Ivermectin inhibits replication of the malignant catarrhal fever virus alcelaphine herpesvirus 1.

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates that is caused by genetically and antigenically related gamma herpesviruses of the genus Macavirus. Infection of the natural host species is efficient and asymptomatic but spread to susceptible hosts is often fatal with clinical signs including fever, depression, nasal and ocular discharge. There is no recognised treatment for MCF but a vaccine for one MCF virus, alcelaphine herpesvirus 1 (AlHV-1), has been described. In this paper we describe the inhibition of AlHV-1 replication and propagation by the anthelminthic drug ivermectin. Concentrations of 10 µM or greater led to significant reductions in both copy number and viable titre of virus tested in culture medium, with little replication detected at over 20 µM ivermectin. In the absence of alternative treatments, further testing of ivermectin as a candidate antiviral treatment for MCF may therefore be justified.

Prospective cohort study reveals unexpected aetiologies of livestock abortion in northern Tanzania.

Livestock abortion is an important cause of productivity losses worldwide and many infectious causes of abortion are zoonotic pathogens that impact on human health. Little is known about the relative importance of infectious causes of livestock abortion in Africa, including in subsistence farming communities that are critically dependent on livestock for food, income, and wellbeing. We conducted a prospective cohort study of livestock abortion, supported by cross-sectional serosurveillance, to determine aetiologies of livestock abortions in livestock in Tanzania. This approach generated several important findings including detection of a Rift Valley fever virus outbreak in cattle; high prevalence of C. burnetii infection in livestock; and the first report of Neospora caninum, Toxoplasma gondii, and pestiviruses associated with livestock abortion in Tanzania. Our approach provides a model for abortion surveillance in resource-limited settings. Our findings add substantially to current knowledge in sub-Saharan Africa, providing important evidence from which to prioritise disease interventions.

Development of a recombinant ELISA for ovine herpesvirus 2, suitable for use in sheep.

The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5'-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins. An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle. This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.

Analysis of immune responses to attenuated alcelaphine herpesvirus 1 formulated with and without adjuvant.

The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These observations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain.

Bovine viral diarrhoea virus loses quasispecies diversity rapidly in culture.

Bovine viral diarrhoea (BVD) is an important disease of cattle, with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in persistently infected (PI) animals is, therefore, essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from the serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250 bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this pestivirus A genotype 1a field strain within serum and after culture. We report the distribution and diversity of over 800 SNPs and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples.

Immune-associated traits measured in milk of Holstein-Friesian cows as proxies for blood serum measurements.

Previous work has highlighted that immune-associated (IA) traits measurable in blood are associated with health, productivity, and reproduction in dairy cows. The aim of the present study was to determine relationships between IA traits measured in blood serum and those simultaneously measured in milk as well as their association with disease phenotypes. All animals were Holstein-Friesian cows from the Langhill research herd (n = 546) housed at the SRUC Dairy Research Centre in Scotland. Milk and serum samples were collected on 20 separate occasions between July 2010 and March 2015 and analyzed by ELISA for haptoglobin (Hp), tumor necrosis factor-α (TNF-α), and natural antibodies binding keyhole limpet hemocyanin (NAbKLH) and lipopolysaccharide (NAbLPS). Data were analyzed using mixed linear models that included pedigree information. Analyses revealed positive phenotypic correlations between milk and serum NAb (0.59 ≤ r ≤ 0.77), Hp (r = 0.37), and TNF-α (r = 0.12). Milk and serum NAb were also found to have a strong genetic correlation (0.81 ≤ r ≤ 0.94) and were genetically correlated with cow lameness (0.66 and 0.79 for milk NAbKLH and serum NAbLPS, respectively). Clinical mastitis was found to be phenotypically correlated with both milk and serum Hp (0.09 ≤ r ≤ 0.23). Serum Hp was also strongly genetically correlated with other cellular IA traits such as percent NKp46+ (a natural killer cell marker; 0.35) and percent peripheral blood mononuclear cells (PBMC; -0.90). Similarly, genetic correlations were found to exist between serum TNF-α and percent NKp46+ (0.22), percent PBMC (0.41), and percent lymphocytes (0.47). Excluding serum Hp, all milk and serum IA traits were repeatable, ranging from 0.11 (milk Hp) to 0.43 (serum NAbLPS). Between-animal variation was highest in milk and serum NAb (0.34-0.43) and significant estimates of heritability were also observed in milk and serum NAb (0.17-0.37). Our findings show that certain IA traits, such as NAbKLH and NAbLPS, found in milk and serum are strongly correlated and highlight the potential of using routinely collected milk samples as a less invasive and cost-effective source of informative data for predictive modeling of animal IA traits.

