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Identification of Rodent Species That Infest Poultry Houses in Mafikeng, North West Province, South Africa

Identification of Rodent Species That Infest Poultry Houses in Mafikeng, North West Province,... Hindawi International Journal of Zoology Volume 2019, Article ID 1280578, 8 pages https://doi.org/10.1155/2019/1280578 Research Article Identification of Rodent Species That Infest Poultry Houses in Mafikeng, North West Province, South Africa 1 1 1 2 Tsepo Ramatla , Nthabiseng Mphuthi, Kutswa Gofaone, MoetiO.Taioe, 3 1 Oriel M. M. Thekisoe, and Michelo Syakalima Department of Animal Health, School of Agriculture, Faculty of Natural and Agricultural Science, Mafikeng Campus, North West University, Private Bag X2046, Mmabatho, 2735, South Africa Center forConservationScience,NationalZoologicalGardensofSouthAfrica, SouthAfrican NationalBiodiversity Institute, PO Box 754, Pretoria, 0001, South Africa Unit for Environmental Sciences and Management, North West University, Potchefstroom Campus, Private Bag X6001, Potchefstroom 2520, South Africa Correspondence should be addressed to Tsepo Ramatla; ra21205450@gmail.com Received 1 November 2018; Accepted 25 March 2019; Published 18 April 2019 Academic Editor: Hynek Burda Copyright © 2019 Tsepo Ramatla et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rodents cause serious adverse eeff cts on farm production due to destruction of food, contamination of feed, and circulation of diseases. eTh extent of damage or the diseases spread will depend on the type of rodents that invade the farm. This study was conducted in order to find out the species of rodents that infest poultry farms around Mafikeng, North West Province of South Africa.Thestudy waspartofabroaderproject that wasinvestigating Salmonella vectors in the poultry farms around the province. eTh study trapped 154 rodents from selected farms and used the Cytochrome oxidase subunit 1 (COI) and the Cytochrome b (Cyt- b) barcoding genes for species identification. Two rodent pest species, namely, Rattus rattus (the black rat) and Rattus tanezumi (the Asian Rat/Asian House Rat) were identified. A total of 99 (64.3%) were identified as Rattus rattus and 55 (35.7%) were Rattus tanezumi. Between the two target genes, Cyt-b gene was only able to identify 40 (25.97%) of the total samples while COI was more efficient and amplified all the samples and thus was a better target gene for this kind of identification. The two rat species identified are known vectors of serious diseases; thus their presence should be regarded as an indication of high risk for diseases. Despite having been detected in the country before, finding R. tanezumi as the second largest rat species in the area was unexpected since this species is known to be indigenous to Asia. 1. Introduction mulium. eTh genus has some of the most adaptable rodents in theworld andmostofthemhavetheiroriginsinAsiawhere Rodents are relatively small mammals belonging to the order they migrated from to other parts of the world following Rodentia that includes porcupines, rats, mice, squirrels and the development of agriculture which provided food and marmots [1]. eTh y are famously known to cause huge losses shelter for their survival. Their intricate association with tostoredfood,crops,andproperty andalsototransmitmany farms makes them very important vectors of pathogens some pathogens that cause diseases of humans and animals [2]. of which are zoonotic. For instance, the brown rat is famous as The house mouse ( Mus musculus), roof rats (Rattus rattus), a carrier of gastrointestinal helminths and mites responsible and the brown rat (R. norvegicus) are the three main species for Plague, the black rat is a carrier of trematode species, of rodents usually found worldwide [3]. eTh genus Rattus is cestode species, and Salmonella spp., and the Asian rat is a one of the most common rodents found in poultry houses source of gastrointestinal helminths [4–7]. worldwide. Identifying the rodent species in a farm set-up is, The genus Rattus consists mainly of Black rat (R. rattus), therefore, important in determining the specific rat species’ Norway rat (R. norvegicus), Asian rat (R. tanezumi), and R. risk to diseaseaswellasother adverseeeff ctsinafarm. 2 International Journal of Zoology MAFIKENG Figure 1: Map of Africa showing the Mafikeng sampling area in the North West Province of South Africa. Unfortunately, rodents are not very easy to distinguish by Molema district (Figure 1). The city lies between 25 and 28 C the routine methods available that use physical attributes and South of the Equator and 22 and 28 C longitude east of so molecular identification has been oer ff ing the best option the Greenwich meridian. It shares an international border for identification. Molecular identification can be achieved with the Republic of Botswana in the North and is 260 km by anumberofmethods butDNAbarcoding, whichisa West of Johannesburg. Mafikeng is built on the open veld taxonomic method that uses a short genetic marker in an at an elevation of 1500 m along the banks of the Upper organism’s DNA to identify it to a particular species, has been Molopo River. Climatic conditions of the province differ found easy and particularly effective for this purpose [8]. eTh signicfi antly from West to East. eTh Western region receives target gene used for barcoding is the COI gene which is a very less than 300 mm of rain per annum, the central region common gene among species and has