Detection and identification of blood-borne infections in goats in Nigeria using light microscopy and polymerase chain reaction

Author(s): Anise N Happi*, Deborah M. Buba, Paul E Oluniyi and Kazeem Akano Haemoparasitisms in animals are known to impose substantial economic burdens on owners. In Nigeria, most laboratories utilize only Light Microscopy (LM) for their diagnosis. Hence there is a need to have an update assessment of haemoparasitism of goat in Nigeria using molecular investigation. Using LM, blood samples from a total of 173 goats in Ibadan were screened ... Abstract View Full Article View DOI: 10.17352/ijvsr.000060


Introduction
Blood-borne parasitic diseases rank as the most important disease factor hindering small ruminants' production in tropical and subtropical regions. In goats, these diseases are not well characterized, although small ruminant farming is the main livestock resource in some rural regions in Nigeria and Africa. The benefi ts derived from sheep and goats in the tropics are signifi cantly below the expected. This is mainly due to reduced production associated to numerous factors, of which disease is the most important [1]. Goats in Sub-Saharan Africa are infected with a wide variety of parasites most importantly vector-borne haemoparasites [2,3]. Haemoparasitic infections have been reported in sheep in the Southwestern parts of Nigeria [4,5] and were attributed to suitable environmental conditions appropriate for the survival of vectors of the diseases [5]. Tick-Borne Diseases (TBDs) serve as limitation to livestock production in many developing countries of the world as they are responsible for increased morbidity and mortality resulting in reduced production of meat, milk and other livestock by-products [6][7][8]. Although economic losses caused by haemoparasitic diseases are known to be high worldwide [9,10], the direct losses caused by the haemoparasite infections are due to ill-health, premature slaughter, rejection of some body parts at meat inspection and death [11], while indirect losses could be attributed to lower production, quarantine measures, tick control, vaccination [12] and medications.
Haemoparasitism creates limitation to improved productivity, animal health [13] and economic gain [14][15][16][17] [18][19][20] and these have been attributed to various reasons ranging from the parasite similarity to some cell structures, other parasites, artifacts due to faulty processing of slides, level of parasitaemia, and the competency of the microscopist [19]. On the other hand, Polymerase Chain Reaction (PCR) provides a more accurate diagnosis owing to its higher sensitivity and specifi city, and it is fast becoming an inevitable facility for routine diagnosis given the large repertoire of haemoparasitic diseases with the need for adequate/specifi c therapy. Thus, it is now known that the latter method is unavoidable, particularly in the tropical African practice as there is a strong need for its integration in routine screening for accurate and prompt diagnosis with consequent successful control and reduction in economic loss.
Diagnosis of some diseases may also be inferred from host cell response and clinicopathological changes associated with the disease particularly in endemic areas. Various studies have also reported various changes in the biochemical and cellular constituents of blood imposed by haemoparasites [21,22]. Cytological diagnosis has been in the forefront of diagnostic techniques because of its availability as it is rapid, safe, cost-effective [23], easy to carry out and non-invasive.
Cytopathology has been used in the diagnosis of many infectious and non-infectious diseases most especially in the detection and identifi cation of common microorganisms in various cytologic specimens [24]. Given that literatures on cytopathological changes associated with these endemic haemoparasitic infections in small ruminants are relatively scanty, this study sought to reinvestigate haemoparasitic infections in small ruminants using PCR and sequencing, evaluate the haematological parameters and to record the morphological changes in blood cells associated with those parasites for an update in the hemoparasite types circulating in the studied area and, to understand their haemocytological changes for effective control and prevention.

Animals, blood and tissue sample collection
Blood and tissue (liver, spleen and mesenteric lymph node) samples were collected randomly within 6

Haematological evaluation
Full blood count, haemoglobin concentration, and total plasma protein were determined from each blood sample using standard haematological techniques [25][26][27] and as previously described by Happi, et al. [18].

Light microscopic detection of haemoparasites
Giemsa stained thin blood and buffy coat smears were screened for haemoparasites under x1000 magnifi cation (oil immersion) with an Olympus microscope according to the characteristic already described and also reported by Happi, et al. [18].

Erythrocytological evaluation of samples
Giemsa stained thin blood smears were examined under light microscopy at x1000 magnifi cation with immersion oil for red blood cell abnormalities. Quantitation of different erythrocyte abnormalities were recorded over 200 red blood cells. Morphological abnormalities were reported in semi quantitative fashion as previously described [28]. Results of the semi quantitative count of the erythrocyte morphological abnormalities were converted using reference guide [28], to give the descriptive clinical interpretation of the morphological abnormalities or erythrocytological diagnosis associated with the haemoparasitic infections. Percentage animal in each group with erythrocytopathological changes was also estimated.