Estimating genetic and phenotypic parameters of cellular immune-associated traits in dairy cows.

Data collected from an experimental Holstein-Friesian research herd were used to determine genetic and phenotypic parameters of innate and adaptive cellular immune-associated traits. Relationships between immune-associated traits and production, health, and fertility traits were also investigated. Repeated blood leukocyte records were analyzed in 546 cows for 9 cellular immune-associated traits, including percent T cell subsets, B cells, NK cells, and granulocytes. Variance components were estimated by univariate analysis. Heritability estimates were obtained for all 9 traits, the highest of which were observed in the T cell subsets percent CD4+, percent CD8+, CD4+:CD8+ ratio, and percent NKp46+ cells (0.46, 0.41, 0.43 and 0.42, respectively), with between-individual variation accounting for 59 to 81% of total phenotypic variance. Associations between immune-associated traits and production, health, and fertility traits were investigated with bivariate analyses. Strong genetic correlations were observed between percent NKp46+ and stillbirth rate (0.61), and lameness episodes and percent CD8+ (-0.51). Regarding production traits, the strongest relationships were between CD4+:CD8+ ratio and weight phenotypes (-0.52 for live weight; -0.51 for empty body weight). Associations between feed conversion traits and immune-associated traits were also observed. Our results provide evidence that cellular immune-associated traits are heritable and repeatable, and the noticeable variation between animals would permit selection for altered trait values, particularly in the case of the T cell subsets. The associations we observed between immune-associated, health, fertility, and production traits suggest that genetic selection for cellular immune-associated traits could provide a useful tool in improving animal health, fitness, and fertility.

Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen.

Assessment of the rabbit as a wildlife reservoir of bovine viral diarrhea virus: serological analysis and generation of trans-placentally infected offspring.

Eradication of bovine viral diarrhea virus (BVDV) is ongoing in many European countries and is based on removal of persistently infected (PI) cattle. In this context, low-level risks, including alternative reservoirs of infection, may become more important as the number of BVDV-free herds increases. Alternative reservoirs include livestock, such as sheep and goats, as well as wildlife, including deer and rabbits. Due to the extensive nature of the beef industry in Scotland, where an eradication program started in 2010, contact between cattle and alternative reservoir hosts is common. Seroprevalence to BVDV in rabbit populations can be high. In addition, rabbits can be infected with BVDV by natural routes, indicating that they could be a wildlife reservoir of infection. We analyzed the potential risk to livestock from rabbit populations in the UK by two approaches. First, ∼260 serum samples from free-ranging wild rabbits in Scotland and northern England were tested for BVDV-specific antibodies by ELISA. Only three samples exhibited low level BVDV-specific reactivity, suggesting that BVDV infection of rabbits was not frequent. Second, rabbits were challenged with BVDV at day 7 or 12 of pregnancy. This did not lead to any clinical signs in the infected animals or obvious increases in abortion or stillbirth in the infected dams. Samples from the dams, placental material and ∼130 offspring were tested by BVDV-specific RT-PCR and antibody ELISA. Positive PCR results in the placentas and in the tissues and body fluids of rabbits up to 10 days old showed that trans-placental infection of rabbits with BVDV had occurred. Many of the offspring had BVDV-specific antibodies. These data support the view that a wildlife reservoir of BVDV in rabbit poses a small but non-zero risk of re-infection for BVDV-free cattle herds. Rabbits are susceptible to infection with BVDV but only a small proportion of free-living rabbits in the UK appear to have been infected.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

Identification of immuno-reactive capsid proteins of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.

The effect of the TLR9 ligand CpG-oligodeoxynucleotide on the protective immune response to alcelaphine herpesvirus-1-mediated malignant catarrhal fever in cattle.

