Morphology, epidemiology, and phylogeny of Babesia: An overview.

Babesiosis is a tick-borne hemoprotozoan disease of domestic and wild animals. The disease is caused by various species of Babesia and some species of Babesia have also zoonotic significance. The parasite in vertebrate hosts' remains in erythrocytes and the morphology of Babesia spp. is not uniform in all vertebrate hosts. With the advancement of science, particularly the use of molecular techniques made it easy to study the evolution of parasites and thereby reclassifying Babesia spp. as per their phylogeny and to establish the relation of one isolate of Babesia spp. with isolates throughout the world. An attempt also made in this communication to enlighten the readers regarding relationship of one isolate of Babesia spp. of a particular area to another isolate of Babesia spp. of that area or other parts of the world and phylogenetic classification of Babesia spp. was also discussed. It has been concluded that as the study on Babesia is complex in nature so monitoring of the infection with the use of modern techniques is very much needed to control the infection. Second, more research work on phylogenetic relationship of Babesia spp. isolated from different hosts is needed, particularly in India to know the evolution of Babesia spp. of a particular area, as it has great importance to study the trans boundary diseases of animals.

INTRODUCTION

In the year 1888, Babes investigated outbreaks of diseases with symptoms of hemoglobinuria in cattle in Romania and was the first to discover piroplasm in the blood of cattle.[1] Initially, he thought it to be a bacterium that had named as Hematococeus bovis and later it was changed to Babesia bovis. After 5 years, Smith and Kilborne demonstrated the causative organism of “Texas Fever” (babesiosis) as Pyrosoma bigeminum (Babesia bigemina).[2] After that the first demonstration of transovarian transmission of Babesia through its tick vector – a finding of historic significance was done.[3] Babesiosis was discovered gradually in various parts of the world with its zoonotic potential particularly in the tropical and subtropical countries along with its economic impact.[45] The first confirmed case of human fatal babesiosis caused by Babesia divergens was recorded in 1956.[6] Since then, zoonotic significance of babesiosis that too as a potentially life threatening zoonotic infection in human came out.[7] There are several species of Babesia which causes human infections throughout the world[8] but major one is Babesia microti particularly in North America.[9] Babesiosis is transmitted during blood feeding by infected ticks. The disease is considered as the most economically important tick-borne disease in tropical and subtropical areas.[10] If we take the cattle babesiosis in respect of economic impact then we could see losses occurs due to mortality, decreased milk or meat production, abortions, reduction of draft power, cost under the head of control measures, including increased cost of management to maintain ill animals. An annual loss of 16.9, 5.1, 5.4, 6.8, 21.6, 19.4, 57.2, 3.1, and 0.6 million US dollars in Australia, Kenya, Zimbabwe, Tanzania, South Africa, China, India, Indonesia, and Philippines, respectively, have been estimated due to babesiosis and anaplasmosis.[2] From India it is reported that due to clinical babesiosis in a crossbred cattle decreased milk production could be noticed for 30 days and during this period total loss of 51.6 L of milk has been estimated.[11]

MORPHOLOGY

On the basis of morphology, babesias are divided into two groups – small babesias (1.0–2.5 μm long) which included B. bovis, Babesia gibsoni, B. microti, Babesia rodhaini, etc., and large babesias (2.5–5.0 μm long) which included B. bigemina, Babesia caballi, Babesia Canis, etc., The orientation of the parasite in the red blood cells (RBCs) depends on its size because large pyriform parasites meet at their pointed ends at an acute angle to each other and small forms make an obtuse angle to each other.[12] More than 100 species of Babesia have been identified that are infecting many mammalian and some avian species.[7] The following are the morphology of different Babesia spp. of animals according to their hosts.

CATTLE AND BUFFALOES

B. bigemina - large form (4.5 μm × 2.0 μm) of Babesia. The parasites are characteristically pear shaped. Round (2–3 μm in diameter) oval or irregularly shaped form may also be found.

B. bovis - small form (2.0 μm × 1.5 μm) of Babesia. Slightly larger than B. divergens, vacuolated signet ring forms are particularly common.[13]

B. divergens - small form (1.5 μm × 0.4 μm) of Babesia. Generally remained as paired form, superficially lie on the RBC, stout and pyriform or circular forms may be found.

B. major - large form (3.2 μm × 1.5 μm) of Babesia. Pyriform bodies, the angle between the organism is <90°. Round forms with a diameter of about 1.8 μm are also available.

CANINE

Canine babesiosis is caused by two species of Babesia viz; B. canis and B. gibsoni, which are morphologically differentiated on the basis of their size.

