Introduction
Goat farming is an activity with a significant socioeconomic impact on families and offers livelihoods, sources of employment, and economic income1,2. Mexico has an approximate population of 8’840,467 goats, and Sinaloa accounts for approximately 2.05 % of this (192,833)3. The importance of goats is more evident in arid, semi-arid, and desert areas due to their ability to take advantage of the vegetation characteristic of these regions, which makes them the main species for the production of meat, milk, and cheese in the aforementioned areas, which in turns makes them an important source of food and income for marginal producers4; likewise, they are livestock primarily managed by women and children5.
Goats are hosts to various parasites, such as lice. These ectoparasites belong to the class Insecta and order Phthiraptera. They are divided into four suborders: Anoplura (suckers), Rhynchophthirina, Amblycera, and Ischnocera (chewers)6,7. Lice are one of the least-studied ectoparasites compared to others, such as ticks or mosquitoes; most studies focus on highlighting the dermatological problems caused by their bites, evident in production animals, where these parasites are usually perceived more as a nuisance than as a significant threat8. Lice are obligate and permanent ectoparasites of birds and mammals and depend on their hosts for survival9. These parasites negatively affect animals, causing discomfort such as pruritus, skin lesions10,11, anemia, allergic reactions12,13, weight loss, nutritional deficiencies, reduced vigor14,15, growth retardation14, and secondary bacterial infections; they are potential vectors of infectious agents, such as Anaplasma spp and Bartonella spp, and induce a state of immunosuppression, which promotes the appearance of diseases8.
Morphological studies of lice in goats were carried out in Ethiopia16, India17,18, Pakistan19, and Mexico8,20-22, identifying the presence of Bovicola caprae (Ischnocera, Trichodectidae) and Linognathus africanus (Anoplura, Linognathidae). In addition, several studies used cox1 gene sequencing to molecularly characterize lice populations in different animal species23,24. The prevalence and diversity of lice infestation in goats are influenced by multiple risk factors, such as management systems, animal density, hygiene in production units, and the physiological state of the animals, among others19,25,26. In the state of Sinaloa, there is no information on ectoparasites and associated risk factors that increase their presence in goats. This study aimed to identify the species of lice present in goats and to analyze risk factors associated with their infestation in various municipalities of the state of Sinaloa, Mexico.
Material and methods
Study area
The study was conducted in the municipalities of Mocorito, Culiacán, Navolato, and Elota, located in the state of Sinaloa, which occupies an area of 57,365.4 km2, representing 2.9 % of Mexico’s territory. The average annual temperature is 25.9 °C, with a maximum temperature of 30.4 °C in June and July, and a minimum temperature of 20.6 °C in January; the average relative humidity is 68 %, with a maximum of 81 % in September and a minimum of 51 % in April; the average annual rainfall is 658.66 mm. The state’s climate is characterized as semi-dry, very warm, with rainfall in summer, according to the Köppen climate classification modified by García27.
Study type and sample size
The study was cross-sectional, descriptive, and observational. The sampling unit was the livestock production unit (LPU). A total of 62 goats were sampled, which were distributed across 7 LPUs: 5 extensive (71.43 %) and 2 intensive (28.57 %). The LPUs lacked a schedule for deworming against endo- and ectoparasites. In a personal communication with the owners, the last deworming was performed a year ago, and no clinical signs attributable to the presence of lice or other parasites were observed. The samplings were carried out as follows: two LPUs in the municipality of Culiacán (10 and 16 goats, respectively), two in Navolato (5 and 9 goats), two in Mocorito (10 and 6 goats), and one in Elota (6 goats).
Sample collection and lice identification
The animals were examined using the fur-separation method described by Lewis28. The lice collected were deposited in plastic jars containing 70 % ethyl alcohol; one container was used for each positive animal, and each was labeled with the corresponding information (identification, sex, and age). The identification of the specimens was conducted in the Parasitology Laboratory of the Faculty of Veterinary Medicine and Zootechnics (FMVZ, for its acronym in Spanish) of the Autonomous University of Sinaloa (UAS, for its acronym in Spanish). The collection of data on the LPU (types of facilities, animal density, production system, and floor type).
