Occurrence of ticks and tick-borne mixed parasitic microbiota in cross-bred cattle in District Lahore, Pakistan

Ocorrência de carrapatos e microbiota parasitária mista transmitida por carrapatos em bovinos mestiços no distrito de Lahore, Paquistão

Abstract

The present study was focused on the incidence of ticks and tick-borne diseases (TTBD) in cross-bred cattle (Friesian x Sahiwal) of two farms (n = 2548) in district Lahore, Pakistan. We collected total of 572 ticks (adults and nymphs) and blood samples (10 ml) for microscopic i.e., blood smear test - Giemsa Stain (BST) and molecular analysis; Reverse Line Blot-General Primer-PCR (RLB-PCR) and Specie Specific Primer PCR (SP-PCR) from infested cattle (n = 100) from months of April to September. Results: The tick specie identified was Rhipicephalus microplus at both farms, with significant difference in infestations rate amongst both farms (p< 0.0001). The cross-bred cattle having higher ratio of Friesian blood and lower ratio of Sahiwal blood were mostly infested by ticks (p < 0.0458) and haemoparasites (p <0.474) and vice versa. The SP-PCR showed higher number of haemoparasites infection than BST, which revealed 16% T. annulata (p < 0.0001 and k value 0.485, 0.0001), 51% B. bigemina (p < 0.0001 and k value 0.485, 0.0001) and 15% A. marginale (p < 0.001 and k value 0.207, 0.001), respectively. The single infection with B. bigemina was 34% (n = 34/100) and A. marginale 6% (n = 6/100). The double infection with T. annulata/B. bigemina was 8% (n = 8/100) and B. bigemina/A. marginale 1% (n = 1/100). Whereas the triple infection with T. annulata/B. bigemina/A .marginale was 8% (n = 8/100). The phylogenetic study of isolated sequence of T. annulata revealed close homology to isolates from Iran (87%), B. bigemina to isolates from Cuba (94 to 100%) and A. marginale with isolates from Pakistan (99 to 98%).

Keywords:
Rhipicephalus microplus; polymerase chain reaction; Theileria annulata; Babesia bigemina; Anaplasma marginale

Resumo

O presente estudo foi enfocado na incidência de carrapatos e doenças transmitidas por carrapatos (TTBD) em bovinos mestiços (Friesian x Sahiwal) de duas fazendas (n = 2.548) no distrito de Lahore, Paquistão. Foram coletados 572 carrapatos (adultos e ninfas) e amostras de sangue (10 ml) para microscopia, ou seja, esfregaço sanguíneo - coloração de Giemsa (BST) e análise molecular; Reverse Line Blot-General Primer-PCR (RLB-PCR) e Specific Primer PCR (SP-PCR) -, de bovinos infestados (n = 100) nos meses de abril a setembro. Resultados: A espécie de carrapato identificada em ambas as fazendas foi Rhipicephalus microplus, com diferença significativa na taxa de infestação nos dois locais (p < 0,0001). Os bovinos mestiços Friesian, com maior proporção de sangue, e Sahiwal, com menor proporção de sangue, foram principalmente infestados por carrapatos (p < 0,0458) e hemoparasitos (p < 0,474), e vice-versa. O SP-PCR mostrou maior número de infecção por hemoparasitos do que a BST, revelando 16% de Theileria annulata (p < 0,0001; k valor 0,485; 0,0001), 51% de Babesia bigemina (p < 0,0001; k valor 0,485; 0,0001) e 15% de Anaplasma marginale (p < 0,001; valor de k 0,207; 0,001). A infecção única com B. bigemina foi de 34% (n = 34/100), e com A. marginale, de 6% (n = 6/100). A dupla infecção com T. annulata/B. bigemina foi de 8% (n = 8/100), e com B. bigemina/A. marginale, de 1% (n = 1/100). Já a tripla infecção com T. annulata/B. bigemina/A. marginale foi de 8% (n = 8/100). O estudo filogenético da sequência isolada de T. annulata revelou estreita homologia com isolados do Irã (87%), de B. bigemina com isolados de Cuba (94 a 100%) e de A. marginale com isolados do Paquistão (98 a 99%).

Palavras-chave:
Rhipicephalus microplus; reação em cadeia da polimerase; Theileria annulata; Babesia bigemina; Anaplasma marginale

1. Introduction

Ticks are known for their negative impact on animal and human health through infestation and are capable of transmitting a wide range of pathogens including protozoan, viruses and bacteria. The ticks during summer exceed all other arthropod parasites in the number and varieties of diseases (Castro and Newman, 2003). Ticks are widely distributed in different ecological and geographical regions of Pakistan and reported to transmit bovine theileriosis, babesiosis and anaplasmosis in livestock. The Sahiwal breed exhibits a high degree of resistance to ticks (Ashfaq and Razzak, 2000). The exotic livestock breed is highly susceptible to ticks and tick-borne diseases even kept in very controlled environment (Young et al., 1988). The overall prevalence of Theileria annulata, Babesia bigemina and Anaplasma marginale in a cattle population can be as 22.0%, 19.33% and 10.6%, respectively (El-Dakhly et al., 2020). T. annulata can be found in both erythrocytes and lymphocytes of their host and is transmitted by ticks of Hyalomma (H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum and H. marginatum marginatum (Sayin et al., 2003). B. bigemina is presently considered as one of the most important constraints in production of livestock and transmitted by Rhipicephalus ticks (Makenov et al., 2021) and mainly infects the red blood cells (Ristic, 1981). A. marginale is caused by a group of obligate intracellular bacteria and are transmitted by Rhipicephalus ticks (Yan et al., 2020; Galay et al., 2021), and observed within erythrocytes (Jurkovic et al., 2020). These haemoparastes can be seen in thick and thin smears of blood prepared with Giemsa’s stain (Darghouth et al., 1996). The PCR test is more specific and sensitive in the diagnosis of tick-borne diseases including Theileriosis, Babesiosis and Anaplasmosis. The subject study has been carried out to find the prevalence of ticks and tick-borne diseases (TTBD) in various crosses of cross-bred cattle (Friesian x Sahiwal) in two mid-sized commercial farms in Lahore, Pakistan.

2. Materials and Methods

2.1. Animal data

During field study, the reference population of cross-bred cattle (Friesian x Sahiwal) was selected from two mid-sized commercial farms of city of Lahore, Pakistan (31.5204° N, 74.35887° E). These farms are named as Centre Farm (CF) and Branch Farm (BF). These farms are in the heart of city and dependent on each other on various animal management and veterinary practices. CF is a milk producing farm and holds large no of animals (n = 1459). BF is basically rearing and breeding farm (n = 1089) and holds heifers and dry adult cattle. The breeding policy of these farms are to cross the Friesian breed (sire) with Sahiwal breed (dam) to achieve blood and genetic ratio between 50-95% Friesian blood in offspring to get max potential of milk production as well as to avoid the disease proneness of pure exotic cattle. Hence, six types of offspring (Friesian x Sahiwal) are found at these farms and termed in ratios as 1/2 (50:50), 5/8 (62.5:37.5), 3/4 (75:25), 7/8 (87.5:12.5), 15/16 (93.75:6.25) and 31/32 (96.875:3.125), respectively. These farms follow a strict regime of management and veterinary practices.

2.2. Tick collection

The ticks for this study was collected from April to September. The data of animals found infested with ticks were also recorded. These included the age, type, breed, month of sampling, number and location of ticks on each animal. Ticks removed and placed in tubes containing 70% ethanol. The ticks were examined under stereo microscope (Olympus SZ40, Japan) and morphologically identified (Estrada-Peña et al., 2004).

2.3. Blood collection

Screening of animals found infested by ticks were done by thick blood smear examination and PCR. Ten milliliter blood collected from juggler vein of cattle in disposable syringes and transferred five milliliter each to two vacutainers (EDTA) under aseptic condition.

2.4. Thick blood smears

The dried blood smears stained by Giemsa’s Staining Technique studied under microscope for presence of protozoans (Soulsby, 1982; Moretti et al., 2010).

2.5. PCR tests

The blood collected in vacutainers stored at -20°C in freezer till further processing. The DNA extraction of blood was carried out by using DNA extraction kit (GeneAll® Type G, ExGene) as per manufacturer's protocol (Handbook for DNA purification version 3.3). The DNA extracted were placed in -70°C freezer till further processing. The concentration of DNA was estimated by Spectrophotometric analysis. In this procedure 10 µl of DNA was mixed with 90 µl of autoclaved distilled water. The quantity of DNA was calculated by using 260 nm and 280 nm wavelength ratio. The primers used in subject study is as shown in Table 1.

Table 1
Primer used in PCR tests along with target gene and predicted amplicon size.