B. canis - large form (4–5 μm long) of Babesia. Pyriform in shape, pointed one end, and round other. In a single RBC, multiple infection that is, more than one organism up to 16 may be found [Figure 1].

Figure 1

Babesia canis within red blood cell in Giemsa stained blood smear of a dog

B. gibsoni - small form (1.5–2.5 μm) of Babesia. Lack usual pyriform shapes, trophozoites are annular or oval; signet ring forms may occur [Figure 2].

Figure 2

Babesia canis within red blood cell in Giemsa stained blood smear of a dog

OVINE AND CAPRINE

B. motasi - large form (2.5–4.5 μm) of Babesia, organisms are pyriform.

B. ovis - small form (1.0–2.5 μm in length) of Babesia, maximum are round, remain at the margin of the RBC.

Babesia foliate - small form of Babesia. Leaf-shaped, located more centrally in RBC.

Babesia taylori - small form (1.5–2.0 μm long) of Babesia, ovoid to round in shape, multiple infections up to 16 parasites in a RBC may be seen.

EQUINES

B. caballi - large form of Babesia. Commonly occur as pair. Pyriform and measures 2.5–4 μm long. Round or oval forms with 1.5–3.0 μm in diameter may be found.

Babesia equi - small form (2.0 μm) of Babesia. The piroplasms characteristically form a Maltese cross of four organisms.

B. equi is also named as Theileria equi due to its preerythrocytic development as recorded in other Theileria species.[14] The differences and similarities between these organism have been worked out using molecular techniques and 18S rDNA sequence analysis reclassified recently B. equi as T. equi.[15] Among two species of infecting equines, B. equi (T. equi) considered as more pathogenic and widespread than B. caballi and there is no confirmed report regarding the occurrence of B. caballi from India.[16] On the contrary, B. caballi is more prevalent in Southern African countries than T. equi.

SWINE

Babesia trautmanni - large form of Babesia, 2.5–4.5 μm × 2 μm in diameter. Frequently occurs in pairs, but oval, amoeboid, and ring forms may also be found. Multiple infection in single RBC (up to 4 parasites) may be found.

FELINE

Babesia cati - large form of Babesia and Babesia felis - small form of Babesia. Babesia sp. in a cat from Kerala with a mean size of 2.4 μm × 1.3 μm has been reported.[17] This Babesia was found to present in RBC as single, in pairs, or multiple up to eight trophozoites within RBC. The large form of Babesia, B. cati from Indian wild cat was reported in Madras.[18]

EPIDEMIOLOGY

The epidemiology of babesiosis in general depended on several parameters such as availability of host, presence of ticks that act as vector of transmission of infections, presence of parasites within vectors, as well as hosts and environmental condition. These parameters are responsible for spread of infections. Absence of any one parameter will discontinue the spread of infections. As regarding epidemiology of babesiosis, a state of “Endemic Stability” where the relationship between host, parasite, vector, and environment remained in such a way that clinical disease occur rarely or not at all,[19] can also be taken into consideration. The parasite Babesia itself is the weakest point of this system of spread of infection as it needs both vectors and host for its survival,[20] and thus dependent on them. The second weakest point is the vector which depends on host and finally the host which support these two parameters to spread the infection but is not dependent on these two parameters.

HOST

One important fact is that Babesia spp. are not strictly host specific that might be a reason of ever increasing list of new species. Babesia infection in host depending on age of the host and inverse age resistance like young animals are less and older animals are more susceptible to infection. Breeds are also important for getting infection where Bos indicus are more resistant than Bos taurus. Besides, low grade immunity could be observed in previously infected animals. Several reports on occurrence of babesiosis in cattle in India are available.[2122232425262728] From North-Eastern region of India, B. bigemina infections in 3.6% cattle using polymerase chain reaction (PCR) has been reported.[29] Lower prevalence of B. bigemina has been reported from different parts of India both by conventional (Giemsa staining) and molecular methods. By conventional method, 0.48–2.7% prevalence rate has been reported from Punjab,[3031] Kerala,[32] and Karnataka.[33] By using PCR, prevalence of 0.66% and 2.43% has been reported from Kerala[32] and Punjab,[30] respectively. In India, in case of cattle, B. bigemina infection predominates over B. bovis infection and very few reports of B. bovis are available.[33] From Pakistan 9% cattle were found as positive for Babesia spp. infections out of which 6% were found infected with B. bigemina and 3% were found infected with B. bovis after examination of stained blood smear. In the same study, PCR detected 20% positive animals with a distribution of 13% with B. bigemina and 7% with B. bovis.[34]

