If you gotta start somewhere, why not here - A vaccine for Cytauxzoon felis

One major group that has parasites of medical and veterinary importance is the Subclass Hematozoa of the phylum Apicomplexa. This subclass has 2 orders, one of which has the infamous intraerythrocytic genera - Plasmodium, Leucocytozoon and Hemoproteus, and the other has the Piroplasm genera Theileria and Babesia. (see my previous post to see the relationship between these)

Parasites and vectors - Two worlds collide

While Theileria is the more famous of the parasites in the Family Theileriidae, a new cousin parasite was identified in 1976 in domestic cats in the southern US. Cytauxzoon felis, whose definitive host is the Lynx rufus (Bobcat), causes an acute, lethal disease in Felis domesticus . Few survive. Fantastic reviews on the clinical manifestation are found here and here. Cytauxzoon is vector borne and carried by the lone star tick Amblyomma americanum. Transmission is indirect and prevention has relied on disrupting the life cycle at the tick stage. There have been no traditional vaccines so far, because the piroplasms cannot be grown in culture. It has been found previously that of the three life cycle stages of the parasite, viz., sporozoites, schizonts and merozoites, protective immunity is seen with the schizont stage. Cats that had survived the schizogenous phase of the disease were the rare ones that survived a challenge with the organism again. Merozoites do not protect against infection.   

(Source : CDC ; http://www.cdc.gov/stari/disease/)

If we gotta start somewhere, I say here

In a recent article published in PLoS One in Oct 2013, Tarigo J. et al. have used the molecular genomics approach to expedite the identification of a new protein antigen to serve as a vaccine candidate. They started with blood from a cat that had died of C.felis. They isolated the parasitic merozoites from the erythrocytes (RBCs) and sequenced the 9.1 megabase genome using Roche's 454 pyrosequencing. 

Next Generation Sequencing is all the rage these days. Sanger's first generation method is near-obsolete.But, you can still get a short product sequenced (using the method) for as low as $5. Roche's 454 pyrosequencing, Illumina's reversible terminator sequencing, ABI's SOLiD sequencing by ligation and Helicos' single molecule sequencing , which are/were the second gen, are being supplanted slowly even as I write by third generation technologies, viz., PacBio Real Time single molecule sequencing, Complete Genomics' combined proanchor hybridization and ligation, and Life Tech's Ion Torrent sequencing. (Hui, 2014).

Now, if you have never been a part of a genome sequencing project, you could be under the incorrect impression that the 454 sequencer spits out  perfectly ordered nucleotide data that can be uploaded to GenBank and analysed at leisure. The reality is, however, disturbingly far from such an assumption. You typically get about a hundred thousand (or more) short 'reads' as output. Imagine someone cutting up the front page of a newspaper into individual alphabets and then asking you to reassemble the little pieces into coherent words, sentences, paragraphs and sections. You would think that the person was crazy because the task is near impossible manually. What if you were given a software that could do it for you? With reasonable error, the software could probably do it. Such software that handle genomic data are called assemblers. They put nucleotide data together into cogent contigs de novo, i.e. they form sentences in a new language that they do not know. Some of the most popular of these (for 454 and other platforms) are Newbler, Edena, EULER- SR, Gapcloser, Oases, Ray, SOAPdenovo, Vcake, velvet and wgs. For C.felis genome assembly, the authors used Newbler (Tarigo, 2013; Kumar, 2010)

To study genes that code proteins, transcribed messenger RNA is usually utilized to construct a cDNA library. Traditional cDNA library construction involves a polyT primer sequence and two PCR runs to make double stranded cDNA, followed by cloning into vectors. Improved methods have now appeared on the scene including Clontech's SMARTer PCR cDNA synthesis kit, which is a single step reverse transcription reaction using a 'Switching Mechanism At the 5' end of the RNA Template'. SMART, indeed! The cDNA library was then sequenced to generate ESTs or Expressed Sequence Tags using the 454 platform and Newbler. ESTs, in the newspaper analogy, would be the equivalent of the first and the last few words of a sentence. Since the complete genome was available, ESTs were used to map protein-coding regions back onto the full length genome using other software. In this paper, GeneMark-ES 2.5 was used. Other software that can do the same are Apollo, Augustus, CEGMA, ESTScan, Eval, FrameDP,  Genethreader and PASA. These are basic annotation software that model gene structures onto genome data. After the first proteome was computed, a second predicted proteome was created using GlimmerHMM. The data was then used to run BLAST searches on NCBI, focusing specifically on the already deposited transcriptomes of Theileria parva, Babesia bovis and Plasmodium falciparum.

 Basic Local Alignment Search Tool for proteins (BLASTp) was then used to predict shared genes. The authors accepted 60% similarities across at least 30% of the query sequence as matches. They identified the cf67 gene on the genome of C.felis, which is syntenic to p67/SPAG-1/BOV57 on Theileria parva. Going back to the newspaper analogy, syntenous genes are equivalent to words that occur at the same place in a sentence. For example, cf67 and p67 are like the fourth word, on a headline spanning ten words, from two different newspapers - they are located at the same spot but are not the same. In the case of cf67 and p67, the three genes/ words preceding them were conserved/the same.The gene was then directionally cloned into a plasmid vector, and the product was used to perform in vitro transcription and translation reactions. The His tagged translation products were purified using Ni-NTA magnetic agarose beads and their quality assessed using western blot analysis with anti-His antibodies. Another western blot was carried out with serum from cats that had been infected but had survived the infection. There was a high degree of immunoreactivity observed with the sera against the C terminal and the whole ORF clones of cf67. This makes cf67 a great candidate antigen for immunological studies. 

