Uninfected Calves Research Articles

201 Transcriptome analysis of bovine abomasal mucosa following experimental infection with Ostertagia ostertagi

Abstract Ostertagia ostertagi is a cattle stomach nematode that causes significant disease; however, little is known about host responses to infection. Due to the emergence of drug resistance in these parasites, there is a need for alternative treatments. To understand host responses to O. ostertagi infection in the gastric mucosa, gene expression in fundic and pyloric mucosa was compared between infected and uninfected calves for 21 d post-infection. The number of differentially expressed genes (DEGs) increased over time with more upregulated than downregulated DEGs at each time point. Fundic mucosa had more DEGs than pyloric mucosa across all time points. The largest changes occurred between d 7 to 9 and 10 post-infection. Most DEGs are associated with immunity, cellular reorganization, cell migration, and proliferation. Tuft/epithelial cell response to the infection was atypical, lacking key cytokines. Numerous genes associated with T cell exhaustion were upregulated. The data collectively indicate that O. ostertagi infection elicited host immune responses, yet also suggest parasite-induced immunosuppression is present. This may explain why cattle are slow in developing protective immunity to O. ostertagi. Understanding mechanisms of parasite evasion will facilitate the rational design of protective vaccines against complex nematode parasites.

Behavioural response to gastrointestinal parasites of yearling dairy calves at pasture

ABSTRACT Aims To investigate the association between gastrointestinal parasites (GIP) and animal behaviour in dairy calves under New Zealand pastoral conditions, using animal-mounted, accelerometer-based sensors. Methods Thirty-six, 5–6-month-old, Friesian-Jersey, heifer calves fitted with animal activity sensors to track behaviour were randomly allocated to one of two treatment groups. Half the animals were challenged with an oral dose of 20,000 larvae of Ostertagia ostertagi and Cooperia oncophera once a week for 3 weeks and half were unchallenged. Five weeks after the last dose, seven infected and nine uninfected animals were treated with an oral anthelmintic (AHC) and data collected for a further week. Accelerometer data were classified into minutes per day eating, ruminating, in moderate–high activity or in low activity. Live weight and faecal egg counts (FEC) were recorded weekly over the study period. All animals co-grazed a newly sown pasture not previously grazed by ruminants and were moved every week to fresh grazing. Treatment status was blinded to those managing the animals which were otherwise treated identically. Results Complete behavioural records were available from 30/36 calves, (13 challenged and 17 unchallenged). Before treatment with AHC, FEC increased in infected and un-treated calves over the study, while uninfected animals maintained a near zero FEC. There was no difference in live weight gain between the two groups over the study period. Bayesian, multinomial regression predicted differences in animal behaviour between infected and uninfected animals that were not treated with AHC over the 7 weeks following initial infection. Parasitised calves not treated with AHC were less active and spent up to 6 (95% highest density interval (HDI) = 1–11) minutes/day less in low level activity and up to 15 (95% HDI = 7–20) minutes/day less in moderate to high level activity. They ruminated up to 9 (95% HDI = 2–15) minutes/day more and ate up to 10 (95% HDI = 2–19) minutes/day more than control calves that were not treated with AHC. The effect of AHC on time spent in each behaviour differed between infected and uninfected calves and increased the coefficient of dispersion of the behavioural data. Conclusions and clinical relevance Small differences in animal behaviour can be measured in calves with GIP. However, to use this to target treatment, further validation studies are required to confirm the accuracy of behavioural classification and understand the complex drivers of animal behaviour in a dynamic and variable pasture-parasite–host environment.

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates.

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 109CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate.