In India, the situation of canine babesiosis is not clear[35] except few reports.[363738394041] The prevalence of canine babesiosis in Assam has been reported as 21.7% without mentioning the species.[38] The incidence reported from Northern part of India varied between 0.66% and 8.9%[3742] while from Southern India 11.6% prevalence of hemoprotozoa and among hemoprotozoa B. canis and B. gibsoni were recorded as 3.9% and 84.9%, respectively.[39] A total of 44 (39.63%) dogs were diagnosed as positive for Babesia infections after microscopic examination from North-Eastern region of India.[43] Among these, B. canis infection was diagnosed in 5 dogs (4.50%) and B. gibsoni infection in 39 (35.13%) dogs microscopically in Giemsa stained blood smears. Molecular diagnosis using PCR detected 63 (56.75%) dogs positive for Babesia infection. Single infection with B. canis was found in 9 (8.10%) dogs while B. gibsoni alone was detected in 3 (2.70%) dogs. Mixed infections by both these species were detected in 51 (45.94%) dogs. Overall, PCR detected 54 (48.64%) dogs as B. gibsoni and 60 (54.05%) dogs as B. canis positive. Equine babesiosis due to T. equi has been reported from India.[44454647]

PARASITE

The parasite Babesia itself is essential for causing the disease babesiosis as well as the transmitting agent. For infection to the vectors, parasitemia in the host and for infection to host, parasitemia in vectors is essential. But, there is no direct relation between the level of parasitemia in the host and the tick infection rate.[48] The study on interaction between Babesia microplus ticks and Babesia spp. infections revealed no detrimental effect of the parasite on naturally infected B. microplus and pathogenic effect of Babesia on ticks need not to be considered.[20]

VECTOR

All species of Babesia are naturally transmitted from animal to animal through the bites of ticks and within ticks’ transovarian transmission (transmission of infection through eggs from mother ticks), and stage-to-stage transmission (transmission of infection from egg to larvae to nymph to adult) occurs. Ticks are widely distributed throughout the world particularly in tropical and subtropical countries, and 80% of the world cattle are affected with ticks and ticks borne diseases.[49]

The ticks of genera Boophilus, Rhipicephalus, Hemaphysalis, Hyalomma, and Ixodes acts as vector for transmission of B. bigemina and ticks of genera Boophilus, Rhipicephalus, and Ixodes are responsible for transmission of B. bovis. The presence of B. microplus ticks in B. bigemina infected animals reported earlier.[50] In Pakistan, in cattle, the prevalence of Hyalomma tick was highest followed by Boophilus, Hemaphysalis, and Rhipicephalus.[34]

Babesia spp. in dogs are transmitted by ticks Rhipicephalus sanguineus, Hemaphysalis longicornis, Hemaphysalis leachi, and Dermacentor marginatus.[51] Other modes of transmission are through blood transfusion[52] and through the transplacental transmission as reported.[53]

T. equi infection is transmitted among equines through Ixodid ticks of the genera Hyalomma, Rhipicephalus, and Dermacentor. In donkeys and horses of India, Hyalomma species have been found as a potential vector for transmission of the infections.[14] Reservoir of B. caballi infection is ticked rather than horses because transovarian transmission occurs in ticks with B. caballi (except Rh. evertsi evertsi), and infections persist in ticks throughout several generations. But, the reservoir of B. equi infection are horses rather than ticks as only stage-to-stage transmission occurs in ticks with B. equi infections.[54] Besides transmission of infection through ticks, transplacental transmission of T. equi from carrier mother to foals has been reported from Punjab.[16] Intra-uterine transmission of equine babesiosis has also been reported.[55]

With the advancement of molecular diagnosis using PCR, now-a-days several authors detected the presence of Babesia organism in ticks. In a study, PCR-based detection of bovine Babesia spp. in ticks R. sanguineus and R. bursa has been done successfully.[10] B. bigemina DNA in ticks by DNA hybridization using a nonradioactive probe has been detected[56] and it is opined that this newly developed DNA probe could be used in epidemiological study of the disease. The frequency of B. bovis and B. bigemina infection in B. microplus engorged female ticks and eggs collected from cattle reared in an area of endemic babesiosis was carried out using PCR and nested PCR[57] and the infectivity of B. bigemina was recorded as higher to ticks fed on calves (56.2%) than cows (15.9%). Another interesting finding of the study was the significantly higher frequency of infection with B. bigemina (56.2%) than B. bovis (4.7%) in female engorged ticks collected from calves. Using nested-PCR similar findings of frequency of B. bigemina was higher in female ticks collected from calves (24.4%) than those collected from cows (9.4%) reported.[58] The frequency of Babesia spp. infections in engorged female B. microplus ticks and eggs collected from calves showed more frequency of infections in calves than engorged ticks collected from cows. The hatching rates of B. microplus larvae has been reported to lower in samples collected from calves than from cows particularly in those where Babesia spp. was detected in egg samples.[59]