The authors also found that cf67 is conserved in samples of C.felis from eight states in the southern US, with very little variation at the C terminal region. The cf67 was expressed in the schizogony stage. This was determined with insitu hybridization using a negative sense riboprobe directed against cytoplasmic mRNA in the schizont stage.Thus, the authors were able to identify a highly conserved sequence that was transcribed into an antigenic protein at the lifestage that could be targeted by the immune system to provide protective immunity. The heavy reliance on bioinformatics and in silico genomics is remarkable.

In a related review, Sirskyj et al., labelled such new innovative uses of bioinformatics for vaccine design as 'vaccinomics'. Conventional methods often miss proteins that are expressed at certain life cycle stages, but not at others, and so are potentially missed and overlooked as vaccine candidates. Software are available to predict immunodominant T and B cell epitopes. But despite the best predictions, field and clinical trials have not always been successful (Sirskyj,2011).

There is hope in the distance

Translational medicine techniques, to make the jump form epitope prediction to commercially viable vaccine, are still lacking across the veterinary and human medical and research fields. Although safety and efficacy are big concerns with conventional cell derived vaccines, they are not so with recombinant protein vaccines. Immune responses are slightly more controlled with the latter. The biggest concerns however arise from the degradation of the vaccine (either the insert or the vector) inside the body, lack of an effective strong immunity and delivery routes. These issues are more easily overcome in the veterinary field than in the human field because of sanctions on testing of vaccine candidates on animals. So far, the licensed recombinant vaccines available in the US for animals include the gI and gX deleted DIVA vaccine for pseudorabies in pigs and 11 poultry vaccines including fowlpox vectored vaccines for avian influenza, ND, Avian encephalomyelitis, ILT and Mycoplasma gallisepticum; Marek's disease virus vectored vaccine for ND, ILT deletion mutants of Salmonella typhimurium and deletion mutant of ILTV expressing HA from avian influenza.Vaccines for companion animals include canarypox vectored canines vaccines against Lyme disease, CDV and rabies; canary pox vectored feline vaccines against FeLV and rabies. Vaccines for horses include canary pox vectored vaccines against west nile virus and equine influenza. The newly identified vaccine candidate antigen for Cytauxzoon felis needs a lot of dedicated labwork ex silico before it can be made into a commercial vaccine for your pet. But in silico predictions are a great way to start.  

References :

Tarigo JL, Scholl EH, McK Bird D, Brown CC, Cohn LA, Dean GA, Levy MG, Doolan 
DL, Trieu A, Nordone SK, Felgner PL, Vigil A, Birkenheuer AJ. A novel candidate
vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics. PLoS 
One. 2013 Aug 20;8(8):e71233

Kumar S, Blaxter ML. Comparing de novo assemblers for 454 transcriptome data. 
BMC Genomics. 2010 Oct 16;11:571. 

Hui P. Next generation sequencing: chemistry, technology and applications. Top
Curr Chem. 2014;336:1-18.

Sirskyj D, Diaz-Mitoma F, Golshani A, Kumar A, Azizi A. Innovative
bioinformatic approaches for developing peptide-based vaccines against
hypervariable viruses. Immunol Cell Biol. 2011 Jan;89(1):81-9.

A midsummer nightmare - Cyclospora

In the mid summer of 2013, 631 people across 25 states in the US became ill with diarrhea, anorexia, low grade fever, nausea, and other non specific gastrointestinal symptoms. About 49 of these patients had symptoms severe enough to warrant hospitalization. A mad scramble of trace-back investigations in Iowa and Nebraska traced the outbreak to two famous restaurant chains and the consumption of salads that originated outside the country. A curious coccidian parasite called Cyclospora cayetanensis was identified as the etiological agent.

Before we analyse the associated investigation, let us look at the parasite itself :

Cyclospora was first recognized in the early 1980s in patients with enteric disease. 'Enteric disease' is an elegant medical term that encompasses the whole spectrum of clinical signs (nausea, diarrhea, abdominal pain, cramps etc) that are manifested by pathological changes in the small and large intestines. Cyclospora was initially misdiagnosed as everything ranging from artifacts to blue green alga, with the range in between including Cryptosporidium, Isospora, Coccidian-like body, cyanobacterium-like body, and unidentified flagellate. Reports in 1995 put an end to speculation by characterizing the SSU rRNA gene and concluding that the parasite was related to Eimeria , which as you all know is a Coccidian apicomplexan.