DNA extraction
DNA extraction and purifi cation from blood obtained from each animal was performed using the QIAamp DNA blood mini kits (QIAGEN Inc. 27220 Turnberry, Lane, Valencia, USA) following the manufacturer's protocol. DNA templates were extracted from 126 randomly selected samples.

Polymerase chain reaction for detection of haemoparasites
Using a modifi ed protocol [29], the DNA templates were fi rst amplifi ed in 2 series of PCR reactions using 2 sets of selected primer pairs [viz: RLB-F2 (5ꞌ-GAC ACA GGG AGG TAG TGA CAA G-3ꞌ) and RLB-R2 (5ꞌ-CTA AGA ATT TCA CCT CTG ACA GT-3ꞌ)] with an amplicon size of approximately 400bp of the 18S rRNA gene spanning V4 hypervariable region [30], to detect Babesia/Theileria spp, and 16S8FE (5ꞌ-GGA ATT CAG AGT TGG ATC MTG GYT CAG -3ꞌ) and B-GA1B (5ꞌ-CGG GAT CCC GAG TTT GCC GGG ACT TYT TCT -3ꞌ) with an amplicon size of approximately 500bp of the 16S rRNA gene spanning the hypervariable V1 region of the genera to detect Anaplasma / Ehrlichia [31]. The PCR was done using the touch down program described by Schouls, et al. [31] and modifi ed by Berggoetz, et al. [32], to minimize non-specifi c amplifi cation. In addition, PCR detection of hemotropic mycoplasma (hemoplasma) was performed on 91 randomly selected DNA samples from the 173 goats by a protocol described by Nishizawa, et al. [33], using the primer set HEMOF (5'-ATATTCCTACGGGAAGCAGC-3') and HEMOR (5'-ACCGCAGCTGCTGGCACATA-3') [34] with the PCR condition previously described [19] [19]. Two positive samples for hemoplasma, anaplasma/ ehrlichia and, babesia /theileria species each, obtained from the cow [19] were used as positive controls in all PCR reactions while double distilled water was used as negative control.
After amplifi cation, 5μl of each amplicon was resolved by electrophoresis on a 1% agarose gel and visualized under ultraviolet trans-illuminator light at 325nm wavelength.

DNA sequenc i n g a nd phylogenetic analysis for haemoparasite identifi cation
Randomly selected PCR amplicons from each genus (2 from anaplasma/ehrlichia, 2 from Babesia/theileria and 10 from hemotropic mycoplasma) were purifi ed using a Qiaquick PCR purifi cation kit (QIAGEN) and sent for sequencing by Eton Following BLASTn analysis, the sequence identifi ed as Hemotropic mycoplasma spp. by BLAST analysis was aligned with sixteen (16) established mycoplasma species while the sequence identifi ed as Pseudomonas sp. was aligned with sixteen (16) established pseudomonas species using MAFFT v7.388 [36], with further adjustment made manually as necessary in Geneious [35]. Two neighbor-joining trees [37] were generated also in Geneious from a distance matrix corrected for nucleotide Citation: Happi  substitutions by the Tamura-Nei model [38]. The trees were viewed and manually edited using FigTree (http://tree.bio. ed.ac.uk/software/fi gtree/) [39].

Data analysis
Data were double entered serially using codes assigned to individual animal and were analyzed using GraphPad Prism All tests were two-tailed and statistical signifi cance was set at P <0.05.

Comparison of haemoparasites detected by LM and PCR
Overall, the detection rate by PCR was signifi cantly higher   age, gender and breed of haemoparasite-infected and noninfected goats (Table 1). In addition, these factors were similar in goats infected with the various haemoparasites.

Blood cell abnormalities
Overall, blood cell abnormalities were observed in 38 goats

Cytopathology of lymph nodes, spleens and livers associated with haemoparasitic infections in goats
The cytopathological changes observed in the spleens,    The lymph nodes of 35.3% haemoparasite infected animals showed lymphoid hyperplasia while 28.3% revealed an acute lymphandenitis, and 33.3% had normal lymph node. Lymphomatous lymph node was recorded in one case of A/E positive goat. In addition, a morula was observed in the lymph node of a goat positive for A/E with a few mitotic lymphocytes and binucleated plasma cells. Other changes in tissue smear were erythrophagocytosis and haemosiderosis in the lymph node of B/T+A/E infected goat. In most aparasitaemic goats, the spleen, lymph node and liver showed normal cytological morphology.