Primer	Primer Sequence	Target Genome	Predicted amplicon size	References
Primer Set-A Forward Reverse	Reverse Line Blot (RLB) General primer 5ˊ- GAC ACA GGG AGG TAG TGA CAA G - 3ˊ 5ˊ- CTA AGA ATT TCA CCT CTG ACA GT - 3ˊ	18s rRNA gene	430 bp	Gubbels et al., 1999
Primer Set-B Forward Reverse	T. annulata specific 5ˊ- CAA ATG AGC TTC TGG GGA GC - 3ˊ 5ˊ- TTC CTG CCA TTG CCA AAA GTC - 3ˊ	Cytochrome b gene	475 bp	Bilgic et al., 2010
Primer Set-C Forward Reverse	B. bigemina specific 5ˊ- GAC GAA TCG GAA AAG CCA CG - 3ˊ 5ˊ- AGA GGG ACT CCT GTG CTT CA - 3ˊ	18s rRNA gene	321 bp	Umber et al., 2020
Primer Set-D Forward Reverse	B.bovis specific 5ˊ- AAT ATG GGT TGG GCA ATG CG - 3ˊ 5ˊ- CCA CCC AAA ACA AGA GCA ACT - 3ˊ	Cytochrome b gene	269 bp	Umber et al., 2020
Primer Set-E Forward Reverse	A. marginale specific 5ˊ- CCT TAT GGG GTG GGC TAC AC - 3ˊ 5ˊ- CCC GAG AAC GTA TTC ACC GT - 3ˊ	16s rRNA gene	178 bp	Designed in current study

The PCR analysis of samples were done by using ready to use Master Mix of Bioshop®, Canada. The concentration used for tests were 10 µl Master Mix, 2 µl each forward and reverse primers, 4 µl denucleased water and 2 µl DNA extract of samples. Amplification of 20 µl samples were performed for specified number of cycles in a thermocycler SimpliAmp™, Applied Biosystems, Thermo Fisher Scientific. Known positive and negative samples were also included in each PCR test. The condition of PCR cycles for each primer set is as shown in Table 2. The amplified products of each PCR run (6 µl) was examined on 1% (w/v) agarose gel added with SYBR® Safe DNA Gel Stain, Invitrogen, Thermo Fisher Scientific, USA and subsequently photographed using a GelDoc system of BioRad, USA and Digital Camera.

Table 2
The conditions of PCR used for each primer set.

PCR Protocols for Heamoparasites
Primers	Cycles Number	Initial Denaturing	Denaturing	Annealing	Extension	Final Extension
RLB	40	95°C for 4 min	94°C for 35 seconds	51°C for 35 seconds	72°C for 35 seconds	72°C for 10 min
T.annulata	35	95°C for 4 min	95°C for 30 seconds	58°C for 30 seconds	72°C for 1 min	72°C for 10 min
B.bigemina	37	95°C for 1 min	95°C for 30 seconds	57°C for 30 seconds	72°C for 30 seconds	72°C for 5 min
B.bovis	40	94°C for 5 min	94°C for 5 seconds	60°C for 45 seconds	72°C for 45 seconds	72°C for 10 min
A.marginale	35	95°C for 5 min	94°C for 30 seconds	55°C for 30 seconds	72°C for 1 min & 30 seconds	72°C for 5 min

2.6. Phylogenetic studies

For each samples found positive on gel electrophoresis the gel extracted and DNA purified using GeneJET Genomic DNA purification kit by Thermo Scientific, Lithuania, using manufacturer’s guidelines. The purified DNA along with primers were submitted to 1st BASE DNA Sequencing Company, Singapore for DNA sequencing and identification of protozoan. The processed sequences were then compared with the already published sequences in the National Centre for Biotechnology Information (NCBI) using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to get the specific identity of individual organism/ protozoan. The current reference sequences were downloaded from NCBI. For T. annulata the evolutionary history was inferred by using the Maximum Likelihood method and Tamura 3-parameter model (Tamura, 1992). The tree with the highest log likelihood (-1566.88) created. The percentage of trees in which the associated taxa clustered together were shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and Biological Neighbour Joining (BioNJ) algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 13 nucleotide sequences. There was a total of 903 positions in the final dataset. Evolutionary analyses were conducted in Molecular Evolutionary Genetics Analysis across Computing Platforms software (MEGA X) (Kumar et al., 2018). In case of B. bigemina, the evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree was created. The tree drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree (Felsenstein, 1985). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980) and are in the units of the number of base substitutions per site. This analysis involved 18 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 705 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The evolutionary history of A. marginale was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree created. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980) and are in the units of the number of base substitutions per site. This analysis involved 11 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There was a total of 175 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

2.7. Statistical analysis

Statistical analysis performed by using the software program of SPSS text analysis @2004 SPSS 25.0 (California, USA) for windows as well as GraphPad Prism version 5.0 (La, Jolla, USA). The prevalence of tick was calculated by number of animals found infested with ticks divided by total number of animals and numbers in each age group of animals. The Chi-square and One-Way ANOVA tests were used to compare the prevalence of ticks and protozoan in each farm, age group, season of collection and breed. A p-value of <0.05 was considered as statistically significant.

3. Results

3.1. Prevalence of ticks

A total of 572 ticks (including 292 males, 235 females and 45 nymphs) of R. microplus were collected from 3.92% (100/2548) cattle in this study. Interestingly, no other tick genus was found at both farms. At CF 3.63% (53/1459) and at BF 4.31% (47/1089) cattle were found infested, hence statistically significant difference in tick infestation (p < 0.0001). The animals belonging to all age groups were found infested in both farms. The overall prevalence of ticks in both farms in calves was 0.51% (n = 13/2548), heifers 1.26% (n = 32/2548) and adults 2.16% (n = 55/2548). However, at CF 5.3% (13/245) of calves and 3.53% (40/1131) of adult cattle, whereas in BF 3.9% (32/820) of heifers and 5.57% (15/269) adults were found infested. No heifer in CF (n = 83) was found infested during the study. There was a significant difference of tick infestation between three age groups (p < 0.0001). The month-wise distribution of infested animals for April was 9% (n= 9/100), May 11% (n = 11/100), June 17% (n= 17/100), July 24% (n = 24/100), August 25% (n = 25/100) and September 14% (n = 14/100). There was significant difference statistically between month of infestation between three animal groups (p < 0.0001).

3.2. Tick Infestation and Genetic makeup of animals

The number of cross-bred cattle (Friesian x Sahiwal) having higher concentration of Friesian blood and lower concentration of Sahiwal blood were mostly infested by ticks both in CF and BF. The cattle with genetic makeup of 7/8 were highly infested at 34% (n = 34/100) followed by 15/16 at 38% (n = 38/100) and 31/32 at 12% (n = 12/100) infestation rates, whereas animals with 1/2 at 2% (n = 2/100), 5/8 at 8% (n = 8/100) and 3/4 at 6% (n = 6/100) were least infested by ticks. The same pattern was observed in calves, heifers and adults. Statistically significant difference was found (p < 0.0458) (see Figure 1).

Figure 1
Prevalence of ticks in various groups of cross=bred (Friesian x Sahiwal) cattle.

3.3. Blood smear and PCR test

The erythrocytes on blood smears test (BST) of animals infested with ticks (n = 100) were examined for presence of intracellular piroplasm/organism of the Theileria, Babesia and Anaplasma species. The results revealed incidence of theileriosis as 4% (n = 4/100), babesiosis 25% (n = 25/100) and anaplasmosis 2% (n = 2/100). The RLB-PCR test for 18s rRNA gene amplified 430bp size band as depicted in agarose gel electrophoresis (see Figure 2). The RLB-PCR showed 59% (n = 59/100) infection rate (CF n = 29/100 and BF n = 30/100) of haemoparasites. The SP-PCR for T. annulata (cytochrome b gene), B. bigemina (18s rRNA gene) and A. marginale (16s rRNA gene) produced PCR product of 475bp, 321 bp and 175 bp, respectively (see Figure 3A, 3B and 3C). The SP-PCR showed that 57% cattle (57/100; CF n = 29/53 and BF n = 28/47) showed presence of haemoparasites. The incidence of T. annulata was found 16% (n = 16/100; CF n = 10/53 and BF n = 6/47), B. bigemina at 51% (n = 51/100; CF n = 23/53 and BF n = 28/47) and A. marginale 15% (n = 15/100; CF n = 12/53 and BF n = 3/47). It is interesting to note that in RLB-PCR 15.2% (n = 9/59) positive cases were not confirmed by SP-PCR for presence of specific protozoan. Moreover, 11.9% (n = 7/59) negative cases in RLB-PCR showed presence of protozoan in SP-PCR. The statistical difference between BST and SP-PCR for T. annulata was significant (p < 0.01 and k value 0.252, 0.001). The B. bigemina infection detected by BST and SP-PCR was also statistically significant (p < 0.0001 and k value 0.485, 0.0001). As for as A. marginale was concerned significant statistical difference (p < 0.001 and k value 0.207, 0.001) was found between BST and SP-PCR. There was no significant statistical difference between RLB-PCR test and SP-PCR tests. The infected calves, heifers and adults by T. annulata (n = 16/100) detected by SP-PCR were 15.4% (2/13), 3.1% (1/32) & 23.6% (13/55), respectively, which was significant statistically (p < 0.0421). The B. bigemina (n = 51) infected 38.4% (5/13), 65.6% (21/32) & 45.5% (25/55) number of calves, heifers and adults, respectively, and statistically non-significant (p value 0.120). The calves, heifers and adult infected by A. marginale (n = 15/100) were 30.7% (4/13), 6.2% (2/32) and 16.4% (9/55) in numbers, respectively, which was also statistically non-significant (p value 0.103).

Figure 2
RLB specific primer PCR detection of DNA in cross-bred Cattle (Friesian x Sahiwal). L1 100bp ladder. L2 & L3 negative control, L4 PCR positive control. L6, L8, L9, L11, L12, L13, L15, L16, L17, L18, L19 positive for protozoan specific to primers.