Cyclospora sps. are parasites of a wide range of snakes, rodents and mammals, not necessarily in that order, and they appear to be monoxenic (that is to say, they complete all parts of their lifecycle in one host). As is tradition, the oocysts are of diagnostic importance and are fairly typical. They have two sporocysts with two sporozoites each. Each of the sporozoites is folded in two. Diagnostic tests that detect the parasite are in fact designed to recognize this stage. These include the classic ones including the acid fast staining (picture below) with bright field microscopy, modified safranin staining, trichrome staining and the unconventional UV epifluorescence microscopy. Jejunal biopsies and PCR amplification of target genes can detect other stages besides the oocyst stage, which is shown below.


(Source : http://phil.cdc.gov/Phil/details.asp )

A typical Coccidian life cycle
Oocysts are excreted unsporulated in the feces of the infected host. They sporulate in 7 -15 days and become infectious. The sporulated oocysts ultimately come to be found on the surfaces of green leafy vegetables and  fruits. Upon ingestion by a new host, the oocysts excyst, infect the columnar epithelial cells of the proximal intestines (viz, duodenum and jejunum) and undergo merogony to produce two types of meronts that differentiate and undergo gametogony. The macro and microgametocytes fuse to form oocysts that are excreted.

(Source : http://www.cdc.gov/parasites/cyclosporiasis/biology.html)

Public health concerns
Evaluating the parasite from the perspective of the epidemiological triad brings out the following:

Parasite factors : Long sporulation time (7-15 days), which precludes direct person-to-person transmission; resistance to common disinfectants including chlorine; surface adhesins confer strong attachment to fresh produce.

Host factors : So far, Cyclospora cayetanensis seems to infect only humans, although oocysts have been isolated in fowl and dogs in endemic countries. But, the exact role of intermediate and transport hosts is yet to be elucidated, as is also the role of Cyclospora as an opportunistic pathogen. Undoubtedly, infections in immunocompromised patients are symptomatic, especially when the immunocompromise is due to infection with that devious Lentivirus called the Human Immunodeficiency Virus. A 2003 study in Venezuela reported a high percentage of asymptomatic carriers among the other major target population viz. infants.

Animal models have been unsuccessful so far.

Environmental factors : Microenvironmental factors that dictate sporulation are not entirely unknown. Temperature between 22C and 32C is needed for sporulation, which confines the circum-annual distribution to the tropics. This also means that the hemisphere experiencing summer at any given time is more prone to outbreaks than its antipodes. Exact seasonality and geographical distribution are under study. This predominantly tropical disease, has led to outbreaks in temperate regions as well due to the  importation of fresh produce. Travelers to endemic areas tend to pick up the infection due to sheer lack of knowledge of the existence of a parasite of this sort. A broad knowledge base and smart travel skills have become more important than ever. Cases in the United States are sporadic, of which some are acquired internationally and others domestically.

The map below shows cases that were acquired domestically between 1997 and 2008 :


(Source : cdc.gov/mmwr/preview/mmwrhtml/ss6002a1.htm)

The 2013 outbreak

The summer 2013 outbreak peaked between the 16th and 22th of June, and the second spike occurred between the 30th of June and the 6th of July, with 174 and 111 cases respectively.The five-year incidence mean for the same two weeks are 6.8 and 5.6 cases respectively.

The FDA conducted an Environmental Assessment as part of the response to the outbreak. Investigations were conducted between the 12th and the 19th Aug, 2013, more than five weeks after the last date that patients had eaten at one of the restaurants affected ( i.e. July 2, 2013). (Read the FDA report to obtain restaurant names). The salad mix supplied to the two restaurants was produced by T Farms, in a neighboring country. The salad had contained iceberg lettuce, romaine lettuce, green leaf, red cabbage and carrots, of which only the latter was processed at a different facility.   

The processing facility was first investigated in true Sherlock style. Construction and design of the plant,
water processing system, sanitary water system, receipt of raw ingredients, employee health and distribution systems were assessed. These were considered potential sources of the parasite. Infrastructural items and washwater obtained after vegetable washing were systematically cleared and proven negative by standard lab techniques (microscopy and PCR) as the source of the infection.

The source of raw ingredients were assessed in the "ranches" on which they were grown. A thorough examination of the employees working on these ranches and their medical records proved that they were not the source of infection. An assessment of the wells and farms in the vicinity of the ranches proved that they were not sources of infection either. Topography of the land and the rainfall patterns indicated that those geological/meteorological entities did not contribute to the outbreak. The EA team deemed other things on the ranches to be fairly in order and suggested a few minor modifications to the irrigation systems, such as the installation of back flow pumps to wells and the changing of valves on sinks that could potentially lead to cross-contamination.

The final conclusion offered is, " the FDA has not been able to definitively determine how or at what point in the supply chain Cyclospora cayetanensis contaminated the salad mix associated with the outbreak."

Before you criticize or condemn the lack of an expected conclusion, remember that epidemiological investigations are not easy. They do not always conclude successfully. That is, what you want to find is not what you will find. This is true of the biological sciences in general. Hypotheses have to be assessed and changed as one proceeds with data collection. To steadfastly hold onto a pretentious hypothesis and to try and fit the data to your hypothesis is obviously the hallmark of one working with a closed mind. As for Cyclospora, the matter rests for now, atleast until the next food associated outbreak.