been fairly conserved around 550 mm per annum, while the Eastern and South over generations [9, 10]. Another gene commonly used is the Eastern regions receive over 600 mm per annum [12]. Cytochrome b gene which is also a very good discriminatory gene for species identification [8, 11]. es Th e two genes were, 2.2. Collection of Samples. Alistofpoultry farmsinthe therefore, used in this study to identify rodents in poultry Mafikeng area was compiled using the Department of Agri- houses from selected farms around Mafikeng, North West culture records. A few farms in the north, south, east, and Province of South Africa. west were randomly selected, the farmers were approached, and those that agreed were included in the study. Rodents were captured using Sherman rat traps [13] baited with 2. Materials and Methods peanut butter plus cheese and placed where the rats regularly 2.1. Study Area. eTh study was carried out in Mafikeng, visit. The traps were checked each morning during three the North West Province of South Africa. eTh North West consecutive days. The target number of rats was between Province is referred to as one of the biggest agricultural 150 and 200 based on previous studies [2, 5]. Live rats production areas in South Africa, with some of the largest were euthanized humanely using chloroform inhalation [14]. cattle herds in the country found at Stellaland (Vryburg) Their surface was disinfected with 70% ethyl alcohol before and mixed crop farming land. The province is also the dissection. Dissection of the abdominal cavity was done using second largest chicken producer in South Africa at 21.3% aer ft a surgical blade, a pair of forceps, and kidneys were harvested Western Cape with 21.9% (SAPA, 2014). The province has four and placed in 4 C until processing. Extra care was taken in districts, namely, Bojanala Platinum, Ngaka Modiri Molema, order to avoid cross-contamination by using new disposable Dr Ruth Segomotsi Mompati, and Dr Kenneth Kaunda. This utensils like scalpels, forceps, petri-dishes, and gloves for study was conducted around Mafikeng in The Ngaka Modiri each sample. Aer ft collecting the samples, carcasses were International Journal of Zoology 3 placed in carcass containers located within designated carcass for Biotechnology Information (NCBI) to identify sequences refrigerators/freezers in the post mortem room and then with high similarity (38). One direction sequencing was done. incinerated. 2.7. Phylogenetic Analysis. Gene sequences obtained from all positively tested amplicons were edited using BioEdit [17] to 2.3. DNA Extraction. DNA was extracted from tissues (kid- remove any degenerate base pairs and then saved as FASTA ney) using a QIAamp DNA Blood and Tissue Kit [Qiagen, format. To conrfi m sequences obtained from CO1 and Cyt- Hilden, Germany (No. 69504)]. The procedure was per- b analysis, the nucleotide basic local alignment search tool formed according to protocols provided by the manufactur- (BLASTn) was used. Only gene sequences with 97% to 100% ers. The DNA extracted was stored at −80 Cuntil analysis by similarity match score were considered as significant. PCR. The phylogenetic tree was constructed to illustrate the evolutionary relationships among Rattus spp. Multiple align- 2.4. Evaluation of the Quantity and Quality of Isolated DNA. ments of the sequences were carried out by MAFFT program The amount of DNA extracted from the samples was deter- 6.864 against corresponding nucleotide sequences retrieved mined by spectrophotometry with a NanoDrop ND-1000 sys- from Gen-Bank. Evolutionary distance matrices were gen- tem (NanoDrop Technologies, Inc., Wilmington, DE, USA). erated [18]. eTh aligned Cyt-b sequences were used to con- The purity of DNA was determined spectrophotometrically struct a phylogenetic tree as implemented in the MEGA 7 from the ratio of absorbance at 260 and 280 nm (A /A ). 260 280 package and the neighbor-joining (NJ) and distance matrix A ratio of between 1.7 and 2 indicates an excellent quality of methods were used [18]. A bootstrap confidence analysis was DNA. performed with 1000 replicates. A putative chimeric sequence was identified using the Chimera Buster 1.0 software. Manip- 2.5. PCR for Amplicfi ation of CO1 and Cyt-b Genes of Captured ulation and tree editing was carried out using Tree View Rats. For rat species identification, PCR was used targeting [17]. the CO1 and Cyt-b genes of the rats following published For CO1 analysis, multiple and pairwise alignments were protocols [8, 15]. eTh n fi al reaction mixture was 25 𝜇land done by ClustalW on Mega 7 [19]. Subsequently, the evolu- consisted of 2𝜇loftemplateDNA,8.5 𝜇ldoubledistilled tionary history was inferred based on the Hasegawa-Kishino- water, 2X Dream Taq GreenPCR Master Mix(2XDream Yano model [20] with 1000 bootstrap support values. eTh Taq Green bueff r, 4 mM MgCl , 0.4 mM) of each dNTP percentage of trees in which the associated taxa clustered and 1 unit/𝜇lofthermostable Taq polymerase (Thermo together is shown next to the branches. Initial trees for the Scientific, USA), the primer mix contained 10 𝜇M of each heuristic search were obtained automatically by applying oligonucleotide primer. Neighbor-Join and BioNJ algorithms to a matrix of pair- To amplify the 750 bp product of COI, the primers used wise distances estimated using the Maximum Composite were BatL5310 (5 -CCT ACT CRG CCA TTT TAC CTA TG- Likelihood (MCL) approach and then selecting the topology 󸀠 󸀠 3 )and R6036R(5 -ACT TCT GGG TGT CCA AAG AAT with superior