Figure 3
A: PCR detection of T. annulata in cross-bred Cattle (Friesian x Sahiwal). L1 100bp ladder. L2 negative control, L3 PCR positive control. L4 & L5, positive samples. B: PCR detection of B. bigemina in cross-bred Cattle (Friesian x Sahiwal). L1 100bp ladder. L2 PCR positive control. L3, L4 & L5 positive samples. L8 negative control. C: PCR detection of A. marginale in cross-bred Cattle (Friesian x Sahiwal). L1 100bp ladder. L2 PCR negative control. L3 positive control. L5, L6, L7 & L8 positive samples.

Interestingly, single, double and triple protozoan infection were found in same cattle. The SP-PCR confirmed single infection with B. bigemina as 34% (n = 34/100) and A. marginale 6% (n = 6/100). The double infection with T. annulata/B. bigemina was 8% (n = 8/100) and B. bigemina/A. marginale 1% (n = 1/100). The triple infection with T. annulata/B. bigemina/A. marginale was noted as 8% (n = 8/100). It is very interesting to note that T. annulata was not found as a single infection. A total no of 82 protozoans were found in infected cross-bred cattle (57/100). In our study the month wise incident of three protozoans detected by SP-PCR for April was 11% (11/100), May 9% (9/100), June 10% (10/100), July 20% (20/100), August 22% (22/100) and September 10% (10/100), respectively. As for T. annulata the rate of infection was almost constant from April 3% (3/100), May 3% (3/100), June 3% (3/100), July 5% (5/100), August zero (0/16) and September 2% (2/100), respectively. The same pattern was also observed in A. marginale, having rate of infection in April 3% (3/100), May 2% (2/100), June 3% (3/100), July 2% (2/100), August 2% (2/100) and September 3% (3/100), respectively. However, in case of B. bigemina the wet months of July at 13% (13/100) and August at 20% (20/100) had highest incident percentage. The incident of protozoa for months of April was 5% (5/100), May 4% (4/100) June 4% (4/100) and September 5% (5/100), respectively. There was significant difference statistically in months and incidence of haemoparasites (p < 0.029).

3.4. PCR and genetic makeup of animals

It was noted that the cattle having higher concentration of Friesian blood and lower concentration of Sahiwal blood were much more infected and vice versa. The highest number of infected animals were of 7/8 group at 32% (n = 32/100) followed by 15/16 at 30% (n = 30/100), 31/32 at 9% (n = 9/100), 5/8 at 7% (n = 7/100), 3/4 at 3% (n = 3/100) and 1/2 1% (n = 1/100). There was significant difference amongst the various crosses of cattle and overall incident of protozoan (p < 0.0474). It was found that B. bigemina infection in blood sample (n=100) tested by SP-PCR was higher in crosses of 7/8 (18%), 15/16 (19%) and 31/32 (7%) than in 5/8 (4%), 1/2 (1%) and 3/4 (2%). The same pattern was almost found in T. annulata infected cattle i.e. 7/8 (8%), 15/16 (4%) followed by 5/8 (2%), 31/32 (1%) and 3/4 (1%) while in crosses of 1/2 no infection was detected. In case of A. marginale the crosses of 1/2 and 3/4 were free of infections and 7/8 (6%) and 15 (7%) had highest followed by 5/8 (1%) and 31/32 (1%) (see Figure 4). However, for individual protozoans no significant difference was found between various blood ratio (Friesian x Sahiwal) and infection of haemoparasites.

Figure 4
Prevalence of protozoans in various groups of cross-bred (Friesian x Sahiwal) cattle.

3.5. Phylogenetic studies

The level of nucleotide variation and phylogenetic position of partial sequence of T. annulata revealed in this study was compared with the available sequences. The phylogenetic tree showed four major clusters. The first cluster consist of our isolated sequences of cytochrome b gene (OL456214 & OL420757) grouped with isolates from Pakistan (MW354913 & MW354915), India (MZ665960 &MN89344) and Turkey (MK032846), with bootstrap value of 44%. The second cluster was formed by Chinese isolates (MG735208 & MG735209) with bootstrap value of 50%. The third and fourth cluster was formed by Iranian isolates (JQ308837) with bootstrap value of 87% and Spanish isolates (DQ287958 & DQ402154) with bootstrap value of 72%, respectively. Our isolated cytochrome b gene partial sequences were closely related to isolates from Iran and Spain and to a lesser extent to isolates from Pakistan, India, Turkey and China (see Figure 5).

Figure 5
Phylogenetic analysis of T. annulata based on Cytochrome b gene.

The phylogenetic tree analysis for B. bigemina showed that our isolated sequence (OL376658) are most homologous (94-100%) to isolates from Cuba. The isolates from Egypt (MH796638, MH796639 & MH796640) have 88% homology with our sequence. Likewise, current isolates from USA (MH047819, MH050356, MH050357, MH050358, & MH050387 are closer (74 to 84% similarity) to our isolates as compared to isolates from South Africa (MH257710 - 23), which has a homology value of 34 to 73%. Hence, the partial isolated sequence (18s rRNA) in this study has higher homology value to isolates from Cuba, followed by isolates from USA and South Africa, respectively (see Figure 6).

Figure 6
Phylogenetic analysis of B. bigemina based on 18s rRNA gene. Scale bar set at 0.05.

The phylogenetic analysis of partial sequence of 16s rRNA gene of A. marginale isolated in our study revealed three major clusters. The first cluster consists of our sequence (OL407062) grouped with isolates from Pakistan (MK680804, MK680805, MK680806 & MK680807) with a high bootstrap value of 99% homology. The second cluster with high bootstrap value of 98% homology was isolate from USA (DQ000617). The third cluster from China (OM065781), KSA (AB916498) and South Africa (AF414873) showed moderate homology of 62% bootstrap value. All our isolates showed 99 to 98% similarity with isolates from Pakistan and USA and lesser homology to isolates from China, KSA and South Africa (see Figure 7).

Figure 7
Phylogenetic analysis of A. marginale based on 16s rRNA gene.

4. Discussion

TTBD infect cattle in wide ecological and geographical regions of Pakistan (Karim et al., 2017; Atif et al., 2022). The exotic livestock breed is highly susceptible to tick and tick-borne diseases even kept in very controlled environment (Young et al., 1988). In the present study it was found that ticks infested 3.92% of total cattle herd. Previous studies have reported that about 28.2% (Rasul and Akhtar, 1975) and 47.9% (Ghafar et al., 2020b) cattle can be infested with ticks. The lower tick infestation in present study may be attributed to (i) better management practices, (ii) use of acaricides in both farms and (iii) organized and commercial farming.

Moreover, the prevalence of ticks in various age group of exotic and crossbred cattle is highly debatable and scientists have reported wide range of incidence rates depending on ecological regions, quality of farming practices and control measures. In this study we found 5.3% (13/245) of calves, 3.9% (55/1400) of adults and 3.9% (32/820) of heifers infested with ticks, which is much lower as reported by other researcher (Ahmed et al., 2012) who reported 85% cross-bred calves and 73.2% cross-bred adult infested with R. microplus. In another study highest infestation in young calves (47.99%), growing (41.19%), heifer (39.82%) and adult cattle (35.35%) were found (Das and Parasit, 1994). In a recent study it was reported that incident of ticks is higher in calves (i.e.</=1year of age) (55%) than in young animals (i.e. up to 3 years of age) (39%) and adults (48%) (Ghafar et al., 2020b). The reason for low tick infestation in adult animals may be attributed to acquired immunity due to repeated exposure (Tabor et al., 2017) and more attendance to adult cattle as well as in comparison less care to calves (Singh and Rath, 2013; Burrow et al., 2019).

The infestation rate of ticks increases with the progression of summer season. In present study the infestation was highest in wet and hotter, while lower infestation was noted in beginning of summer season and end of summer in September. This is in line with other researchers (Castro and Newman, 2003; Asmaa et al., 2014; Ali et al., 2019) who reported the same pattern of tick infestation.

In our knowledge it is the first study that has established the correlation between tick infestation and various genetic/blood ratio of cross-bred cattle (Friesian x Sahiwal). The cattle having higher concentration of Friesian blood and lower concentration of Sahiwal were among the most infested. Conversely, the animals having lower concentration of Friesian blood and higher concentration of Sahiwal blood were less infested. In a study from Pakistan Rehman et al. (2017) has reported that intensity of tick infestation is significantly lower in indigenous animals compared to exotic and cross-bred cows. In another study it has been reported that tick infestation is higher in crossbred cattle (72%) as compared to their pure breed (61%) (Ahmed et al., 2012). The cross-bred cattle with higher exotic blood and lower indigenous blood makes the animal prone to tick infestation and vice versa, which is proof of resistance/immunity of indigenous cattle towards tick infestation even in cross-bred animals.

Carrier animals, infected with protozoans, mostly asymptomatic, are an important source of infection in cattle farms. The diagnosis of these animals is very difficult through conventional methods; hence use of more sensitive and specific tests is imperative to avoid spread of disease, control of mortality and minimize economic losses (Altay et al., 2008). It has been reported by Irvin (1987) that in case of mixed infection with Theileria and Babesia, confusion may arise to differentiate these species solely on the basis of the morphology, developmental stages, hence under field conditions it is imperative to reach on exact diagnosis.

PCR test is used in detection of clinical and subclinical cases of Theileria, Babesia and Anaplasma (El-Ashker et al., 2015). In the study we carried out, the BST revealed lower incidence of haemoparasites. The RLB-PCR detected higher infection rate than BST. The SP-PCR was also more sensitive and specific in detection of haemoparasites. The BST and PCR were used by Durrani and Kamal (2008) in Pakistan for detection of Theileria and Babesia species in field samples, and concluded that PCR is much more sensitive and specific than BST. The same result has been reported by Nourollahi-Fard et al. (2015), in which BST detected much lower infection of T. annulata than PCR.