References :

Environmental Assessment: 2013 Cyclosporiasis outbreak in Iowa and Nebraska – Findings and Recommendations
http://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ucm375732.htm

Phylum defining feature : The apical complex

The Phylum Apicomplexa is a large and uniquely diverse collection of unicellular, eukaryotic organisms that share amongst other common features, one that is capable of conferring the honor of being placed in this phylum, and one from which this blog derives its name . Ladies and gentlemen, let me introduce (cue fanfare ♪♫♪), the Apical Complex.

Ontologically, the apical complex is a constituent cellular component, found at the anterior end of the mature parasite. It is made up of various cytoskeletal components and membrane bound organelles, and among the latter , we find the micronemes, rhoptries, polar rings and conoids. (All that is standard textbook fare).

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(Source: http://tolweb.org/tree/ToLimages/zoite-tolv1.png; under the Creative Commons License)

Zooming in closer on Toxoplasma gondii, an archetypical apicomplexan, as Hu et al. have done in their paper , we find that the conoid is spirally arranged, which naturally allows for its role in invasion. The conoid is associated with the preconoidal rings, the polar rings and intraconoid microtubules. The polar rings serve as an anchor for microtubules that run from the anterior end of the organism to the posterior end, right under the pellicle. These, because of their location, are called subpellicular microtubules. Microtubule numbers vary across the phylum.When viewed under a phase contrast microscope, the entire apical complex appears as a single dark spot on the anterior end of the parasite.

The conoid is quite remarkable in that although it is made up of alpha-beta tublulins that are similar to the ones found in mammals, the tubulins form an unique ribbon-like polymer. When calcium levels in the cell rise, the conoid along with the associated upper polar ring protrudes beyond the base of the lower polar ring. This is shown in these figures on PubMed, in which alpha-tublin was modified with yellow fluorescent protein labelling.

The micronemes are involved in attachment and penetration. The proteins, named MICs, in these organelles are secreted, before the exocytosis of the rhoptry proteins. Before they get to their compartment, these proteins which have a classic hydrophobic amino terminal signal peptide move through the endoplasmic reticulum and exit the network at the golgi complex. They additionally have two conserved motifs at the C terminus - the first being a SYHYY and the second, an acidic series, EIEYE. These are important in folding, sorting and the spanning of the membrane. There are also distinct adhesive domains in the middle.

As soon as the tachyzoite attaches to the host cell, there is a marked increase in calcium levels in the parasitic cell, as calcium from the acidocalcisomes is released. This triggers the activation of at least two calcium dependant protein kinases that cause further signal cascades, and ultimately the release of micronemal proteins. The adhesive domains are involved in the adhesion and further invasion may take place.

Rhoptry proteins, reviewed in an article in Nature Reviews Microbiology,2008 , by Boothroyd, is conserved in its presence across the phylum and yet is varied in its architecture and numbers. There are 12 in the tachyzoite stages of Toxoplasma, each 2 to 3 micrometers in length. They appear to have compartments when studied with IFA. Protein compartmentalization post-transcriptionally occurs by understudied mechanisms. Rhoptries are even visible under a phase contrast microscope and appear to be related to exosomes. Of the 29 proteins identified in the rhoptries, 24 ROP proteins localize in the bulb region and 5 RON proteins localize in the neck region, with 28 newly recognized proteins yet to be allotted designations/names. Some of the ROP proteins are enzymes like kinases, proteases and phosphatases, that do not have orthologs in related genera. The, one gene- one protein, RON proteins however, have orthologs in related extant sps, although their exact functions are yet to be fully elucidated.  The rhoptries also seem to possess lipids rich in cholesterol. These are suspected to play a role during invasion, by forming vesicles around secreted proteins and ultimately fusing with the parasitophorous vacuole (PV).

During cellular invasion, the rhoptry contents are released. Some of the RON proteins are involved in the formation of the moving junction, which forms the physical interface, that is , the point of contact during invasion. The ROP proteins, after their release, localize in the PV or the PV membrane (PVM) or inside the host cell (including the nucleus), with many other targets being yet unknown. ROP1 is released in the PV as a vesicle and later moves to the membrane. ROP2 and its associates are putative integral PVM proteins that recruit the host mitochondria to the cytoplasmic face of the PVM. PP2C-hn and ROP16 contain typical NLS (Nuclear Localization Signal) sequences that cause them to move to the nucleus. All these molecules seem to play a key role in the biology of Toxoplasma sps.

The exact mechanism of conservation of the apical complex across the phylum seems to be a curious thing, as the phylum seems to be inhabited by protozoans that are as different, from each other in their pathogenesis and habitat, as the night is from the day. It certainly is true that the use of the apical complex is not always the same in all species, and certainly not all have the above described mechanism of Toxoplasma gondii. Thus, the Toxoplasma model, despite being the best that we have got right now, gives us only a glimpse of the preeminence of the great phylum-defining feature. It surely is our starting point. Where we will yet reach from here is unknown . Will we ravel the marvels of the pathogenesis of, say Cyclospora, by studying this feature? Will we bring to light the reason for the bizzare behaviour of the other Apicomplexans ? We can only hope so, for now, because, we have miles to sail in our research before we reach the shores of understanding .