For morphology identification, some specimens were clarified with 20 % KOH for 24 h. Subsequently, they were washed in distilled water for 24 h and dehydrated using ascending alcoholic solutions (40 %, 70 %, and 96 %) for 10 min at each concentration; the lice were then placed in clove oil for 24 h. Finally, they were mounted on a slide with Xylol29,30, labeled, and observed under an optical microscope for identification using dichotomous keys31.
DNA extraction and PCR-based molecular detection of lice
Mixtures of 10 lice of the same species were prepared to extract DNA using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations. The DNA samples were stored at -80 °C until use. The quality and estimated concentration of the DNA samples were checked with the NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The PCR mixture was prepared at a final reaction volume of 25 μL with 12.5 μL of GoTaq® Master Mix (Madison, WI, USA), 1 μL of each oligonucleotide (10 nM), 7 μL of DNA, and 3.5 μL of nuclease-free water. The oligonucleotides used for PCR were the following: F: 5’-ATGGAGTTGTCTGAAGCGGG-3’ and R: 5’-AAGCCCAAATCCGGGAAGAA-3’, which amplify a 645 bp product, for B. caprae; and F: 5’-CCCGGATTTGGGGTTATTTCC-3’ and R: 5’-AAGGTGGCCAGTCAACTGAA-3’ for L. africanus, with a 236 bp amplicon product. The PCR conditions were as follows: an initial denaturation at 95 °C for 5 min, followed by 35 denaturation cycles at 95 °C for 45 sec, annealing at 55-56 °C (B. caprae and L. africanus, respectively) for 45 sec, and extension at 72 °C for 45 sec. A final extension step was performed at 72 °C for 10 min. The amplification products were subjected to electrophoresis with 2 % agarose gel stained with GelRed Nucleic Acid Gel Stain, 10,000X (Biotium, Fremont, San Francisco, USA). Amplification-positive products were sequenced using the Sanger method at Macrogen Inc., Republic of Korea.
Phylogenetic analysis
Consensus sequences of the mitochondrial cox1 gene of B. caprae and L. africanus were generated in the UGENE v.47.0 software, which enabled to assemble and edit the sequences. To confirm the molecular identification of the lice, the consensus sequences were compared with those available in the NCBI database using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Sequences of the mitochondrial cox1 gene from B. caprae and L. africanus stored in GenBank (Table 1) were collected and aligned with MAFFT (https://mafft.cbrc.jp/alignment/server/). The nucleotide substitution model was selected using the JModelTest v.2.1.10 software. Phylogenetic reconstruction was performed using the maximum likelihood inference method with IQ-TREE in Galaxy, and branch support was evaluated using 1,000 Bootstrap replicates (https://usegalaxy.org).