log likelihood value. eTh rate variation model CA-3 ). The PCR conditions were one initial denaturation allowedforsome sitestobeevolutionarilyinvariable([+I], step of 94 C for 2 min, subjected to 35 cycles, denaturation 69.02% sites). eTh tree is drawn to scale, with branch lengths ∘ ∘ at 94 Cfor30sec, annealingat60 C for 30 sec, and the first measured in the number of substitutions per site. ∘ ∘ extension at 72 C for 1 min and a final extension step of 72 C for 5 min with the holding temperature at 4 C[15]. Ethics Committee Approval. Prior to the commencement To amplify the 762 bp product of Cyt-b gene, the primers of the study, the research proposal was approved based used were RGu2L (CAG CAT TTA ACT GTG ACT AAT on Animal Research Ethics Committee (NWU-00274-18- GAC) and RCb9H (TAC ACC TAG GAG GTC TTT AAT A5) guidelines by North West University Research Ethics TG), with the following PCR conditions used: 94 Cfor 3min, Regulatory Committee (NWU-RERC). ∘ ∘ ∘ 35 cycles of 94 Cfor 30sec, 60 Cfor 30sec, 72 Cfor 1min, followed by a final extension at 72 C for 5 min, and cooling ∘ 3. Results down to 4 C and storage [16]. The PCR amplicons in both PCR reactions were analysed 3.1. Rodent’s Identification. A total of 154 rat samples were by electrophoresis in 1% (w/v) agarose gel containing ethid- captured and examined using Cyt-b and COI genes to identify −1 ium bromide (10𝜇gml ) then viewed under UV light at the rodent species. Of the 154 rodents, 99 (64.3%) were 420 nm wavelength. A ChemiDoc Imaging System (Bio-Rad identified by the COI gene primers as Rattus rattus and the ChemiDocTM MP Imaging System, UK) was used to capture rest 55 (35.7%) were Rattus tanezumi (Table 1). Figure 2 shows theimageusingGeneSnap(version6.00.22)software. how the fragments appeared on a gel after amplification with of the COI gene primers. 2.6. Sequencing. Seventeen micro liters of all positive PCR Using the Cyt-b gene primer only 40 samples were ampli- products were sent for sequencing at Inqaba Biotechni- fied from a total of 154 as follows: Rattus rattus 26 (16.9%) cal Industries (Pty) Ltd in Pretoria, South Africa. The and Rattus tanezumi 14 (9.1%) (Table 1). Many samples did acquired sequence was aligned against GenBank data not show any amplification products for Cyt-b as shown in base using Basic Local Alignment Search Tool (BLAST) Figure 3. For the samples which were not ampliefi d, the PCR (www.ncbi.nlm.nih.gov/BLAST) from the National Center was unsuccessful in three repetitions. 4 International Journal of Zoology Table 1: Number of rodents from different farms that were identified using both CO1 and Cyt-b gene. Farm No. of rats Species Cytochrome oxidase 1 Cytochrome b (i) Rattus rattus 19 14 A25 (ii) Rattus tanezumi 62 (i) Rattus rattus 3 − B3 (ii) Rattus tanezumi −− (i) Rattus rattus 15 4 C21 (ii) Rattus tanezumi 6 − (i) Rattus rattus 10 1 D17 (ii) Rattus tanezumi 72 (i) Rattus rattus 46 10 E68 (ii) Rattus tanezumi 22 5 (i) Rattus tanezumi 14 1 H20 (ii) Rattus rattus 61 Total 154 154 40 M1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 189 750 bp COI 750 bp 250 bp Figure 2: PCR amplification of COI gene. Lane M: molecular weight marker (1kb). Lanes 1-19 are COI gene fragments from DNA extracted from Rodents. 3.2. Phylogeny of R. rattus and R. tanezumi. Phylogeny of R. 4. Discussion rattus and R. tanezumi for both Cytochrome Oxidase I (COI) The primary objective of the study was to document and Cytochrome b (Cyt-b) genes was inferred using sequences rodent species that invade poultry houses in the North-West derived from this study as well as those downloaded from Province in particular and in South Africa in general. This GenBank from different countries around the world. In informationisimportant foreconomicaswellasdisease risk both tree topologies monophyly between R. rattus and R. assessment [21, 22]. We were therefore able to show that of tanezumi was well supported with high bootstrap support the 154 rodents, 99 (64.3%) were R. rattus and the other 55 values. Figure 4 represents the tree topology inferred for (35.7%) were R. tanezumi.The R. rattus (the Black rat) was Cyt-b gene. Hylomyscus simus wasusedasanout-group and the most dominant species and it has been known to be in three major clades were derived from the analysis. The clades SouthAfricafor many years[23,24].Itisanimportant rat consisted of the Rattus rattus clade, R. tanezumi clade, and species because it is the most damaging invasive rodent in R. norvegicus clade, respectively. All three derived clades had the world [1]. Furthermore, the rat has also been known as well supported bootstrap values. a vector of diseases infectious to humans [24]. eTh rat is a For the COI tree topology, Hylomyscus simus and carrier of trematode species, cestode species, and nematode Micromy enthrotis were used as out-groups (Figure 5). In this species [5]. A study by Reusken et al. [4] also implicated phylogenetic tree, a monophyletic clade between R. rattus and theseratsasvectors spreading Coxiella burnetii.Itmay also R. tanezumi was again supported with high bootstrap support carry important protozoa which are mainly dangerous for values. eTh observations made from the two phylogenetic immune compromised patients [1]. Bacterial pathogens like trees do confirm that the samples collected in this study are Salmonella that are important to both humans and livestock indeed R. rattus and R. tanezumi, respectively. have also been isolated from R. rattus in many countries, i.e., Japan, [25], Reunion Island [26], Pakistan [27], and Canada 3.3. Accession Numbers Obtained from GenBank. The rep- [14]. In the phylogenetic tree it clustered well with the species resentative sequences obtained in this study have been found in India, Jordan, Tanzania, and other South African deposited to the GenBank database with assigned accession studies. The rat’s occurrence, especially in livestock farms, is numbers ranging from MK645246 to MK645295. therefore a significant health risk factor. International Journal of Zoology 5 M1 1 2 3 4 5 6 7 8 9 10 11 123 762 bp 750 bp 250 bp Figure 3: Lane M: molecular weight marker (1kb). Lanes 1, 2, 3, 5, 10, 11, 12, 13: amplified genes for Cyt-b. Lanes 4, 6, 7, 8, 9 are samples that were not amplified. Rattus rattus MNWP001 Rattus rattus MNWP002 Rattus rattus MNWP003 Rattus rattus MNWP004 Rattus rattus MNWP005 DQ439834 Rattus rattus AB762715 Rattus rattus EF186469 Rattus rattus 83 HE820119 Rattus rattus AB752979 Rattus rattus AB752982 Rattus rattus Rattus tanezumi MNWP033 Rattus tanezumi MNWP034 Rattus tanezumi MNWP035 Rattus tanezumi MNWP036 FJ842265 Rattus tanezumi AB753003 Rattus tanezumi AB753001 Rattus tanezumi FJ842279 Rattus norvegicus GU592988 Rattus norvegicus DQ439842 Rattus norvegicus 69 DQ439840 Rattus norvegicus DQ212188 Hylomyscus simus 100 JX893876 Hylomyscus simus Figure 4: Neighbour-joining tree from the Cyt-b gene. eTh analysis involved 24 nucleotide sequences whereby 9 sequences were derived from this study and the remaining from the NCBI database with 1000 replicates. All positions containing gaps and missing data were eliminated. There were a total of 608 positions in the final dataset. 6 International Journal of Zoology Rattus rattus MNWP001 Rattus rattus MNWP002 Rattus rattus MNWP003 Rattus rattus MNWP040 95 Rattus rattus MNWP010 HM217729 Rattus rattus JF827644 Rattus rattus EF186584 Rattus rattus EF186585 Rattus rattus HM217722 Rattus rattus Rattus tanezumi MNWP061 Rattus tanezumi MNWP067 Rattus tanezumi MNWP068 Rattus tanezumi MNWP069 AB752807 Rattus tanezumi AB752810 Rattus tanezumi AB752808 Rattus tanezumi AB752816 Rattus tanezumi 99 JQ667697 Hylomyscus simus JQ667692 Hylomyscus simus KF999093 Micromys erythrotis KF999094 Micromys erythrotis Figure 5: Phylogenetic tree based on distance matrix analysis of COI gene sequences from R. rattus and R. tanezumi based on the Hasegawa- Kishino-Yano model with 1,000 bootstrap support values. The analysis involved 22 nucleotide sequences whereby 9 sequences were derived from this study and the remaining from the NCBI database. All positions containing gaps and missing data were eliminated. er Th e were a total of 598 positions in the final dataset. The other Rattus species found in this study, R. tanezumi, to its high degree of phylogenetic species differentiation as has also been detected in South Africa before [6, 28]. R. compared to other mtDNA genes [33]. Our study conrfi med tanezumi was first identified in Limpopo Province [24]; these ndings fi and recommends its superior usage over Cyt-b. however to the best of our knowledge, this is the first report of its detection in the North West Province of South Africa. 5. Conclusion What is also significant was the finding that it is the second most dominant species in the poultry farms meaning it is The study established that the two rodent species found in gettingmoreprolicfi andinvasiveall over thecountry.This poultry houses around Mafikeng, North West province of rat species is predominantly found in Asia and wherever it South Africa were R. tanezumi and R. rattus.Thefinding is discovered it will always have its origins from Asia hence that R. tanezumi isthesecondmostprominent ratspecies in the name the Asian House rat. It is a carrier of hantaviruses these farms was unexpected because the rat species was rfi st [29] and it has also been found to carry important mites reported in South Africa not so long ago and that the species as well as helminths [6, 7]. Apart from diseases, the rat is not indigenous to Africa but Asia. It is thus important plays an important role by causing serious damage to field to expect it to increase and become a prominent species in crops, destroying food stores, and also causing infrastructural the years to come. eTh study also conrfi med that cytochrome damage [21, 30, 31]. It is obvious, therefore, that its increasing oxidase I (COI) genes serve as a reliable and more precise presence in South Africa brings with it these negative traits in target for identification of these rodent species. thefarmand humanenvironment. Of thetwo mitochondrialDNA genesusedfor this study, Data Availability cyt-b and COI, the Cyt-b could only amplify 40 (25.97%) samples out of 154. However, COI gene ampliefi d all the eTh sequences data used to support the n fi dings of this study samples thus demonstrating that COI is a better gene for this are available from the corresponding author upon request. purpose. The effectiveness of COI gene in species classifica- tion has also been reported before in wildlife [9], nematodes [32],reedbuck[10],birds [33],rodents [15],and lepidoptera Conflicts of Interest [34]. eTh COI genes have been the most frequent methods used for species identification in animal biological studies due The authors declare no conflicts of interest. International Journal of Zoology 7 Acknowledgments northwest,” Northwestern Naturalist,vol.89, no.3,pp.171–180, This work was supported by the funds made available by [14] C. G. Himsworth, E. Zabek, A. 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Identification of Rodent Species That Infest Poultry Houses in Mafikeng, North West Province, South Africa