References :

1. Boothroyd JC, Dubremetz JF. Kiss and spit: the dual roles of Toxoplasma rhoptries. Nat Rev Microbiol 2008;6:79-88.

2. Hu K, Roos DS, Murray JM. A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J Cell Biol 2002;156:1039-1050.

3. Soldati D, Dubremetz JF, Lebrun M. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int J Parasitol 2001;31:1293-1302.

Oo-complexity - a note on the Eimeria oocyst

As is well known among poultry professionals, subclinical coccidiosis is a huge challenge to the industry. Reinfection in poultry houses is the single most important route of disease sustenance in the population, especially in the deep-litter system. Caged and raised cage systems, although much more effective in keeping other infections low, do not completely isolate the birds from infective stages. Excellent husbandry practices and prophylactic medication are the mainstays of infection-reduction plans. To say that current control strategies are not perfect is an understatement. All that can be done is being done and yet we are getting nowhere. Given the production parameters on high throughput farms, the sheer number of birds raised poses a big economic challenge. Cost of control adds to the variable production cost and raises the cost of production per unit of meat/eggs. The biggest problem with coccidiosis control lies in our inability to control of the oocysts of the parasitic genus, Eimeria.

Eimeria is an obligate protozoan parasite that belongs to the phylum Apicomplexa. The lifecycle is complex and monoxenous (sexual and asexual development takes place in the same definitive host). The oocysts that are formed at the end of sexual development are shed in the feces. They sporulate in the environment and are infectious to susceptible birds.

(Source : http://www.ars.usda.gov/Main/docs.htm?docid=11018; Creative Commons License)

Host and site specificity is maintained during infection. The variability in the virulence of the parasite allows it to cause either hemorrhagic enteritis or malabsorptive enteritis. Now, if you have ever taken a college course in Protozoology, you might already know all that. You might also know that appropriate temperature and oxygen conditions are needed for sporulation, at the end of which the infectious oocyst contain four sporocysts with two sporozoites each (Eimeria sps.). But, let us move in a little deeper, beyond textbook material. What makes the parasite tick (no pun intended)? How is such a complex lifecycle so effortlessly maintained?

The answer lies in the utter complexity of the oocyst .

(Source : http://www.ars.usda.gov/Main/docs.htm?docid=11018; Creative Commons License)

Epidemiologically, it has long been recognized that altering the transmission stage of a pathogen can adversely affect it's survivability. This lends credence to the idea that changing the temperature, pH , oxygen availability etc among other environmental factors changes oocyst viability and transmissibility.

The oocyst is quite resistant, to dessication and hence destruction. In terms of parasitic survival, the oocyst occupies a marvellous functional niche. It can be favorably compared to the eggs of birds with the oocyst wall performing the same function as the calcareous shell, providing strength , protection and stability.
Equivalent to hatching is the process of excystation, by which infective sporozoites are released at the predilection site. The oocyst wall is sturdy enough to resist extreme challenges like the action of detergents (to which most bacteria are susceptible), mechanical force, enzyme action and strong chemical reagents such as bleach and potassium dichromate. Sodium hypochlorite at various strengths removes parts of the oocyst wall.

  In a recent study, Jenkins et al., compared the "Differing susceptibilities of Eimeria acervulina, E. maxima and E. tenella oocysts to dessication". The group concluded that Eimeria maxima oocysts displayed the highest resistance to dessication and were able to survive even in dry litter material. (Jenkins et al., 2013)

The protein rich oocyst wall starts out as electron dense wall forming bodies (WFB) in the endoplasmic reticulum and cytoplasm of the macrogamete. The veil forming bodies, WFB1 and 2 , are sequentially released after 'fertilization' of the macrogamete by the microgamete, to form the bilayered oocyst wall. [The layers are separated by a zone that spans 40 nm, (allowing the outer wall to be stripped away by bleach)] .
The wall forming bodies are chock full of proproteins that ultimately form the wall after post-translational modifications viz. N and C terminal modifications. Biochemical analysis of the wall proteins, using the combined gas chromatography- mass spectrometry technique, by Mai et al. revealed that the walls are mainly made up of proteins in which five amino acids predominate , viz. alanine , proline, valine, aspartic acid and isoleucine. Others have found that tyrosine also predominates and is important in dityrosine-protein cross linking. Lipids and carbohydrates contribute a little to the wall, with the predominant moieties being palmitic acid, stearic acid, oleic acid, linoleic acid, behenic acid, lignoceric acid, cholestane and cholesterol. (Mai et al., 2009)

So , what exactly confers the complexity ? It seems to me that the more we learn about the oocyst wall, the more we understand that there is more that we do not understand. Some questions remain. How do the dityrosine protein crosslinkings provide such enormous strength? What genes encode these proteins? Are there multiple genes that control these functions? Is the nature of the oocyst similar across the apicomplexans? Is it in anyway similar to egg walls of multicellular eukaryotic parasites? Comparative molecular parasitology might be the only way to answer these questions that deal with the very nature of the fabric of life of the oocyst.


References : 

1. Belli SI, Smith NC, Ferguson DJ. The coccidian oocyst: a tough nut to crack! Trends Parasitol 2006;22:416-423.

2. Jenkins MC, Parker C, O'Brien C, et al. Differing susceptibilities of Eimeria acervulina, Eimeria maxima, and Eimeria      

    tenella oocysts to desiccation. J Parasitol 2013;99:899-902.