The RLB test, using oligonucleotide probes can be used to detect low level of parasitaemia to simultaneously identify Theileria and Babesia species, and is very effective and practical tool. In present study, instead of hybridization on probe, the RLB primer was used initially to amplify the DNA fragments in blood samples of infested cattle, and demonstrated on agarose gel, subsequently. The RLB-PCR positive samples can be then subjected to SP-PCR for exact diagnosis of protozoan species. Many researchers around the world have used RLB assay for diagnosis of Theileria and Babesia species. (Gubbels et al., 1999; Sparagano et al., 2000; García-Sanmartín et al., 2006; Iqbal et al., 2013). It is interesting to note that in present study various positive cases in RLB-PCR were not confirmed by SP-PCR for presence of specific protozoan. Moreover, few negative cases in RLB-PCR showed presence of protozoan in SP-PCR. It can be hypothesized that RLB-PCR may have detected DNA samples of some novel protozoan species, for which the specific primer was not used in SP-PCR in our study. This opens up a new research avenue to optimize the RLB-PCR to detect a larger number of protozoans, simultaneously. The negative samples of RLB-PCR, which were revealed positive in SP-PCR points out for need of further optimization of procedures, chemicals, equipment as well as minimize human errors.

In present study the overall incident of T. annulata in tested blood samples by SP-PCR (Cytochrome b gene) was 16%, which was highest in adults 23.6% (13/55), whereas in calves 15.4% (2/13) and heifers 3.1% (1/32), respectively. The infection rate in both farms were almost the same. It is in agreement with the study of Yamchi and Tavassoli (2016) who reported 15.94% tested samples positive for T. annulata in cattle. It has also been reported in a study that infection of B. bigemina is higher among the age groups of 2-7 years in cross-bred animals (Velusamy et al., 2014). In a study carried out in Khyber-Pakhtunkhwa, Pakistan, Ullah et al. (2021) reported that 23.7% of cattle were found infected with T. annulata, however, theileriosis was found to be higher in young animals as compared to adults cross-bred cattle. Another research from Pakistan reported over all T. annulata prevalence of 21%, with cross-bred cattle most susceptible (28%) followed by Sahiwal breed (19%) (Parveen et al., 2021). The lower incident rate of infection in our study is in line with Calleja-Bueno et al. (2017) who reported that incident of T. annulata was lower in farms, which are being given anti-protozoal drugs at least once a year. However, the reported incident of T. annulata in various studies has remarkable differences, which may be as high as 74% (Amiri et al., 2021) or 46.2% (Zeb et al., 2020) or as low as 8% (Rashid et al., 2018). The differences in infection rate may be due to study design, diseased vs healthy vs carrier animals, geographical location, age/numbers of cattle at farm, management, use of anti-protozoal drugs, season and genetic makeup of cattle. It has been mentioned earlier in our study that only ticks of R. microplus was found on infested animals. The absence of Hyalomma ticks raises questions about detection of T. annulata by BST and SP-PCR in understudied cattle. On scrutiny of farms animal data, it was revealed that these animals were shifted from other farms as well as purchased from local market to diversify the gene pool to increase milk production. It is assumed that these cattle were the carrier of T. annulata at time of transfer and/or purchase.

Carrier cattle also infected with Babesia are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for diagnosis and epidemiological studies (Fahrimal et al., 1992). For PCR the organism-specific primers derived from18S rRNA gene of B. bigemina can be used (Laha et al., 2015). In our study the SP-PCR showed overall infection of B. bigemina at 51% of tested blood of apparently healthy animals infested with ticks of R. microplus. The highest infection was 65.6% in heifers, followed by adults at 45.5% and calves at 38.4%. Our findings are in line with Ganzinelli et al. (2020) who reported high infection rate of 54.7% by B. bigemina in purebred cattle using nPCR. These findings are also consistent with Oliveira-Sequeira et al. (2005) who found that B. bigemina was present in 92.6% of calves and in 84% of cows. The presence of ticks on calves and cattle is directly correlated with high infection rates of B. bigemina, which give substance to our findings. However, in contrast the overall positive rates of B. bigemina was also reported as low as 23.6% in cattle (Otgonsuren et al., 2020). Chaudhry et al. (2010) from Pakistan reported 18% positive B. bigemina cases in apparently healthy cattle in a farm that were posing threat for the healthy herd population. The difference in infection rate in various studies may be attributed towards different geographical location, animal management and genetic makeup, control measures and use of anti-protozoal drugs.

In our study the overall incidence of A. marginale on tested blood samples by SP-PCR was 15%. The calves showed higher infection rate of 30.7% followed by adults at 16.4% and heifers 6.2%. It is in agreement with study of Zafar et al. (2022) who reported prevalence of A. marginale from 9-17% in tested blood samples from two southern districts (Lodhran and Dera Ghazi Khan) of Punjab, Pakistan. Our findings are also in agreement with results of Atif et al. (2022) who reported the highest prevalence of A. marginale in cattle of less than 1 year old (32.84%) while the lowest prevalence (6.45%) was in animals aged between 1 and 2 years of age. In another study from Khyber-Pakhtunkhwa, Pakistan showed that on PCR (16S rRNA gene) A. marginale is found to be 16.3% prevalent in cattle of various areas; with breed and acaricidal treatment as significant determinants (Zeb et al., 2020). The sampling sites, vector species, breeds and breeding system as well as the climatic conditions effect the prevalence of A. marginale (Bursakov and Kovalchuk, 2019). As reported in various studies, the prevalence of A. marginale has shown to be vary from 3-41% in Pakistan (Afridi and Ahmad, 2005; Farooqi et al., 2018; Turi et al., 2018; Ashraf et al., 2021; Atif et al., 2022). A. marginale has also been reported from various countries of the world including India (18.48- 45.2%) (Singh et al., 2012; Kumar et al., 2019), Egypt (20%), (Selim et al., 2021), Sri Lanka (32.7%) (Zhyldyz et al., 2019), Iran (38.6%) (Noaman and Shayan, 2010), Algeria (39.4%) (Rjeibi et al., 2018), Brazil (57.5%) (Garcia et al., 2022) and USA (82%) (Hairgrove et al., 2015).

In our study single, double and triple protozoan infection were found in tick infested cattle. The co-infection of protozoans in cattle is not very uncommon, and has been reported in Pakistan as well as by researchers all around the globe. In a study carried out by Atif et al. (2022), based on duplex PCR, the overall prevalence of the two concurrent tick-borne pathogens T. annulata and A. marginale was found to be 19.79%. In another study in Peshawar, Pakistan, out of 68 positive cases, 12 samples (4.21%) were harbouring single infection. Remaining 26 blood samples showed mixed infection of A. marginale with A. centrale 4.21%, B.bovis with A.centrale 3.50%, T. annulata with A. marginale 0.70% and T. parva with A. marginale was recorded in 0.70% cases, respectively (Afridi and Ahmad, 2005). A researcher from Kenya reported that more than 50% of the positive samples were infected with at least two haemoparasites, which generally belonged to different genus, and 29 different types of mixed infections were noted and some cattle concurrently harbouring up to five pathogens (Adjou Moumouni et al., 2015). The researchers from all around the globe reported co-infection with two/three protozoans ranging from 10.46% (Ganguly et al., 2020), 15.1% (Zhou et al., 2016), 17.9% (Jirapattharasate et al., 2017), 19% (Bursakov and Kovalchuk, 2019) and 20.0% (El-Dakhly et al., 2020) infection percentage.

In our study the wet months of summer showed highest percentage of protozoan infection. Our observations are supported by Atif et al. (2022) who reported that incidence of all ticks and tick-borne pathogens are higher in summer, followed by spring, autumn, and winter. The Okafor et al. (2018) summed up that summer/hotter months has higher incident of A. marginale than spring/winter/colder months of the year. In a study Ashraf et al. (2021) reported highest prevalence of A. marginale was observed during autumn (18.3%) followed by summer (9.7%) and winter season (7.1%). Our findings are also in agreement with Jaimes-Duenez et al. (2018) who reported higher values of infection of Babesiosis during the wet season and late wet season. Siddique et al. (2020) found high incidence in summer (23.41%) followed by autumn (20.47%), spring (17.77%) and winter (7.29%), respectively.

To best of our knowledge, this research is the only study which reported that cross-bred cattle (Friesian x Sahiwal) having various genetic/blood concentration, contributed from sire (Friesian) and dam (Sahiwal), has different level of vulnerability to heamoparasites. It was found that cross-bred cattle having higher concentration of Friesian blood and lower concentration of Sahiwal blood are generally more susceptible to infections of haemoparasites and vice versa. It is very interesting finding and in line with many researcher who have reported that imported breed and their cross-breds are much more vulnerable to protozoan infections than local/indigenous breeds (Fivaz et al., 1992; Ashraf et al., 2021; Atif et al., 2022). In Pakistan influx of imported animals has increased drastically in last few decades, and these are bred with local cattle to get their cross-bred offspring for better genetic potential and disease resistance. However, the challenges of adaptability and disease proneness of these imported animals and their cross-bred offspring were always a point of concern in farming sector. There are studies which generally suggest that indigenous/cross-bred are better to purebred cattle in resistance to tick/protozoan infection (Asmaa et al., 2014; Rehman et al., 2017) and protozoan infection (Siddique et al., 2020). With confidence it can be said that no study has been carried out in Pakistan on suitability of various genetic makeup of cross-bred (Friesian x Sahiwal) cattle in local environment. Our study presents the first evidence/glimpse that cross-bred animals of various genetic/blood makeup vary in their immunity level towards TTBD. On basis of this research the farmers may be suggested to preserve the blood level of cross-bred cattle (Friesian x Sahiwal) between ratio of 1/2 (50:50) to 3/4 (75:25) to get maximum milk production capacity of exotic genes as well as benefit from better potential/resistance against TTBD of indigenous blood. Further research, basing on wide geographical region and data, is required to be carried out to reach on conclusive outcome basing on concrete scientific evidence.