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Copyright © 2019 Tsepo Ramatla et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi International Journal of Zoology Volume 2019, Article ID 1280578, 8 pages https://doi.org/10.1155/2019/1280578 Research Article Identification of Rodent Species That Infest Poultry Houses in Mafikeng, North West Province, South Africa 1 1 1 2 Tsepo Ramatla , Nthabiseng Mphuthi, Kutswa Gofaone, MoetiO.Taioe, 3 1 Oriel M. M. Thekisoe, and Michelo Syakalima Department of Animal Health, School of Agriculture, Faculty of Natural and Agricultural Science, Mafikeng Campus, North West University, Private Bag X2046, Mmabatho, 2735, South Africa Center forConservationScience,NationalZoologicalGardensofSouthAfrica, SouthAfrican NationalBiodiversity Institute, PO Box 754, Pretoria, 0001, South Africa Unit for Environmental Sciences and Management, North West University, Potchefstroom Campus, Private Bag X6001, Potchefstroom 2520, South Africa Correspondence should be addressed to Tsepo Ramatla; ra21205450@gmail.com Received 1 November 2018; Accepted 25 March 2019; Published 18 April 2019 Academic Editor: Hynek Burda Copyright © 2019 Tsepo Ramatla et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rodents cause serious adverse eeff cts on farm production due to destruction of food, contamination of feed, and circulation of diseases. eTh extent of damage or the diseases spread will depend on the type of rodents that invade the farm. This study was conducted in order to find out the species of rodents that infest poultry farms around Mafikeng, North West Province of South Africa.Thestudy waspartofabroaderproject that wasinvestigating Salmonella vectors in the poultry farms around the province. eTh study trapped 154 rodents from selected farms and used the Cytochrome oxidase subunit 1 (COI) and the Cytochrome b (Cyt- b) barcoding genes for species identification. Two rodent pest species, namely, Rattus rattus (the black rat) and Rattus tanezumi (the Asian Rat/Asian House Rat) were identified. A total of 99 (64.3%) were identified as Rattus rattus and 55 (35.7%) were Rattus tanezumi. Between the two target genes, Cyt-b gene was only able to identify 40 (25.97%) of the total samples while COI was more efficient and amplified all the samples and thus was a better target gene for this kind of identification. The two rat species identified are known vectors of serious diseases; thus their presence should be regarded as an indication of high risk for diseases. Despite having been detected in the country before, finding R. tanezumi as the second largest rat species in the area was unexpected since this species is known to be indigenous to Asia. 1. Introduction mulium. eTh genus has some of the most adaptable rodents in theworld andmostofthemhavetheiroriginsinAsiawhere Rodents are relatively small mammals belonging to the order they migrated from to other parts of the world following Rodentia that includes porcupines, rats, mice, squirrels and the development of agriculture which provided food and marmots [1]. eTh y are famously known to cause huge losses shelter for their survival. Their intricate association with tostoredfood,crops,andproperty andalsototransmitmany farms makes them very important vectors of pathogens some pathogens that cause diseases of humans and animals [2]. of which are zoonotic. For instance, the brown rat is famous as The house mouse ( Mus musculus), roof rats (Rattus rattus), a carrier of gastrointestinal helminths and mites responsible and the brown rat (R. norvegicus) are the three main species for Plague, the black rat is a carrier of trematode species, of rodents usually found worldwide [3]. eTh genus Rattus is cestode species, and Salmonella spp., and the Asian rat is a one of the most common rodents found in poultry houses source of gastrointestinal helminths [4–7]. worldwide. Identifying the rodent species in a farm set-up is, The genus Rattus consists mainly of Black rat (R. rattus), therefore, important in determining the specific rat species’ Norway rat (R. norvegicus), Asian rat (R. tanezumi), and R. risk to diseaseaswellasother adverseeeff ctsinafarm. 2 International Journal of Zoology MAFIKENG Figure 1: Map of Africa showing the Mafikeng sampling area in the North West Province of South Africa. Unfortunately, rodents are not very easy to distinguish by Molema district (Figure 1). The city lies between 25 and 28 C the routine methods available that use physical attributes and South of the Equator and 22 and 28 C longitude east of so molecular identification has been oer ff ing the best option the Greenwich meridian. It shares an international border for identification. Molecular identification can be achieved with the Republic of Botswana in the North and is 260 km by anumberofmethods butDNAbarcoding, whichisa West of Johannesburg. Mafikeng is built on the open veld taxonomic method that uses a short genetic marker in an at an elevation of 1500 m along the banks of the Upper organism’s DNA to identify it to a particular species, has been Molopo River. Climatic conditions of the province differ found easy and particularly effective for this purpose [8]. eTh signicfi antly from West to East. eTh Western region receives target gene used for barcoding is the COI gene which is a very less than 300 mm of rain per annum, the central region common gene among species and has been