Enterocytozoon bieneusi in fecal samples from calves and cows in Austria

Enterocytozoon bieneusi is an obligate intracellular pathogen that infects livestock, companion animals, and wildlife and has the potential to cause severe diarrhea especially in immunocompromised humans. In the underlying study, fecal samples from 177 calves with diarrhea and 174 adult cows originating from 70 and 18 farms, respectively, in Austria were examined for the presence of E. bieneusi by polymerase chain reaction targeting the Internal Transcribed Spacer 1 (ITS1) region. All positive samples were further sequenced for genotype determination. Overall, sixteen of the 351 (4.6%) samples were positive for E. bieneusi, two of the 174 samples from cows (1.2%) and 14 of the 177 samples from calves (7.9%). In total, four genotypes, J (n = 2), I (n = 12), BEB4 (n = 3), and BEB8 (n = 1), were identified. The uncorrected p-distance between the four ITS1 lineages (344 bp) ranges from 0.3% to 2.9%. The lineages differ by 1 bp (I and J), 2 bp (J and BEB4), and 3 bp (I and BEB4), respectively, and BEB8 differs by 7 to 10 bp from the latter three lineages. Two of the E. bieneusi-positive calves showed an infection with two different genotypes. E. bieneusi occurred significantly more often in calves > 3 weeks (8/59) than in calves ≤ 3 weeks (6/118), respectively (p = 0.049). Calves with a known history of antimicrobial treatment (50 of 177 calves) shed E. bieneusi significantly more often than untreated calves (p = 0.012). There was no statistically significant difference in E. bieneusi shedding in calves with or without a medical history of antiparasitic treatment (p = 0.881). Calves showing a co-infection with Eimeria spp. shed E. bieneusi significantly more often than uninfected calves (p = 0.003). To our knowledge, this is the first report of E. bieneusi in cattle in Austria. Cattle should be considered as a reservoir for human infection since potentially zoonotic E. bieneusi genotypes were detected.

Haemaphysalis longicornis: the life-cycle on dogs and cattle, with confirmation of its vector status for Theileria orientalis in Australia

The intracellular protozoal parasite Theileria orientalis ikeda has rapidly spread across South-eastern Australia since 2006, causing deaths and production losses in cattle. The 3-host “bush tick” Haemaphysalis longicornis (Neumann) appears the principal biological vector in the endemic regions. To generate sufficient numbers of ticks to produce stabilate for infection to confirm vector competency and for acaricide trials, the optimal conditions and stage-specific intervals for the generational life-cycle of H.longicornis was defined on two dogs and two steers. To determine whether H.longicornis was a definitive host for Theileria orientalis, nymphal stages were fed on a steer infected with T.orientalis and moulted adults were permitted to feed for 3 days on an uninfected calf prior to harvest. Subsequent detection of infection after inoculation of four naïve calves with stabilate produced from ground-up adult ticks or dissected salivary glands confirmed H.longicornis as one final (definitive) host for T.orientalis in Australia.

Associations between Theileria orientalis Ikeda type infection and the growth rates and haematocrit of suckled beef calves in the North Island of New Zealand

AIMS: The principle aim of this study was to examine the association between infection with Theileria orientalis Ikeda type and growth rates of suckled beef calves on four beef farms. In addition, associations between calf sex, sampling time, and individual farm and T. orientalis Ikeda type infection intensity and haematocrit (HCT) were investigated.METHODS: The study was a prospective longitudinal study in which 240 calves from four purposively selected beef farms in the North Island of New Zealand were blood sampled and weighed in late spring, mid-summer and early autumn. Two farms were from high-risk (A and B) and two from low-risk (C and D) tick areas. Blood samples were analysed to determine HCT, and the number of T. orientalis Ikeda type organisms/µL of blood (infection intensity) using a quantitative PCR assay. A calf was defined as infected if >415 organisms/µL were detected in a blood sample. Linear mixed models were used to examine associations between infection intensity, mean daily liveweight gain (MDG), HCT, calf sex and time of sampling on the four farms.RESULTS: On Farms A and B nearly all calves were infected at each sampling time, on Farm C <30% were infected at any sampling and on Farm D infection prevalence increased from 32 to 79% between late spring and early autumn. On Farms C and D, from mid-summer to early autumn, mean MDG was 0.127 (95% CI=0.072–0.183) kg/day less for infected than uninfected calves (p<0.001). On all farms MDG was negatively associated with infection intensity for mid-summer and early autumn sampling times (p=0.037). The relationship between time of sampling and infection intensity varied between farms (p<0.001), and between male and female calves (p=0.018). Females had a higher infection intensity than males at the mid-summer and early autumn samplings. The association between HCT and infection intensity varied with sampling time and farm (p=0.018). There was a strong negative association between infection intensity and HCT at the late spring sampling, but in mid-summer there was no association, and in early autumn only a weak association.CONCLUSIONS AND CLINICAL RELEVANCE: This study has shown that beef farmers in the North Island of New Zealand should be concerned about the welfare effects and economic impacts of T. orientalis Ikeda type infection in suckled beef calves.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with β-catenin and a β-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased β-catenin-dependent transcription and cell survival. β-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/β-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/β-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency.IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/β-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and β-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.