| Geographical origin | Species identification | GenBank access number |
|---|---|---|
| Bovicola spp | ||
| Iran | B. caprae | MZ817000 |
| China | B. caprae | MF927687 |
| India | B. caprae | PQ602608 |
| Iran | B. caprae | OK135724 |
| Iraq | B. caprae | PV155639 |
| China | B. caprae | MH001178 |
| Australia | Damalinia meyeri | JN122004 |
| Linognathus spp | ||
| Mexico | L. africanus | OQ927402 |
| Iran | L. stenopsis | ON455010 |
| India | L. africanus | PQ602609 |
| Norway | Linognathus spp | OP686438 |
| Norway | Linognathus spp | OP686439 |
| Canada | Linognathus spp | OP686441 |
| Canada | Linognathus spp | OP686445 |
| Canada | Linognathus spp | OP686442 |
| Norway | Linognathus spp | OP686440 |
| Canada | Linognathus spp | OP686444 |
| Norway | L. setosus | OP686449 |
| Canada | L. setosus | OP686450 |
| Iraq | L. africanus | PV162853 |
| Mexico | L. africanus | OQ927403 |
| Iraq | L. africanus | PP598894 |
| Pakistan | L. africanus | OP948899 |
| EU | L. spicatus | HM171436 |
| United Kingdom | L. ovillus | EU375761 |
| Japan | Canestrinia spp | LC576438 |
Statistical analysis
A positive case was defined as a host in which at least one louse specimen was found. The frequency of infestation was estimated as the ratio of positive animals to the total number of animals sampled32. Information on positivity was used for the analysis of risk factors, regardless of sex, which was carried out in two stages: 1) The data were dichotomized, summarized in contingency tables (2 x 2), and analyzed in Chi-square tests for each of the risk factors. 2) Positive risk factors (P<0.05) were included in a multivariate logistic regression analysis33. The general model was as follows:
Where: values of xi = (x1,.....,x
p
) predictor variables p; exp is the base of natural logarithms 2.71828;
In the marginal multivariate logistic regression model, only variables with significant results (P<0.05) were included. Statistical analyses were performed in R version 4.4.034, and the OddsPlotty35 package was used to estimate the degrees of association [odds ratio (OR)] and 95 % confidence intervals, as well as the estimators of the regression model parameters.
Results
Of the seven LPUs sampled, 85.71 % (6/7) were positive for the presence of the ectoparasite. Of a total of 62 goats sampled, 65 % (40/62) tested positive for at least one species of louse. In total, 547 specimens of lice belonging to two species were collected: Bovicola caprae, of the suborder Ischnocera (chewers), family Bovicoliidae (Figure 1), and Linognathus africanus, of the suborder Anoplura (suckers), family Linognathidae (Figure 2). These species had relative frequencies of 93.60 % (512/547) and 6.40 % (35/547), respectively. The types of infestations observed by LPU were the following: single (B. caprae), in one LPU in Culiacán (81 lice), one in Mocorito (3), and two in Navolato (53, 0); and mixed (B. caprae and L. africanus), in one LPU in Culiacán (129, 1), one in Elota (153, 1), and one in Mocorito (93, 33). Regarding the sex of the specimens, B. caprae showed a predominance of females (82.61 %, 423/512) over males (17.38 %, 89/512). In L. africanus, females accounted for 77.14 % (27/35) and males for 22.85 % (8/35).
The identification of the lice species was also confirmed by PCR, obtaining amplifications of 540-560 bp for B. caprae (four sequences) and 230-250 bp for L. africanus (eight sequences). The consensus sequence of the mitochondrial cox1 gene of B. caprae (PV761107) showed 99.50-99.80 % similarity by BLASTn analysis with sequences from China (MF927687), India (PQ602608, LC768866-LC768868), Iran (MZ817000, OK135716-OK135724), and Iraq (PV155639). On the other hand, the sequence of L. africanus (PV761108) showed 99-100 % similarity with sequences from Malta (OP658950), Mexico (OQ927402, OQ927403), Pakistan (OP948899), and Peru (EU375760).
The above results were corroborated by phylogenetic analysis. The B. caprae sequence was grouped in the described clade with B. caprae sequences from China (MF927687), India (PQ602608), and Iran (MZ817000) (Figure 3). In the case of L. africanus, it was grouped in the specific clade with sequences from Peru (EU375760), Mexico (OQ927402, OQ927403), and Iraq (PP598894) (Figure 4).