Molecular characterization of haemoparasites
The fi eld isolate of A/E sequenced amplicons (accession number: MH553089.1) fell in the same clade and clustered closely with an Anaplasma ovis sequence obtained from a sheep in Kenya with whom it also shared a high pairwise identity (100%) (Figure 3). While in our hemotropic mycoplasma isolates, a sequence shared 100% homology with the published Mycoplasma ovis strain Kamosika3 and Mycoplasma wenyonii (MK484136) from the NCBI database. On the phylogenetic tree, that Mycoplasma wenyonii clustered into the same clade with Mycoplasma wenyonii (HM 538191.1. obtained from a buffalo in China) ( Figure 4). Incidentally, two of the hemoplasma sequence from our study shared high (96 and 98.5 %) homology with Pseudomonas fl uorescens (MH725635) (only the one with 98.5% homology was submitted) from the NBCI database ( Figure 5). Among the two Babesia /Theileria spp sequenced one showed 99% homology with Theileria velifera from the NCBI database and the other one had low grade and low pairwise percentage insuffi cient to include in our fi ndings.

Discussion
Haemoparasites are endemic in sheep and goats in tropical and subtropical regions of the world [40]. However, information about their abundance and distribution in Southern Nigeria (Ibadan) is limited. In addition, species differentiations is challenging among some haemoparasites using LM. The little information available on the prevalence has been obtained using LM [41][42][43][44][45][46]. The hemoparasites commonly reported in small ruminants were Anaplasma, Babesia, Theileria [41][42][43][44], and scanty reports of Eperythrozoon species [41] and Trypanosoma species [43,45] Figure 3: Phylogenetic tree showing evolutionary relationship between the anaplasma sequence from this study (coloured blue) and fi fteen (15) established anaplasma species obtained from the NCBI database. Phylogeny was inferred with a neighbor-joining method (Saitou and Nei, 1987) from a distance-matrix corrected for nucleotide substitutions using the Tamura-Nei method (Tamura and Nei, 1993). Bootstrap values (90% and above) are shown on the nodes.

Figure 4:
Phylogenetic relationship between the sequence from this study (coloured blue) and sixteen (16) other hemoplasmas. Phylogeny was inferred with a neighborjoining method (Saitou and Nei, 1987) from a distance-matrix corrected for nucleotide substitutions using the Tamura-Nei method (Tamura and Nei, 1993). Bootstrap values (90% and above) are shown on the nodes.

