Douglas C. Hodgins

Mannheimia haemolytica and bovine respiratory disease.

Abstract Mannheimia haemolytica is the principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in all neonatal calves. A commensal of the nasopharynx, M. haemolytica is an opportunist, gaining access to the lungs when host defenses are compromised by stress or infection with respiratory viruses or mycoplasma. Although several serotypes act as commensals, A1 and A6 are the most frequent isolates from pneumonic lungs. Potential virulence factors include adhesin, capsular polysaccharide, fimbriae, iron-regulated outer membrane proteins, leukotoxin (Lkt), lipopolysaccharide (LPS), lipoproteins, neuraminidase, sialoglycoprotease and transferrin-binding proteins. Of these, Lkt is pivotal in induction of pneumonia. Lkt-mediated infiltration and destruction of neutrophils and other leukocytes impairs bacterial clearance and contributes to development of fibrinous pneumonia. LPS may act synergistically with Lkt, enhancing its effects and contributing endotoxic activity. Antibiotics are employed extensively in the feedlot industry, both prophylactically and therapeutically, but their efficacy varies because of inconsistencies in diagnosis and treatment regimes and development of antibiotic resistance. Vaccines have been used for many decades, even though traditional bacterins failed to demonstrate protection and their use often enhanced disease in vaccinated animals. Modern vaccines use culture supernatants containing Lkt and other soluble antigens, or bacterial extracts, alone or combined with bacterins. These vaccines have 50–70% efficacy in prevention of M. haemolytica pneumonia. Effective control of M. haemolytica pneumonia is likely to require a combination of more definitive diagnosis, efficacious vaccines, therapeutic intervention and improved management practices.

Factors associated with morbidity, mortality, and growth of dairy heifer calves up to 3 months of age

Calfhood disease is an important problem on many dairy operations that can have substantial effects on heifer survival and productivity, and has economic and welfare impacts. Neonatal calf diarrhea (NCD) and bovine respiratory disease (BRD) are the most common causes of morbidity and mortality in young dairy cattle. The objective of this observational study was to investigate factors associated with the risks of morbidity and mortality, and with growth, in commercial dairy heifers calves. A total of 2874 heifer calves from 19 commercial dairy farms in Minnesota and Ontario were enrolled at 1-7 days of age and followed for approximately 3 months. Using cut-points of serum total protein of 5.2 and 5.7 g/dl, the incidences of failure of transfer of passive immunity (FTPI) were 11 and 32%, respectively. A cut-point of 5.7 g/dl was the most predictive of BRD before 5 weeks of age (sensitivity=40%, specificity=69%). The positive predictive value was poor (PPV=18%), but the negative predictive value was good (NPV=87%). A cut-point of 5.2g/dl was most predictive of death before 5 weeks of age (sensitivity=27%, specificity=89%, PPV=5%, NPV=98%). Serum total protein during the first week of life was a poor predictor of NCD. Over 23% of calves were treated for diarrhea. Risk factors were weight at enrollment, other diseases before 2 weeks of age, and an interaction between season of birth and herd-level incidence of NCD. Almost 22% of calves were treated at least once for BRD. Factors associated with an increased risk of BRD included herd-level incidence of BRD, season of birth, navel dipping, other diseases before 2 weeks of age, failure of transfer of passive immunity, and manual control of temperature in pre-weaning housing. Administration of supplemental antibody products at birth was associated with a reduced incidence of BRD. Overall mortality was 3.5%. Risk of mortality was increased by treatment for BRD and other diseases. The mean average weight gain was 0.95 kg/day (range: 0.11-1.62 kg/day; SD=0.2). Twinning status, FTP, treatment for NCD or other diseases, and month of birth influenced body weight. This study illustrated relationships among various diseases, mortality, and growth. Furthermore, it demonstrated the importance of colostrum for protection against BRD and improved growth performance, while bringing into question the optimal method of determining failure of transfer of passive immunity.

Intranasal Administration of 2/6-Rotavirus-Like Particles with Mutant Escherichia coli Heat-Labile Toxin (LT-R192G) Induces Antibody-Secreting Cell Responses but Not Protective Immunity in Gnotobiotic Pigs

ABSTRACT We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutantEscherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.

Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication.

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A ‘pat7’ motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.

Passive and active components of neonatal innate immune defenses.