We wished to determine the effect of of CpG ODN adjuvant on the magnitude and duration of protective immunity against alcelaphine herpesvirus-1 (AlHV-1) malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of cattle. Immunity was associated with a mucosal barrier of virus-neutralising antibody. The results showed that CpG ODN included either with emulsigen adjuvant and attenuated AlHV-1 (atAlHV-1) or alone with atAlHV-1 did not affect the overall protection from clinical disease or duration of immunity achieved using emulsigen and atAlHV-1. This is in contrast to other similar studies in cattle with BoHV-1 or cattle and pigs with various other immunogens. In addition to this, several other novel observations were made, not reported previously. Firstly, we were able to statistically verify that vaccine protection against MCF was associated with virus-neutralising antibodies (nAbs) in nasal secretions but was not associated with antibodies in blood plasma, nor with total virus-specific antibody (tAb) titres in either nasal secretions or blood plasma. Furthermore, CpG ODN alone as adjuvant did not support the generation of virus-neutralising antibodies. Secondly, there was a significant boost in tAb in animals with MCF comparing titres before and after challenge. This was not seen with protected animals. Finally, there was a strong IFN-γ response in animals with emulsigen and atAlHV-1 immunisation, as measured by IFN-γ secreting PBMC in culture (and a lack of IL-4) that was not affected by the inclusion of CpG ODN. This suggests that nAbs at the oro-nasal-pharyngeal region are important in protection against AlHV-1 MCF.

Analysis of the genetic diversity of ovine herpesvirus 2 in samples from livestock with malignant catarrhal fever.

In order to define better virus isolates from animals with malignant catarrhal fever (MCF), segments of three genes of ovine herpesvirus-2 were amplified from diagnostic samples representing MCF cases with a range of clinical presentations in cattle, including head and eye, alimentary and neurological. The variation within each gene segment was estimated by DNA sequencing, which confirmed that the newly-annotated Ov9.5 gene was significantly more polymorphic than either of the other loci tested (segments of ORF50 and ORF75), with alleles that differed at over 60% of nucleotide positions. Despite this, the nine Ov9.5 alleles characterised had identical predicted splicing patterns and could be translated into Ov9.5 polypeptides with at least 49% amino acid identity. This multi-locus approach has potential for use in epidemiological studies and in charactering chains of infection. However there was no association between specific variants of OvHV-2 and the clinical/pathological presentation of MCF in the cattle analysed.

Experimental infection of rabbits with bovine viral diarrhoea virus by a natural route of exposure.

Bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle that can naturally infect a wide range of even-toed ungulates. Non-bovine hosts may represent reservoirs for the virus that have the potential to hamper BVDV eradication programs usually focused on cattle. Rabbits are very abundant in countries such as the United Kingdom or Australia and are often living on or near livestock pastures. Earlier reports indicated that rabbits can propagate BVDV upon intravenous exposure and that natural infection of rabbits with BVDV may occur but experimental proof of infection of rabbits by a natural route is lacking. Therefore, New Zealand White rabbits were exposed to a Scottish BVDV field strain intravenously, oro-nasally and by contaminating their hay with virus. None of the animals showed any clinical signs. However, the lymphoid organs from animals sacrificed at day five after exposure showed histological changes typical of transient infection with pestivirus. Most organ samples and some buffy coat samples were virus positive at day five but saliva samples remained negative. Development of antibodies was observed in all intravenously challenged animals, in all of the nebulised group and in four of six animals exposed to contaminated hay. To our knowledge this is the first report of BVDV propagation in a species other than ruminants or pigs after exposure to the virus by a natural route. However, to assess the role of rabbits as a potential reservoir for BVDV it remains to be determined whether persistent infection caused by intra-uterine infection is possible and whether BVDV is circulating in wild rabbit populations.

A novel spliced gene in alcelaphine herpesvirus 1 encodes a glycoprotein which is secreted in vitro.

Herpesviruses often contain cryptic, spliced genes that are not obvious from the initial in silico annotation. Alcelaphine herpesvirus 1 (AlHV-1) contains 72 annotated ORFs but there are also a number of gaps between these that may have protein-coding potential. Comparative analysis of coding potential between AlHV-1 and the related ovine herpesvirus 2 (OvHV-2) revealed a putative novel spliced gene that we have termed A9.5. Analysis of cDNA clones from AlHV-1-infected cells revealed three overlapping clones corresponding to A9.5 and the coding sequence was confirmed by reverse transcription PCR of RNA from AlHV-1-infected cattle tissues. The A9.5 gene was predicted to encode a secreted glycoprotein with molecular mass 19 kDa. Empirical analysis showed that a recombinant haemagglutinin-tagged A9.5 fusion protein was secreted from transfected cells and had a molecular mass of 45 kDa, which was reduced to 20 kDa by endoglycosidase F treatment, confirming that A9.5 was a secreted glycoprotein. In situ RNA hybridization showed that A9.5 was expressed in cells associated with malignant catarrhal fever (MCF) lesions in infected cattle. Detailed analysis of the available OvHV-2 sequences revealed an homologous gene (Ov9.5) with conserved splicing signals and predicted amino acid sequence features in both sequenced isolates of this related virus. We have therefore identified a novel spliced gene in two related macaviruses that is expressed in MCF lesions. Future work will determine its importance for the pathogenesis of disease.