Figure 5:
Phylogenetic relationship of the sequence from this study (colored red) together with a sequence from a previous study we carried out on cattle (colored green) and sixteen (16) other pseudomonas species. Phylogeny was inferred with a neighbor-joining method (Saitou and Nei, 1987) from a distance-matrix corrected for nucleotide substitutions using the Tamura-Nei method (Tamura and Nei, 1993). Bootstrap values (90% and above) are shown on the nodes. Citation: Happi  On the whole, the study disclosed high infection rates (62.7%) in the goat population by both LM and PCR. The higher prevalence in the sampled goats could be attributed to the abundance of the vectors involved in the transmission of these parasites due to suitable environmental conditions appropriate for their survival [5] and the higher sensitivity (PCR) method used [47][48][49][50]. The overall prevalence rate recorded during this study in small ruminants is higher than anyone ever reported in Nigeria [41][42][43][44][45][46][51][52][53]. In addition the 7 haemoparasite species recorded are more numerous than the previously reported ones [41][42][43][44][45][46][51][52][53][54][55].
The numerous haemoparasite types observed are as a result of vectors common to most types of haemoparasitism recorded (except T. brucei.) and the environmental, and ecologic factors, which favor presence of tick population in the study area, as well as the combine methods of detection (LM and PCR).
These results revealed that Hemotropic mycoplasma spp is the most prevalent blood parasite (51.6%). This is also the fi rst time hemotropic mycoplasma is reported as the most abundant haemoparasites hosted by small domestic ruminants in Nigeria and Africa as a whole.
Anaplasma ovis, T. brucei, and Mycoplasma ovis (formerly Eperithrozoon ovis) have been the haemoparasites most reported in small ruminants worldwide [59,62], including Nigeria [41,51], causing signifi cant economic loss among farmers. A report of Mycoplasma wenyonii (common hemotropic mycoplasma of bovine) infection with clinical manifestations in sheep has been published in Turkey [63]. However, in Nigeria it has been reported only in cattle [19]. Theileria velifera also known to infect bovine and buffalos in many countries [64][65][66] has also recently been reported from cattle in Ibadan, Nigeria [19]. This cross species infections may be because The report of Borrelia theileri infection in Nigeria is very rare and has been reported in cattle in 1927 and sheep in 1967 [67]. It has recently been reported in sheep in Ghana [2] while Borrelia burgdorferi the cause of a zoonosis called "Lyme's Disease" is commonly found in animals such as cow, and has been reported in sheep, horses, rats and birds in USA (Winscosin), China, and Europe [68][69][70]. Borrelia burgdorferi infection is a common fi nding in Wisconsin, while Borrelia theileri has been identifi ed in cattle, sheep and horses in South Africa. In Ghana, the Borrelia specie found was not characterized. However, due to the movement of many animals from South Africa to Nigeria, the Borrelia spp herewith recorded could be similar to the South African reported parasite. In addition, given the morphology of the borrelia observed during this study, Borrelia theileri is suggested to be the specie found, although was observed in goat. there are a few reports of blood cell apoptosis in T. brucei [71][72][73], Plasmodium spp [74,75], T. gondii [76,77], T. cruzi [78][79][80] and E. histolytica [81] infected hosts, where they are described  [47,84,85], who associated anaplasmosis with degeneration and necrosis of the hepatocytes, secondary to anaemic hypoxia. However, subacute hepatitis was also observed in the liver of T. brucei infected goats in this study and this is in contradiction with the fi ndings of [86] who reported necrosis of hepatocytes only.
Trypanosoma infection in this study was associated with hyperplasia of the spleen. This is in agreement with the fi ndings of [87][88][89][90], but different from the fi ndings of Uche and John [88], who reported degenerative changes in Citation: Happi  the spleen of trypanosoma infected animals. Hyperplasia is an indication of an immunological reaction and increased production of immunoglobulin. It has been established that babesia and theileria are associated with hyperplasia of the spleen and lymph node in animals [91]. This is similar with the fi ndings from this study, although some of the goats infected with Babesia/Theileria also showed splenitis. Other changes in tissue smear such as erythrophagocytosis by macrophage and haemosiderosis in the lymph node of B/T+A/E have been reported by Anosa and Kaneko [89], in deer mice infected with trypanosome spp, [90] and in the spleen of T. brucei infected Wistar rats and goats [72,73]. The Binucleated and mitotic lymphocytes with numerous plasma cells recorded in the lymph nodes of goats infected with A/E and B/T have also been reported in the lymph nodes of T. brucei infected Wistar rats and goats [72,73]. This implies an active or responsive lymph node to parasitic or non-parasitic stimulations.
A sequence from our study shared highest homology (100%) with Mycoplasma ovis and Mycoplasma wenyonii from the NCBI database and on the phylogenetic tree it fell in the Wenyonii (or Haemominutum) cluster. Previous studies have shown that haemoplasmas are divided into two phylogenetic clusters, Wenyonii (Haemominutum) and Hemofelis [92,93].
Incidentally, a sequence from our study shared high homology (98.5%) with several species of Pseudomonas particularly Pseudomonas fl uorescens from the NCBI database. In a recent study, the fi rst report of P. fl uorescens in cattle in Nigeria and Africa was made [19]. This might imply that P. fl uorescens is more prevalent in Nigerian animals than aforethought and further investigative studies need to be carried out to determine their prevalence and pathogenicity in infected hosts.
Interestingly also, on the phylogenetic tree, the sequence from this study shared a most recent common ancestor with the sequence from our previous study and a 99% pairwise identity implying the organism might have found its way into the other animal species from a common source which maybe while grazing on plants or drinking water. This is because many species of pseudomonas are commonly found in soil, water or plant surfaces and have been known to either cause infection in plants/plant products or been used as biological control of plant pathogens [94][95][96][97][98][99][100][101][102].

Conclusion
Haemoparasitic infections are common in small ruminants in Nigeria. The striking disparity in the LM and PCR methods of detection also highlights the need to consider at least light microscopy and PCR detection methods for haemoparasite diagnosis as they may provide more accurate results. The fi rst documentation of hemotropic mycoplasma species as the most prevalent haemoparasite types in infected goats in the studied area is presented. In addition, the demonstration of Mycoplasma wenyonii, Theileria velifera and Pseudomonas fl uorescens infections in the small ruminants here presented is unusual and suggest species cross over. Herein, the results also show that the haemocytopathology and cytopathology of spleen, liver and lymph node during haemoparasitic infection is substantially limited in respect to specifi c diagnosis hence aetiologic diagnosis cannot be made from blood cell morphologic alteration or tissue response. Studies gear towards the investigation of their pathogenicity in the novel host, their origin, diversity and factor of transmission are envisaged for effective prevention and control of these potentially zoonotic organisms.