Regarding the evaluated factors, the Chi-square tests showed significant differences in five of the seven factors (Table 2): facilities, hygiene, density, production system, and floor type. However, in the marginal logistic regression model, only two of these factors remained significant (P<0.05): density and the extensive production system (Table 3).
| Risk factor | N | Positive samples | Frequency % (CI 95%) | OR (CI 95%) | P 1 |
|---|---|---|---|---|---|
| Facilities: | 0.004 | ||||
| Good-Fair | 62 | 20 | 50 (34.51-65.49) | 3.16(1.44-6.97) | |
| Poor | 20 | 90.9 (78.84-98.88) | |||
| Age: | 0.28 | ||||
| 1-2 | 62 | 20 | 55 (33.20-76.80) | 1.35(0.78-2.34) | |
| 3-5 | 42 | 69.05 (55.07-83.03) | |||
| Sex: | 0.55 | ||||
| Female | 62 | 5 | 55.56 (21.20-86.30) | 1.25(0.61-2.55) | |
| Male | 35 | 66.04 (53.29-78.79) | |||
| Hygiene: | 0.04 | ||||
| Good | 62 | 20 | 56.52 (42.20-70.85) | 2.32(1.05-5.14) | |
| Poor | 20 | 87.50 (61.65-98.45) | |||
| Density: | 0.004 | ||||
| Appropriate | 62 | 20 | 50 (34.51-65.49) | 3.16(1.44-6.97) | |
| Inappropriate | 20 | 90.91 (78.84-98.88) | |||
| Production system: | 0.006 | ||||
| Intensive | 62 | 19 | 90.48 (69.62-98.88) | 3.01(1.36-6.63) | |
| Extensive | 21 | 51.22 (35.91-66.52) | |||
| Floor: | 0.02 | ||||
| Concrete | 62 | 35 | 35.71 (10.61-60.81) | 2.20(1.17-4.14) | |
| Dirt | 5 | 72.92 (60.35-85.49) | |||
| Risk factor | β estimator | Standard error | OR (CI 95%) | P-value |
|---|---|---|---|---|
| Density: | 0.001 | |||
| Appropriate | 2.6 | 0.63 | 13.07 (3.77-45.30) | |
| Inappropriate | ||||
| Production system: | 0.001 | |||
| Extensive | 2.57 | 0.63 | 13.41 (3.88-46.63) | |
| Intensive | ||||
| Constant | 2.28 | 0.53 | 0.001 | |
Discussion
Lice are common ectoparasites of small ruminants; their diversity and distribution are poorly documented, creating a critical knowledge gap worldwide and in various regions of Mexico8. The results of this study show the presence of B. caprae and L. africanus lice in goats from different municipalities in Sinaloa, with frequencies of 93.60 % and 6.40 %, respectively. These results suggest an endemic lice infestation in goats in the region. Both species have already been identified morphologically in other regions of the world with different frequencies. For instance, in Hawassa, Ethiopia16, various ectoparasites were collected, of which 6.25 % (12/192) were goat lice; of these, 66.66 % (8/12) were identified as Bovicola spp, and 33.33 % (4/12) as Linognathus spp. In Pakistan19, 804 ectoparasites were collected in goats, of which 9.42 % (76/804) were lice, 34.21 % (26/76) corresponded to Bovicola spp, 28.94 % (22/76) to Linognathus spp, and 36.84 % (28/76) to Haematopinus spp. In India, two studies were conducted in order to identify ectoparasites present in goats, among which lice species were detected; the first was carried out by Ajith et al17, who sampled three different districts. In the districts of Dehradun and Tehri Garhwal, 75.00 % (156/208) of the ectoparasites collected were lice, of which 84.61 % corresponded to B. caprae and 15.38 % to L. africanus. In the district of Bareilly, Uttar Pradesh, 31.25 % (35/112) of ectoparasites were lice, with frequencies of 5.71 % (2/35) and 94.28 % (33/35), respectively. The second study was conducted in Rampur by Rashmi and Saxena18, who collected a total of 3,123 goat lice, of which 59.78 % (1,867/3,123) were B. caprae and 40.21 % (1,256/3,123) were L. africanus.