3. Mai K, Sharman PA, Walker RA, et al. Oocyst wall formation and composition in coccidian parasites. Mem Inst        

   Oswaldo Cruz 2009;104:281-289.

An Apicomplexan by any other name ..... is still a Protozoan

Frank E. Cox, of the London School of Hygiene and Tropical Medicine, makes the most unflattering accusation in his article "Systemics of Parasitic Protozoa". He asserts that 'parasitologists' have completely ignored the relationships revealed by 'protozoologists', between and among non-parasitic protozoan groups and that they "continue to be embedded in the classifications of the 1980s" (Cox, 2002). Ooh, ouch ! I feel that such a great wrong must soon be corrected. So, let us get right to it !

The scientific community has moved on from Whittaker's Five Kingdom classification system that you and I learnt in school. Cavalier-Smith, in 1998, proposed a "Revised Six Kingdom System" in which all botanical life was classified into Kingdoms Fungi, Plantae and Chromista, zoological species into Protozoa and Animalia, while Bacteria were awarded their own kingdom (Cavalier-Smith,1998). In an earlier paper, he proposed 18 phyla under the Kingdom Protozoa, of which Phylum Apicomplexa was but one.

Other classification styles developed before this time, rule that the three phyla Apicomplexa, Dinozoa (Dinoflagellates) and Ciliophora (Ciliates like Paramecium) must be grouped together under the monophyletic group Alveolata.  All members have a subsurface alveoli, microtubules, mitophores and mitochondria with ampulliform or tubular cristae. (Adl et al.,2005)

Cavalier-Smith proposed that the phylum Apicomplexa be further subdivided into two subphyla : Apicomonada and Gamontozon, the latter of which is divided into "infraphylum" Sporozoa (whose members have nine singelet centrioles, complete conoids and conidial rings and the general presence of oocysts and sporocysts in their life cycles) and infraphylum Hematozoa (whose members have a more primitive centriole and lack the presence of oocysts and sporocysts, instead undergoing merogony in vertebrate erythrocytes and gametogony in arthropod guts). (Cavalier-Smith,1993)

In his letter, Cox suggests that based on new analysis Apicomonada has lost its supposed relatedness to the other Apicomplexans (which certainly is true, because it had initially contained only mollusc parasites), and so Sporozoa must be restored to Phylum status (Cox, 2002). This suggestion was apparently not widely accepted, as proved by latter scientific articles that still call the phylum Apicomplexa. Textbooks including popular ones like 'Georgis' Parasitology for Veterinarians' get around this dilemma by adding 'Sporozoa' in parentheses after 'Apicomplexa'.

The traditional grouping of the Apicomplexans, on the basis of phenotype, host, tissue and vector, has been under four broad categories : Coccidians, Gregarines, Haemosporidians and Piroplasms.Each of the groups are defined below:

Coccidia : "host specific, intracellular parasites of the intestines and other organs, of vertebrates with alternating asexual and sexual phases of development resulting in the production of environmentally resistant oocysts in the feces of definitive hosts" (Barta , 2009)

Gregarines : " extra/intra cellular protozoan parasites with large mature gamonts that develop extracellularly with most exhibiting syzygy in their developmental cycles" (Barta , 2009)

Hemosporidians : "obligate heteroxenous blood parasites that undergo sexual development in Dipteran flies and asexual development in vertebrate host" (Dimitrov, 2013)

Piroplasms : Pleomorphic, heteroxenous with an incomplete apical complex; lacks an oocyst stage and flagella (Adl,2005)

Molecular phylogenetic analysis has only added to the confusion by suggesting corrections to canonical taxonomic nomenclature, that is more often not followed by others save the original authors of scientific papers. In the midst of this melee, Cryptosporidium occupies an unique phylogenetic niche. It does not exhibit the cellular vampirism exhibited by the dinoflagellates and gregarines, does not possess variant surface proteins and lacks an apicoplast (a genome- containing plastid-like organelle, homologous to chloroplasts). Analysis of the small subunit ribosomal RNA suggests that the genus is more closely related to the Archigregarines than the Coccidians, but still forms its own clade. With the Archigregarines, Cryptosporidium shares these important characters : monoxenous life cycle, oocyts with four sporozoites, a usual location in the host gastrointestinal tract and extracellular gamonts or trophozoites. (Barta , 2006)

The Tree of Life Web Project has the following "hypothetical tree" for the phylum Apicomplexa showing the major branches viz. clades:

(http://tolweb.org/onlinecontributors/app?service=external/ViewImageData&sp=46943 ; Creative Commons License) 

At long last, we come to the real problem. The real question. If taxonomy is so inconstant, how will we/one study diversity and (dare I say it) evolution? There has been a clamour for a taxonomic scheme that will reflect phylogeny.

With this in mind, Morrison D. in a paper titled "Prospects for elucidating the phylogeny of Apicomplexa" lists five important changes that need to occur before any useful, directed progress can be made.