ENVIRONMENT

Climatic data such as environmental temperature, humidity, and rainfall of a particular area are responsible for transmission of babesiosis and opined that the important factors of the presence of B. bigemina in cattle in hilly region of Meghalaya might be due to the spread of infected ticks under favorable climatic conditions, which has been changed in Meghalaya as a whole due to global warming that results in the spread of ticks and babesiosis.[60] It has been mentioned that the longevity of larvae and the number of annual generations of B. microplus are influenced by the climate.[20]

PHYLOGENY

As discussed earlier, morphologically babesias are two groups – large and small depending on their size. Recent literature reported morphological classifications are also related to phylogenetic characterizations that are based on small subunit-ribosomal RNA gene (18S rRNA) sequences. As per phylogenetic clusters, small babesias are more related to Theileria spp than the large, with the exception of B.divergens which is genetically related to large babesias.[79] Recently, 18S rRNA gene for phylogenetic analysis of Babesia has been used[61] and accordingly piroplasms were divided into five distinct clades – (i) Babesia spp. from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis, and oher Babesia spp. from cattle. (ii) B. canis and B. gibsoni from canines together with B. divergens and Babesia odocoilei, (iii) B. microti group with B. rodhaini, B. felis, Babesia leo, B. microti, and Theileria annae (iv) Western US Theileria – like group, containing Babesia conradae (v) Theileria-group containing all Theileria spp. from bovines.[27]

B. canis has been identified from the blood of three cats by molecular diagnosis.[61] The feline genotype of B. canis as B. canis subsp. presentii has been proposed.[62] So, it has been opined that the larger forms of Babesia in cats might be a feline genotype of canine Babesia sp.[17] Partial sequencing of 18S rRNA gene demonstrated that in cat with Babesia like symptoms was infected with B. canis canis.[63]

Worldwide there are three different sub species of B. canis named as B. canis rossi, B. canis canis, and B. canis vogeli that have been recognized on the basis of geographical distribution of vector tick, differences in pathological and clinical syndrome, antigenic property, and molecular analysis.[64] Similarly, recent molecular analyses have revealed three morphologically similar but genotypically distinct small Babesia of which B. gibsoni Asia type is endemic to Asia, North America, North and East Africa.[65] Among the three sub species of B. canis, B. canis vogeli is predominant in Brazil.[66] B. canis is considered to be virulent in dogs that are found in Europe and B. rossi that are only reported from Africa is highly virulent[35] whereas B. vogeli which is worldwide distributed are considered as mildly virulent.[67] B. canis vogeli has been reported from India using PCR.[68] Sequence and phylogenetic analysis of 18S rRNA and ITS 1 sequences of 15 small form Babesia positive canine blood samples collected from India revealed thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype.[69] They have also found that another two isolates were close together with B. orientalis, Babesia sp. (Kashi 1 isolate), and B. occultans of bovines. In a study, Indian isolates of B. bigemina showed genetic variation among themselves. In dogs of Northwest Spain, a B. microti-like piroplasm named as B. annae (T. annae) has been found.[70]

Two B. bigemina isolates of North India showed highest similarity (64%) whereas two South India isolates showed 50% similarity and isolates from these two region showed only 41% similarity.[71] The Indian B. bigemina sequences were found to be closely related with the cognate gene nucleotide sequences of B. bigemina from Argentina and Kenya where 99.1–99.9% and 99.0–99.7% nucleotide identity was observed, respectively. Distant relationship of these Indian organisms was observed with few cognate gene sequences from China where more than 7% divergence was observed in the distance matrix.[29] B. bigemina isolates from yak of Arunachal Pradesh has been shown to have a close relation with isolates from China, Mexico, Australia, and Zimbabwe.[72]

CONCLUSIONS

Many epidemiological factors are involved for causing babesiosis in vertebrate hosts and the study on babesiosis is complex in nature involving genetic composition and resistance of hosts, climatic conditions, tick infections, and infection rate in hosts. Monitoring of the infection with the use of modern techniques is very much needed to control the infection. The parasites Babesia have a wide host range and very little work has been done in India regarding phylogenetic relationship of Babesia spp. isolated from different hosts. More research work in this respect is needed to know the evolution of Babesia spp. of a particular area as it has great importance to study the trans boundary diseases of animals.