To confirm the results of PCR, sequencing of isolates of T. annulate, B. bigemina and A. marginale was performed. The level of nucleotide variation and phylogenetic position of partial sequence of T. annulata revealed that our isolated sequences (cytochrome b gene) are closely related to isolates from Iran and Spain and to a lesser extent to isolates from China and least similar to Pakistan, India and Turkey. In case of B. bigemina our partial isolated sequence (16s RNA gene) in this study has higher homology to isolates from Cuba followed by USA and lesser homology to South Africa, respectively. The phylogenetic analysis of partial sequence (16s rRNA gene) of A. marginale isolated in current study showed higher similarity with isolates from Pakistan and USA and lesser homology to isolates from China, KSA and South Africa, respectively. The phylogenetic studies by various scientists from Pakistan has reported various homology level of their isolates with partial sequences of other isolates all around the world (Ghafar et al., 2020a; Atif et al., 2022). Further studies are required to map out the complete epidemiological profiles of these haemoparasites in local population of cross-bred cattle in various demographical regions of Pakistan.

5. Conclusion

Ticks of genus R. microplus infect the cross-bred cattle (Friesian x Sahiwal) of all ages, and most active during the hotter and humid season of the year. The crosses of cattle possessing higher genetic level of foreign breed are most susceptible to ticks infestation and vice versa. The PCR test was highly sensitive and specific as compared to conventional blood smear test for diagnosis of haemoparasites. RLB-PCR is a reliable and rapid test for screening of cattle suffering from blood parasites. Amongst the major haemoparasites transmitted by ticks the B. bigemina was most prevalent followed by T. annulata and A. marginale. The haemoparasites infected all animal age groups, prevalent in hot and humid weather and diagnosed mostly in crosses of cross-bred cattle with higher level of foreign blood. Single and double and triple infection of haemoparasites were also noted in animals.