1. Taxon sampling
Anthropocentric research driven by economics and the relative veterinary-medical interest of the parasite has resulted in small, biased sample-data that do not adequately define the boundaries of taxa. Genebank is chock full of Plasmodium, Cryptosporidium, Theileria, Babesia and Toxoplasma sequences, whilst the Eimeria, Sarcocystis, Isospora and Gregarina have few to no representatives. Morrison also states that outgroups and basal taxa must be better studied to help classify already known species into clades.

The accusation of skewed sampling seems quite true. A search on NCBI revealed that as of today (6 Jan 2014), only 47 Apicomplexan species (of the many thousands that exist) have had their complete genome sequenced. All these are virulent parasites of animals/man.

2. Multiple molecular data sets
Frequently, trees are constructed on the basis of one gene (Quite simply because whole genomes are not available to play around with). Such trees, however, are not a true reflection of the relationship between species and clades. Among the Apicomplexans, the 18rRNA gene is most studied. But this gene is most prone to variation in terms of copy number. Other genes commonly studied are the HSP70, Actin, and a few mitochondrial genes. Morrison suggests that analyses be made with multiple gene sets, making sure that both nuclear and organellar genes (the latter show maternal inheritance) are included. Organellar genes can include mitochondrial genes and apicoplast (when they exist) genes.

3. Phylogentic analyses
Sequence alignment and tree building using the default parameters built into bioinformatic tools is naive, reproaches Morrison. Artifacts may arise from sequence length variation due to indel events, compositional variations like AT content and will not be resolved by multiple analyses (neighbor-joining, max-likelihood etc). Data must not violate the assumptions of the many analyses.

4. Reinterpretation of homologies
The thing about new knowledge is that it must agree or disagree completely or in degrees with old knowledge. Often reinterpretation is essential if we intend to get something worthwhile out of all our research efforts at all. A great example cited by Morrison, and which I spoke about in my previous post is the ineffectiveness of anti-coccidials on Cryptosporidium, easily explained by the genus not belonging to Coccidia.

5. Directed Data Collection
"The collection of pertinent data for the Apicomplexa can be best described as haphazard, which is unlikely to be of much practical value phylogenetically", laments Morrison. He proposes the formation of an informal group which would more likely be able to reach a consensus, over an autocratic formal group or a lackadaisical large group.(Morrison, 2008). I agree with him in that, for any progress from these mires of ignorance, directed data collection is essential.


Some, with adequate reason, have no interest at all in the systemics of taxonomy. Others are absolutely enthralled by the nuances of biological nomenclature. I, being one of the latter, have tried to understand the complexity of the system and have briefly presented the above the way I have understood it. Undoubtedly, the above is not absolute and is subject to change. But, change we will  (our understanding and even our knowledge base) when the time comes. Till then, I'll leave you here on the shores of (the land of ) Systemics. So long. Farewell !


References :

1. Adl SM, Simpson AG, Farmer MA, et al. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005;52:399-451.

2. Barta JR, Thompson RC. What is Cryptosporidium? Reappraising its biology and phylogenetic affinities. Trends Parasitol 2006;22:463-468.

3. Cavalier-Smith T. Kingdom protozoa and its 18 phyla. Microbiol Rev 1993;57:953-994.

4. Cavalier-Smith T. A revised six-kingdom system of life. Biol Rev Camb Philos Soc1998;73:203-266.

5. Cox FE. Systematics of the parasitic Protozoa. Trends Parasitol 2002;18:108.

6. Moore RB, Oborník M, Janouskovec J, et al. A photosynthetic alveolate closely related to apicomplexan parasites. Nature 2008;451:959-963.

7. Dimitrov, D., Valkiunas, G., Zehtindjiev, P., Ilieva, M., & Bensch, S. (2013). Molecular characterization of haemosporidian parasites (Haemosporida) in yellow wagtail (Motacilla flava), with description of in vitro ookinetes of Haemoproteus motacillae. Zootaxa, 3666(3), 369–381.

8. Morrison DA. Prospects for elucidating the phylogeny of the Apicomplexa.Parasite. 2008;15(3):191-6.

9. Šlapeta, Jan and Victoria Morin-Adeline. 2011. Apicomplexa Levine 1970. Sporozoa Leucart 1879. Version 18 May 2011. http://tolweb.org/Apicomplexa/2446/2011.05.18 in The Tree of Life Web Project

Cryptosporidiosis in dogs : Add another agent to the differential diagnosis list, why don't you?

I started my research for this post under the assumption that I was soon going to be buried under a ton of clinical articles and good-practice reviews about Cryptosporidium infections in dogs. I came away bitterly disappointed at the utter lack of clinical pointers.I had fully expected a well defined, pathognomonic diagnostic finding , to have been developed and published under the aegis of one Veterinary Association or the other. Unfortunately, that does not seem to be the case.

Ordinarily, when a case presents with diarrhea, a vet would try to narrow down the possible causes, . A list called the "Differential Diagnosis List" is (mentally) made which is conveniently divided into infectious and non infectious etiological agents that could have caused the clinical sign(s). Agents are eliminated based on clinical evidence and the results of the tests at the clinician's disposal. 'Cryptosporidiosis' is listed among the glorified ranks of Tritrichomonas foetus, Giardia sps, Balantidium coli and metazoans under the broad category of 'Parasites that cause diarrhea' in that definitive guide of lists, called "Differential Diagnosis in Small Animal Medicine"(edited by Gough A.). Listing the disease and not the agent is still a thing of great curiosity to me. On the list that runs six pages, Cryptosporidium has scored a spot. If Cryptosporidium caused diarrhea in dogs frequently, it would earn a firm place on the DD list of  every veterinarian.