Potential Role for a β-Catenin Coactivator (High-Mobility Group AT–Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BoHV-1) is abundantly expressed in latently infected sensory neurons. Although the LR gene encodes several products, ORF2 appears to mediate important steps during the latency-reactivation cycle because a mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency in calves. We recently found that the Wnt/β-catenin signaling pathway is regulated during the BoHV-1 latency-reactivation cycle (Y. Liu, M. Hancock, A. Workman, A. Doster, and C. Jones, J Virol 90:3148-3159, 2016). In the present study, a β-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was detected in significantly more neurons in the trigeminal ganglia of latently infected calves than in those of uninfected calves. Consequently, we hypothesized that HMGA1 cooperates with ORF2 and β-catenin to maintain latency. In support of this hypothesis, coimmunoprecipitation studies demonstrated that ORF2 stably interacts with a complex containing β-catenin and/or HMGA1 in transfected mouse neuroblastoma (Neuro-2A) cells. Confocal microscopy provided evidence that ORF2 was relocalized by HMGA1 and β-catenin in Neuro-2A cells. ORF2 consistently enhanced the ability of HMGA1 to stimulate β-catenin-dependent transcription, suggesting that interactions between ORF2 and a complex containing β-catenin and HMGA1 have functional significance. An ORF2 stop codon mutant, an ORF2 nuclear localization mutant, or a mutant lacking the 5 protein kinase A or C phosphorylation sites interfered with its ability to stimulate β-catenin-dependent transcription. Since the canonical Wnt/β-catenin signaling pathway promotes neurogenesis (synapse formation and remodeling) and inhibits neurodegeneration, interactions between ORF2, HMGA1, and β-catenin may be important for certain aspects of the latency-reactivation cycle.IMPORTANCE The lifelong latency of bovine herpesvirus 1 (BoHV-1) requires that significant numbers of infected sensory neurons survive infection and maintain normal functions. Consequently, we hypothesize that viral products expressed during latency cooperate with neuronal factors to maintain latency. Our studies revealed that a β-catenin coactivator, high-mobility group AT-hook 1 protein (HMGA1), was readily detected in a subset of trigeminal ganglion neurons in latently infected calves but not in uninfected calves. A viral protein (ORF2) expressed in latently infected neurons interacted with β-catenin and HMGA1 in transfected cells, which resulted in the nuclear localization of β-catenin. This interaction correlated with the ability of ORF2 to stimulate the coactivator functions of HMGA1. These findings are significant because the canonical Wnt/β-catenin signaling pathway promotes neurogenesis and inhibits neurodegeneration.

Severe otitis and pneumonia in adult cattle with mixed infection of Mycoplasma bovis and Mycoplasma agalactiae

Respiratory disease and otitis were seen on a northern Italian farm in weaned bulls obtained from multiple sources. Mycoplasma bovis was consistently identified in nasal swabs, tonsil, ears, eyes and...

Targeted silencing of the Aquaporin 2 gene of Rhipicephalus (Boophilus) microplus reduces tick fitness.