References

• ADJOU MOUMOUNI, P.F., ABOGE, G.O., TERKAWI, M.A., MASATANI, T., CAO, S., KAMYINGKIRD, K., JIRAPATTHARASATE, C., ZHOU, M., WANG, G., LIU, M., IGUCHI, A., VUDRIKO, P., YBANEZ, A.P., INOKUMA, H., SHIRAFUJI-UMEMIYA, R., SUZUKI, H. and XUAN, X., 2015. Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya. Parasites & Vectors, vol. 8, no. 1, pp. 496. http://dx.doi.org/10.1186/s13071-015-1106-9 PMid:26420543.
  » http://dx.doi.org/10.1186/s13071-015-1106-9
• AFRIDI, Z.K. and AHMAD, I., 2005. Incidence of anaplasmosis, babesiosis and theileriosis in dairy cattle in Peshawar, Pakistan. Pakistan: Sarhad Journal of Agriculture.
• AHMED, S., NUMAN, M., MANZOOR, A.W. and ALI, F.A., 2012. Investigations into Ixodidae ticks in cattle in Lahore, Pakistan. Veterinaria Italiana, vol. 48, no. 2, pp. 185-191. PMid:22718335.
• ALI, A., KHAN, M.A., ZAHID, H., YASEEN, P.M., QAYASH KHAN, M., NAWAB, J., UR REHMAN, Z., ATEEQ, M., KHAN, S. and IBRAHIM, M., 2019. Seasonal dynamics, record of ticks infesting humans, wild and domestic animals and molecular phylogeny of Rhipicephalus microplus in Khyber Pakhtunkhwa Pakistan. Frontiers in Physiology, vol. 10, pp. 793. http://dx.doi.org/10.3389/fphys.2019.00793 PMid:31379587.
  » http://dx.doi.org/10.3389/fphys.2019.00793
• ALTAY, K., AYDIN, M.F., DUMANLI, N. and AKTAS, M., 2008. Molecular detection of Theileria and Babesia infections in cattle. Veterinary Parasitology, vol. 158, no. 4, pp. 295-301. http://dx.doi.org/10.1016/j.vetpar.2008.09.025 PMid:19008048.
  » http://dx.doi.org/10.1016/j.vetpar.2008.09.025
• AMIRI, M.S., YAGHFOORI, S. and RAZMI, G., 2021. Molecular Detection of Theileria annulata among dairy cattle and vector ticks in the Herat Area, Afghanistan. Archives of Razi Institute, vol. 76, no. 1, pp. 79-85. PMid:33818960.
• ASHFAQ, M. and RAZZAK, W., 2000. Prevalence of Theileria annulata infection in cross bred cattle in Faisalabad. Pakistan Veterinary Journal, vol. 57, pp. 131-136.
• ASHRAF, S., PARVEEN, A., MUHAMMAD AWAIS, M., GILLANI, Q., AKTAS, M., OZUBEK, S. and IQBAL, F., 2021. A Report on Molecular Detection and Phylogenetic Evaluation of Anaplasma marginale in ticks and blood samples collected from cattle in District Layyah in Punjab (Pakistan). Current Microbiology, vol. 78, no. 1, pp. 274-281. http://dx.doi.org/10.1007/s00284-020-02256-0 PMid:33125524.
  » http://dx.doi.org/10.1007/s00284-020-02256-0
• ASMAA, N.M., ELBABLY, M.A. and SHOKIER, K.A., 2014. Studies on prevalence, risk indicators and control options for tick infestation in ruminants. Beni-Suef University Journal of Basic and Applied Sciences, vol. 3, no. 1, pp. 68-73. http://dx.doi.org/10.1016/j.bjbas.2014.02.009
  » http://dx.doi.org/10.1016/j.bjbas.2014.02.009
• ATIF, F.A., ABBAS, R.Z., MEHNAZ, S., QAMAR, M.F., HUSSAIN, K., NAZIR, M.U., ZAMAN, M.A., KHAN, A.U. and SAID, M.B., 2022. First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab, Pakistan. Tropical Animal Health and Production, vol. 54, no. 2, pp. 155. http://dx.doi.org/10.1007/s11250-022-03158-y PMid:35362760.
  » http://dx.doi.org/10.1007/s11250-022-03158-y
• BILGIC, H.B., KARAGENÇ, T., SHIELS, B., TAIT, A., EREN, H. and WEIR, W., 2010. Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals. Veterinary Parasitology, vol. 174, no. 3-4, pp. 341-347. http://dx.doi.org/10.1016/j.vetpar.2010.08.025 PMid:20880635.
  » http://dx.doi.org/10.1016/j.vetpar.2010.08.025
• BURROW, H.M., MANS, B.J., CARDOSO, F.F., BIRKETT, M.A., KOTZE, A.C., HAYES, B.J., MAPHOLI, N., DZAMA, K., MARUFU, M.C., GITHAKA, N.W. and DJIKENG, A., 2019. Towards a new phenotype for tick resistance in beef and dairy cattle: a review. Animal Production Science, vol. 59, no. 8, pp. 1401-1427. http://dx.doi.org/10.1071/AN18487
  » http://dx.doi.org/10.1071/AN18487
• BURSAKOV, S.A. and KOVALCHUK, S.N., 2019. Co-infection with tick-borne disease agents in cattle in Russia. Ticks and Tick-Borne Diseases, vol. 10, no. 3, pp. 709-713. http://dx.doi.org/10.1016/j.ttbdis.2019.03.004 PMid:30878569.
  » http://dx.doi.org/10.1016/j.ttbdis.2019.03.004
• CALLEJA-BUENO, L., SAINZ, A., GARCIA-SANCHO, M., RODRIGUEZ-FRANCO, F., GONZALEZ-MARTIN, J.V. and VILLAESCUSA, A., 2017. Molecular, epidemiological, haematological and biochemical evaluation in asymptomatic Theileria annulata infected cattle from an endemic region in Spain. Ticks and Tick-Borne Diseases, vol. 8, no. 6, pp. 936-941. http://dx.doi.org/10.1016/j.ttbdis.2017.08.006 PMid:28887101.
  » http://dx.doi.org/10.1016/j.ttbdis.2017.08.006
• CASTRO, A. and NEWMAN, R.M., 2003. The benefit-cost analysis of disease control programs. In: P. Lessard and B.D. Perry, eds. Investigation of disease outbreaks and impaired productivity Philadelphia: W.B. Saunders.
• CHAUDHRY, Z.I., SULEMAN, M., YOUNUS, M. and ASLIM, A., 2010. Molecular detection of Babesia bigemina and Babesia bovis in crossbred carrier cattle through PCR. Pakistan Journal of Zoology, vol. 42, no. 2
• DARGHOUTH, M.E., BOUATTOUR, A., BEN MILED, L. and SASSI, L., 1996. Diagnosis of Theileria annulata infection of cattle in Tunisia: comparison of serology and blood smears. Veterinary Research, vol. 27, no. 6, pp. 613-621. PMid:8956476.
• DAS, S.S. and PARASIT, J., 1994. Prevalence of ixodid tick infestation on farm animals in Pantnagar, Tarai of Uttar Pradesh. Appl Anim Biol, vol. 3, no. 1, pp. 31-73.
• DURRANI, A.Z. and KAMAL, N., 2008. Identification of ticks and detection of blood protozoa in Friesian cattle by polymerase chain reaction test and estimation of blood parameters in district Kasur, Pakistan. Tropical Animal Health and Production, vol. 40, no. 6, pp. 441-447. http://dx.doi.org/10.1007/s11250-007-9117-y PMid:18575972.
  » http://dx.doi.org/10.1007/s11250-007-9117-y
• EL-ASHKER, M., HOTZEL, H., GWIDA, M., EL-BESKAWY, M., SILAGHI, C. and TOMASO, H., 2015. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray. Veterinary Parasitology, vol. 207, no. 3-4, pp. 329-334. http://dx.doi.org/10.1016/j.vetpar.2014.12.025 PMid:25591406.
  » http://dx.doi.org/10.1016/j.vetpar.2014.12.025
• EL-DAKHLY, K.M., ARAFA, W.M., SOLIMAN, S., ABDEL-FATAH, O.R., WAHBA, A.A., ESTEVE-GASENT, M.D. and HOLMAN, P.J., 2020. Molecular detection, phylogenetic analysis, and genetic diversity of Theileria annulata, Babesia bigemina, and Anaplasma marginale in Cattle in Three Districts of Egypt. Acta Parasitologica, vol. 65, no. 3, pp. 620-627. http://dx.doi.org/10.2478/s11686-020-00189-z PMid:32207056.
  » http://dx.doi.org/10.2478/s11686-020-00189-z
• ESTRADA-PEÑA, A., BOUATTOUR, A., CAMICAS, J.L. and WALKER, A.R., 2004. Ticks of domestic animals in the Mediterranean region Spain: University of Zaragoza.
• FAHRIMAL, Y., GOFF, W.L. and JASMER, D.P., 1992. Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA. Journal of Clinical Microbiology, vol. 30, no. 6, pp. 1374-1379. http://dx.doi.org/10.1128/jcm.30.6.1374-1379.1992 PMid:1624551.
  » http://dx.doi.org/10.1128/jcm.30.6.1374-1379.1992
• FAROOQI, S.H., IJAZ, M., RASHID, M.I., NABI, H., ISLAM, S., AQIB, A.I., HUSSAIN, K., KHAN, A., RIZVI, S.N.B., MAHMOOD, S., MEHMOOD, K. and ZHANG, H., 2018. Molecular epidemiology of bovine Anaplasmosis in Khyber Pakhtunkhwa, Pakistan. Tropical Animal Health and Production, vol. 50, no. 7, pp. 1591-1598. http://dx.doi.org/10.1007/s11250-018-1599-2 PMid:29740781.
  » http://dx.doi.org/10.1007/s11250-018-1599-2
• FELSENSTEIN, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution; International Journal of Organic Evolution, vol. 39, no. 4, pp. 783-791. http://dx.doi.org/10.1111/j.1558-5646.1985.tb00420.x PMid:28561359.
  » http://dx.doi.org/10.1111/j.1558-5646.1985.tb00420.x
• FIVAZ, B.H., DE WAAL, D.T. and LANDER, K., 1992. Indigenous and crossbred cattle--a comparison of resistance to ticks and implications for their strategic control in Zimbabwe. Tropical Animal Health and Production, vol. 24, no. 2, pp. 81-89. http://dx.doi.org/10.1007/BF02356949 PMid:1305338.
  » http://dx.doi.org/10.1007/BF02356949
• GALAY, R.L., LLANETA, C.R., MONREAL, M., ARMERO III, A.L., BALUYUT, A.B.D., REGINO, C.M.F., SANDALO, K.A.C., DIVINA, B.P., TALACTAC, M.R., TAPAWAN, L.P., MOJARES, M.C.L., ALVAREZ, C.R., MAGO, E.R., ENCARNACION, N.D., ANDOH, M. and TANAKA, T., 2021. Molecular Prevalence of Anaplasma marginale and Ehrlichia in domestic large ruminants and Rhipicephalus (Boophilus) microplus ticks from Southern Luzon, Philippines. Frontiers in Veterinary Science, vol. 8, pp. 746705. http://dx.doi.org/10.3389/fvets.2021.746705 PMid:34722706.
  » http://dx.doi.org/10.3389/fvets.2021.746705
• GANGULY, A., MAHARANA, B.R. and GANGULY, I., 2020. Pentaplex PCR assay for rapid differential detection of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi in cattle. Biologicals, vol. 63, pp. 81-88. http://dx.doi.org/10.1016/j.biologicals.2019.10.011 PMid:31708375.
  » http://dx.doi.org/10.1016/j.biologicals.2019.10.011
