Ahmed Abd-Elfattah

Vertical transmission of Tetracapsuloides bryosalmonae (Myxozoa), the causative agent of salmonid proliferative kidney disease.

The freshwater bryozoan, Fredericella sultana, is the main primary host of the myxozoan endoparasite, Tetracapsuloides bryosalmonae which causes proliferative kidney disease (PKD) of salmonid fish. Because spores that develop in bryozoan colonies are infectious to fish, bryozoans represent the ultimate source of PKD. Bryozoans produce numerous seed-like dormant stages called statoblasts that enable persistence during unfavourable conditions and achieve long-distance dispersal. The possibility that T. bryosalmonae may undergo vertical transmission via infection of statoblasts has been the subject of much speculation since this is observed in close relatives. This study provides the first evidence that such vertical transmission of T. bryosalmonae is extensive by examining the proportions of infected statoblasts in populations of F. sultana on two different rivers systems and confirms its effectiveness by demonstrating transmission from material derived from infected statoblasts to fish hosts. Vertical transmission in statoblasts is likely to play an important role in the infection dynamics of both bryozoan and fish hosts and may substantially contribute to the widespread distribution of PKD.

Fate of Tetracapsuloides bryosalmonae (Myxozoa) after infection of brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in salmonids. We assessed differences in intensity of T. bryosalmonae infection between brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss from the clinical phase of infection onwards. Specific pathogen-free fish were exposed to T. bryosalmonae spores under controlled laboratory conditions and sampled at 6, 8, 10, 12, 14, and 17 wk post exposure (wpe), and the transmission of T. bryosalmonae from infected fish to the bryozoan Fredericella sultana was observed. Parasite load was determined in fish kidneys by quantitative real-time PCR (qRT-PCR), and parasite stages were detected in kidney, liver, and spleen tissues at different time points by immunohistochemistry. T. bryosalmonae was successfully transmitted from infected brown trout to F. sultana colonies but not from infected rainbow trout. Body length and weight of infected brown trout did not differ significantly from control brown trout during all time points, while length and weight of infected rainbow trout differed significantly compared to controls from 10 to 17 wpe. qRT-PCR revealed that parasite load was significantly higher in kidneys of brown trout compared with rainbow trout. Immunohistochemistry showed high numbers of intra-luminal stages (sporogonic stages) in kidneys of brown trout with low numbers of pre-sporogonic stages. Sporogonic stages were not seen in kidneys of rainbow trout; only high numbers of pre-sporogonic stages were detected. Numbers of pre-sporogonic stages were low in the spleen and liver of brown trout but high in rainbow trout. These data confirmed that there are differences in the development and infection progress of T. bryosalmonae between brown trout and rainbow trout.

Identification of differentially expressed genes of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) in response to Tetracapsuloides bryosalmonae (Myxozoa).

Tetracapsuloides bryosalmonae Canning et al., 1999 (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. We have shown previously that the development and distribution of the European strain of T. bryosalmonae differs in the kidney of brown trout (Salmo trutta) Linnaeus, 1758 and rainbow trout (Oncorhynchus mykiss) Walbaum, 1792, and that intra-luminal sporogonic stages were found in brown trout but not in rainbow trout. We have now compared transcriptomes from kidneys of brown trout and rainbow trout infected with T. bryosalmonae using suppressive subtractive hybridization (SSH). The differentially expressed transcripts produced by SSH were cloned, transformed, and tested by colony PCR. Differential expression screening of PCR products was validated using dot blot, and positive clones having different signal intensities were sequenced. Differential screening and a subsequent NCBI-BLAST analysis of expressed sequence tags revealed nine clones expressed differently between both fish species. These differentially expressed genes were validated by quantitative real-time PCR of kidney samples from both fish species at different time points of infection. Expression of anti-inflammatory (TSC22 domain family protein 3) and cell proliferation (Prothymin alpha) genes were upregulated significantly in brown trout but downregulated in rainbow trout. The expression of humoral immune response (immunoglobulin mu) and endocytic pathway (Ras-related protein Rab-11b) genes were significantly upregulated in rainbow trout but downregulated in brown trout. This study suggests that differential expression of host anti-inflammatory, humoral immune and endocytic pathway responses, cell proliferation, and cell growth processes do not inhibit the development of intra-luminal sporogonic stages of the European strain of T. bryosalmonae in brown trout but may suppress it in rainbow trout.

Establishment of medium for laboratory cultivation and maintenance of Fredericella sultana for in vivo experiments with Tetracapsuloides bryosalmonae (Myxozoa)

The freshwater bryozoan Fredericella sultana (Blumenbach) is the most common invertebrate host of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Culture media play an important role in hatching of statoblasts and maintaining clean bryozoan colonies for Malacosporea research. We developed a novel culture medium, Bryozoan Medium C (BMC), for the cultivation and maintenance of F. sultana under laboratory conditions. Statoblasts of F. sultana were successfully hatched to produce transparent-walled, specific pathogen-free (SPF) colonies that were maintained >12 months in BMC at pH 6.65. Tetracapsuloides bryosalmonae was successfully transmitted from infected brown trout, Salmo trutta L., to newly hatched F. sultana colonies in BMC, then from the infected bryozoan to SPF brown trout. This study demonstrated the utility of BMC (pH 6.65) for hatching statoblasts, long-term cultivation of clean and transparent bryozoan colonies and maintenance of the Tetracapsuloides bryosalmonae life cycle in the laboratory for molecular genetic research and other studies such as host–parasiteinteraction.