Identification of immune traits correlated with dairy cow health, reproduction and productivity.

Detailed biological analyses (e.g. epidemiological, genetic) of animal health and fitness in the field are limited by the lack of large-scale recording of individual animals. An alternative approach is to identify immune traits that are associated with these important functions and can be subsequently used in more detailed studies. We have used an experimental dairy herd with uniquely dense phenotypic data to identify a range of potentially useful immune traits correlated with enhanced (or depressed) health and fitness. Blood samples from 248 dairy cows were collected at two-monthly intervals over a 10-month period and analysed for a number of immune traits, including levels of serum proteins associated with the innate immune response and circulating leukocyte populations. Immune measures were matched to individual cow records related to productivity, fertility and disease. Correlations between traits were calculated using bivariate analyses based on animal repeatability and random regression models with a Bonferroni correction to account for multiple testing. A number of significant correlations were found between immune traits and other recorded traits including: CD4(+):CD8(+) T lymphocyte ratio and subclinical mastitis; % CD8(+) lymphocytes and fertility; % CD335(+) natural killer cells and lameness episodes; and serum haptoglobin levels and clinical mastitis. Importantly these traits were not associated with reduced productivity and, in the case of cellular immune traits, were highly repeatable. Moreover these immune traits displayed significant between-animal variation suggesting that they may be altered by genetic selection. This study represents the largest simultaneous analysis of multiple immune traits in dairy cattle to-date and demonstrates that a number of immune traits are associated with health events. These traits represent useful selection markers for future programmes aimed at improving animal health and fitness.

Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus.

Studies of the molecular epidemiology of viral diseases are dependent on the analysis of large numbers of samples from infected individuals, and the assembly of relevant sequence databases are a prerequisite to investigate chains of infection. As part of research in support of the Scottish BVDV eradication campaign, we have established a direct RT-PCR method for the high throughput amplification and analysis of the informative 5'-untranslated region of the BVDV genome. Heat-treatment followed by a one-step RT-PCR, performed in 96-well plates, produced sufficient material for sequence analysis from 0.5 µl of serum or plasma. Of 93 samples assayed, only five failed to give full sequence data for the region amplified and these were subsequently successfully analysed in single tube format reactions. This approach improved the speed of analysis, reduced costs, operator time and the potential for contamination, and may allow analysis of samples for which volumes are too low for conventional RNA isolation. It also has the potential for wider application in both human and animal disease research in which high throughput and low cost would increase the size of datasets that can be obtained.

Host gene expression changes in cattle infected with Alcelaphine herpesvirus 1.

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates caused by infection with gamma-herpesviruses of the genus Macavirus. These viruses do not establish a productive infection but instead replicate in a cell-associated fashion in T lymphocytes, leading to systemic immune dysregulation and a generally fatal outcome. Despite significant progress in understanding the pathology of this disease, its pathogenesis remains unclear. To identify genes and pathways affected in clinical MCF, sixteen bovine GeneCHIP microarrays were used to assay RNA from kidney and lymph node of four MCF-affected and four control Bos taurus steers. This is the first expression study of AlHV-1-MCF in the bovine host. Over 250 genes showed significant changes in gene expression in either lymph node or kidney, while expression of 35 genes was altered in both tissues. Pathway and annotation analysis of the microarray data showed that immune response and inflammatory genes were up-regulated in the kidney while proliferation-associated transcripts were additionally increased in the lymph node. The genes that showed the largest expression rises in both diseased tissues included cytotoxic enzymes and pro-inflammatory chemokines. These data are consistent with disease-induced stimulation of inflammatory responses involving interferon-γ, including cytotoxic T cell recruitment and activation in peripheral tissues containing virus-infected cells. However it remains unclear whether the tissue damage in MCF lesions is due entirely to the activity of infected cells or whether uninfected T cells, recruited and activated at lesion sites through the action of infected cells, contribute to the pathogenesis of MCF.