Detection of Borrelia and Babesia species in Haemaphysalis punctata ticks sampled in Southern England.

The distribution and population size of the red sheep tick (Haemaphysalis punctata) are increasing in Northern Europe, and in the United Kingdom reports of human biting by this species have increased in recent years. To assess the risk of tick-borne disease (TBD) transmission to humans and livestock by H. punctata, ticks sampled from sites in Southern England were screened using PCR for either Borrelia species or piroplasms over a three year period, 2018-2020. A total of 302 H. punctata were collected from eight locations. From these, two Babesia species associated with TBD infections in livestock, Babesia major and Babesia motasi, and the human pathogen Borrelia miyamotoi were detected, predominantly from a single location in Sussex. Consequently, the range expansion of this tick across Southern England may impact public and livestock health.

First report of human exposure to Hyalomma marginatum in England: Further evidence of a Hyalomma moulting event in north-western Europe?

Hyalomma marginatum is widely distributed across the Mediterranean, Northern Africa and the Middle East. Current climate conditions in Northern Europe are thought to limit the species' ability to moult to the adult stage. It is a vector of several pathogens of human and veterinary concern, including Crimean-Congo haemorrhagic fever virus, for which it is the primary vector in Europe. Here, we report the first human exposure to a locally acquired adult H. marginatum in England, and the second detection in England of Rickettsia aeschlimannii associated with imported Hyalomma.

Assessment of replication and virulence of attenuated pseudorabies virus in swine.

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.

Comparison of bovine herpesvirus 1 vaccines for rapid induction of immunity.

The purpose of the study reported here was to compare four different vaccine regimens for their efficacy in protecting calves from challenge with bovine herpesvirus 1 (BHV1) at 5 and 14 days after vaccination. Nine experimental groups of five calves each were used to compare four vaccination regimens and a group that received no vaccine. The four vaccine regimens were: a BHV1 intramuscular (IM) vaccine containing both modified live virus (MLV) and killed virus (KV); an MLV intranasal (IN) vaccine; concurrent administration of MLV IN vaccine and MLV IM vaccine; and concurrent administration of MLV IN vaccine and KV vaccine. All vaccine regimens induced solid protection against BHV1 challenge at 14 days after vaccination. All of the vaccine regimens also induced statistically significant (P < .05) protective immunity at 5 days after vaccination; however, there were significant differences (P < .05) in the degree of protection. The best protection induced by 5 days after vaccination was provided by the MLV + KV combination vaccine injected IM and the MLV vaccines given IN and IM concurrently. The MLV + KV combination vaccine administered IM gave significantly (P < .05) better protection by 5 days after vaccination than the MLV vaccine administered IN. Administering a KV BHV1 vaccine IM at the same time as the MLV BHV1 IN vaccine significantly (P < .05) reduced the effectiveness of the IN vaccine for inducing early protective immunity.

Safety and efficacy of an avirulent live Salmonella choleraesuis vaccine for protection of calves against S dublin infection.

OBJECTIVE: To evaluate the safety and efficacy of avirulent live Salmonella choleraesuis strain 54 (SC54) as a vaccine to protect calves against salmonellosis caused by S dublin. ANIMALS: 40 head of clinically normal 3 to 5-week-old male Holstein calves that were culture negative for Salmonella sp. PROCEDURE: Calves were randomly assigned to 4 test groups of 10 calves each. Group 1 received 8.5 x 10(7) colony-forming units (CFU) of SC54 SC. Groups 2 and 3 received 1.13 x 10(9) CFU of SC54, SC and intranasally, respectively. Group 4 received saline solution as a vaccine control. All calves were challenge exposed orally with 1.74 x 10(9) CFU of virulent S dublin 14 days after vaccination. Clinical signs and Salmonella shedding were monitored for 28 days after vaccination. Calves were necropsied, and organs were cultured for Salmonella sp 14 days after challenge exposure. RESULTS: Calves of groups 2 and 3 had slightly high rectal temperature after vaccination. Salmonella dublin challenge exposure resulted in mild clinical signs of salmonellosis. All vaccinated groups had significantly (P < 0.05) lower rectal temperature, fecal shedding of S dublin, and recovery of S dublin from organs after necropsy. SC54 was not recovered from fecal or blood samples collected after vaccination or from injection site samples or organs collected at necropsy. CONCLUSIONS: SC54 given intranasally or SC to calves was safe and significantly (P < 0.05) reduced clinical signs and bacterial shedding after oral challenge exposure with S dublin. CLINICAL RELEVANCE: SC54 has potential as an effective vaccine to aid in prevention of salmonellosis caused by S dublin in calves.

Military pregnancies and adverse perinatal outcome.