Persistence of Tetracapsuloides bryosalmonae (Myxozoa) in chronically infected brown trout Salmo trutta

Proliferative kidney disease (PKD) is a widespread disease of farmed and wild salmonid populations in Europe and North America, caused by the myxozoan parasite Tetracapsuloides bryosalmonae. Limited studies have been performed on the epidemiological role in spread of the disease played by fish that survive infection with T. bryosalmonae. The aim of the present study was to evaluate the persistence of T. bryosalmonae developmental stages in chronically infected brown trout Salmo trutta up to 2 yr after initial exposure to laboratory-infected colonies of the parasites alternate host, the bryozoan Fredericella sultana. Kidney, liver, spleen, intestine, brain, gills and blood were sampled 24, 52, 78 and 104 wk post-exposure (wpe) and tested for T. bryosalmonae by PCR and immunohistochemistry (IHC). Cohabitation trials with specific pathogen free (SPF) F. sultana colonies were conducted to test the viability of T. bryosalmonae. PCR detected T. bryosalmonae DNA in all tissue samples collected at the 4 time points. Developmental stages of T. bryosalmonae were demonstrated by IHC in most samples at the 4 time points. Cohabitation of SPF F. sultana with chronically infected brown trout resulted in successful transmission of T. bryosalmonae to the bryozoan. This study verified the persistence of T. bryosalmonae in chronically infected brown trout and their ability to infect the bryozoan F. sultana up to 104 wpe.

Differential modulation of host genes in the kidney of brown trout Salmo trutta during sporogenesis of Tetracapsuloides bryosalmonae (Myxozoa).

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. In Europe, spores of T. bryosalmonae develop in the kidney of infected brown trout Salmo trutta and are released via urine to infect the freshwater bryozoan Fredericella sultana. The transcriptomes of kidneys of infected and non-infected brown trout were compared by suppressive subtractive hybridization. Differential screening and a subsequent NCBI BLAST analysis of expressed sequence tags revealed 21 transcripts with functions that included cell stress and cell growth, ribonucleoprotein, signal transduction, ion transporter, immune response, hemoglobin and calcium metabolisms. Quantitative real time PCR was used to verify the presence of these selected transcripts in brown trout kidney at sporogonic stages of T. bryosalmonae development. Expression of cold-inducible RNA-binding protein, cyclin-dependent kinase inhibitor 2A, prothymosin alpha, transforming protein RhoA, immunoglobulin light chain and major histocompatibility complex class I were up-regulated significantly in infected brown trout. Expression of both the hemoglobin subunit beta and stanniocalcin precursor were down-regulated significantly in infected brown trout. This study suggests that cell stress and cell growth processes, signal transduction activities, erythropoiesis and calcium homeostasis of the host are modulated during sporogonic stages of parasite development, which may support the sporogenesis of T. bryosalmonae in the kidney of brown trout.

Cystoisospora suis - A Model of Mammalian Cystoisosporosis

Cystoisospora suis is a coccidian species that typically affects suckling piglets. Infections occur by oral uptake of oocysts and are characterized by non-hemorrhagic transient diarrhea, resulting in poor weight gain. Apparently, primary immune responses to C. suis cannot readily be mounted by neonates, which contributes to the establishment and rapid development of the parasite, while in older pigs age-resistance prevents disease development. However, the presence of extraintestinal stages, although not unequivocally demonstrated, is suspected to enable parasite persistence together with the induction and maintenance of immune response in older pigs, which in turn may facilitate the transfer of C. suis-specific factors from sow to offspring. It is assumed that neonates are particularly prone to clinical disease because infections with C. suis interfere with the establishment of the gut microbiome. Clostridia have been especially inferred to profit from the altered intestinal environment during parasite infection. New tools, particularly in the area of genomics, might illustrate the interactions between C. suis and its host and pave the way for the development of new control methods not only for porcine cystoisosporosis but also for other mammalian Cystoisospora infections. The first reference genome for C. suis is under way and will be a fertile ground to discover new drugs and vaccines. At the same time, the establishment and refinement of an in vivo model and an in vitro culture system, supporting the complete life cycle of C. suis, will underpin the functional characterization of the parasite and shed light on its biology and control.