In Mexico, three studies have been carried out to identify lice in goats. The first was conducted by Ballados-Gonzalez et al8, who collected 563 lice from goats and sheep; of these, 12.07 % (68/563) came from goats, 32.35 % were identified as B. caprae, collected in Hidalgo, and 67.64 % were identified as L. africanus, obtained from two municipalities in the state of Veracruz: Tuxpan and Veracruz. The other two studies were carried out in Coahuila: González-Álvarez21 reported a frequency of 15.38 % (2/13) for B. caprae and 84.61 % (11/13) for L. africanus; and Lozoya-Saldana et al20 recorded a frequency of 47.51 % (67/141) of B. caprae and 43.26 % (61/141) of L. africanus. The difference in frequency between countries and regions can be influenced by various factors, such as age, sex, types of facilities, animal density, production system, and climate, among other factors8,17,36. B. caprae is a louse that feeds on skin and hair debris, without the need to pierce the host’s skin. In contrast, L. africanus feeds on blood, so it needs to stay in areas with thin skin rich in capillaries, which limits its mobility. These differences explain why B. caprae reaches higher abundance; it does not depend on specialized conditions or limited areas, favoring its proliferation in extensive systems where there is frequent contact between animals37,38.
The phylogenetic tree placed the sequence of the mitochondrial cox1 gene of B. caprae (PV761107) in the same clade and subgroup as MF927687 (China) and MZ817000 (Iran); the nodes showed a low support value (bootstrap = 40 % and 20 %, respectively), which indicates a close phylogenetic relationship. Nonetheless, the BLASTn analysis showed high similarities with MF927687 (99.77 %) and MZ817000 (99.53 %), suggesting a considerable genetic identity, although not sufficiently divergent to generate robust clades in the phylogenetic analysis.
As for the phylogenetic tree of the mitochondrial cox1 gene of L. africanus (PV761108), it was grouped in the same clade as sequences reported in Peru (EU375760), Mexico (OQ927402, OQ927403), and Iraq (PP598894) within a subclade with a bootstrap value of 97 %. This result is consistent with the BLASTn analysis, which showed similarities ranging from 99.58 % to 100 % with these sequences, reinforcing the molecular identification results and providing greater taxonomic precision for the lice present in goats.
One of the main factors identified was the high population density, where goats housed in these conditions (less than 1.5 m2 per adult goat) presented a 13.07 times higher risk of lice infection (P<0.01), showing consistency with their behavior, using direct contact as the main route of transmission17,19,25. The second factor with significant presence was the production system (P<0.001), in which goats under extensive production systems had a 13.41 times higher risk of infestation. This is similar to what was reported by Syamsul et al36 in Kelantan, Malaysia, where they mentioned that extensive production systems had a high prevalence due to a higher risk of exposure, which is due to the limited care of animals and the use of grazing, practices that are mainly used in extensive production systems. The factors of sex and age of the goats showed 34.19 % in females, 5.8 % in males, 12.9 % in individuals younger than 3 yr, 18.06 % in three-year-old individuals, and 9.03 % in individuals older than 4 yr; however, the presence of lice was not associated with the sex and age of the goats. This coincides with a study conducted by Ajith et al17, who found no significant association between the factors, as the LPUs participating in the study did not have pens separated by age or sex, and all the goats lived in the same space, which facilitated the spread of lice.
As for the majority presence of B. caprae compared to L. africanus, it may be due to its inability to generate irritative processes; these feed on body secretions and dead skin cells, which does not cause irritation, in contrast to L. africanus. In addition, B. caprae is a species of chewing louse most widely distributed in the Americas, with records in various parts of the United States, Cuba, Argentina, Brazil, Guyana, Colombia, and Costa Rica, as well as Mexico8.
Conclusions and implications
The lice species identified by morphological and molecular analysis were B. caprae and L. africanus; their presence confirms their endemicity in four municipalities of the state of Sinaloa. Likewise, a high herd density and an extensive production system are factors that predispose their presence in the goats of the LPUs. This study provides a basis for future research, among which the implication of diseases these ectoparasites could transmit to goats from Sinaloa stands out.