OBJECTIVE: To identify significant risk factors for an adverse outcome in active-duty military women. METHODS: A prospective study of 105 pregnancies and their outcome. RESULTS: The data revealed that: (1) single women more than married personnel had cesarean births when compared with forceps and vacuum (P < 0.03) or spontaneous vaginal delivery (P < 0.04); and (2) active-duty women who gained < 25 pounds during pregnancy developed preterm labor more often (P < 0.05). CONCLUSIONS: Risk factors for these adverse outcomes remain unknown.

Factors adversely affecting pregnancy outcome in the military.

This prospective study was undertaken to identify the significant risk factors associated with adverse pregnancy outcome in active-duty women. The deliveries of 300 consecutive pregnancies of active-duty women were assessed for maternal-fetal outcome. The risk factors evaluated were: marital status, parity, race, smoking and alcohol consumption while pregnant, maternal weight gain during pregnancy, maternal height, and educational level. Two-thirds of these women were junior enlisted personnel (rank E-4 or below) and worked under demanding job conditions over which they had little control. Increased age as a risk factor was associated with a significant increase in pregnancy-associated complications of cesarean birth, operative vaginal delivery, pregnancy-induced hypertension, preterm labor, maternal transport for fetal indications, intrauterine growth restriction, intrauterine fetal death, postpartum hemorrhage, placenta previa, and 5-minute Apgar scores < 7 (p = 0.039). In gravidas more than 65 inches in height with a weight gain more than 42 pounds, there was a significant increase in the complications of pregnancy (p = 0.022). Interactions of these risk factors yielded a significant age (p = 0.025), maternal height (p = 0.007), and height times weight gain interaction (p = 0.006) association with pregnancy complications. The risk factors of advancing maternal age and tall stature with a maternal weight gain of more than 42 pounds are associated with increased pregnancy complications of active-duty women.

Relationship of hemolysis buffer structure, pH and ionic strength to spontaneous contour smoothing of isolated erythrocyte membranes.

Isolated human erythrocyte membranes crenate when suspended in isotonic medium, but can use MgATP to reduce their net positive curvature, yielding smooth discs and cup forms that eventually undergo endocytosis. An earlier report from this laboratory (Patel, V.P. and Fairbanks, G. (1981) J. Cell Biol. 88, 430-440), has described a phenomenon of ATP-independent shape change in which ghosts prepared by hemolysis and washing in synthetic zwitterionic buffers crenated at 0 degree C, but underwent conversion to smooth discs and cups when warmed in the absence of MgATP. We have further explored the effect of the hemolysis condition on the requirement for ATP in ghost shape change. 25 hemolysis buffers were applied at 10 mM (pH 7.4, 0 degree C). Eight anionic buffers with relatively high ionic strength (e.g., phosphate and diethylmalonic acid (DMA] yielded ghosts requiring ATP for shape change, while two cationic buffers (Bistris and imidazole) and ten synthetic zwitterionic buffers (e.g., Tricine and Hepes) with lower ionic strength produced ghosts that smoothed spontaneously at 30 degrees C. Hemolysis at intermediate ionic strength yielded mixed populations in which spontaneous smoothing was expressed in all-or-none fashion. Maximal ATP-independent shape change was induced by hemolysis at pH 7.3-7.7, while ATP was required after hemolysis at pH less than or equal to 7.1 even when the ionic strength at hemolysis was low. Ghosts requiring ATP could be converted to ATP independence by washing at low ionic strength, but ATP independence could not be reversed readily by washing at high ionic strength. Exposure to low ionic strength at pH greater than 7.1 presumably changes membrane organization in a way that alters the temperature dependence of tensions within the bilayer or skeleton of the composite membrane.

Inhibition of erythrocyte membrane shape change by band 3 cytoplasmic fragment.

The ATP-dependent transformation of crenated white human erythrocyte ghosts into smoothed disc and cup forms is inhibited by the soluble 40-45-kilodalton (kDa) cytoplasmic portion of the major transmembrane protein, band 3. The band 3 fragment was prepared by chymotryptic treatment of inverted vesicles stripped of peripheral proteins. When present at greater than or equal to 0.2 mg per mg membrane protein (ie, greater than or equal to 2 mol fragment per mol endogenous band 3), the fragment significantly reduced the rate of shape change but did not alter the proportion of membranes that were ultimately converted into smoothed forms (greater than 90%). The inhibitory activity of the fragment could not be attributed to contamination of the fragment preparation by actin or proteolytic enzymes. ATP-independent shape transformation was not inhibited. The band 3 fragment may compete with endogenous, intact band 3 for an association with the spectrin-actin network required for ATP-dependent smoothing of crenated membranes.

The lateral mobility and surface distribution of Lyt-1, Lyt-2 and Lyt-3 on mouse thymocytes.