However, allow me make a statement that seems to defy all clinical logic, but is firmly within the grasp of commonly understood parasitology knowledge : Cryptosporidium canis, C. parvum and C. meleagridis  (all of which have been found in dogs) do not always seem to produce "clinical" disease in dogs. They are typical parasites, carried along for the journey, which do not really harm the host. They seem to favour featuring in multiple etiology dramas, playing the roles of supporting parasites, neither sufficient nor necessary, over the role of protogonist parasites that direct the ebb and flow of the disease.

To treat this "disease" in dogs with the commonly available coccidiostats would be utterly silly and would cast a clinician in poor light , because the parasite is technically not a Coccidian to begin with. The taxonomy tempests have not been stilled on the displacement of Cryptosporidium from the Coccidian Apicomplexans.

Now, for a brief review of clinical research.
In 2000, the "Dog genotype" was validated by phylogenetic analysis of 18S and HSP70 as the new species, Cryptosporidium canis . (Morgan et al., 2000). Publications have implicated C.canis as the causal agent in both canine and human patients, including a case of a HIV infected human patient who harboured the canine genotype. (Pieniazek et al., 1999) But, how clinically relevant these are, is highly debatable

In the first infection report of Cryptosporidiosis in a week old pup, published in 1983, the authors include this disclaimer, "Cryptosporidia may have played a role in the enteritis seen in this pup, but further studies are needed to establish its pathogenecity". That is, the fact that they saw organisms that resembled Cryptosporidium could have been a happy coincidence, or in strict Pathology terms, "an incidental finding" that masked an insidious underlying cause. (Wilson et al., 1983) It has been established since that "Morphology is not a reliable tool for delineating species within Cryptosporidium" (Fall et al., 2003). Thus, it is impossible to know, which species of Cryptosporidium had infected the pup or if there had been another agent , say CPV that had coinfected it.

In a paper published in the Journal of American Animal Hospital Association in 1999, Willard and Bouley describe the fatal case of an immunocompromised 8 wk old dog that was coinfected with Cryptosporidium and other Coccidia. Which came first - the immunocompromise or the infection - is unknown.

Titilincu et al., in a paper titled "Prevalence of Cryptosporidium Spp. in asymptomatic dogs by ELISA and risk factors associated with infection" , published in Lucrari Stiiniifice Medicina Veterinara Vol XLIII (I) , in 2010 maintain that 53% of the 374 sampled dogs were positive on the ELISA, but were clinically asymptomatic. The positive animals when serotyped, were found to be infected with C.canis.

In a longitudinal study (from birth to 1 year of age) published in Acta Veterinaria Scandinavica in 2007 by Hamnes et al., Cryptosproridium was found by immunofluorescent staining in 44.1% of the 290 sampled animals from Norway. Since they used a direct FITC conjugated MAb against C.parvum, the samples identified were of course exclusively C.parvum. Again, the glaringly obvious point in the study is the complete lack of clinical data to support a clinical infection.

Despite the above mentioned studies, the jury is still out on the importance of Cryptosporidium sps. as a pathogenic agent in dog diarrhea, and we are left to ponder on these questions that will still haunt us:
1. Does Cryptosporidium canis actually cause disease in dogs?
2. What percentage of the cases harbour other enteric pathogens?
3. Do detection methods matter? Is it better to perform a PCR on a clinical sample than to perform a ZN staining on it ?
4. Should all cases that are positive using PCR be prescribed coccidiostats irrespective of clinical status ?
5. Should we add the agent to our DD list just to be safe ?

While some answers are obvious, others require a fair bit of clinical and lab research. To assume that answers will be obtained without both working together is obviously foolish.

References :
Morgan UM, Xiao L, Monis P, et al. Cryptosporidium spp. in domestic dogs: the "dog" genotype. Appl Environ Microbiol 2000;66:2220-2223.

 Pieniazek NJ, Bornay-Llinares FJ, Slemenda SB, et al. New cryptosporidium genotypes in HIV-infected persons. Emerg Infect Dis 1999;5:444-449.

Fall A, Thompson RC, Hobbs RP, et al. Morphology is not a reliable tool for delineating species within Cryptosporidium. J Parasitol 2003;89:399-402.
Wilson RB, Holscher MA, Lyle SJ. Cryptosporidiosis in a pup. J Am Vet Med Assoc1983;183:1005-1006.

A change of direction

The end of the semester is in sight. I had maintained this blog for the MICR756 class and had focused my attention on the 'Public Health Aspects of a complex Apicomplexan disease', viz. Malaria. Over the course of the semester, I found that I liked maintaining a blog and that I actually liked this sort of unofficial scientific writing.

So, from January, the blog will move to a new URL : apicomplexity.blogspot.com and will include topics starring other less known (and according to me, more interesting) apicomplexans such as Cryptosporidium, Isospora etc.

Thank you, all, for your patience. A special thanks to Dr.Nathan Fisher for the idea of a blog .