fairly conserved around 550 mm per annum, while the Eastern and South over generations [9, 10]. Another gene commonly used is the Eastern regions receive over 600 mm per annum [12]. Cytochrome b gene which is also a very good discriminatory gene for species identification [8, 11]. es Th e two genes were, 2.2. Collection of Samples. Alistofpoultry farmsinthe therefore, used in this study to identify rodents in poultry Mafikeng area was compiled using the Department of Agri- houses from selected farms around Mafikeng, North West culture records. A few farms in the north, south, east, and Province of South Africa. west were randomly selected, the farmers were approached, and those that agreed were included in the study. Rodents were captured using Sherman rat traps [13] baited with 2. Materials and Methods peanut butter plus cheese and placed where the rats regularly 2.1. Study Area. eTh study was carried out in Mafikeng, visit. The traps were checked each morning during three the North West Province of South Africa. eTh North West consecutive days. The target number of rats was between Province is referred to as one of the biggest agricultural 150 and 200 based on previous studies [2, 5]. Live rats production areas in South Africa, with some of the largest were euthanized humanely using chloroform inhalation [14]. cattle herds in the country found at Stellaland (Vryburg) Their surface was disinfected with 70% ethyl alcohol before and mixed crop farming land. The province is also the dissection. Dissection of the abdominal cavity was done using second largest chicken producer in South Africa at 21.3% aer ft a surgical blade, a pair of forceps, and kidneys were harvested Western Cape with 21.9% (SAPA, 2014). The province has four and placed in 4 C until processing. Extra care was taken in districts, namely, Bojanala Platinum, Ngaka Modiri Molema, order to avoid cross-contamination by using new disposable Dr Ruth Segomotsi Mompati, and Dr Kenneth Kaunda. This utensils like scalpels, forceps, petri-dishes, and gloves for study was conducted around Mafikeng in The Ngaka Modiri each sample. Aer ft collecting the samples, carcasses were International Journal of Zoology 3 placed in carcass containers located within designated carcass for Biotechnology Information (NCBI) to identify sequences refrigerators/freezers in the post mortem room and then with high similarity (38). One direction sequencing was done. incinerated. 2.7. Phylogenetic Analysis. Gene sequences obtained from all positively tested amplicons were edited using BioEdit [17] to 2.3. DNA Extraction. DNA was extracted from tissues (kid- remove any degenerate base pairs and then saved as FASTA ney) using a QIAamp DNA Blood and Tissue Kit [Qiagen, format. To conrfi m sequences obtained from CO1 and Cyt- Hilden, Germany (No. 69504)]. The procedure was per- b analysis, the nucleotide basic local alignment search tool formed according to protocols provided by the manufactur- (BLASTn) was used. Only gene sequences with 97% to 100% ers. The DNA extracted was stored at −80 Cuntil analysis by similarity match score were considered as significant. PCR. The phylogenetic tree was constructed to illustrate the evolutionary relationships among Rattus spp. Multiple align- 2.4. Evaluation of the Quantity and Quality of Isolated DNA. ments of the sequences were carried out by MAFFT program The amount of DNA extracted from the samples was deter- 6.864 against corresponding nucleotide sequences retrieved mined by spectrophotometry with a NanoDrop ND-1000 sys- from Gen-Bank. Evolutionary distance matrices were gen- tem (NanoDrop Technologies, Inc., Wilmington, DE, USA). erated [18]. eTh aligned Cyt-b sequences were used to con- The purity of DNA was determined spectrophotometrically struct a phylogenetic tree as implemented in the MEGA 7 from the ratio of absorbance at 260 and 280 nm (A /A ). 260 280 package and the neighbor-joining (NJ) and distance matrix A ratio of between 1.7 and 2 indicates an excellent quality of methods were used [18]. A bootstrap confidence analysis was DNA. performed with 1000 replicates. A putative chimeric sequence was identified using the Chimera Buster 1.0 software. Manip- 2.5. PCR for Amplicfi ation of CO1 and Cyt-b Genes of Captured ulation and tree editing was carried out using Tree View Rats. For rat species identification, PCR was used targeting [17]. the CO1 and Cyt-b genes of the rats following published For CO1 analysis, multiple and pairwise alignments were protocols [8, 15]. eTh n fi al reaction mixture was 25 𝜇land done by ClustalW on Mega 7 [19]. Subsequently, the evolu- consisted of 2𝜇loftemplateDNA,8.5 𝜇ldoubledistilled tionary history was inferred based on the Hasegawa-Kishino- water, 2X Dream Taq GreenPCR Master Mix(2XDream Yano model [20] with 1000 bootstrap support values. eTh Taq Green bueff r, 4 mM MgCl , 0.4 mM) of each dNTP percentage of trees in which the associated taxa clustered and 1 unit/𝜇lofthermostable Taq polymerase (Thermo together is shown next to the branches. Initial trees for the Scientific, USA), the primer mix contained 10 𝜇M of each heuristic search were obtained automatically by applying oligonucleotide primer. Neighbor-Join and BioNJ algorithms to a matrix of pair- To amplify the 750 bp product of COI, the primers used wise distances estimated using the Maximum Composite were BatL5310 (5 -CCT ACT CRG CCA TTT TAC CTA TG- Likelihood (MCL) approach and then selecting the topology 󸀠 󸀠 3 )and R6036R(5 -ACT TCT GGG TGT CCA AAG AAT with superior log