Fluorescein-conjugated monoclonal antibodies were used to investigate the properties of the Lyt-1, Lyt-2 and Lyt-3 antigens on the murine lymphocyte cell surface. Monoclonal antibody to Lyt-1 patched and capped on the thymocyte cell surface, whereas an Fab fragment of the antibody gave a uniform distribution of fluorescence on most cells and diffused freely in the plane of the membrane. Fluorescence photobleaching measurements showed that the intact antibody was relatively immobile on the cell surface, whereas the Fab fragment was freely mobile. Monoclonal antibody to Lyt-2 or Lyt-3 (which are linked to one another by disulfide bonds) gave a uniform distribution of fluorescence and was mobile on the cell surface. When cells were incubated with antibodies to Lyt-2 and Lyt-3 simultaneously, cells appeared patched and the lateral mobility of the antibodies was greatly reduced. Treatment of cells with either sodium azide or 2-mercaptoethanol followed by N-ethyl maleimide to reduce and block sulfhydryl groups inhibited the patch formation caused by simultaneous incubation with both Lyt-2 and Lyt-3 antibodies.

Evaluation of IgG molecules, Fab' fragments and IgG-horseradish peroxidase conjugates as surface labels for freeze-etched membranes.

Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti-SRBC IgG, antigen-binding fragments of IgG (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labelled ghosts were deep-etched and replicated with platinum-carbon to visualize their surface features in the transmission electron microscope. The surfaces of control ghosts contain a very low number of 'background' particles (42 +/- 8 particles/micron 2) that vary in size from 4.5 to 25 nm. After labelling with whole IgG the density of surface particles (average diameter 12.3 nm) increases dramatically to 480 +/- 54 per micron 2. Fab'-labelled ghosts exhibited both significantly fewer (87 +/- 14 particles/micron 2) and smaller (average diameter 9.8 nm) surface particles. Ghosts labelled with IgG-HRPO conjugates possessed 590 +/- 45 particles/micron 2 with an average diameter of 15.3 nm. When these ghosts were incubated with diaminobenzidine and hydrogen peroxide the average size but not the density of the particles increased. Based on these and other observations we conclude that an organic surface marker for freeze-etched membranes has to have a diameter of greater than 15 nm if it is to be consistently seen over extended areas and against the background granularity of the surface of a red blood cell ghost. Somewhat better resolution may be expected if markers consisting of inorganic crystals with a distinct shape and coupled to Fab' fragments can be made.

IgG molecules, Fab' fragments and IgG-peroxidase conjugates as markers in replicas of deep-etched, labeled cell surfaces.

Sheep red blood cell (SRBC) ghosts were incubated with preparations of anti-SRBC IgG, antigen-binding fragments (Fab') or IgG coupled to horseradish peroxidase (HRPO). Frozen samples of the labeled ghosts were deep-etched and replicated with platinum-carbon to visualize their surface features in the transmission electron microscope. An analysis of both size and density of surface "particles" observed on labeled ghosts was performed to evaluate which markers could be practically used to label these membranes. It was concluded that in this system a marker must exhibit a diameter of greater than 150 A (the apparent size of IgG-HRPO conjugates) to be consistently seen over extensive surface areas and to be distinguished from the background granularity of the SRBC ghost surface. This rules out the use of Fab' or IgG as markers in this system.

Immunospecific labeling of mouse lymphocytes in the scanning electron microscope.

Bone marrow-derived (B) and thymus-derived (T) Balb/c mouse lymphocytes were identified in the scanning electron microscope (SEM) by the immunospecific attachment of one of several kinds of large-molecular-weight markers distinguishable in SEM. These markers (tobacco mosaic virus, keyhole limpet hemocyanin, bushy stunt virus, and bacteriophage T4) could be modified with hapten groups and linked with anti-hapten antibody, in an indirect (sandwich) scheme, to hapten-modified anti-cell-surface antibody bound to the cell surface. Hapten-modified antibodies to B cell antigens (goat anti-mouse-immunoglobulin) or to T cell antigens (rabbit anti-mouse brain) were employed to identify these two lymphoid cell types in unfractionated spleen, mesenteric lymph node, bone marrow, and thymus cell populations. The topography of B cells was always indistinguishable from that of T cells. No surface features were found to be unique to either cell type. In suspension, the majority of B and T cells had one or no microvilli regardless of the tissue source of the labeled cells. Cells in suspension that had microvilli (usually 10% of the total cell population) were always unlabeled. However, after cell contact with a glass surface, approximately half of both the B and T cell population had a villous topography.

Hapten-sandwich labeling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy.

A hapten-sandwich procedure has been used for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy. Antihapten antibody was used to link hapten-modified tobacco mosaic virus, bushy stunt virus, or hemocyanin to hapten-modified human erythrocytes. The antihapten antibody bridge was also used to link the hapten-virus marker to hapten-modified antibodies against mammary tumor virus on mouse mammary tumor cells, or against immunoglobulin receptors on mouse splenic lymphocytes. In all cases, labeling was highly specific. With this technique, it is possible to (a) compare morphological features of cells bearing differing cell surface antigens, and (b) examine the arrangement of specific antigenic sites on a cell surface or their distribution relative to membrane structures such as microvilli.