPV-antibodies findings to latitude or characteristic topographical and ecological peculiarities of the study area was found. Although the causative agent is still unknown we believe that orthopox viruses probably have a ubiquitous presence among red foxes.

A longitudinal study to characterize the distribution patterns of Mycobacterium avium ssp. paratuberculosis in semen, blood and faeces of a naturally infected bull by IS 900 semi-nested and quantitative real-time PCR.

Johne's disease is caused by Mycobacterium avium ssp. paratuberculosis (MAP) and has been recognized as an important bacterial infection in ruminants. Although MAP has been detected in semen and within the reproductive organs of bulls, the bacterial distribution and shedding patterns are currently not well characterized. Our investigation was performed to detect and quantify MAP in faeces, semen and blood samples repeatedly drawn from a naturally infected but asymptomatic 18-month-old German Simmental breeding bull candidate over a period of 3 years (June 2007-November 2010). Qualitative and quantitative polymerase chain reaction (PCR) techniques were used to correlate the presence and matrix-specific amounts of MAP. In total, 65 sampling dates were selected. Mycobacterium avium ssp. paratuberculosis was detected intermittently in all matrices with MAP-free intervals of up to 18 weeks by an IS900 semi-nested PCR. The number of MAP-positive results from semen and blood samples was higher than from faecal samples. A quantitative polymerase chain reaction detected the highest MAP contents in faeces (10(3) -10(6) MAP/g), while lower amounts were found in semen and blood samples (10(2) -10(5) MAP/ml). Although no significant agreement was calculated between the presence of MAP in faeces and blood, a statistically significant positive correlation between its occurrence in semen and blood was determined (r = 0.38, P < 0.05, n = 29). The present study contributes to a more detailed understanding of MAP distribution patterns in faeces, semen and blood of a subclinically infected breeding bull candidate. It highlights the possible role of breeding bulls as a source of MAP transmission and indicates the need for further monitoring and hygienic measures to prevent the spread of the infection via semen.

Establishment of a model of Streptococcus iniae meningoencephalitis in Nile tilapia (Oreochromis niloticus).

Streptococcus iniae is an invasive pathogen causing meningitis and other lesions in various fish species. Furthermore, S. iniae is an emerging zoonotic agent that causes cellulitis in man. The aims of this study were to establish an intraperitoneal infection model for S. iniae in Nile tilapia (Oreochromis niloticus) and to develop a new histopathological scoring system to reflect the degree and extent of inflammation as well as the presence of necrosis in the brain and eye. Intraperitoneal administration of 10(6) colony-forming units (CFU) led to 80% mortality and numerous fish developing clinical signs of central nervous system dysfunction. Microscopical examination of four regions of the brain (olfactory bulb, cerebellum, cerebrum and optical lobe) and the eye revealed the presence of lymphohistiocytic leptomeningitis, meningoencephalitis and endophthalmitis. Lesions were dominated by macrophages that often contained intracellular bacteria. Necrosis was recorded in some cases. Bacteriological screening revealed that multiple organs, including brain and eye, were infected with S. iniae and S. iniae colonized the scales and gills in high number. S. iniae was detected in tank water during the first week post infection, suggesting that infected tilapia might shed up to 3 × 10(7) CFU of S. iniae within 24 h. A multiplex polymerase chain reaction allowed confirmation of the challenge strain by detection of the virulence factors simA, scpI, cpsD, pgi, pgm and sagA.

Generalized fatal Cowpox virus infection in a cat with transmission to a human contact case.

A 4-month-old female domestic shorthair cat was infected by a virus of the Poxvirus family. The animal developed a severe pneumonia and generalized ulcerating lesions of the skin. Histologically, typical eosinophilic intracytoplasmic inclusion bodies indicative of an Orthopoxvirus (OPV) infection were present. The lung showed grey-white to haemorrhagic nodular lesions with a central zone of complete necrosis of alveolar and bronchial tissue. Electron microscopy from skin and lung nodules revealed typical square-shaped OPV particles. Cultivation of the virus on chorio-allantoic membranes of embryonated chicken eggs resulted in haemorrhagic plaques. Restriction enzyme analysis, PCR and sequencing of the D8L gene identified the OPV isolate as a typical Cowpox virus. It was transmitted by the cat to a human contact person who developed a local nodular dermatitis at the inoculation site in association with signs of general infection and had an increase of OPV-specific neutralizing antibodies in paired serum samples.

"Recreational" drug abuse associated with failure to mount a proper antibody response after a generalised orthopoxvirus infection.

Infections with orthopoxviruses usually lead to cross-protection among all species of the family. This has been a prerequisite for successful eradication of smallpox. Here we report the rare case of a 17-year-old male, who survived a generalised cowpox virus infection of unusual severity but surprisingly did not show a proper seroconversion. Only a very weak antibody production was observed in early and late serum samples, which initially appeared to be cowpox virus specific in immunofluorescence. No neutralising antibodies were detected and in Western blotting antibody specificity was restricted to the orthopoxvirus H3L protein only. The patient had been hospitalised for alcohol and cannabis intoxication 2 months prior to the orthopoxvirus infection and high levels of cannabinoids have been found repeatedly in the urine and upon one occasion also benzodiazepines. As these substances are known to interfere with antibody production and no immunodeficiencies were detected, drug-induced immunosuppression can be suspected as the most likely cause. Therefore a possible link between "soft" drug use and sufficient immunosuppression to warrant alterations in vaccine policies using live virus vaccines like smallpox vaccine should be further studied.