BackgroundTicks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms of osmoregulation. Members of the AQP family are critical for blood-feeding arthropods considering the extreme osmoregulatory changes that occur during their feeding. We investigated the pattern of expression of a newly identified AQP2 gene of R. microplus (RmAQP2) in different tick tissues and stages. We also examined in vivo the biological implications of silencing expression of RmAQP2 silencing during tick feeding on either uninfected or B. bovis-infected cattle.MethodsIn silico gene analyses were performed by multiple alignments of amino acid sequences and topology prediction. Levels of RmAQP2 transcripts in different tick tissues and stages were analyzed by reverse transcriptase quantitative PCR. Patterns of expression of RmAQP2 protein were investigated by immunoblots. Gene silencing was performed by RNA interference and in vivo functional analyses carried out by feeding ticks on either uninfected or B. bovis-infected cattle.ResultsRmAQP2 transcripts were found in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged females; however, of all tick tissues and stages examined, RmAQP2 protein was found only in salivary glands of partially engorged females. RmAQP2 silencing significantly reduced tick fitness and completely abrogated protein expression. The effect of RmAQP2 silencing on fitness was more pronounced in females fed on a B. bovis-infected calf than in ticks fed on an uninfected calf and none of their larval progeny survived.ConclusionsCollectively, considering the gene expression and tick fitness data, we conclude that RmAQP2 is critical for tick blood feeding and may be a suitable candidate target for the development of novel strategies to control R. microplus and tick-borne parasites.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1226-2) contains supplementary material, which is available to authorized users.

Acute phase proteins as local biomarkers of respiratory infection in calves.

BackgroundCumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci.ResultsIntra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP—and decreasing albumin concentrations in blood, both verifying a systemic acute phase response.Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]—and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection.ConclusionIn conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.

Hyperproliferative action of bovine papillomavirus: genetic and histopathological aspects.

The bovine papillomavirus (BPV) causes papillomas that regress spontaneously, but can also progress to malignancy. This study evaluated the role of BPV in oncogenesis. Twenty-four samples from uninfected calves and the papillomas of BPV infected cattle were subjected to molecular diagnosis, as well as histopathological and immunohistochemical analyses. The comet assay (CA) was used to evaluate the clastogenic potential of BPV. The results confirmed the presence of BPV-2, 3, 5, and 9 in infected samples. Histopathological analysis revealed acanthosis, koilocytosis, hypergranulosis, hyperkeratosis, and transformed fibroblasts.E7 and L1 BPV proteins were detected in the epithelium, as well as in the connective tissues, indicating productive infection at different sites. CA results showed that BPV-2, 5, and 9 exhibit the same level of clastogenicity. These findings support the oncogenic action of BPV in establishing a favorable microenvironment for oncogenesis.

Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves

The purpose of this study was to monitor local cytokine responses to Dictyocaulus viviparus in calves during primary infection and re-infection. Bronchoalveolar lavage fluid (BALF) was collected weekly from experimentally infected calves and interleukin-2 (IL-2), IL-4, IL-5, IL-10 and IFN-γ mRNA expression was quantified in BALF cells. The major finding was a prominent transient increase in IL-4 mRNA expression, compared with that of uninfected calves, observed in BALF cells collected 2-3weeks post-primary D.viviparus infection. At 2weeks post-infection, macroscopic worms were also first observed in BALF. Calves re-infected after 10weeks were partially immune which was evident at slaughter 5weeks post-infection as a lower worm burden than in previously naïve calves infected at the same time. IL-4 mRNA expression in BALF cells 2weeks post-re-infection was increased compared with that of uninfected animals but not as high as that of primarily infected calves. BALF cell expression of the other cytokines tested for was not as clearly effected by the D.viviparus infection. It seems likely that the strong IL-4 response observed during primary infection reflects an innate response to the worms that may initiate an ensuing Th2 response, which confers protective immunity.

Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle

Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n = 5) and uninfected cows (n = 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease.

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis

BackgroundCattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis.MethodsA group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated.ResultsThe subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls.ConclusionCollectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Analysis of Babesia bovis infection-induced gene expression changes in larvae from the cattle tick, Rhipicephalus (Boophilus) microplus

BackgroundCattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world’s tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis.MethodsTotal RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR.ResultsTicks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf.ConclusionOur experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis. Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.