• GANZINELLI, S., BENITEZ, D., GANTUYA, S., GUSWANTO, A., FLORIN-CHRISTENSEN, M., SCHNITTGER, L. and IGARASHI, I., 2020. Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina. Transboundary and Emerging Diseases, vol. 67, no. S2, pp. 159-164. http://dx.doi.org/10.1111/tbed.13435 PMid:31880063.
  » http://dx.doi.org/10.1111/tbed.13435
• GARCIA, A.B., JUSI, M.M.G., FRESCHI, C.R., RAMOS, I.A.S., MENDES, N.S., BRESSIANINI DO AMARAL, R., GONCALVES, L.R., ANDRE, M.R. and MACHADO, R.Z., 2022. High genetic diversity and superinfection by Anaplasma marginale strains in naturally infected Angus beef cattle during a clinical Anaplasmosis outbreak in southeastern Brazil. Ticks and Tick-Borne Diseases, vol. 13, no. 1, pp. 101829. http://dx.doi.org/10.1016/j.ttbdis.2021.101829 PMid:34798528.
  » http://dx.doi.org/10.1016/j.ttbdis.2021.101829
• GARCÍA-SANMARTÍN, J., NAGORE, D., GARCÍA-PÉREZ, A.L., JUSTE, R.A. and HURTADO, A., 2006. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays. BMC Veterinary Research, vol. 2, no. 1, pp. 16. http://dx.doi.org/10.1186/1746-6148-2-16 PMid:16684356.
  » http://dx.doi.org/10.1186/1746-6148-2-16
• GHAFAR, A., CABEZAS-CRUZ, A., GALON, C., OBREGON, D., GASSER, R.B., MOUTAILLER, S. and JABBAR, A., 2020a. Bovine ticks harbour a diverse array of microorganisms in Pakistan. Parasites & Vectors, vol. 13, no. 1, pp. 1. http://dx.doi.org/10.1186/s13071-019-3862-4 PMid:31900233.
  » http://dx.doi.org/10.1186/s13071-019-3862-4
• GHAFAR, A., GASSER, R.B., RASHID, I., GHAFOOR, A. and JABBAR, A., 2020b. Exploring the prevalence and diversity of bovine ticks in five agro-ecological zones of Pakistan using phenetic and genetic tools. Ticks and Tick-Borne Diseases, vol. 11, no. 5, pp. 101472. http://dx.doi.org/10.1016/j.ttbdis.2020.101472 PMid:32723634.
  » http://dx.doi.org/10.1016/j.ttbdis.2020.101472
• GUBBELS, J.M., DE VOS, A., VAN DER WEIDE, M., VISERAS, J., SCHOULS, L., DE VRIES, E. and JONGEJAN, F., 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. Journal of Clinical Microbiology, vol. 37, no. 6, pp. 1782-1789. http://dx.doi.org/10.1128/JCM.37.6.1782-1789.1999 PMid:10325324.
  » http://dx.doi.org/10.1128/JCM.37.6.1782-1789.1999
• HAIRGROVE, T., SCHROEDER, M.E., BUDKE, C.M., RODGERS, S., CHUNG, C., UETI, M.W. and BOUNPHENG, M.A., 2015. Molecular and serological in-herd prevalence of Anaplasma marginale infection in Texas cattle. Preventive Veterinary Medicine, vol. 119, no. 1-2, pp. 1-9. http://dx.doi.org/10.1016/j.prevetmed.2015.02.006 PMid:25732914.
  » http://dx.doi.org/10.1016/j.prevetmed.2015.02.006
• IQBAL, F., KHATTAK, R., OZUBEK, S., KHATTAK, M., RASUL, A. and AKTAS, M., 2013. Application of the reverse line blot assay for the molecular detection of Theileria and Babesia sp. in sheep and goat blood samples from Pakistan. Iranian Journal of Parasitology, vol. 8, no. 2, pp. 289-295. PMid:23914243.
• IRVIN, A.D. 1987. Characterization of species and strains of Theileria. In: J. R. Baker and R. Muller, eds, Advances in Parasitology London: Academic Press, vol. 26, pp. 145-197. http://dx.doi.org/10.1016/S0065-308X(08)60296-1
  » http://dx.doi.org/10.1016/S0065-308X(08)60296-1
• JAIMES-DUEÑEZ, J., TRIANA-CHAVEZ, O., HOLGUIN-ROCHA, A., TOBON-CASTANO, A. and MEJIA-JARAMILLO, A.M., 2018. Molecular surveillance and phylogenetic traits of Babesia bigemina and Babesia bovis in cattle (Bos taurus) and water buffaloes (Bubalus bubalis) from Colombia. Parasites & Vectors, vol. 11, no. 1, pp. 510. http://dx.doi.org/10.1186/s13071-018-3091-2 PMid:30208941.
  » http://dx.doi.org/10.1186/s13071-018-3091-2
• JIRAPATTHARASATE, C., ADJOU MOUMOUNI, P.F., CAO, S., IGUCHI, A., LIU, M., WANG, G., ZHOU, M., VUDRIKO, P., EFSTRATIOU, A., CHANGBUNJONG, T., SUNGPRADIT, S., RATANAKORN, P., MOONARMART, W., SEDWISAI, P., WELUWANARAK, T., WONGSAWANG, W., SUZUKI, H. and XUAN, X., 2017. Molecular detection and genetic diversity of bovine Babesia spp., Theileria orientalis, and Anaplasma marginale in beef cattle in Thailand. Parasitology Research, vol. 116, no. 2, pp. 751-762. http://dx.doi.org/10.1007/s00436-016-5345-2 PMid:28028631.
  » http://dx.doi.org/10.1007/s00436-016-5345-2
• JURKOVIĆ, D., MIHALJEVIC, Z., DUVNJAK, S., SILAGHI, C. and BECK, R., 2020. First reports of indigenous lethal infection with Anaplasma marginale, Anaplasma bovis and Theileria orientalis in Croatian cattle. Ticks and Tick-Borne Diseases, vol. 11, no. 5, pp. 101469. http://dx.doi.org/10.1016/j.ttbdis.2020.101469 PMid:32723641.
  » http://dx.doi.org/10.1016/j.ttbdis.2020.101469
• KARIM, S., BUDACHETRI, K., MUKHERJEE, N., WILLIAMS, J., KAUSAR, A., HASSAN, M.J., ADAMSON, S., DOWD, S.E., APANSKEVICH, D., ARIJO, A., SINDHU, Z.U., KAKAR, M.A., KHAN, R.M.D., ULLAH, S., SAJID, M.S., ALI, A. and IQBAL, Z., 2017. A study of ticks and tick-borne livestock pathogens in Pakistan. PLoS Neglected Tropical Diseases, vol. 11, no. 6, pp. e0005681. http://dx.doi.org/10.1371/journal.pntd.0005681 PMid:28650978.
  » http://dx.doi.org/10.1371/journal.pntd.0005681
• KIMURA, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, vol. 16, no. 2, pp. 111-120. http://dx.doi.org/10.1007/BF01731581 PMid:7463489.
  » http://dx.doi.org/10.1007/BF01731581
• KUMAR, N., SOLANKI, J.B., VARGHESE, A., JADAV, M.M., DAS, B., PATEL, M.D. and PATEL, D.C., 2019. Molecular assessment of Anaplasma marginale in Bovine and Rhipicephalus (Boophilus) microplus tick of endemic tribal belt of Coastal South Gujarat, India. Acta Parasitologica, vol. 64, no. 4, pp. 700-709. http://dx.doi.org/10.2478/s11686-019-00041-z PMid:30915720.
  » http://dx.doi.org/10.2478/s11686-019-00041-z
• KUMAR, S., STECHER, G., LI, M., KNYAZ, C. and TAMURA, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, vol. 35, no. 6, pp. 1547-1549. http://dx.doi.org/10.1093/molbev/msy096 PMid:29722887.
  » http://dx.doi.org/10.1093/molbev/msy096
• LAHA, R., MONDAL, B., BISWAS, S.K., CHAND, K., DAS, M., SARMA, D., GOSWAMI, A. and SEN, A., 2015. Detection of Babesia bigemina infection in cattle from north-eastern India by polymerase chain reaction and its genetic relatedness with other isolates. Tropical Animal Health and Production, vol. 47, no. 3, pp. 633-636. http://dx.doi.org/10.1007/s11250-015-0769-8 PMid:25663024.
  » http://dx.doi.org/10.1007/s11250-015-0769-8
• MAKENOV, M.T., TOURE, A.H., KORNEEV, M.G., SACKO, N., PORSHAKOV, A.M., YAKOVLEV, S.A., RADYUK, E.V., ZAKHAROV, K.S., SHIPOVALOV, A.V., BOUMBALY, S., ZHURENKOVA, O.B., GRIGOREVA, Y.E., MOROZKIN, E.S., FYODOROVA, M.V., BOIRO, M.Y. and KARAN, L.S., 2021. Rhipicephalus microplus and its vector-borne haemoparasites in Guinea: further species expansion in West Africa. Parasitology Research, vol. 120, no. 5, pp. 1563-1570. http://dx.doi.org/10.1007/s00436-021-07122-x PMid:33788020.
  » http://dx.doi.org/10.1007/s00436-021-07122-x
• MORETTI, A., MANGILI, V., SALVATORI, R., MARESCA, C., SCOCCIA, E., TORINA, A., MORETTA, I., GABRIELLI, S., TAMPIERI, M.P. and PIETROBELLI, M., 2010. Prevalence and diagnosis of Babesia and Theileria infections in horses in Italy: a preliminary study. Veterinary Journal (London, England), vol. 184, no. 3, pp. 346-350. http://dx.doi.org/10.1016/j.tvjl.2009.03.021 PMid:19394253.
  » http://dx.doi.org/10.1016/j.tvjl.2009.03.021
• NOAMAN, V. and SHAYAN, P., 2010. Comparison of microscopy and PCR-RFLP for detection of Anaplasma marginale in carrier cattle. Iranian Journal of Microbiology, vol. 2, no. 2, pp. 89-94. PMid:22347555.
• NOUROLLAHI-FARD, S.R., KHALILI, M. and GHALEKHANI, N., 2015. Detection of Theileria annulata in blood samples of native cattle by PCR and smear method in Southeast of Iran. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, vol. 39, no. 2, pp. 249-252. http://dx.doi.org/10.1007/s12639-013-0333-2 PMid:26064010.
  » http://dx.doi.org/10.1007/s12639-013-0333-2
• OKAFOR, C.C., COLLINS, S.L., DANIEL, J.A., HARVEY, B., COETZEE, J.F. and WHITLOCK, B.K., 2018. Factors associated with seroprevalence of bovine Anaplasmosis in Texas. Veterinary Parasitology. Regional Studies and Reports, vol. 14, pp. 32-40. http://dx.doi.org/10.1016/j.vprsr.2018.08.004 PMid:31014734.
  » http://dx.doi.org/10.1016/j.vprsr.2018.08.004
• OLIVEIRA-SEQUEIRA, T.C.G., OLIVEIRA, M.C.S., ARAUJO JUNIOR, J.P. and AMARANTE, A.F.T., 2005. PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle. International Journal for Parasitology, vol. 35, no. 1, pp. 105-111. http://dx.doi.org/10.1016/j.ijpara.2004.09.002 PMid:15619521.
  » http://dx.doi.org/10.1016/j.ijpara.2004.09.002
• OTGONSUREN, D., SIVAKUMAR, T., AMGALANBAATAR, T., ENKHTAIVAN, B., NARANTSATSRAL, S., TUVSHINTULGA, B., ZOLJARGAL, M., MUNKHGEREL, D., DAVKHARBAYAR, B., BAATARJARGAL, P., DAVAASUREN, B., MYAGMARSUREN, P., BATTSETSEG, B., BATTUR, B. and YOKOYAMA, N., 2020. Molecular epidemiological survey of Babesia bovis, Babesia bigemina, and Babesia sp. Mymensingh infections in Mongolian cattle. Parasitology International, vol. 77, pp. 102107. http://dx.doi.org/10.1016/j.parint.2020.102107 PMid:32205192.