likelihood value. eTh rate variation model CA-3 ). The PCR conditions were one initial denaturation allowedforsome sitestobeevolutionarilyinvariable([+I], step of 94 C for 2 min, subjected to 35 cycles, denaturation 69.02% sites). eTh tree is drawn to scale, with branch lengths ∘ ∘ at 94 Cfor30sec, annealingat60 C for 30 sec, and the first measured in the number of substitutions per site. ∘ ∘ extension at 72 C for 1 min and a final extension step of 72 C for 5 min with the holding temperature at 4 C[15]. Ethics Committee Approval. Prior to the commencement To amplify the 762 bp product of Cyt-b gene, the primers of the study, the research proposal was approved based used were RGu2L (CAG CAT TTA ACT GTG ACT AAT on Animal Research Ethics Committee (NWU-00274-18- GAC) and RCb9H (TAC ACC TAG GAG GTC TTT AAT A5) guidelines by North West University Research Ethics TG), with the following PCR conditions used: 94 Cfor 3min, Regulatory Committee (NWU-RERC). ∘ ∘ ∘ 35 cycles of 94 Cfor 30sec, 60 Cfor 30sec, 72 Cfor 1min, followed by a final extension at 72 C for 5 min, and cooling ∘ 3. Results down to 4 C and storage [16]. The PCR amplicons in both PCR reactions were analysed 3.1. Rodent’s Identification. A total of 154 rat samples were by electrophoresis in 1% (w/v) agarose gel containing ethid- captured and examined using Cyt-b and COI genes to identify −1 ium bromide (10𝜇gml ) then viewed under UV light at the rodent species. Of the 154 rodents, 99 (64.3%) were 420 nm wavelength. A ChemiDoc Imaging System (Bio-Rad identified by the COI gene primers as Rattus rattus and the ChemiDocTM MP Imaging System, UK) was used to capture rest 55 (35.7%) were Rattus tanezumi (Table 1). Figure 2 shows theimageusingGeneSnap(version6.00.22)software. how the fragments appeared on a gel after amplification with of the COI gene primers. 2.6. Sequencing. Seventeen micro liters of all positive PCR Using the Cyt-b gene primer only 40 samples were ampli- products were sent for sequencing at Inqaba Biotechni- fied from a total of 154 as follows: Rattus rattus 26 (16.9%) cal Industries (Pty) Ltd in Pretoria, South Africa. The and Rattus tanezumi 14 (9.1%) (Table 1). Many samples did acquired sequence was aligned against GenBank data not show any amplification products for Cyt-b as shown in base using Basic Local Alignment Search Tool (BLAST) Figure 3. For the samples which were not ampliefi d, the PCR (www.ncbi.nlm.nih.gov/BLAST) from the National Center was unsuccessful in three repetitions. 4 International Journal of Zoology Table 1: Number of rodents from different farms that were identified using both CO1 and Cyt-b gene. Farm No. of rats Species Cytochrome oxidase 1 Cytochrome b (i) Rattus rattus 19 14 A25 (ii) Rattus tanezumi 62 (i) Rattus rattus 3 − B3 (ii) Rattus tanezumi −− (i) Rattus rattus 15 4 C21 (ii) Rattus tanezumi 6 − (i) Rattus rattus 10 1 D17 (ii) Rattus tanezumi 72 (i) Rattus rattus 46 10 E68 (ii) Rattus tanezumi 22 5 (i) Rattus tanezumi 14 1 H20 (ii) Rattus rattus 61 Total 154 154 40 M1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 189 750 bp COI 750 bp 250 bp Figure 2: PCR amplification of COI gene. Lane M: molecular weight marker (1kb). Lanes 1-19 are COI gene fragments from DNA extracted from Rodents. 3.2. Phylogeny of R. rattus and R. tanezumi. Phylogeny of R. 4. Discussion rattus and R. tanezumi for both Cytochrome Oxidase I (COI) The primary objective of the study was to document and Cytochrome b (Cyt-b) genes was inferred using sequences rodent species that invade poultry houses in the North-West derived from this study as well as those downloaded from Province in particular and in South Africa in general. This GenBank from different countries around the world. In informationisimportant foreconomicaswellasdisease risk both tree topologies monophyly between R. rattus and R. assessment [21, 22]. We were therefore able to show that of tanezumi was well supported with high bootstrap support the 154 rodents, 99 (64.3%) were R. rattus and the other 55 values. Figure 4 represents the tree topology inferred for (35.7%) were R. tanezumi.The R. rattus (the Black rat) was Cyt-b gene. Hylomyscus simus wasusedasanout-group and the most dominant species and it has been known to be in three major clades were derived from the analysis. The clades SouthAfricafor many years[23,24].Itisanimportant rat consisted of the Rattus rattus clade, R. tanezumi clade, and species because it is the most damaging invasive rodent in R. norvegicus clade, respectively. All three derived clades had the world [1]. Furthermore, the rat has also been known as well supported bootstrap values. a vector of diseases infectious to humans [24]. eTh rat is a For the COI tree topology, Hylomyscus simus and carrier of trematode species, cestode species, and nematode Micromy enthrotis were used as out-groups (Figure 5). In this species [5]. A study by Reusken et al. [4] also implicated phylogenetic tree, a monophyletic clade between R. rattus and theseratsasvectors spreading Coxiella burnetii.Itmay also R. tanezumi was again supported with high bootstrap support carry important protozoa which are mainly dangerous for values. eTh observations made from the two phylogenetic immune compromised patients [1]. Bacterial pathogens like trees do confirm that the samples collected in this study are Salmonella that are important to both humans and livestock indeed R. rattus and R. tanezumi, respectively. have also been isolated from R. rattus in many countries, i.e., Japan, [25], Reunion Island [26], Pakistan [27], and Canada 3.3. Accession Numbers Obtained from GenBank. The rep- [14]. In the phylogenetic tree it clustered well with the species resentative sequences obtained in this study have been found in India, Jordan, Tanzania, and other South African deposited to the GenBank database with assigned accession studies. The rat’s occurrence, especially in livestock farms, is numbers ranging from MK645246 to MK645295. therefore a significant health risk factor. International Journal of Zoology 5 M1 1 2 3 4 5 6 7 8 9 10 11 123 762 bp 750 bp 250 bp Figure 3: Lane M: molecular weight marker (1kb). Lanes 1, 2, 3, 5, 10, 11, 12, 13: amplified genes for Cyt-b. Lanes 4, 6, 7, 8, 9 are samples that were not amplified. Rattus rattus MNWP001 Rattus rattus MNWP002 Rattus rattus MNWP003 Rattus rattus MNWP004 Rattus rattus MNWP005 DQ439834 Rattus rattus AB762715 Rattus rattus EF186469 Rattus rattus 83 HE820119 Rattus rattus AB752979 Rattus rattus AB752982 Rattus rattus Rattus tanezumi MNWP033 Rattus tanezumi MNWP034 Rattus tanezumi MNWP035 Rattus tanezumi MNWP036 FJ842265 Rattus tanezumi AB753003 Rattus tanezumi AB753001 Rattus tanezumi FJ842279 Rattus norvegicus GU592988 Rattus norvegicus DQ439842 Rattus norvegicus 69 DQ439840 Rattus norvegicus DQ212188 Hylomyscus simus 100 JX893876 Hylomyscus simus Figure 4: Neighbour-joining tree from the Cyt-b gene. eTh analysis involved 24 nucleotide sequences whereby 9 sequences were derived from this study and the remaining from the NCBI database with 1000 replicates. All positions containing gaps and missing data were eliminated. There were a total of 608 positions in the final dataset. 6 International Journal of Zoology Rattus rattus MNWP001 Rattus rattus MNWP002 Rattus rattus MNWP003 Rattus rattus MNWP040 95 Rattus rattus MNWP010 HM217729 Rattus rattus JF827644 Rattus rattus EF186584 Rattus rattus EF186585 Rattus rattus HM217722 Rattus rattus Rattus tanezumi MNWP061 Rattus tanezumi MNWP067 Rattus tanezumi MNWP068 Rattus tanezumi MNWP069 AB752807 Rattus tanezumi AB752810 Rattus tanezumi AB752808 Rattus tanezumi AB752816 Rattus tanezumi 99 JQ667697 Hylomyscus simus JQ667692 Hylomyscus simus KF999093 Micromys erythrotis KF999094 Micromys erythrotis Figure 5: Phylogenetic tree based on distance matrix analysis of COI gene sequences from R. rattus and R. tanezumi based on the Hasegawa- Kishino-Yano model with 1,000 bootstrap support values. The analysis involved 22 nucleotide sequences whereby 9 sequences were derived from this study and the remaining from the NCBI database. All positions containing gaps and missing data were eliminated. er Th e were a total of 598 positions in the final dataset. The other Rattus species found in this study, R. tanezumi, to its high degree of phylogenetic species differentiation as has also been detected in South Africa before [6, 28]. R. compared to other mtDNA genes [33]. Our study conrfi med tanezumi was first identified in Limpopo Province [24]; these ndings fi and recommends its superior usage over Cyt-b. however to the best of our knowledge, this is the first report of its detection in the North West Province of South Africa. 5. Conclusion What is also significant was the finding that it is the second most dominant species in the poultry farms meaning it is The study established that the two rodent species found in gettingmoreprolicfi andinvasiveall over thecountry.This poultry houses around Mafikeng, North West province of rat species is predominantly found in Asia and wherever it South Africa were R. tanezumi and R. rattus.Thefinding is discovered it will always have its origins from Asia hence that R. tanezumi isthesecondmostprominent ratspecies in the name the Asian House rat. It is a carrier of hantaviruses these farms was unexpected because the rat species was rfi st [29] and it has also been found to carry important mites reported in South Africa not so long ago and that the species as well as helminths [6, 7]. Apart from diseases, the rat is not indigenous to Africa but Asia. It is thus important plays an important role by causing serious damage to field to expect it to increase and become a prominent species in crops, destroying food stores, and also causing infrastructural the years to come. eTh study also conrfi med that cytochrome damage [21, 30, 31]. It is obvious, therefore, that its increasing oxidase I (COI) genes serve as a reliable and more precise presence in South Africa brings with it these negative traits in target for identification of these rodent species. thefarmand humanenvironment. Of thetwo mitochondrialDNA genesusedfor this study, Data Availability cyt-b and COI, the Cyt-b could only amplify 40 (25.97%) samples out of 154. However, COI gene ampliefi d all the eTh sequences data used to support the n fi dings of this study samples thus demonstrating that COI is a better gene for this are available from the corresponding author upon request. purpose. The effectiveness of COI gene in species classifica- tion has also been reported before in wildlife [9], nematodes [32],reedbuck[10],birds [33],rodents [15],and lepidoptera Conflicts of Interest [34]. eTh COI genes have been the most frequent methods used for species identification in animal biological studies due The authors declare no conflicts of interest. International Journal of Zoology 7 Acknowledgments northwest,” Northwestern Naturalist,vol.89, no.3,pp.171–180, This work was supported by the funds made available by [14] C. G. Himsworth, E. Zabek, A. 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