Investigating the interactions of Cyprinid herpesvirus-3 with host proteins in goldfish Carassius auratus

Keywords: antibody, electrospray ionization mass spec-trometry, koi herpesvirus disease, protein purification.Cyprinid herpesvirus-3 (CyHV-3) is the aetiologi-cal agent of a serious and deadly disease, termed‘koi herpesvirus disease’ (KHVD), that causes highmortality in koi and common carp Cyprinus carpioL. (Pikarsky et al. 2004; Ilouze et al. 2008). Sincethe initial identification of CyHV-3 in Europe(Bretzinger et al. 1999), the USA and Israel (Hed-rick et al. 2000), and Japan (Sano et al. 2004),there is a great effort to limit the spread ofCyHV-3. Recently, the focus of prevention hasshifted to understanding how the virus propagatesand what carriers exist for the virus. Environmen-tal factors such as mating (Uchii et al. 2011),feeding activities (Kielpinski et al. 2010; Minam-oto et al. 2011; Fournier et al. 2012) and proxim-ity in aquaculture (Dishon et al. 2005) arecontributing factors in the spread of CyHV-3.Earlier studies suggested that immunization withattenuated virus may prevent the spread ofCyHV-3 (Ronen et al. 2003; Perelberg et al.2005), but current studies suggest that CyHV-3spread is due to subclinical carriers (Bergmann K Eide et al. 2011), which can infectna€ive fish (St-Hilaire et al. 2005). Recent studieshave shown that CyHV-3 DNA exists indifferent species of healthy looking Cyprinid fishthat include grass carp Ctenopharyngodon idella,goldfish Carassius auratus and blue back ideLeuciscus idus, as well as in non-Cyprinid fish thatinclude Russian sturgeons Acipenser gueldenstaedtii,Atlantic sturgeons A. oxyrinchus and Ancistrus sp.,suggesting that these fish may be potential carriersfor the CyHV-3 (Bergmann et al. 2009; Kempteret al. 2009). In addition, cohabitation studies haveshown that CyHV-3 can be transferred to goldfishfrom infected koi fish (El-Matbouli, Saleh & Soli-man 2007), but goldfish do not show clinicalsigns of the disease (Bergmann et al. 2010a).The genome of CyHV-3 consists of 295 kB(Aoki et al. 2007) that codes for 156 unique openreading frames (ORFs) that are all transcribed intomRNA transcripts (Ilouze, Dishon & Kotler2012). Mass spectrometry (MS) identified 40CyHV-3 proteins incorporated into mature virions(Michel et al. 2010). Some of the CyHV-3 pro-teins have been further characterized, such asORF81, a component of the viral envelope(Rosenkranz et al. 2008) and genes involved inDNA synthesis (Ilouze et al. 2006; Fuchs et al.2011). Recently, Sunarto et al. (2012) showedthat CyHV-3 transcribes a potential immune sup-pression gene, ORF134, a homolog of interleu-kin-10 (IL-10). CyHV-3 has also been shown tomodulate the innate immune response pathway invitro (Adamek et al. 2012) and in vivo (Adameket al. 2013). Gene expression analysis usingmicroarray chips of two common carp lines thatdiffer in mortality rates during CyHV-3 infectionidentified 581 genes in line ‘R3’ and 107 genes inline ‘K’ that are differentially transcribed duringCyHV-3 infection (Rakus et al. 2012). The eluci-dation of interaction partners is often used to

Antibody and cytokine response to Cystoisospora suis infections in immune-competent young pigs

BackgroundTo date, investigations on the immune response to Cystoisospora suis infections focused on suckling piglets, the age group clinically most affected. Actively immunizing piglets is unfeasible due to their immature immune system and the typically early infection in the first days after birth. Therefore, understanding and possibly enhancing the immune response of immune-competent animals is the prerequisite to develop a passive immunization strategy for piglets which currently rely on very limited treatment options.MethodsTo investigate antibody and cytokine responses of immune-competent animals and the impact of the oral immunization protocol on their immune response, growers with unknown previous exposure to C. suis (10–11 weeks-old) were infected one or three times with different doses (600 and 6000 or 200 and 2000, respectively) of C. suis oocysts, and compared to uninfected controls. Oocyst excretion was evaluated, and blood and intestinal mucus antibody titers were determined by IFAT. Systemic production of Th1, Th2, inflammatory and regulatory cytokines was determined in different immune compartments at mRNA and (after stimulation with a recombinant merozoite-protein) at protein level by PCR and multiplex fluorescent immunoassay, respectively.ResultsInfection generated significantly increased serum IgA and IgG levels against C. suis sporozoites and merozoites, irrespective of infection mode, with IgG against merozoites showing the strongest increase. No clinical signs and only occasional excretion were observed. The systemic cytokine response to C. suis was only weak. Nonetheless, in white blood cells, IL-4, IL-6 and IL-10 mRNA-levels significantly increased after infection, whereas IFN-ɣ, IL-2 and TGF-β expression tended to decrease. In mesenteric lymph nodes (MLN), IL-10 and TNF-α levels were elevated while splenic cytokine expression was unaltered upon infection. Stimulated MLN-derived lymphocytes from infected pigs produced slightly more IL-12 and less IFN-α than controls.ConclusionsAn infection and a subsequent systemic immune response can be induced in immune-competent animals by all evaluated infection models and growers can be used as models to mimic sow immunizations. The immune response to C. suis, although mild and with considerable variation in cytokine expression, was characterized by a Th2-associated and regulatory cytokine profile and antibody production. However, none of the parameters clearly stood out as a potential marker associated with protection. Antibody titers were significantly positively related with oocyst excretion and might thus serve as correlates for parasite replication or severity of infection.