  » http://dx.doi.org/10.1016/j.parint.2020.102107
• PARVEEN, A., ASHRAF, S., AKTAS, M., OZUBEK, S. and IQBAL, F., 2021. Molecular epidemiology of Theileria annulata infection of cattle in Layyah District, Pakistan. Experimental & Applied Acarology, vol. 83, no. 3, pp. 461-473. http://dx.doi.org/10.1007/s10493-021-00595-6 PMid:33599889.
  » http://dx.doi.org/10.1007/s10493-021-00595-6
• RASHID, M., AKBAR, H., RASHID, I., SAEED, K., AHMAD, L., AHMAD, A.S., SHEHZAD, W., ISLAM, S. and FAROOQI, S., 2018. Economic significance of tropical theileriosis on a Holstein Friesian dairy farm in Pakistan. The Journal of Parasitology, vol. 104, no. 3, pp. 310-312. http://dx.doi.org/10.1645/16-179 PMid:29485311.
  » http://dx.doi.org/10.1645/16-179
• RASUL, G. and AKHTAR, A.S., 1975. Survey of hard ticks of livestock in Pakistan. Pak J Anim Sci, vol. 1, no. 4, pp. 7-11.
• REHMAN, A., NIJHOF, A.M., SAUTER-LOUIS, C., SCHAUER, B., STAUBACH, C. and CONRATHS, F.J., 2017. Distribution of ticks infesting ruminants and risk factors associated with high tick prevalence in livestock farms in the semi-arid and arid agro-ecological zones of Pakistan. Parasites & Vectors, vol. 10, no. 1, pp. 190. http://dx.doi.org/10.1186/s13071-017-2138-0 PMid:28420420.
  » http://dx.doi.org/10.1186/s13071-017-2138-0
• RISTIC, M. 1981. Babesiosis. In: M. Ristic and I. McIntyre, eds. Diseases of cattle in the tropics: economic and zoonotic relevance. Dordrecht: Springer Netherlands, pp. 443-468.
• RJEIBI, M.R., AYADI, O., REKIK, M. and GHARBI, M., 2018. Molecular survey and genetic characterization of Anaplasma centrale, A. marginale and A. bovis in cattle from Algeria. Transboundary and Emerging Diseases, vol. 65, no. 2, pp. 456-464. http://dx.doi.org/10.1111/tbed.12725 PMid:29034616.
  » http://dx.doi.org/10.1111/tbed.12725
• SAITOU, N. and NEI, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, vol. 4, no. 4, pp. 406-425. PMid:3447015.
• SAYIN, F., KARAER, Z., DINCER, S., CAKMAK, A., INCI, A., YUKARI, B.A., EREN, H., VATANSEVER, Z., NALBANTOGLU, S. and MELROSE, T.R., 2003. A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata. Veterinary Parasitology, vol. 113, no. 2, pp. 115-121. http://dx.doi.org/10.1016/S0304-4017(03)00045-1 PMid:12695036.
  » http://dx.doi.org/10.1016/S0304-4017(03)00045-1
• SELIM, A., MANAA, E., ABDELHADY, A., BEN SAID, M. and SAZMAND, A., 2021. Serological and molecular surveys of Anaplasma spp. in Egyptian cattle reveal high A. marginale infection prevalence. Iranian Journal of Veterinary Research, vol. 22, no. 4, pp. 288-297. PMid:35126536.
• SIDDIQUE, R.M., SAJID, M.S., IQBAL, Z. and SAQIB, M., 2020. Association of different risk factors with prevalence of Babesiosis in cattle and buffalos. Pakistan Journal of Agricultural Sciences, vol. 57, no. 2, pp. 517-524.
• SINGH, H., JYOTI, M., HAQUE, M., SINGH, N.K. and RATH, S.S., 2012. Molecular detection of Anaplasma marginale infection in carrier cattle. Ticks and Tick-Borne Diseases, vol. 3, no. 1, pp. 55-58. http://dx.doi.org/10.1016/j.ttbdis.2011.10.002 PMid:22309860.
  » http://dx.doi.org/10.1016/j.ttbdis.2011.10.002
• SINGH, N.K. and RATH, S.S., 2013. Epidemiology of ixodid ticks in cattle population of various agro-climatic zones of Punjab, India. Asian Pacific Journal of Tropical Medicine, vol. 6, no. 12, pp. 947-951. http://dx.doi.org/10.1016/S1995-7645(13)60169-8 PMid:24144025.
  » http://dx.doi.org/10.1016/S1995-7645(13)60169-8
• SOULSBY, E.J.L., 1982. Helminths, arthropods and protozoa of domesticated animals. 7th ed. London: Baillier Tindall and Cassel.
• SPARAGANO, O., LORIA, G.R., GUBBELS, M.-J., DE VOS, A.P., CARACAPPA, S. and JONGEJAN, F., 2000. Integrated molecular diagnosis of Theileria and Babesia species of cattle in Italy. Annals of the New York Academy of Sciences, vol. 916, no. 1, pp. 533-539. http://dx.doi.org/10.1111/j.1749-6632.2000.tb05332.x PMid:11193668.
  » http://dx.doi.org/10.1111/j.1749-6632.2000.tb05332.x
• TABOR, A.E., ALI, A., REHMAN, G., ROCHA GARCIA, G., ZANGIROLAMO, A.F., MALARDO, T. and JONSSON, N.N., 2017. Cattle tick Rhipicephalus microplus-host interface: a review of resistant and susceptible host responses. Frontiers in Cellular and Infection Microbiology, vol. 7, pp. 506. http://dx.doi.org/10.3389/fcimb.2017.00506 PMid:29322033.
  » http://dx.doi.org/10.3389/fcimb.2017.00506
• TAMURA, K., 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+ C-content biases. Molecular Biology and Evolution, vol. 9, no. 4, pp. 678-687. PMid:1630306.
• TURI, A., RAHMAN, A., ALI, I., RAFIULLAH, S.A., KHAN, K., SHAH, I., GONDAL, M., RASHID, A., AHMED, S. and WAZIR, I., 2018. Comparative analysis of indirect ELISA and real time PCR for the detection of Anaplasma marginale in buffalo, cattle and sheep in district Peshawar and Lakki Marwat, Pakistan. Asian J. Life Sci, vol. 6, no. 1, pp. 1-6. http://dx.doi.org/10.17582/journal.sajls/2018/6.1.1.6
  » http://dx.doi.org/10.17582/journal.sajls/2018/6.1.1.6
• ULLAH, R., SHAMS, S., KHAN, M.A., AYAZ, S., AKBAR, N.U., DIN, Q.U., KHAN, A., LEON, R. and ZEB, J., 2021. Epidemiology and molecular characterization of Theileria annulata in cattle from central Khyber Pakhtunkhwa, Pakistan. PLoS One, vol. 16, no. 9, pp. e0249417. http://dx.doi.org/10.1371/journal.pone.0249417 PMid:34529664.
  » http://dx.doi.org/10.1371/journal.pone.0249417
• UMBER, R., SHAFQAT, S., MATIULLAH, K., IMRAN, R., HAROON, A. and ANEELA, D., 2020. Identification of 23 kD immunogen from native antigens of Babesia bigemina in splenectomized calf. International Journal of Agriculture and Biology, vol. 24, pp. 1788-1794.
• VELUSAMY, R., RANI, N., PONNUDURAI, G., HARIKRISHNAN, T., ANNA, T., ARUNACHALAM, K., SENTHILVEL, K. and ANBARASI, P., 2014. Influence of season, age and breed on prevalence of haemoprotozoan diseases in cattle of Tamil Nadu, India. Veterinary World, vol. 7, no. 8, pp. 574-578. http://dx.doi.org/10.14202/vetworld.2014.574-578
  » http://dx.doi.org/10.14202/vetworld.2014.574-578
• YAMCHI, J.A. and TAVASSOLI, M., 2016. Survey on infection rate, vectors and molecular identification of Theileria annulata in cattle from North West, Iran. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, vol. 40, no. 3, pp. 1071-1076. http://dx.doi.org/10.1007/s12639-014-0636-y PMid:27605839.
  » http://dx.doi.org/10.1007/s12639-014-0636-y
• YAN, Y., JIANG, Y., TAO, D., ZHAO, A., QI, M. and NING, C., 2020. Molecular detection of Anaplasma spp. in dairy cattle in southern Xinjiang, China. Veterinary Parasitology. Regional Studies and Reports, vol. 20, pp. 100406. http://dx.doi.org/10.1016/j.vprsr.2020.100406 PMid:32448523.
  » http://dx.doi.org/10.1016/j.vprsr.2020.100406
• YOUNG, A.S., GROOCOCK, C.M. and KARIUKI, D.P., 1988. Integrated control of ticks and tick-borne diseases of cattle in Africa. Parasitology, vol. 96, no. 2, pp. 403-432. http://dx.doi.org/10.1017/S0031182000058388 PMid:3287285.
  » http://dx.doi.org/10.1017/S0031182000058388
• ZAFAR, S.N., KHAN, A., NIAZ, S., AKTAS, M., OZUBEK, S., FAROOQ, M., ADIL, M.M., ZAJAC, Z., IQBAL, F., ALHIMAIDI, A.R. and SWELUM, A.A., 2022. Prevalence of Anaplasma marginale in cattle blood samples collected from two important livestock regions in Punjab (Pakistan) with a note on epidemiology and phylogeny of parasite. Saudi Journal of Biological Sciences, vol. 29, no. 3, pp. 1515-1520. http://dx.doi.org/10.1016/j.sjbs.2021.11.020 PMid:35280590.
  » http://dx.doi.org/10.1016/j.sjbs.2021.11.020
• ZEB, J., SHAMS, S., DIN, I.U., AYAZ, S., KHAN, A., NASREEN, N., KHAN, H., KHAN, M.A. and SENBILL, H., 2020. Molecular epidemiology and associated risk factors of Anaplasma marginale and Theileria annulata in cattle from North-western Pakistan. Veterinary Parasitology, vol. 279, pp. 109044. http://dx.doi.org/10.1016/j.vetpar.2020.109044 PMid:32032840.
  » http://dx.doi.org/10.1016/j.vetpar.2020.109044
• ZHOU, M., CAO, S., SEVINC, F., SEVINC, M., CEYLAN, O., MOUMOUNI, P.F.A., JIRAPATTHARASATE, C., LIU, M., WANG, G., IGUCHI, A., VUDRIKO, P., SUZUKI, H. and XUAN, X., 2016. Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey. Ticks and Tick-Borne Diseases, vol. 7, no. 1, pp. 126-134. http://dx.doi.org/10.1016/j.ttbdis.2015.09.008 PMid:26492823.
  » http://dx.doi.org/10.1016/j.ttbdis.2015.09.008
• ZHYLDYZ, A., SIVAKUMAR, T., IGARASHI, I., GUNASEKARA, E., KOTHALAWALA, H., SILVA, S.S.P. and YOKOYAMA, N., 2019. Epidemiological survey of Anaplasma marginale in cattle and buffalo in Sri Lanka. The Journal of Veterinary Medical Science, vol. 81, no. 11, pp. 1601-1605. http://dx.doi.org/10.1292/jvms.19-0242 PMid:31548475.
  » http://dx.doi.org/10.1292/jvms.19-0242

Publication Dates

• Publication in this collection
  09 Dec 2022
• Date of issue
  2022

History

• Received
  08 Aug 2022
• Accepted
  23 Oct 2022