Abstract Innate immune defenses are crucial for survival in the first days and weeks of life. At birth, newborns are confronted with a vast array of potentially pathogenic microorganisms that were not encountered in utero. At this age, cellular components of the adaptive immune system are in a naïve state and are slow to respond. Antibodies received from the dam are essential for defense, but represent a finite and dwindling resource. Innate components of the immune system detect pathogen-associated molecular patterns (PAMPs) on microorganisms (and their products) by means of pattern-recognition receptors (PRRs). Soluble mediators of the innate system such as complement proteins, pentraxins, collectins, ficolins, defensins, lactoferrin, lysozyme etc. can bind to structures on pathogens, leading to agglutination, interference with receptor binding, opsonization, neutralization, direct membrane damage and recruitment of additional soluble and cellular elements through inflammation. Cell-associated receptors such as the Toll-like receptors (TLRs) can activate cells and coordinate responses (both innate and adaptive). In this paper, accumulated knowledge of the receptors, soluble and cellular elements that contribute to innate defenses of young animals is reviewed. Research interest in this area has been intermittent, and the literature varies in quantity and quality. It is hoped that documentation of the limitations of our knowledge base will lead to more extensive and enlightening studies.

Vaccination of neonates: problem and issues.

Many serious infectious diseases occur early in life; efficacious vaccination of neonates has been a longstanding goal in both human and veterinary medicine. Efforts to immunize in the first weeks of life, in various species, have had limited success in general. This has been attributed to a combination of immaturity of the neonatal immune system and interference by maternal antibodies. Most studies of neonatal immune responsiveness have been carried out in neonatal mice, or by examination of cellular components of human umbilical cord blood. Both approaches have their limitations. The current review describes factors, including corticosteroids, complement proteins, cytokines, maternal lymphocytes and antibodies, which may influence immune responses of neonates, comparing data from studies of domestic animals and humans. Neonates are highly dependent on passive (maternal) antibodies for protection against a wide range of pathogens. These maternal antibodies have been noted to interfere with active immune responses to many, but not all, vaccines. Various theories have been proposed to explain this phenomenon, including epitope masking, clearance of immune complexes and FcγRII mediated regulation of B cells. Remarkably, many studies examining the effects of passive antibodies on immune responses of adults, have demonstrated immune enhancing effects. The evidence for enhancing and suppressive effects of passive antibodies on antigen uptake, processing and regulation of lymphocyte responses is reviewed. Since maternal antibodies (as present in neonates) differ in subisotypes and affinity from the passive antibodies often used in experimental systems, here is a need for better experimental models investigating the effects of bona fide maternal antibodies on immune responses of neonates (not adult surrogates). Vaccines can be optimized for use in neonates - by making better use of existing vaccine technologies and by harnessing the potential of recent immunological and technological advances.

Modulation by colostrum-acquired maternal antibodies of systemic and mucosal antibody responses to rotavirus in calves experimentally challenged with bovine rotavirus

Abstract The effect of colostral maternal antibodies (Abs), acquired via colostrum, on passive protection and development of systemic and mucosal immune responses against rotavirus was evaluated in neonatal calves. Colostrum-deprived (CD) calves, or calves receiving one dose of pooled control colostrum (CC) or immune colostrum (IC), containing an IgG1 titer to bovine rotavirus (BRV) of 1:16,384 or 1:262,144, respectively, were orally inoculated with 105.5 FFU of IND (P[5]G6) BRV at 2 days of age. Calves were monitored daily for diarrhea, virus shedding and anti-BRV Abs in feces by ELISA. Anti-rotavirus Ab titers in serum were evaluated weekly by isotype-specific ELISA and virus neutralization (VN). At 21 days post-inoculation (dpi), all animals were euthanized and the number of anti-BRV antibody secreting cells (ASC) in intestinal and systemic lymphoid tissues were evaluated by ELISPOT. After colostrum intake, IC calves had significantly higher IgG1 serum titers (GMT=28,526) than CC (GMT=1195) or CD calves (GMT<4). After BRV inoculation, all animals became infected with a mean duration of virus shedding between 6 and 10 days. However, IC calves had significantly fewer days of diarrhea (0.8 days) compared to CD and CC calves (11 and 7 days, respectively). In both groups receiving colostrum there was a delay in the onset of diarrhea and virus shedding associated with IgG1 in feces. In serum and feces, CD and CC calves had peak anti-BRV IgM titers at 7 dpi, but IgA and IgG1 responses were significantly lower in CC calves. Antibody titers detected in serum and feces were associated with circulation of ASC of the same isotype in blood. The IC calves had only an IgM response in feces. At 21 dpi, anti-BRV ASC responses were observed in all analyzed tissues of the three groups, except bone marrow. The intestine was the main site of ASC response against BRV and highest IgA ASC numbers. There was an inverse relationship between passive IgG1 titers and magnitude of ASC responses, with fewer IgG1 ASC in CC calves and significantly lower ASC numbers of all isotypes in IC calves. Thus, passive anti-BRV IgG1 negatively affects active immune responses in a dose-dependent manner. In ileal Peyer’s patches, IgM ASC predominated in calves receiving colostrum; IgG1 ASC predominated in CD calves. The presence in IC calves of IgG1 in feces in the absence of an IgG1 ASC response is consistent with the transfer of serum IgG1 back into the gut contributing to the protection of the intestinal mucosa.

Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines

Triple-layered virus-like particles (VLPs) were produced in a baculovirus expression system from the two prevalent bovine rotavirus (BRV) serotypes, IND (P[5]G6) and 2292B (P[11]G10). Five groups of pregnant cows were inoculated intramuscularly and intramammarily with IND VLPs [BRV RF VP2, and IND VP4, 6, and 7, 250 microg per dose], 2292B VLPs [RF VP2, Cr VP4 (P[11]), and 2292B VP6 and 7, 250 microg per dose], combined IND/2292B VLPs (125 microg each VLP per dose), inactivated IND BRV (5x10(7)PFU per dose, pre-inactivation), or cell supernatant (mock-controls) in incomplete Freunds adjuvant. Serum, colostrum and milk were collected and tested for isotype-specific antibodies, and homologous and heterologous neutralizing antibodies (VN) to BRV by ELISA and VN tests, respectively. After vaccination, the IgG1 and homologous VN geometric mean antibody titers (GMTs) to BRV in serum of vaccinated groups were significantly (P<0.05) higher than in the mock-controls through postpartum day (PPD) 30. In colostrum, the IgG1 and IgA, and the homologous and heterologous VN GMTs of the IND VLP, 2292B VLP, combined IND/2292B VLP and the inactivated IND groups were significantly enhanced compared to the mock-controls, except for the heterologous VN GMTs in the inactivated IND group. However, the VLP vaccine groups had significantly higher homologous and heterologous VN GMTs than the inactivated IND group. The VN GMTs of the IND/2292B VLP group were statistically similar to the homologous VN GMTs of the IND or 2292B VLP groups, although the IgG1 GMT was lower. In milk, the IgG1 and homologous VN GMTs of the VLP groups were significantly higher than the inactivated IND or the mock-control groups through PPD30. However, the heterologous and homologous VN GMTs of inactivated IND group were statistically similar to the mock-control group at PPD0 and 30, respectively. These results demonstrate that the BRV antibody titers in serum, colostrum and milk are significantly enhanced by the use of triple-layered VLPs and inactivated IND vaccines, but significantly higher antibody responses were observed in the VLP vaccinated cows. The combined IND/2292B VLP vaccine induced comparable VN responses to BRV in serum, colostrum and milk compared to those induced by the individual IND or 2292B VLP vaccines, suggesting that at least two different serotypes can be mixed to confer maximum antibody responses to the incorporated serotypes.

Homotypic and Heterotypic Serum Isotype-Specific Antibody Responses to Rotavirus Nonstructural Protein 4 and Viral Protein (VP) 4, VP6, and VP7 in Infants Who Received Selected Live Oral Rotavirus Vaccines

Homotypic and heterotypic serum isotype-specific antibody responses to rotavirus enterotoxin nonstructural protein (NSP)-4, independent neutralization antigens viral protein (VP)-4 and VP7, and group A rotavirus common antigen VP6 were analyzed by an immunocytochemistry assay in infants who received 1 of several live oral rotavirus vaccines. Significant serum immunoglobulin (Ig) A and IgG antibody responses to homotypic and/or heterotypic NSP4s of genotype [A], [B], or [C] were detected after vaccination. The magnitude of antibody responses to homotypic and heterotypic NSP4s was not significantly different, irrespective of the NSP4 genotype of the administered vaccine strain. In addition, there were no significant differences between IgA antibody responses to homotypic and heterotypic VP7s. In contrast, IgA antibody responses to VP4 were predominantly homotypic. IgA antibody responses to VP7 were lower in magnitude than those to VP4 but were comparable to those to NSP4. Antibody titers to homotypic and/or heterotypic NSP4s were positively correlated with those to VP6 before and after vaccination.

Carbohydrate-based Clostridium difficile vaccines

Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface.