Introduction

Bovine leukemia virus (BLV), a virus of the Retroviridae family, Deltaretrovirus genus, is closely related to human T-lymphocyte leukemia virus 1 (HTLV-1) (Schwartz et al. 1994; Aida et al. 2013). BLV has a widespread prevalence and severe economic losses in the cattle industry worldwide (Bartlett et al. 2020). It is the etiological agent of Enzootic Bovine Leucosis (EBL), a disease that causes neoplastic lesions in cattle (Gillet et al. 2007; Aida et al. 2013). EBL has been classified by the World Organization for Animal Health (WOAH) as one of the diseases that could significantly affect global trade (Polat et al. 2017). As soon as BLV infects a host, it integrates a DNA intermediate as a provirus, both randomly and permanently into the host cell’s genome (Schwartz et al. 1994; Saito et al. 2022). BLV infection is characterized by a long incubation period and a variety of clinical outcomes. The majority of BLV-infected cattle are subclinical in the aleukemic stage, although, digestive disturbance, weight loss, weakness, reduced meat, and milk production, shortened longevity, and immune suppression may occur (Aida et al. 2013; Bartlett et al. 2013; Frie and Coussens 2015; Norby et al. 2016; Polat et al. 2017). About one-third of infected cattle can induce the persistent lymphocytosis stage, which is characterized by non-malignant polyclonal expansion of B-lymphocytes. However, only 1–5% develop B-cell lymphoma within different organs such as the spleen, liver, heart, abomasum, uterus, lymph nodes, and spinal cord resulting in wide varieties of clinical signs after a long latency (Burny et al. 1988; Gillet et al. 2007; Aida et al. 2013). Carcass condemnation at slaughter commonly occurs in cattle with lymphosarcoma in some countries such as Japan as it is judged unfit for human consumption (White and Moore 2009; Kobayashi et al. 2014).

BLV proviral load (PVL) is represented by the number of integrated copies of the retroviral genome into the host genome (Kettmann et al. 1979; Tajima et al. 1998). The level of BLV PVL is strongly correlated with BLV infection capacity (Jimba et al. 2010; Sato et al. 2018) and EBL progression (Somura et al. 2014; Ohno et al. 2015; Lo et al. 2020). BLV-infected cattle with high PVL (HPVL) are suggested to increase the risk of BLV transmission via direct contact with BLV-free animals. However, cows with low PVL (LPVL) are at low risk of BLV infection for BLV-free animals (Juliarena et al. 2016). There is evidence for the presence of BLV provirus in the milk, nasal mucus, and saliva of dairy cows with PVL > 10,000, 14,000, and 18,000 copies/105 blood cells, respectively (Yuan et al. 2015; Watanuki et al. 2019). These cattle could spread BLV through milk, nasal discharge, and saliva (Hopkins and DiGiacomo 1997; Yuan et al. 2015; Watanuki et al. 2019; Barzegar et al. 2021; Nakatsuchi et al. 2022). BLV transmission varies greatly by geography and agricultural system (Bartlett et al. 2020). It can be transmitted by blood-sucking insects in addition to the other iatrogenic procedures involving the transfer of infected blood between animals (i.e., dehorning, ear tattooing, rectal palpation, and needle reuse), and is responsible for disease propagation in a herd (Hopkins and DiGiacomo 1997). Transmission may occur transplacentally from an infected dam to the fetus, intrapartum by contact with infected blood, or postpartum from the dam to the calf through ingestion of infected milk (Hopkins and DiGiacomo 1997).

BLV genome consists of four structural and enzymatic genes namely; gag, pro, pol, and env, regulatory genes tax and rex, and accessory genes R3 and G4, and is flanked by two identical long terminal repeats (LTRs). BLV env gene is transcribed as an envelope glycoprotein (Env) complex that comprises, gp30 transmembrane (TM) protein and gp51 surface glycoprotein (SU), which is essential in the virus entry into the host cell (Gillet et al. 2007; Matsuura et al. 2019). Up to date, 12 BLV genotypes have been identified globally based on the phylogenetic analysis of env-gp51 sequences after a recent detection of BLV genotype-12 in Kazakhstan (Sultanov et al. 2022). BLV has invaded all continents and is currently distributed everywhere (Polat et al. 2017). Recent investigations in most nations outside of Europe have reported continuous increases in BLV prevalence in both beef and dairy cattle (Bartlett et al. 2020), although, about 20 European countries have successfully eradicated the BLV and achieved the BLV-free status (More et al. 2017).

In Egypt, few studies identifying BLV provirus in cattle were conducted. Our group has characterized the BLV genotypes 1 and 4 circulating in Egyptian dairy (Hamada et al. 2020) and beef cattle (Metwally et al. 2020) for the first time. Another study detected BLV genotype-1 in one sample from cattle blood (Selim et al. 2021). Furthermore, two serological surveys of anti-BLV antibodies were performed in cattle samples from a limited geographical region of Egypt (Zaher and Ahmed 2014; Selim et al. 2021). BLV national control strategies have been neglected in Egypt because the disease is present in an asymptomatic form in most animals (Selim et al. 2021). The BLV distribution throughout a wide area of Egypt is poorly investigated and the level of BLV PVL as a potential risk factor among Egyptian cattle has not been previously analyzed. Therefore, this study aimed to detect the BLV PVL among dairy and beef cattle from Southern, Middle, and Northern areas in Egypt using the BLV-CoCoMo-quantitative polymerase chain reaction (qPCR)-2 method. We also aimed to analyze the influence of some risk factors on BLV prevalence among Egyptian cattle. Moreover, the correlations between the BLV PVL levels in the infected animal’s blood and the investigated risk factors were targeted.

Materials and methods

Ethical approval

All animals were handled by the regulation of the Animal Ethics Committee at the Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt, and by the regulation of RIKEN, Japan in strict accordance with good animal practice following the guidelines of RIKEN. Permission of the farm owners was obtained for sample collection. The study was reviewed and approved by the Research Code of Ethics (RCOE-DMU) at the Damanhour University (DMU/VetINF-2022-/0151), and by the RIKEN Animal Experiments Committee (approval number H29-2-104).

Cattle population

A total number of 433 (266 dairies and 167 beef) apparent healthy cattle from 24 cattle farms and four abattoirs located in six Egyptian provinces, distributed in Southern (Qena and Luxor), Middle (Cairo and Fayoum), and Northern (Beheira, and Damietta) areas of Egypt were randomly selected (Fig. 1). The sample size from each farm and/or abattoir was detected by Thrusfield’s formula (Thrusfield 2018) considering the expected BLV prevalence of 15.8% as reported by Zaher and Ahmed (Zaher and Ahmed. 2014). The blood samples from cattle in each farm and/or abattoir (about 10%) were randomly collected for investigation, while the total samples’ number from each province (Table 1) was based on the owner’s permission. These animals included various cattle breeds such as the Egyptian Native breed (n = 28), Holstein breed (n = 125), and the Mixed breed (n = 280), the crossbreed between foreign breeds and Native cattle, and included cattle of both sexes (188 males and 245 females) of different ages, < 2 years (n = 48), 2–5 years (n = 210), and > 5 years (n = 175). Regarding farm size, a number of 141 blood samples were collected from cattle in 21 small-size farms (< 200 heads per farm), and 125 blood samples were collected from cattle in three large-size farms (> 200 heads per farm), while 167 bulls were samples from abattoirs of unknown farm size. The investigated cattle appeared in good health, and none of them had any obvious signs of lymphadenitis or tumors as examined by the veterinarians in different farms and abattoirs.

Fig. 1
figure 1

The geographical map of Egypt shows the location of the six provinces, where the samples were collected in this study. In the North, Damietta is indicated by purple color and Beheira by blue. In the Middle, Cairo is indicated by green color and Fayom by brown. In the South, Qena is indicated by yellow color and Luxor by red. Numbers on the left side indicate the climatic conditions [Average annual temperature °C (T) and humidity % (H)] in the North (Blue), Middle (Yellow), and South (Red), which obtained from https://jssa.journals.ekb.eg/ (Accessed, 6 June 2023)

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Table 1 Characteristics of the tested cattle and the prevalence of bovine leukemia virus in cattle from six different provinces in Egypt
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Blood sampling and DNA extraction

Blood samples were collected into a glass tube containing K2 EDTA anticoagulant using the coccygeal vein puncture procedure and then transported immediately into the laboratory. Genomic DNA was extracted from 300 µl whole blood using the Wizard Genomic DNA Purification Kit (Promega; Madison, WI, USA), according to the manufacturer’s instructions. The concentration, quality (A260/280), and purity (A260/230) of extracted DNA samples were measured using NanoDrop One Spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA). DNA samples of concentration > 200 ng/µl, quality > 1.8, and purity > 2 were subjected to investigation, while samples of low quality were re-extracted. DNA samples were diluted in nuclease-free water to a final concentration of 30 ng/µl for PCR experiments.

Evaluation of BLV PVL using CoCoMo-qPCR-2

BLV PVL was estimated using the CoCoMo-qPCR-2 method (RIKEN Genesis; Kanagawa, Japan), as described previously (Jimba et al. 2010; Takeshima et al. 2015). This study used a commercial CoCoMo-qPCR-2 kit (RIKEN Genesis; Kanagawa, Japan) to detect the PVL according to the manufacturer’s instructions. Briefly, a 183-bp sequence of the BLV LTR regions was amplified in a reaction mixture containing THUNDERBIRD Probe qPCR Mix (Toyobo; Tokyo, Japan), using the degenerate primer pairs, CoCoMo FRW and CoCoMo REV, and a 15 bp 6-carboxyfluorescein (FAM)-labeled LTR probe. To normalize the viral genomic DNA level within the host cellular genome, a 151-bp sequence of BoLA-DRA was amplified using the primer pairs, DRA-FW and DRA-RW, and a FAM-labeled DRA probe, as previously described (Takeshima et al. 2015). The reaction was carried out in 96-well plates according to the manufacturer’s guidelines on a LightCycler® 480 Instrument II (Roche Diagnostics; Basel, Switzerland). The PVL was calculated using the equation (number of BLV-LTR copies/number of BoLA-DRA copies) × 105 cells.

Statistical analysis

BLV prevalence among the tested cattle was determined by counting. The statistical analysis was performed using SPSS version 25 (IBM, New York, USA). The significanceof the investigated risk factors (age, sex, breed, production type, farm size, and location) and the differences in BLV PVL between the cattle groups were analyzed, and the Odds ratios (ORs) and 95% confidence intervals (CI) were calculated. BLV PVL was analyzed by the Kruskal-Wallis test for age, breed, farm size, and location, while the Mann-Whitney U test was used for sex and production type. Additionally, a logistic regression (LR) model analysis was performed to identify the most important risk factor (s) related to BLV infection. The investigated risk factor’s P-values for entry into or removal from the LR model were < 0.20. The LR model fitted with BLV infection as the outcome variable (negative: 0, positive: 1), and included effects of four variables; breed (3 categories: Native, Mixed, and Holstein), production type (2 categories: beef and dairy), farm size (3 categories: small, large, and abattoir), and location (6 categories: Qena, Damietta, Beheira, Fayoum, Luxor, and Cairo). The fit of the LR model was estimated using the Hosmer–Lemeshow goodness-of-fit test. Moreover, the risk influence of age (3 categories: > 2, 2–5, and < 5 years) on the occurrence of HPVL (2 categories: LPVL < 1000: 0, and HPVL ≥ 1000: 1) was evaluated using the LR model in BLV-positive cases which were dichotomized at a level of 1000 copies/105 cells (Farias et al. 2017). A p-value of < 0.05 was considered statistically significant. An OR greater than 1 indicated an increased risk of outcomes (BLV infection or occurrence of HPVL), and an OR less than 1 indicated a decreased risk of outcomes (BLV infection or occurrence of HPVL). The figures were constructed in a Graph Pad Prism version 7 (GraphPad Software, San Diego, California USA).

Results

Regarding the prevalence of BLV, samples were collected from different provinces representing the various Egyptian geographic and climatic regions. Cattle samples were collected either from animal farms or abattoirs, seeking more inclusive results and interpretation. The total BLV prevalence in cattle from the six investigated Egyptian provinces was 24.2% (105/433) (Table 1). In the case of the southern region, positive samples were recorded only in cattle from Qena province (12.9%; 23/178), while no positive samples have been detected from Luxor (0/16) (Table 1). Samples from Fayoum and Cairo provinces representing the middle region demonstrated a prevalence of 7.7% (1/13) and 77.4% (41/53), respectively (Table 1). Herein, the highest prevalence was detected in Damietta (100%; 40/40) with no positive cases in Beheira province (0/133), both provinces are located in northern Egypt (Table 1). These results demonstrated the distribution of BLV among cattle from various geographical areas in Egypt.

Risk factors analysis was conducted to assess the influence of age, sex, breed, production type, farm size, and location on the prevalence of BLV in cattle in Egypt. The findings indicated that the tested variables were not identified as risk factors except for the breed, farm size, and location factors (Table 2). The prevalence of BLV in cattle < 2 years old (18.7%), set as a reference group was lower than that in cattle 2–5 years old (23.3%, OR = 1.3, 95% CI: 0.6–2.9, p = 0.492), and also than the prevalence in older cattle > 5 years old (26.9%, OR = 1.6, 95% CI: 0.7–3.5, p = 0.251) but not significant in comparison. Moreover, the positivity of BLV was higher in bulls (26.1%) than that in cows (22.9%, OR = 0.8, 95% CI: 0.5–1.3, p = 0.440). While in the case of breed-wise effect, the Mixed cattle breed, set as reference the prevalence was significantly lower against the Holstein breed (36.0%, OR = 2.4, 95% CI: 1.5–3.8, p = 0.0003), but not against the Native breed of cattle tested (21.4%, OR = 1.1, 95% CI: 0.4-3, p = 0.785). Also, the type of production was investigated without recording statistically significant differences among the dairy cows (21.8%) and beef cattle (28.1%, OR = 1.4, 95% CI: 0.9–2.2, p = 0.134). On the contrary, the size of the farm was recorded as a potential risk factor as the highest significant prevalence was found in large-size farms (> 200 heads per farm) (36.0%, OR = 5.3, 95% CI: 2.7–10.4, p < 0.0001), followed by cases from abattoirs (28.1%, OR = 3.9, 95% CI: 1.9–7.5, p < 0.0001) compared to the reference small-size farms (< 200 heads per farm) (9.2%). Regarding the location of the cattle tested, Qena province, where the highest number of samples showed a prevalence of 12.9%, was taken as a reference. A significantly higher prevalence was detected in cattle from Cairo (77.4%, OR = 23, 95% CI: 10.6–50, p < 0.0001), and Damietta (100%, OR = Infinity, p < 0.0001), while a non-significant decrease in prevalence was reported in cattle from Fayoum (7.7%, OR = 0.5, 95% CI: 0.07–4.5, p = 0.7).

Table 2 Results of the univariable analysis of the risk factors associated with the prevalence of bovine leukemia virus among cattle in Egypt
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The results of the LR model showed the risk effect of four significant variables (breed, type of production, farm size, and location) on the BLV infection (Table 3). Considering the OR, an OR equal to 1 shows no effect; an OR greater than 1 shows the variable in question increases the odds of the outcome (BLV infection); and an OR less than 1 indicates the variable in question decreases the odds of the outcome (BLV infection). Based on the OR evaluation, the Holstein cattle are predicted to be infected with BLV 1.582 (95% CI:1.041–3.04, p = 0.007) times more likely compared to the Native cattle, although, the Mixed breed was not significant (95% CI: 0.106–2.206, p = 0.40). However, beef cattle are predicted to be infected with BLV 1.088 (95% CI: 1.035–3.327, p = 0.0001) times more likely compared to dairy cattle. Additionally, the probability of BLV infection in cattle kept in large farms is 1.26 (95% CI: 1.134–1.503, p = 0.0001) times more likely compared to cattle kept in small farms, while the cattle samples from abattoirs were of non-significant values (OR = 1.44, 95% CI: 0.174–2.361, p = 0.154) when compared to cattle from small farms. Regarding location, cattle from Damietta and Cairo are predicted to be infected with BLV 1.43 (95% CI: 1.005–2.043, p = 0.0001) and 1.16 (95% CI: 1.01–1.41, p = 0.0001) more likely compared to cattle from Qena, respectively. However, cattle from Beheira, Fayoum, and Luxor were of non-significant values (p = 0.994, 0.998, and 0.998 respectively) when compared to cattle from Qena.

Table 3 Detailed estimates from the logistic regression (LR) model of risk factors associated with the prevalence of bovine leukemia virus among cattle in Egypt
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The frequency of cattle carrying LPVL or HPVL levels which were classified at a level of 1000 copies per 105 white blood cells was estimated for each category of the risk variables (age, sex, breed, type of production, farm size, and location) (Table 4). In total, the 105 positive cattle samples, in which even one copy is considered positive during interpretation were included. The data indicated HPVL in 21% (22/105), while 79% (84/105) of the tested samples showed LPVL. The mean PVL for all positive cattle was 8651.6 copies / 105 cells. However, in this study, the correlation of PVL with the clinical picture could not be established. Cattle aged < 2 years showed a percentage of 11.1% were carrying HPVL (mean PVL = 138.2), and 2.1% of cattle aged 2–5 years showed HPVL (mean PVL = 140.2), while 42.6% of cattle > 5 years were carrying HPVL (mean PVL = 19,177). Females showed a higher frequency of HPVL (35.7%; mean PVL = 16,099) than males (4.1%; mean PVL = 160.1). The Native cattle showed 16.7% with HPVL (mean PVL = 195.5), and only 1.9% of Mixed cattle showed HPVL (mean PVL = 130.9), while 44.4% of Holstein cattle were carrying HPVL (mean PVL = 20,027). Dairy cows showed a higher frequency of HPVL (34.5%; mean PVL = 15,545) than beef cattle (4.3%; mean PVL = 166.1). Regarding farm size, none of the animals from small farms showed HPVL (0%; mean PVL = 31.08), while cattle from large farms showed the highest frequency of HPVL (44.4%; mean PVL = 20,027), and samples from abattoirs showed HPVL in 4.3% (mean PVL = 166.1). Additionally, cattle from Damietta showed the highest frequency of HPVL (50%; mean PVL = 22,522) compared to cattle from Cairo showed HPVL (4.9%, mean PVL = 173.5), while none of the cattle from Qena showed HPVL (0%, mean PVL = 43.8). Moreover, only one positive sample from Fayoum was detected and carried LPVL (433 copies/105 cells).

Table 4 Descriptive statistics for infected cattle by BLV PVL levels for each categorical variable
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In addition, the above-mentioned factors (age, sex, breed, type of production, farm size, and location) have been analyzed as risk factors considering the PVL instead of the BLV prevalence (Fig. 2). A significantly higher PVL was recorded in older cattle > 5 years old than those of 2–5 years old (p < 0.0001) and cattle < 2 years old (p < 0.0001). Moreover, the lower PVL in cattle < 2 years old compared to 2–5 years old cattle was statistically significant (p = 0.04) (Fig. 2A). Regarding sex, cows were more vulnerable to BLV than bulls as indicated by statistically higher PVL in female cattle than males (p = 0.0008) (Fig. 2B). Similarly, to those obtained using prevalence, a breed-wise effect significantly affected the PVL in the current study. The Holstein breed demonstrated a higher PVL than those in the Mixed cattle breed (p < 0.0001) and Native cattle (p < 0.0001), while no significant difference has been reported between the Native and Mixed breeds (p = 0.13) (Fig. 2C). Consistently to those recorded to sex-wise effect, a highly significant difference was obtained among dairy (cows) and beef cattle (bull) as dairy cattle had a higher PVL (p = 0.0053) (Fig. 2D). Also, cattle housed in large-size farms showed significantly higher PVL than those bred in small-size farms (p < 0.0001), and sporadic cases from abattoirs (p < 0.0001), and also significantly lower in small-size farms compared to cases from abattoirs (p = 0.02) (Fig. 2E).

Fig. 2
figure 2

The influence of the risk factors, (A) age, (B) sex, (C) breed, (D) production type, (E) farm size and (F) location on the BLV PVL (log10 copies / 105 cells) among the infected Egyptian cattle. Black bars show the median of log10PVL and 95% confidence interval values. Significant differences within the different risk factors were calculated using SPSS version 25, then the figure was constructed using Graph Pad Prism version 7. A P < 0.05 were considered statistically significant. The comparison of p-values of BLV PVL between the groups was estimated by Kruskal-Wallis with Dunn’s multiple comparisons test (age, breed, farm size, and location) and Mann-Whitney U Test (sex and production type), and was indicated above the error bars

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Regarding PVL levels in the cattle tested from different locations (Fig. 2F), samples from Damietta showed the highest PVL, while a significant decrease in PVL in cattle from Cairo (p < 0.0001), and Qena (p < 0.0001) in comparison was recorded. Furthermore, a significant increase in PVL levels in cattle from Cairo compared to Qena (p = 0.018) was shown.

The estimates from the LR model which analyzed the risk of age on the occurrence of HPVL among the infected cattle were shown (Table 5). The data revealed that the probability of carrying HPVL in cattle > 5 years is 1.27 (95% CI: 1.03–2.09, p < 0.001) times more likely compared to cattle < 2 years old. However, the probability of carrying HPVL in cattle 2–5 years compared to cattle < 2 years old was not significant (p = 0.999). Such results regarding PVL have made a valuable addition by correlating the BLV prevalence with PVL as an indicator of the severity of the infection.

Table 5 Estimates from the logistic regression (LR) model of the age associated with BLV PVL in the infected cattle
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Discussion

The geography of Egypt relates to two continents, North Africa and Southwest Asia. It has an area of 1,002,450 km2 which is divided into 28 provinces. Approximately, 5% of the total area is cultivable regions, which support more than 100 million humans and 18 million animals, of them about 5 million cattle heads (FAO stat. 2018). The majority of Egyptian cattle are reared for dairy purposes however male calves and infertile females are reared for beef production (Abdi et al. 2020). The updated statistical reports recorded a gradual decrease in the number of cattle in Egypt due to outbreaks of infectious diseases (FAO stat, 2020). Little is known about the impact of BLV infection in both the dairy and beef cattle industries due to the lack of BLV screening on a large scale and its asymptomatic form in most animals (Suarez Archilla et al. 2022). In this study, firstly, we performed a random screening of BLV among dairy and beef animals from six Egyptian provinces distributed in different climatic and geographical regions. Secondly, the risk factors influencing BLV infection were analyzed. Finally, the correlations between the BLV PVL levels in the infected animal’s blood and the analytic risk factors were estimated.

This study reported a 24.2% total BLV prevalence among the cattle tested by using the BLV-CoCoMo- qPCR-2 assay. Noticeably, this BLV molecular prevalence was relatively high compared to the previous serological evidences that reported a 15.8% infection rate in cattle from Alexandria, Kafr-Elsheikh, and Menofia (Zaher and Ahmed. 2014), and 20.8% in Menofia and Qalyubia (Selim et al. 2019). This data is in accordance with the theory that BLV prevalence is increasing in many countries all over the world (Bartlett et al. 2020). Another reason for the higher prevalence in this study could be the method of BLV diagnosis as we used the BLV-CoCoMo- qPCR-2 method which is able to detect even one copy of the integrated BLV provirus in the host genome (Takeshima et al. 2015). This method was highly effective in the detection of BLV in cattle from different countries (Polat et al. 2016; Moe et al. 2020; Lo et al. 2020), and it can detect new infections, before the development of antibodies to BLV (Takeshima et al. 2015). It was proven to be an effective method for monitoring the progression of BLV-induced disease and allowed us to evaluate the BLV infection from a different viewpoint compared with classical serological methods (Jimba et al. 2012). Additionally, the study area and sample population of cattle investigated in this study were different from the previous studies, which included animals only from the north of Egypt. Our study was more comprehensive and represented more different geographical and climatic localities of Egypt in the Southern, Middle, and Northern. The geography might be also a reason for a highly variable BLV prevalence between the examined provinces. The climatic changes could play an important role in the BLV distribution everywhere as the blood-sucking insects are important route of BLV transmission in some parts of the world in addition to iatrogenic routes in other regions (Bartlett et al. 2020). In this context, the climate of Egypt is very confusing as in the southern regions, the climate is very dry, hot, and rarely rains, however, humid in the middle, and cold and rainy in the northern regions. This could be a reason for the high prevalence of BLV in Damietta which is located on the branch of the River Nile and includes many ponds, canals, and rice fields where the flies prefer to inhabit (Selim et al. 2020b). Furthermore, Damietta is a coastal province, where an important seaport for the entry of imported animals from worldwide to Egypt is present. However, this study did not investigate whether the entry of cattle could be a risk factor for BLV infection in this region because in this province we investigated only a single large dairy farm. Therefore, we think about a future thorough screening of BLV in such an important area. In contrast, Selim and colleagues did not find significant associations between the BLV prevalence and the studied geographic regions in the Nile Delta of Egypt because of the similar geographic nature (Selim et al. 2020a, 2021).

Regarding risk factors analysis in this study, the BLV prevalence was greatly influenced by the cattle breeds and the size of comprising farm. Interestingly, higher prevalence was reported among the imported Holstein cattle which reared in large-size farms than Mixed and Native cattle which reared in small farms. Because of the low productivity of milk in the Egyptian Native and Mixed cattle, Egypt imports Holstein cattle from Germany, the Netherlands, and the United States (US) to produce higher quantities of milk for consumers and to improve the genetic characteristics of the Egyptian Native and Mixed cattle via breeding (Abdi et al. 2020). The imported cattle are usually kept in large-size farms comprising high numbers of animals. In this study, Holstein cows tested were imported as pregnant heifers from the US. To our knowledge, the veterinarians in the Egyptian quarantines randomly investigate some imported cattle for the presence of antibodies to BLV using serological methods. However, it might be a new infection before the appearance of antibodies, since the BLV prevalence in cattle in the US is approximately 46.5% as reported in a wide national survey (LaDronka et al. 2018). These findings were supported by Zaghawa and colleagues in 2002, who reported a 50.3% of BLV infection rate after the appearance of BLV symptoms in a dairy herd of Holstein Friesian cattle imported from the US (Zaghawa et al. 2002). Therefore, we believe that a comprehensive study to detect BLV among imported cattle in quarantines based on various techniques could shed more light on the risk of imported cattle from everywhere to spread BLV infection in Egypt. Selim and colleagues indicated that the herd size and housing were potential risk factors for BLV prevalence after a serological screening of cattle from the Nile Delta in Egypt (Selim et al. 2020a, 2021). Additionally, our data agree with the studies that found increased within-herd prevalence of BLV in larger herds compared to small herds (Baumgartener et al. 1975; Haghparast et al. 2012). However, conversely to our data, some studies reported age as another risk factor influencing BLV infection (Nagy et al. 2007; Erskine et al. 2012; Selim et al. 2020a). We think that the influence of age on the BLV infection in this study might be affected by the testing of a low number of samples from cattle < 2 years (n = 48) compared to the previous studies (Nagy et al. 2007; Erskine et al. 2012; Selim et al. 2020a). With regards to beef cattle showed a higher BLV prevalence than dairy cattle in this study, it might be one of the reasons that the beef animals were young ages and originally isolated from dairy mothers to be kept in overcrowded feedlots in Egypt. Furthermore, calves probably get the infection from the milk of infected mothers with high PVL and/or by other horizontal routes of transmission (Yuan et al. 2015; Watanuki et al. 2019). This result supports the reports of the global increase of BLV prevalence everywhere (Bartlett et al. 2020) and predicts a future increase in BLV infection in Egyptian cattle.

BLV-positive samples were dichotomized at a level of 1000 copies/105 cells into LPVL and HPVL as previously described (Farias et al. 2017). In total, our results reported a high BLV PVL in the blood of the BLV-positive cattle (mean = 8651.6 copies/105 cells). However, we did not notice any clinical symptoms of BLV such as lymphadenitis and tumors among the examined animals. This in agreement with the theory proved that the majority of infected animals are asymptomatic carriers (Aida et al. 2013; Polat et al. 2017). Our findings were supported by the results of Lo and colleagues who reported a strong association between the BLV PVL and occurrence of lymphoma in cattle (Lo et al. 2020). They found that asymptomatic cows carried a mean PVL of 9,401 copies/105 cells, while cows induced-lymphoma carried a mean PVL of 99,522 copies/105 cells. Similarly, Kobayashi and colleagues reported a higher PVL in EBL cattle compared to asymptomatic cattle (Kobayashi et al. 2020). In this context, the HPVL reported in Egyptian cattle investigated in this study could be a potential risk factor for disease transmission within the infected farms via multiple routes of BLV transmission (Juliarena et al. 2016). Additionally, previous studies detected the BLV provirus in milk, saliva, and nasal discharge increasing the possibility of BLV transmission horizontally by licking, sneezing, rubbing the nose, or milking (Yuan et al. 2015; Watanuki et al. 2019). Therefore, we think that HPVL reported in cattle from the Damietta dairy farm (mean PVL = 22,522) could be a reason for 100% BLV prevalence in the samples tested.

The association between the risk factors (age, sex, breed, production type, farm size and location) and the BLV PVL levels revealed significant differences within all categories of the infected cattle. Moreover, the effect of age on the occurrence of HPVL was analyzed in detail by the LR model. The older cattle group > 5 years old showed a higher BLV PVL, and they are predicted to carry HPVL 1.27 times more likely than the youngest group. However, no significant difference was ultimately proven in BLV PVL levels between the younger groups of infected cattle. This data is consistent with studies linked the PVL to the longevity of the infected animals as old cows are more likely to develop the malignant form of B-cell lymphoma after a latency (Zaghawa et al. 2002; Gillet et al. 2007; Radostits et al. 2007; Aida et al. 2013; Gutiérrez et al. 2014; Lo et al. 2020). Furthermore, our findings agree with Lo and colleagues who indicated that age was significantly associated with BLV susceptibility and PVL levels (Lo et al. 2020). A combination of sex, breed, production type, and the farm size risk factors effects on the BLV PVL revealed a significant increase in PVL in infected female Holstein dairy cows that were kept in large-size farms than PVL in infected males, Native and Mixed beef cattle that kept in small farms and sporadic cases from abattoirs. However, no significant difference was observed in BLV PVL levels in infected Native and Mixed cattle breeds. As previously reported BLV is likely to spread more in large-sized than small-sized herds (Kobayashi et al. 2010; Haghparast et al. 2012; Hamada et al. 2020; Selim et al. 2020a). Therefore, the importation of cows with high BLV PVL from BLV-endemic countries is considered a potential risk for BLV as it could transmit the infection to BLV-free cows. However, the Native and Mixed cattle that carry LPVL are possibly at fewer risks of BLV transmission (Juliarena et al. 2016). Additionally, other genetic factors in the Native and Mixed cattle could be associated with BLV susceptibility and PVL levels. In this context, some studies referred the BLV susceptibility and the PVL levels to the polymorphism in the bovine leukocyte antigen (BoLA)-DRB3 in the Holstein and Japanese black cattle (Juliarena et al. 2016; Lo et al. 2020). However, it is poorly investigated in the Egyptian breeds.

Despite this study being the first to evaluate the BLV PVL levels in Egyptian cattle, it had two limitations that should be mentioned. First, it was carried out on a few samples from some provinces because of difficulties with the owner’s permission for sampling. Second, some risk factors related to cattle management and BLV transmission were not included because of inadequate information in the farms’ records. Therefore, we believe that a comprehensive nationwide study of BLV infection and PVL across various Egyptian provinces and an analysis of more risk factors could shed more light on the disease’s epidemiology.

Conclusion

In conclusion, this study gives insight into the effect of the risk factors, age, sex, breed, production type, farm size and location of the cattle tested on the prevalence of BLV among Egyptian dairy and beef cattle from different geographical and climatic regions. Holstein breed, beef cattle, large-size farms and cattle from Damietta and Cairo were ultimately proven as the most important risk for BLV infection in Egyptian cattle. Additionally, the BLV PVL levels were absolutely high and greatly variable within the different categories of tested cattle. Females, Holstein, dairy cows, older than 5 years, that were kept in large farms, and cattle from Damietta have maintained the highest BLV PVL levels and could act as a source of BLV transmission to BLV-free animals. Ultimately, older cattle are predicted to carry HPVL 1.27 times more likely compared to younger age. However, the association between the BLV prevalence as well as the BLV PVL and the clinical picture of EBL in Egyptian cattle is needed.