Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Understanding the mechanisms of viral and bacterial coinfections in bovine respiratory disease: a comprehensive literature review of experimental evidence.

Bovine respiratory disease (BRD) is one of the most important diseases impacting the global cattle industry, resulting in significant economic loss. Commonly referred to as shipping fever, BRD is especially concerning for young calves during transport when they are most susceptible to developing disease. Despite years of extensive study, managing BRD remains challenging as its aetiology involves complex interactions between pathogens, environmental and host factors. While at the beginning of the twentieth century, scientists believed that BRD was only caused by bacterial infections ("bovine pasteurellosis"), we now know that viruses play a key role in BRD induction. Mixtures of pathogenic bacteria and viruses are frequently isolated from respiratory secretions of animals with respiratory illness. The increased diagnostic screening data has changed our understanding of pathogens contributing to BRD development. In this review, we aim to comprehensively examine experimental evidence from all existing studies performed to understand coinfections between respiratory pathogens in cattle. Despite the fact that pneumonia has not always been successfully reproduced by in vivo calf modelling, several studies attempted to investigate the clinical significance of interactions between different pathogens. The most studied model of pneumonia induction has been reproduced by a primary viral infection followed by a secondary bacterial superinfection, with strong evidence suggesting this could potentially be one of the most common scenarios during BRD onset. Different in vitro studies indicated that viral priming may increase bacterial adherence and colonization of the respiratory tract, suggesting a possible mechanism underpinning bronchopneumonia onset in cattle. In addition, a few in vivo studies on viral coinfections and bacterial coinfections demonstrated that a primary viral infection could also increase the pathogenicity of a secondary viral infection and, similarly, dual infections with two bacterial pathogens could increase the severity of BRD lesions. Therefore, different scenarios of pathogen dynamics could be hypothesized for BRD onset which are not limited to a primary viral infection followed by a secondary bacterial superinfection.

Interference between avian corona and influenza viruses: The role of the epithelial architecture of the chicken trachea.

Respiratory viral infections are among the major causes of disease in poultry. While viral dual infections are known to occur, viral interference in chicken airways is mechanistically hardly understood. The effects of infectious bronchitis virus (IBV) infection on tissue morphology, sialic acid (sia) expression and susceptibility of the chicken trachea for superinfection with IBV or avian influenza virus (AIV) were studied. In vivo, tracheal epithelium of chickens infected with IBV QX showed marked inflammatory cell infiltration and loss of cilia and goblet cells five days post inoculation. Plant lectin staining indicated that sialic acids redistributed from the apical membrane of the ciliated epithelium and the goblet cell cytoplasm to the basement membrane region of the epithelium. After administration of recombinant viral attachment proteins to slides of infected tissue, retained binding of AIV hemagglutinin, absence of binding of the receptor binding domain (RBD) of IBV M41 and partial reduction of IBV QX RBD were observed. Adult chicken trachea rings were used as ex vivo model to study the effects of IBV QX-induced pathological changes and receptor redistribution on secondary viral infection. AIV H9N2 infection after primary IBV infection was delayed; however, final viral loads reached similar levels as in previously uninfected trachea rings. In contrast, IBV M41 superinfection resulted in 1000-fold lower viral titers over the course of 48 h. In conclusion, epithelial changes in the chicken trachea after viral infection coincide with redistribution and likely specific downregulation of viral receptors, with the extend of subsequent viral interference dependent on viral species.

SARS-CoV-2 superinfection and reinfection with three different strains.

We report a corona virus disease (COVID-19) case with unprecedented viral complexity. In the first severe episode, two different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains (superinfection) were identified within a week. Three months after discharge, the patient was readmitted and was infected in a nosocomial outbreak with a different strain, suffering a second milder COVID-19 episode.

An Embedded Multiscale Modelling to Guide Control and Elimination of Paratuberculosis in Ruminants.

In recent years, multiscale modelling approach has begun to receive an overwhelming appreciation as an appropriate technique to characterize the complexity of infectious disease systems. In this study, we develop an embedded multiscale model of paratuberculosis in ruminants at host level that integrates the within-host scale and the between-host. A key feature of embedded multiscale models developed at host level of organization of an infectious disease system is that the within-host scale and the between-host scale influence each other in a reciprocal (i.e., both) way through superinfection, that is, through repeated infection before the host recovers from the initial infectious episode. This key feature is demonstrated in this study through a multiscale model of paratuberculosis in ruminants. The results of this study, through numerical analysis of the multiscale model, show that superinfection influences the dynamics of paratuberculosis only at the start of the infection, while the MAP bacteria replication continuously influences paratuberculosis dynamics throughout the infection until the host recovers from the initial infectious episode. This is largely because the replication of MAP bacteria at the within-host scale sustains the dynamics of paratuberculosis at this scale domain. We further use the embedded multiscale model developed in this study to evaluate the comparative effectiveness of paratuberculosis health interventions that influence the disease dynamics at different scales from efficacy data.

Ligilactobacillus salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells and Improve Protection Against Intestinal Viral-Bacterial Superinfection.

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-ß expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-ß, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-ß, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

Epidemiology and biology of a herpesvirus in rabies endemic vampire bat populations.

Rabies is a viral zoonosis transmitted by vampire bats across Latin America. Substantial public health and agricultural burdens remain, despite decades of bats culls and livestock vaccinations. Virally vectored vaccines that spread autonomously through bat populations are a theoretically appealing solution to managing rabies in its reservoir host. We investigate the biological and epidemiological suitability of a vampire bat betaherpesvirus (DrBHV) to act as a vaccine vector. In 25 sites across Peru with serological and/or molecular evidence of rabies circulation, DrBHV infects 80-100% of bats, suggesting potential for high population-level vaccine coverage. Phylogenetic analysis reveals host specificity within neotropical bats, limiting risks to non-target species. Finally, deep sequencing illustrates DrBHV super-infections in individual bats, implying that DrBHV-vectored vaccines might invade despite the highly prevalent wild-type virus. These results indicate DrBHV as a promising candidate vector for a transmissible rabies vaccine, and provide a framework to discover and evaluate candidate viral vectors for vaccines against bat-borne zoonoses.

Coinfections and their molecular consequences in the porcine respiratory tract.

Understudied, coinfections are more frequent in pig farms than single infections. In pigs, the term "Porcine Respiratory Disease Complex" (PRDC) is often used to describe coinfections involving viruses such as swine Influenza A Virus (swIAV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine CircoVirus type 2 (PCV2) as well as bacteria like Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Bordetella bronchiseptica. The clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. In this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. Coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. We discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health.

Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy.

BACKGROUND: Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions. RESULTS: Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage. CONCLUSIONS: Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.

In vivo study of viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus coexistence in Senegalese sole (Solea senegalensis).

The effect of IPNV-VHSV coinfection and superinfection on the mortality caused by both viruses in Senegalese sole has been analysed. No effect was observed after coinfection. However, a clear viral interference was recorded between a primary IPNV and a subsequent VHSV infection, which led to a survival increase in the infected sole of 50% points when compared with fish infected only with VHSV. The significantly higher Mx transcriptional values in the fish pre-exposed to IPNV (at least at first days after superinfection) and the increased daily mortality when low Mx transcriptional levels were recorded suggest that Mx may be involved in the protective effect against VHSV infection. However, in fish subjected to VHSV primary/IPNV secondary infection, no interference was observed.

Experimental bluetongue virus superinfection in calves previously immunized with bluetongue virus serotype 8.

The effect of a superinfection with bluetongue virus serotype 1 (BTV1) was evaluated on two groups of four calves. One group received a commercial inactivated BTV serotype 8 (BTV8) vaccine. This group and the non-vaccinated group of calves were challenged twice (4 months apart) with the European BTV8 strain isolated during the 2006-2007 epidemics. Calves were then infected with a BTV1 inoculum which was found to be unexpectedly contaminated by BTV serotype 15 (BTV15). BTV1 and BTV15 single infections were performed on two other groups of three BTV naïve calves. A severe clinical picture was obtained after superinfection with BTV1/BTV15 in both vaccinated and non-vaccinated animals and after challenge with BTV8 in non-vaccinated animals. BTV1 and BTV15 single infection caused only very slight clinical signs. After superinfection and at the viraemic peak, there were an average of above 1000 times more BTV15 genomic copies than BTV1 ones. BTV1 RNA could be detected only in the spleen of one calf whereas BTV15 RNA was found in 15 organs of seven different animals. BTV8 immunization whether it was acquired through vaccination and challenges or challenges alone did not change BTV1 or BTV15 RNA detection in superinfected animals. However in these animals a partial cross neutralization between BTV8 and BTV1 might be involved in the lower BTV1 replication versus BTV15. Infection with different serotypes can occur also in the field. Interference between virus strains, genetic reassortment and cross-protection were considered as mechanisms to explain the clinical outcomes and the other virological and immunological findings in the course of BTV1/BTV15 superinfection.

Anaplasma marginale superinfection attributable to pathogen strains with distinct genomic backgrounds.

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.

Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host.

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek's disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well.

The effect of the time interval between exposures on the susceptibility of chickens to superinfection with Marek's disease virus.

Marek's disease virus (MDV) is ubiquitous within commercial poultry flocks because current vaccines do not prevent MDV infection or transmission. In order for newly-evolved MDV strains to become established within a flock, it seems inevitable that any new strain would need to infect and replicate in chickens previously infected with resident MDV strains. This phenomenon is difficult to detect and there is no clear evidence that it is even possible. Four experiments were performed to demonstrate superinfection and evaluate the effect of time between challenges on the effect of superinfection with the use of two pairs of fully virulent MDV strains that could be discriminated by novel technology: 1) JM/102W and rMd5//38CVI, and 2) rMd5 and rMd5//38CVI. Feather follicle epithelium (FFE), spleen, and tumor samples were collected at single or multiple time points from the same bird to determine the frequency and distribution of each virus present following superinfection, with the use of pyrosequencing and immunohistochemistry. Superinfection was observed in 82 of 149 (55%) FFE samples following short-interval challenge (24 hr) compared to only 6 of 121 (5%) samples following long-interval challenge (13 days), indicating a strong influence of challenge interval. In cases where the first inoculated virus was weak or delayed, the second inoculated virus was detected in 42 of 95 (44%) birds. In tumors from dually challenged birds, the second virus was again present much more often following short-interval challenge (68%) compared to long-interval challenge (11%). Virus mixtures in tumors were less common compared to those in FFE samples. Vaccination with turkey herpesvirus had no significant effect on the virus frequency for either virus pair or challenge time interval, suggesting these conclusions may be applicable to vaccinated chickens in the field. These studies demonstrated superinfection for the first time with two fully virulent MDV strains and suggest that short-interval challenge exposure and/or weak initial exposures may be important factors leading to superinfection--a prerequisite for the establishment of a second virus strain in the population. This model system should be useful to elucidate this important phenomenon further.

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli.

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, gammadelta-TCR, alphabeta1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.

Progression of lesions in the respiratory tract of broilers after single infection with Escherichia coli compared to superinfection with E. coli after infection with infectious bronchitis virus.

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.

Experimental superinfection of a Lesser Malayan mousedeer (Tragulus javanicus) persistently infected with bovine viral diarrhea virus.

A Lesser Malayan mousedeer (Tragulus javanicus), persistently infected with noncytopathogenic bovine viral diarrhea virus (BVDV) type 1f, was experimentally superinfected with a cytopathogenic isolate of BVDV type 1c, which antigenically partially matched the endogenous strain. Within the observational period of 125 days after superinfection, the animal did not demonstrate any clinical signs of the disease and/or significant changes in blood values. Neutralizing antibodies were detected at 35 and 42 days postinfection. The isolate causing the superinfection was found in feces, nasal swabs, and saliva starting from day 29 and at various times postchallenge. Macroscopic or histologic examination did not reveal mucosal disease-like lesions, despite the detection of the cytopathogenic isolate in the salivary gland, rumen, abomasum, kidney, and superficial prescapular lymph node. Results indicate that the cytopathogenic BVDV strain, which was used in the superinfection, persisted in the viremic animal without causing disease within the observation period.

Experimental test for premunition in a lizard malaria parasite (Plasmodium mexicanum).

Premunition in Plasmodium spp. is the prevention of superinfection by novel genotypes entering an already established infection in a vertebrate host. Evidence for premunition was sought for the lizard malaria parasite, P. mexicanum, in its natural host, the fence lizard, Sceloporus occidentalis. Clonal diversity (= alleles for the haploid parasite) was determined with the use of 3 microsatellite markers. Both naturally infected lizards (N = 25) and previously noninfected lizards (N = 78) were inoculated intraperitoneally (IP) with blood from donor infections and followed over a 3-mo period. Compared to the success of clonal establishment in all the naive lizards (78/78 successful), clones entering preexisting infections had a significant disadvantage (9/25 successful). The number of preexisting clones (1-2 vs. 3-4) within recipient infections had no effect on the success of superinfection. Infections that excluded entering novel clones did not have higher initial asexual parasitemia, but had a higher initial density of gametocytes, suggesting they were older. Infections allowing superinfection experienced a higher final parasitemia.