Histochemical and morphometric characterization of broncho-pneumonia in calves caused by infection with Mycoplasma bovis

The aim of this study was to identify morphometric histological features of pneumonia caused by Mycoplasma bovis in calves. Eight three-month-old calves were infected with M. bovis and samples of their lung tissue, three weeks after exposure, compared to samples from four uninfected calves. In the uninfected animals the goblet cells were clustered in the crypt area of the epithelial folds, while in the infected calves they had migrated towards the tips of the folds and were distributed evenly throughout the folds. In infected lung tissue there was goblet cell hyperplasia and metaplasia in the bronchioles and an increased epithelial height. Goblet cell mucin in uninfected calves was acidic, but in infected calves most goblet cells contained neutral mucins. These morphometric and histochemical bronco-epithelial changes may be able to be used as markers of the severity of bovine respiratory mycoplasmosis.

Serological cross-reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle

Seroconversion and cross-reactivity in cattle infected with Anaplasma marginale or a recently described Ehrlichia species (BOV2010 from British Columbia, Canada) were investigated. The study used 76 samples from 20 animals, a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for bovine anaplasmosis, and an indirect fluorescent antibody test (IFAT). Blood smear examination and/or polymerase chain reaction assay were performed to confirm or rule out the presence of Anaplasma or Ehrlichia. Samples comprised 3 groups. Group 1 consisted of 24 samples from 9 cattle naturally infected with Ehrlichia sp. BOV2010. Group 2 had 13 samples from 3 A. marginale-infected cattle from Manitoba, Canada. Group 3 had 39 samples, consisting of 26 from 5 calves experimentally infected with Ehrlichia sp. BOV2010, 10 from 2 calves experimentally infected with A. marginale from cattle (Manitoba) or bison (Saskatchewan), and 3 from an uninfected calf. All samples from cattle naturally or experimentally infected with Ehrlichia sp. BOV2010 or A. marginale were seropositive for A. marginale by both cELISA and IFAT, except 3 calves euthanized at 28 and 33 days post-inoculation (DPI) that did not seroconvert. Antibodies were detected in 2 experimental animals inoculated with Ehrlichia sp. BOV2010, as early as 28 and 33 DPI by the cELISA and IFAT, respectively, and by 42 DPI for both tests. The current study demonstrates that the specificity of the recombinant major surface protein 5 (MSP5) antigen is not restricted to Anaplasma spp., which reduces the utility of the test for serological diagnosis of bovine anaplasmosis in regions where Ehrlichia sp. BOV2010-infected cattle might exist.

Immune responses to haemorrhagic septicaemia (HS) vaccination in Trypanosoma evansi infected buffalo-calves

To assess the immunosuppressive effect of Trypanosoma evansi infection in buffalo-calves on immune responses to heterologous antigen, the study was planned to examine the responses of haemorrhagic septicaemia vaccination in simultaneously and previously (80 days before vaccination) T. evansi-infected buffalo-calves. Eight buffalo-calves were divided into three groups. Buffalo-calves of group A (n = 3) were previously (80 days before primary vaccination with haemorrhagic septicaemia [HS] vaccine) infected with T. evansi (1 x 10(7) tryps.calf(-1); sc) and that of group B (n = 3) were infected with T. evansi (1 x 10(7) tryps.calf(-1); sc) on the day of primary vaccination with HS vaccine. Two healthy uninfected control calves given only HS vaccine were kept in group C. All the buffalo-calves were given a booster dose of vaccine 21 days post-primary vaccination (PPV). Twenty eight days PPV, animals of group A were given trypanocidal quinapyramine prosalt at 6.66 mg kg(-1). Immunosuppressive effect of T. evansi infection was evident from day 7 PPV with HS vaccine. The effect was more pronounced in previously T. evansi-infected buffalo-calves as compared with simultaneously infected buffalo-calves. Group A buffalo-calves appeared to have recovered from the immunosuppressive effect after 28 days post-trypanocidal treatment as observed by humoral and cell-mediated immune responses. Immunosuppressive effect to HS vaccination was observed in T. evansi-infected buffalo-calves, and trypanocidal therapy enabled the calves to mount the responses similar to uninfected controls.